Multivalent vaccine compositions and uses thereof

Abstract

The present invention describes compositions and methods for inducing an immune response against parenteral pathogenic Escherichia coli (ExPEC), thereby providing immune protection from a disease associated with ExPEC. In particular, the present invention describes compositions comprising colibacillus polysaccharide antigens O1, O2, O4, O6, O8, O15, O16, O18, O25 and O75, and further comprising conjugates covalently bound to a carrier protein of O153 or O21 or both O153 and O21, for use in the prevention of invasive ExPEC disease.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and vaccines. In particular, the present invention relates to a composition comprising polysaccharides of Escherichia coli O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75 antigens and further comprising polysaccharides of O153 or O21, or both O153 and O21 antigens. The present invention further relates to the use of such a composition for inducing an immune response against Escherichia coli to prevent or treat Escherichia coli infection, in particular to prevent extraintestinal pathogenic Escherichia coli (ExPEC) disease.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 383,841, filed on November 15, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Reference to electronically submitted sequence listing

[0005] The contents of the electronic sequence listing (004852_206WO1.xml; size: 30,323 bytes; creation date: November 13, 2023) are incorporated herein by reference in their entirety. Background Art

[0006] Extraintestinal pathogenic Escherichia coli (ExPEC), along with commensal E. coli strains, are typically harmless inhabitants of the human intestine. However, ExPEC strains can possess virulence factors that allow them to colonize and infect sites outside the gastrointestinal tract, causing diverse and severe invasive diseases that result in significant annual morbidity, mortality, and costs. ExPEC strains are the most common cause of urinary tract infections (UTIs). They are also contributors to surgical site infections and neonatal meningitis, are associated with abdominal and pelvic infections, and are associated with hospital-acquired pneumonia. They are also occasionally implicated in other extraintestinal infections, such as osteomyelitis, cellulitis, and wound infections. All of these major infection sites can lead to ExPEC bacteremia (Russo et al., 2003). Neonates, the elderly, and immunocompromised patients are particularly susceptible to ExPEC infections, including invasive ExPEC disease (IED).

[0007] Bacterial resistance to antibiotics is a major concern in the fight against bacterial infections, and multidrug-resistant (MDR) Escherichia coli strains are becoming increasingly common.

[0008] O-antigen serotype is based on the chemical structure of O polysaccharide antigen, which is the outer membrane part of lipopolysaccharide (LPS) in Gram-negative bacteria. More than 180 serologically unique Escherichia coli O-antigens have been reported (Stenutze et al., FEMS Microbial Rev. 2006; 30: 382-403), although the vast majority of ExPEC isolates are classified in less than 20 O-antigen serotypes. Full-length Escherichia coli O-antigen is typically composed of approximately 10 to 25 repeating sugar units attached to a highly conserved LPS core structure, and each component is independently synthesized by enzymes encoded primarily in rfb and rfa gene clusters. After O-antigen polymerization, the O-antigen polysaccharide backbone can typically be modified by adding acetyl or glucose residues. These modifications effectively increase the diversity of serotypes by producing serotypes with a common polysaccharide backbone but different antigenicity of the side branches.

[0009] ExPEC infections can be caused by any serotype. Although certain serotypes are overrepresented in ExPEC infections, surface polysaccharides from ExPEC isolates still exhibit considerable antigenic diversity, making the development of surface polysaccharide-based ExPEC vaccines challenging (Russo et al., Vaccine. 2007; 25: 3859-3870). Moreover, some O-antigens may be poorly immunogenic. In addition, according to studies of pneumococcal conjugate vaccines, when multiple serotypes can cause disease, the vaccine composition (such as the selection of serotypes included in the vaccine and the dose level of the included serotypes) may be crucial, because the use of vaccines targeting certain serotypes may potentially increase the carriage of serotypes not included in the vaccine and the disease caused by the serotypes not included, or even increase the carriage of serotypes included in the vaccine but for which immunity is poor (Lipsitch, Emerging Infectious Diseases; 1999, 5: 336-345). Ideally, a vaccine should maximize its beneficial effect in preventing disease caused by the serotypes included in the vaccine, while minimizing the risk of new disease arising from increased carriage of non-vaccine serotypes.

[0010] Efforts to develop vaccines to prevent ExPEC infections have focused on O-antigen polysaccharide conjugates. A 12-valent O-antigen conjugate vaccine was synthesized by extracting and purifying the O-antigen polysaccharide and chemically conjugating it with detoxified Pseudomonas aeruginosa exotoxin A, and tested for safety and immunogenicity in a Phase I clinical study (Cross et al., J. Infect. Dis. (1994) v. 170, pp. 834-40). This candidate vaccine has never been approved for clinical use. Recently, a bioconjugation system in Escherichia coli has been developed, in which both the polysaccharide antigen and the carrier protein are synthesized in vivo and subsequently conjugated in vivo by the activity of the oligosaccharide transferase PglB (a Campylobacter jejuni enzyme) expressed in Escherichia coli (Wacker et al., Proc. Nat. Acad. Sci. (2006) v. 103, pp. 7088-93). This N-linked protein glycosylation system enables the transfer of diverse polysaccharides to carrier proteins within bacteria.

[0011] Bioconjugation has been successfully used to generate conjugate polysaccharides of tetravalent O-antigen vaccine candidates for E. coli (Poolman and Wacker, J. Infect. Dis. (2016) v. 213 (1), pp. 6-13). Decavalent and nine-valent vaccine compositions of bioconjugates of E. coli O-antigen polysaccharides have been previously described (e.g., WO 2020 / 191082, WO 2022 / 058945). The development of a successful ExPEC vaccine requires coverage of the major serotypes, and the presence of further O-antigen modifications in subsets of ExPEC isolates poses a further challenge to covering isolates that exhibit both unmodified and modified LPS. In addition, the immune response to a vaccine composition comprising O-antigens from multiple serotypes may vary depending on the serotype. Therefore, there is a continuing need in the art for vaccines against ExPEC. In particular, there is a need for surface polysaccharide-based ExPEC vaccines that can be safely and efficiently manufactured on an industrial scale and that can be used to provide an effective immune response and preferably provide protection against ExPEC serotypes O153 and / or O21, and preferably also against other serotypes prevalent among ExPEC disease isolates. Summary of the Invention

[0012] In a first aspect, the present invention relates to a composition comprising E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75 antigenic polysaccharides, and wherein the composition further comprises an O21 antigenic polysaccharide or an O153 antigenic polysaccharide or both O21 and O153 antigenic polysaccharides, wherein each of the antigenic polysaccharides is independently covalently linked to a carrier protein. In certain embodiments, the O1 antigen is O1A, the O4 antigen is glucosylated (O4A), the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B. In certain embodiments, the antigenic polysaccharides of the composition of the present invention comprise the structures described in Table 1 herein.

[0013] In certain embodiments, the compositions of the present invention further comprise at least one additional E. coli antigenic polysaccharide covalently linked to a carrier protein.

[0014] In certain embodiments, the E. coli O antigen polysaccharide present in the composition of the present invention consists of: (i) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O153, or (ii) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O21; or (iii) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O153 and O21.

[0015] In certain embodiments, the carrier protein is detoxified exotoxin A (EPA) or CRM of Pseudomonas aeruginosa. 197 Preferably, the carrier protein is EPA. In certain embodiments, the carrier protein comprises 1 to 20 glycosylation consensus sequences having the amino acid sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro, preferably the glycosylation consensus sequence has the amino acid sequence of SEQ ID NO: 1. In certain embodiments, each carrier protein comprises the amino acid sequence of SEQ ID NO: 2.

[0016] In certain embodiments, the E. coli antigenic polysaccharide of the composition is covalently linked to the carrier protein by bioconjugation or chemical conjugation. Preferably, the E. coli antigenic polysaccharide is covalently linked to the carrier protein by bioconjugation. In certain embodiments, the E. coli antigenic polysaccharide is covalently linked to an Asn residue in a glycosylation site in the carrier protein.

[0017] In a second aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a composition as described herein.

[0018] In a third aspect, provided herein is a method of inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject, comprising administering to the subject a composition as described herein or a pharmaceutical composition as described herein. In certain embodiments, the method of inducing an immune response against Escherichia coli limits the severity of invasive ExPEC disease or prevents invasive ExPEC disease in the subject, preferably, wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

[0019] In a fourth aspect, the present invention provides a recombinant prokaryotic host cell for preparing a bioconjugate of an Escherichia coli O153 antigen polysaccharide covalently linked to a carrier protein, the recombinant prokaryotic host cell comprising:

[0020] a. The nucleotide sequence of the rfb gene cluster of the O153 antigen polysaccharide;

[0021] b. a nucleotide sequence encoding the carrier protein, the carrier protein comprising at least one glycosylation site, the glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro, preferably having SEQ ID NO: 1; and

[0022] c. Nucleotide sequence encoding oligosaccharyltransferase PglB.

[0023] In certain embodiments, the E. coli O153 antigenic polysaccharide comprises a structure of formula (O153):

[0024] [→2)-β-D-Ribf-(1→4)-β-D-Galp-(1→4)-α-D-GlcpNAc-(1→4)-β-D-Galp-(1→3)-α-D-GlcpNAc-(1→] n

[0025] wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, preferably from 7 to 25.

[0026] In certain embodiments, the PglB in the prokaryotic host cell used to prepare the bioconjugate of E. coli O153 comprises amino acid mutations N311V, K482R, D483H, and A669V relative to wild-type PglB having the amino acid sequence of SEQ ID NO: 4. The present invention also includes a method for preparing a bioconjugate of an E. coli O153 antigenic polysaccharide covalently linked to a carrier protein, the method comprising culturing a recombinant prokaryotic host cell as described herein to produce the bioconjugate.

[0027] In a fifth aspect, the present invention provides a recombinant prokaryotic host cell for preparing a bioconjugate of an Escherichia coli O21 antigen polysaccharide covalently linked to a carrier protein, the recombinant prokaryotic host cell comprising:

[0028] a. The nucleotide sequence of the rfb gene cluster of the O21 antigen polysaccharide;

[0029] b. A nucleotide sequence encoding the carrier protein, the carrier protein comprising at least one glycosylation site, the glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro, preferably having SEQ ID NO: 1;

[0030] c. a nucleotide sequence encoding oligosaccharyltransferase PglB; and

[0031] d. Nucleotide sequence encoding UDP-glucose 4-epimerase.

[0032] In certain embodiments, the E. coli O21 antigenic polysaccharide comprises a structure of formula (O21):

[0033]

[0034] wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, preferably from 7 to 25.

[0035] In certain embodiments, the PglB in the prokaryotic host cell used to prepare the bioconjugate of E. coli O21 comprises the amino acid mutation N311V relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 4. The present invention also includes a method for preparing a bioconjugate of an E. coli O21 antigenic polysaccharide covalently linked to a carrier protein, the method comprising culturing a recombinant prokaryotic host cell as described herein to produce the bioconjugate. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : IgG response induced by O153-EPA bioconjugate. Sprague Dawley rats were immunized intramuscularly three times with formulation buffer or 3 different doses (0.04 μg, 0.40 μg or 4.00 μg O153-EPA) of O153-EPA conjugate. Serum Ab levels were measured by ELISA on days 0, 14 and 42 after immunization. Individual titers (log10 EC50 titers) and geometric mean titers (GMT) ± 95% CI are shown. The dotted line indicates the threshold above which the dilution curve of the sample has a 4PL fit.

[0037] Figure 2 : IgG response induced by O21-EPA bioconjugate. Sprague Dawley rats were immunized intramuscularly three times with formulation buffer or 3 different doses (0.04 μg, 0.40 μg or 4.00 μg O21-EPA) of O21-EPA bioconjugate. Serum Ab levels were measured by ELISA on days 0, 14 and 42 after immunization. Individual titers (log10 EC50 titers) and GMT ± 95% CI are shown. The dotted line indicates the threshold above which the dilution curve of the sample has a 4PL fit.

[0038] Figure 3 : Functions of antibodies induced by O153-EPA bioconjugate.

[0039] Sprague Dawley rats were immunized intramuscularly three times with formulation buffer or 4.00 μg / dose of the O153-EPA bioconjugate. Antibody-mediated killing of O153 bacteria was measured by OPKA on day 42 post-immunization. Individual opsonization index (OI) values and GMT ± 95% CI are shown. ***p < 0.001, Wilcoxon rank-sum test, with Bonferroni correction for multiple comparisons as required for analysis of ELISA results.

[0040] Figure 4 : Functions of antibodies induced by O21-EPA bioconjugate.

[0041] Sprague Dawley rats were immunized intramuscularly three times with formulation buffer or 4.00 μg / dose of the O21-EPA bioconjugate. Antibody-mediated killing of O21 bacteria was measured by OPKA on day 42 post-immunization. Individual opsonization index (OI) values and GMT ± 95% CI are shown. ***p < 0.001, Wilcoxon rank sum test, with Bonferroni correction for multiple comparisons as needed for analysis of ELISA results.

[0042] Figures 5A-5L Serum IgG responses induced by ExPEC12V vaccines. Sprague Dawley rats were immunized intramuscularly three times with 4 / 8 μg PS / dose of ExPEC12V (4 μg of all O-antigens except O25, which was 8 μg), 0.4 / 0.8 μg PS / dose of ExPEC12V (0.4 μg of all O-antigens except O25, which was 0.8 μg), or formulation buffer. O1A ( Figure 5A )、O2( Figure 5B )、O4( Figure 5C )、O6A( Figure 5D )、O8( Figure 5E )、O15( Figure 5F )、O16( Figure 5G )、O18( Figure 5H )、O21( Figure 5I )、O25B( Figure 5J )、O75( Figure 5K ) or O153( Figure 5L )Specific serum Ab levels.

[0043] Individual titers (EC50 titers) and GMT ± 95% CI are shown. DETAILED DESCRIPTION

[0044] definition

[0045] Various publications, articles, and patents are cited or described in the Background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like included in this specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0046] 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. Otherwise, certain terms cited herein have the meanings as set forth in the specification. It must be noted that, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural references unless the context clearly dictates otherwise.

[0047] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0048] When used in conjunction with a number, the term "about" refers to any number that is within ±10% (eg, ±5 or ±1%) of the cited number.

[0049] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0050] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "commprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprising" may be replaced by the terms "containing" or "including" or, at times, by the term "having" when used herein.

[0051] As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of aspects or embodiments of the present invention, any of the aforementioned terms "comprising," "containing," "including," and "having" may be replaced with the term "consisting of" or "consisting essentially of" to alter the scope of the present disclosure.

[0052] As used herein, the connection term "and / or" between multiple referenced elements should be understood to cover both individual and combined options. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any of these options should be understood to fall within this meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one of these options should also be understood to fall within this meaning and therefore meet the requirements of the term "and / or".

[0053] As used herein, the terms "O polysaccharide", "O-antigen", "O-antigen", "O-antigen polysaccharide", "O-polysaccharide antigen" and the abbreviation "OPS" all refer to the O-antigen of Gram-negative bacteria, which is a component of lipopolysaccharide (LPS) and is specific to each serotype or serotype (sub) of Gram-negative bacteria. O-antigens typically comprise repeating units (RU) of two to seven sugar residues. As used herein, RU is set to be equal to the biological repeat unit (BRU). BRU describes the RU of O-antigen as it is synthesized in vivo. Different serotypes of Escherichia coli express different O-antigens. In E. coli, the gene products involved in the biogenesis of O-antigens are encoded by the rfb gene cluster. Unless otherwise indicated, whenever an O-antigen polysaccharide is mentioned herein, it may refer to the O-antigen polysaccharide of each E. coli serotype and any existing serosubtype thereof, for example, when an O1 antigen polysaccharide is mentioned, it may be an O-antigen polysaccharide of an E. coli serosubtype O1A, O1A1, O1B or O1C, and an O25 antigen polysaccharide may refer to an E. coli O25A or O25B antigen polysaccharide, etc. The corresponding structures (moiety structures or O-units) of RUs of many E. coli serotypes and subserotypes and their O-antigen polysaccharides are provided in Table 1 of WO2020 / 039359, which is incorporated herein by reference.

[0054] As used herein, "rfb cluster" and "rfb gene cluster" refer to a gene cluster encoding the enzymatic machinery capable of synthesizing the O-antigen backbone structure. The term rfb cluster can be applied to any O-antigen biosynthetic cluster, and preferably refers to a gene cluster from Escherichia, particularly Escherichia coli.

[0055] As used herein, the term "O1A" refers to the O1A antigen of Escherichia coli (a serotype of Escherichia coli serotype O1). The term "O2" refers to the O2 antigen of Escherichia coli (a serotype of Escherichia coli serotype O2). The term "O4" refers to the O4 antigen of Escherichia coli (a serotype of Escherichia coli serotype O4). The term "O6A" refers to the O6A antigen of Escherichia coli (a serotype of Escherichia coli serotype O6). The term "O8" refers to the O8 antigen of Escherichia coli (a serotype of Escherichia coli serotype O8). The term "O15" refers to the O15 antigen of Escherichia coli (a serotype of Escherichia coli serotype O15). The term "O16" refers to the O16 antigen of Escherichia coli (a serotype of Escherichia coli serotype O16). The term "O18A" refers to the O18A antigen of Escherichia coli (a serotype of Escherichia coli serotype O18). The term "O25B" refers to the O25B antigen from Escherichia coli (a serosubtype of E. coli serotype O25). The term "O75" refers to the O75 antigen of E. coli (E. coli serotype O75). The term "O153" refers to the O153 antigen of E. coli (E. coli serotype O153). The term "O21" refers to the O21 antigen of E. coli (E. coli serotype O21). As used herein, the term "O4A" refers to the O4 O-antigen of E. coli with a glucose side branch (E. coli serotype O4) (this term was previously described as "O4 Glc+" in WO2020191082 and WO2022 / 058945).

[0056] The structures of several E. coli O-antigen polysaccharides mentioned in this application are shown in Table 1 below. A single repeating unit of each E. coli O-antigen polysaccharide is shown.

[0057] Table 1: Structure of Escherichia coli O-antigen polysaccharide

[0058]

[0059]

[0060] 1 Each n is independently an integer from 1 to 100, such as 1-50, 1-40, 1-30, 1-20 and 1-10, 3-50, 3-40, 5-30, for example at least 5, such as 5-40, for example 7-30, for example 7 to 25, for example 10 to 20, but in some cases can be 1-2.

[0061] All monosaccharides described herein have their common meanings as known in the art. Monosaccharides can have a D or L configuration. If D or L is not specified, the sugar is understood to have a D configuration. Monosaccharides are typically represented by abbreviations known and used in the art. For example, Glc refers to glucose; D-Glc refers to D-glucose; and L-Glc refers to L-glucose. Other common abbreviations for monosaccharides include: Rha, rhamnose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose; Man, mannose; Man3Me, 3-O-methyl-mannose; Gal, galactose; FucNAc, N-acetylfucosamine; and Rib, ribose. The suffix "f" refers to furanose, and the suffix "p" refers to pyranose.

[0062] The terms "RU," "repeat unit," and "repeating unit" used with respect to O-antigen refer to the biological repeating unit (BRU) of the O-antigen as it is synthesized in vivo by cellular machinery (e.g., glycosyltransferases). The number of RUs of the O-antigen may vary depending on the serotype, and in embodiments of the present invention typically varies from about 1-100 RUs, preferably about 1 to 50 RUs, such as 1-50 RUs, 1-40 RUs, 1-30 RUs, 1-20 RUs, and 1-10 RUs, and more preferably at least 3 RUs, at least 4 RUs, at least 5 RUs, such as 3-50 RUs, preferably 5-40 RUs, preferably 5-30 RUs, for example 7-25 RUs, for example 10-20 RUs. However, in some cases, the number of RUs of the O-antigen may be 1-2. The structure of each O-antigen specifically described herein is shown as containing one RU, with the variable "n" representing the number of RUs. In each O-antigen polysaccharide in the bioconjugate of the present invention, n is independently an integer from 1 to 100, such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, preferably at least 3, more preferably at least 5, such as 3 to 50, preferably 5 to 40 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40), more preferably 5 to 30, but in some cases can be 1 to 2. In some embodiments, n is independently an integer from about 7 to 25, for example, about 10 to 20. These values may vary between individual O-antigen polysaccharides in the composition and are provided herein as average values, i.e., if a bioconjugate is described herein as having n independently being an integer between 5 and 40, the composition contains a majority of O-antigen polysaccharides having 5 to 40 repeating units, but may also contain some O-antigen polysaccharides having fewer than 5 repeating units or more than 40 repeating units.

[0063] As used herein, the terms "conjugate" and "glycoconjugate" refer to a sugar or sugar antigen (e.g., oligosaccharides and polysaccharides)-protein conjugate that is linked to another chemical substance, including but not limited to proteins, peptides, lipids, etc. Glycoconjugates can be prepared chemically, for example, by chemical (synthetic) linkage of a protein and a sugar or sugar antigen. The term glycoconjugate also includes bioconjugates.

[0064] As used herein, in the context of administering an O-antigen to a subject in a method according to embodiments of the present invention, the term "effective amount" refers to an amount of an O-antigen sufficient to induce a desired immune effect or immune response in a subject. In certain embodiments, an "effective amount" refers to an amount of an O-antigen sufficient to produce immunity in a subject to achieve one or more of the following effects in the subject: (i) prevent the development or onset of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (ii) prevent the recurrence of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (iii) prevent, reduce, or ameliorate the severity of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (iv) reduce the duration of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (v) prevent ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (x) increase the survival of subjects with ExPEC infection, preferably invasive ExPEC disease; (xi) eliminate ExPEC infection, preferably invasive ExPEC disease; (xii) inhibit or reduce ExPEC replication; and / or (xii) enhance or improve one or more prophylactic or therapeutic effects of another therapy.

[0065] An "effective amount" may vary depending on a variety of factors, such as the subject's physical condition, age, weight, health status, etc.; the route of administration, such as oral or parenteral; the composition administered, such as the target O-antigen, other co-administered O-antigens, adjuvants, etc.; and the specific disease for which immunity is required. When the O-antigen is covalently bound to a protein carrier, the effective amount of the O-antigen is calculated based solely on the O-antigen polysaccharide portion of the conjugate. All concentrations, amounts, and ratios of the conjugates (including bioconjugates) described herein are also calculated solely based on the weight of the O-antigen polysaccharide portion of the conjugate, regardless of the concentration, amount, or ratio of any conjugated carrier protein, unless otherwise stated. For example, administering 16 μg of a particular bioconjugate means that the administered bioconjugate contains 16 μg of the particular O-antigen polysaccharide, and the amount of the conjugated carrier protein is not included in this number. As another example, if a composition is said to comprise a conjugate of O-antigen polysaccharides from serotypes A and B in a ratio of 2:1, this means that in the composition, based on the weight of the conjugated O-antigen polysaccharides, ignoring the weight of the conjugated carrier protein, the concentration or amount of conjugated O-antigen polysaccharide A is twice the concentration or amount of conjugated O-antigen polysaccharide B.

[0066] As used herein, the term "invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED)" is an acute illness concurrent with a systemic bacterial infection, which is microbiologically confirmed by the isolation and identification of E. coli from the blood or other normally sterile body sites, or from the urine of a patient who has signs and symptoms of an invasive disease (systemic inflammatory response syndrome (SIRS), sepsis, or septic shock) and has no other identifiable source of infection. In certain embodiments, IED is an acute illness concurrent with a systemic bacterial infection, which is microbiologically confirmed by (i) the isolation and identification of E. coli from the blood or other normally sterile body sites, or (ii) the isolation and identification of E. coli from the urine of a patient who has life-threatening organ dysfunction due to a dysregulated host response to infection originating from the urinary tract and / or male reproductive organs, and has no other identifiable source of infection.

[0067] IEDs may include, but are not necessarily limited to, urinary tract infection (UTI), surgical site infection, abdominal or pelvic infection, pneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, wound infection, pyelonephritis, prostate biopsy-associated infection (e.g., transrectal ultrasound-guided prostate needle biopsy [TRUS-PNB]-associated infection), urosepsis, meningitis, peritonitis, cholangitis, soft tissue infection, pyomyositis, septic arthritis, endophthalmitis, suppurative thyroiditis, sinusitis, endocarditis, neutropenic fever, and prostatitis (including but not limited to acute bacterial prostatitis).

[0068] In certain preferred embodiments, the IED includes sepsis. In certain preferred embodiments, the IED includes bacteremia. In certain embodiments, the present invention provides a composition according to the present invention for preventing sepsis caused by Escherichia coli. In certain embodiments, the present invention provides a composition according to the present invention for preventing bacteremia caused by Escherichia coli.

[0069] The term "IED event meeting sepsis criteria" refers to an IED case that includes evidence of life-threatening organ dysfunction due to a dysregulated host response to infection. If the Sequential Organ Failure Assessment (SOFA) total score shows an abrupt change of 2 points or more from baseline, the IED case meets sepsis criteria and is considered secondary to an IED. In certain embodiments, the present invention provides a composition according to the present invention for preventing IED meeting sepsis criteria. As used herein, the term "urosepsis" is sepsis caused by infection originating from the genitourinary tract and / or male reproductive organs.

[0070] The term "bacteremic IED" is a case of IED that involves the isolation and identification of E. coli from blood. In certain embodiments, the present invention provides a composition according to the present invention for use in preventing bacteremic IED.

[0071] 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 the antigen present in the composition.

[0072] As used herein, a "composition" comprising more than one E. coli antigenic polysaccharide can be a single pharmaceutical composition comprising more than one E. coli antigenic polysaccharide in the same pharmaceutical composition, or a combination of more than one pharmaceutical compositions comprising more than one E. coli antigenic polysaccharide in separate pharmaceutical compositions. In a preferred embodiment, the composition is a single pharmaceutical composition. In a method of inducing an immune response against E. coli, a "composition" comprising more than one E. coli antigenic polysaccharide can be administered to a subject in need thereof as a single pharmaceutical composition comprising more than one E. coli antigenic polysaccharide, or can be administered to a subject as a combination of separate pharmaceutical compositions. In a preferred embodiment, a single pharmaceutical composition is administered to a subject.

[0073] As used herein, in the context of administering two or more O-antigens or compositions to a subject, the term "combination" or "combination of..." does not limit the order in which the O-antigens or compositions are administered to the subject. For example, a first composition can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second composition to the subject. Preferably, the two or more O-antigens are administered to the subject substantially simultaneously, e.g., within five minutes of each other, and more preferably the two or more O-antigens are administered simultaneously by administering at least two compositions simultaneously, most preferably by administering a single composition comprising the two or more O-antigens.

[0074] As used herein, "subject" or "patient" means any animal, preferably a mammal, most preferably a human, that will be or has been vaccinated with 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, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., most preferably humans. The terms "subject" and "patient" are used interchangeably herein.

[0075] The term "percent (%) sequence identity" or "% identity" describes the number of matches ("hits") of identical amino acids between two or more aligned amino acid sequences compared to the number of amino acid residues that make up the total length of the amino acid sequences. In other words, using alignment, the percentage of identical amino acid residues (e.g., 90%, 95%, 97% or 98% identity) can be determined for two or more sequences when the sequences are compared and aligned for maximum correspondence (as measured using a sequence comparison algorithm known in the art), or when aligned manually and visually. The sequences compared for sequence identity can thus differ by one or more substitutions, one or more additions or one or more deletions of amino acids. Suitable programs for aligning protein sequences are known to those skilled in the art. The percent sequence identity of protein sequences can be determined, for example, using programs such as CLUSTALW, ClustalOmega, FASTA or BLAST, for example using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

[0076] For example, for amino acid sequences, sequence identity and / or similarity can be determined by using standard techniques known in the art, or by inspection, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.); Devereux et al, 1984, Nucl. Acids 14:116; Res. 12:387-395 described best fit sequence program (preferably using default settings). In certain embodiments, the percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and join penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0077] Another example of a useful algorithm is the BLAST algorithm, which is described in: Altschul et al, 1990, J. Mol. Biol. 215:403-410; Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, which is available from Altschul et al, 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values.

[0078] Another useful algorithm is Gapped BLAST reported by Altschul et al, 1993, Nucl. Acids Res. 25:3389-3402.

[0079] It is shown herein that vaccination or immunization with a composition comprising Escherichia coli 01, 02, 04, 06, 08, 015, 016, 018, 025 and 075 antigenic polysaccharides and further comprising 0153 or 021 or 0153 and 021 antigenic polysaccharides, each independently covalently linked to a carrier protein, induces a consistent and robust immune response against all of 01, 02, 04, 06, 08, 015, 016, 018, 025 and 075, as well as 0153 and / or 021, which was not predictable prior to the present invention.

[0080] Thus, in a first aspect, the present invention provides a composition comprising Escherichia coli 01, 02, 04, 06, 08, 015, 016, 018, 025, 075 and 0153 antigenic polysaccharides, wherein each of the antigenic polysaccharides is independently covalently linked to a carrier protein.

[0081] In certain embodiments, the concentration of the 025 and / or 075 antigenic polysaccharide is independently increased relative to the concentration of each of the 01 , 02, 04, 06, 08, 015, 016, 018, and 0153 antigenic polysaccharides.

[0082] In certain embodiments, the weight ratio of the concentration of the 025 antigen polysaccharide to each of the 01, 02, 04, 06, 08, 015, 016, 018, 075, and 0153 antigen polysaccharides is independently about 1.5:1 to about 2.5:1.

[0083] In certain embodiments, the weight ratio of the concentration of the 025 antigenic polysaccharide to each of the 01, 02, 04, 06, 08, 015, 016, 018, 075, and 0153 antigenic polysaccharides is independently about 2:1.

[0084] In certain embodiments, the weight ratio of the concentration of the 025 and 075 antigenic polysaccharides to each of the 01, 02, 04, 06, 08, 015, 016, 018, and 0153 antigenic polysaccharides is independently about 1.5:1 to about 2.5:1.

[0085] In certain embodiments, the weight ratio of the concentration of the 025 and 075 antigenic polysaccharides, independently to each of the 01 , 02, 04, 06, 08, 015, 016, 018, and 0153 antigenic polysaccharides, is about 2:1.

[0086] In certain embodiments, the concentration of E. coli antigen polysaccharide O1:O2:O4:O6:O8:O15:O16:O18:O153:O25:O75 is in a weight ratio of 1:1:1:1:1:1:1:1:1:1:2:2.

[0087] In another aspect, the present invention provides a composition comprising Escherichia coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O21 antigenic polysaccharides, wherein each of the antigenic polysaccharides is independently covalently linked to a carrier protein.

[0088] In certain embodiments, the concentration of the 025 and / or 075 antigenic polysaccharide is independently increased relative to the concentration of each of the 01 , 02, 04, 06, 08, 015, 016, 018, and 021 antigenic polysaccharides.

[0089] In certain embodiments, the weight ratio of the concentration of the 025 antigen polysaccharide to each of the 01, 02, 04, 06, 08, 015, 016, 018, 075, and 021 antigen polysaccharides is independently about 1.5:1 to about 2.5:1.

[0090] In certain embodiments, the weight ratio of the concentration of the 025 antigen polysaccharide to each of the 01 , 02, 04, 06, 08, 015, 016, 018, 075, and 021 antigen polysaccharides is independently about 2:1.

[0091] In certain embodiments, the weight ratio of the concentration of the 025 and 075 antigenic polysaccharides, independently to each of the 01, 02, 04, 06, 08, 015, 016, 018, and 021 antigenic polysaccharides, is about 1.5:1 to about 2.5:1.

[0092] In certain embodiments, the weight ratio of the concentration of the 025 and 075 antigenic polysaccharides, independently to each of the 01 , 02, 04, 06, 08, 015, 016, 018, and 021 antigenic polysaccharides, is about 2:1.

[0093] In certain embodiments, the concentration of E. coli antigen polysaccharide O1:O2:O4:O6:O8:O15:O16:O18:O21:O25:O75 is in a weight ratio of 1:1:1:1:1:1:1:1:1:1:2:2.

[0094] In another aspect, the present invention provides a composition comprising Escherichia coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O153 and O21 antigenic polysaccharides, wherein each of the antigenic polysaccharides is independently covalently linked to a carrier protein.

[0095] In certain embodiments, the concentration of the 025 and / or 075 antigenic polysaccharide is independently increased relative to the concentration of each of the 01 , 02, 04, 06, 08, 015, 016, 018, 0153, and 021 antigenic polysaccharides.

[0096] In certain embodiments, the weight ratio of the concentration of the 025 antigen polysaccharide to each of the 01, 02, 04, 06, 08, 015, 016, 018, 075, 0153, and 021 antigen polysaccharides is independently about 1.5:1 to about 2.5:1.

[0097] In certain embodiments, the weight ratio of the concentration of the 025 antigen polysaccharide to each of the 01, 02, 04, 06, 08, 015, 016, 018, 075, 0153, and 021 antigen polysaccharides is independently about 2:1.

[0098] In certain embodiments, the weight ratio of the concentration of the 025 and 075 antigenic polysaccharides, independently to each of the 01, 02, 04, 06, 08, 015, 016, 018, 0153, and 021 antigenic polysaccharides, is about 1.5:1 to about 2.5:1.

[0099] In certain embodiments, the weight ratio of the concentration of the 025 and 075 antigenic polysaccharides, independently to each of the 01, 02, 04, 06, 08, 015, 016, 018, 0153, and 021 antigenic polysaccharides, is about 2:1.

[0100] In certain embodiments, the concentration of E. coli antigen polysaccharide O1:O2:O4:O6:O8:O15:O16:O18:O21:O153:O25:O75 is in a weight ratio of 1:1:1:1:1:1:1:1:1:1:1:2:2.

[0101] In certain embodiments of the methods according to the present invention, the O1 antigen is O1A, the O4 is glucosylated (O4A), the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B. In certain embodiments, the O1A, O2, glycosylated O4, O6A, O15, O16, O18A, O25B, O75, O21, and O153 antigen polysaccharides comprise the structures of formula (O1A), (O2), (O4A), (O6A), (O15), (O16), (O18A), (O25B), (O75), (O21), and (O153), respectively, as shown in Table 1, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, more preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

[0102] In one embodiment, the O1 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O1 antigen polysaccharide comprises a structure of formula (O1A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O1 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O1 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0103] In one embodiment, the O2 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O2 antigen polysaccharide comprises a structure of formula (O2) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O2 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O2 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0104] In one embodiment, the O4 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O4 antigen polysaccharide is a glucosylated O4 antigen polysaccharide, and in a specific embodiment, it comprises the structure of formula (O4A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O4 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O4 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0105] In one embodiment, the O6 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O6 antigen polysaccharide comprises a structure of formula (O6A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O6 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O6 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0106] In one embodiment, the O8 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O8 antigen polysaccharide comprises a structure of formula (O8) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O8 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O8 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0107] In one embodiment, the O15 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O15 antigen polysaccharide comprises a structure of formula (O15) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O15 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O15 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0108] In one embodiment, the O16 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O16 antigen polysaccharide comprises a structure of formula (O16) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O16 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O16 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0109] In one embodiment, the O18 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O18 antigen polysaccharide comprises a structure of formula (O18A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O18 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O18 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0110] In one embodiment, the O25 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O25 antigen polysaccharide comprises an O25B antigen polysaccharide, and in a specific embodiment, it comprises the structure of formula (O25B) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O25 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O25 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0111] In one embodiment, the O75 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O75 antigen polysaccharide comprises an O75 antigen polysaccharide, and in a specific embodiment, it comprises the structure of formula (O75) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O75 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O75 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0112] In one embodiment, the O153 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O153 antigen polysaccharide comprises an O153 antigen polysaccharide, and in a specific embodiment, it comprises the structure of formula (O153) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O153 antigen polysaccharide is part of a chemical conjugate or a bioconjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O153 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0113] In one embodiment, the O21 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O21 antigen polysaccharide comprises an O21 antigen polysaccharide, and in a specific embodiment, it comprises the structure of formula (O21) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O21 antigen polysaccharide is part of a chemical conjugate or a biological conjugate and is covalently linked to a carrier protein, such as EPA or CRM. 197 In a preferred embodiment, the O21 antigenic polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably by bioconjugation.

[0114] In certain embodiments, the compositions described herein further comprise at least one additional E. coli O-antigen polysaccharide covalently linked to a carrier protein.

[0115] In certain embodiments, the compositions described herein consist essentially of E. coli O-antigen polysaccharides O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, and O153 covalently linked to a carrier protein. In such compositions, other components may optionally be present, but the composition does not contain additional E. coli O-antigen polysaccharides.

[0116] In certain embodiments, the compositions described herein consist essentially of E. coli O-antigen polysaccharides O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, and O21 covalently linked to a carrier protein. In such compositions, other components may optionally be present, but the composition does not contain additional E. coli O-antigen polysaccharides.

[0117] In certain embodiments, the compositions described herein consist essentially of E. coli O-antigen polysaccharides O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O153, and O21 covalently linked to a carrier protein. In such compositions, other components may optionally be present, but the composition does not contain additional E. coli O-antigen polysaccharides.

[0118] In certain embodiments, each carrier protein is independently selected from the group consisting of: detoxified exotoxin A of Pseudomonas aeruginosa (EPA), Escherichia coli flagellin (FliC), CRM 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clot factor A, clot factor B, Escherichia coli heat-labile enterotoxin, detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae hemolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from non-typeable Haemophilus influenzae.

[0119] In one embodiment, the carrier protein is detoxified exotoxin A (EPA) or CRM of Pseudomonas aeruginosa. 197 .CRM 197 The amino acid sequence of the protein is shown in SEQ ID NO:5.

[0120] For preparation of bioconjugates, the carrier protein is preferably encoded in the host cell, for example on a plasmid.

[0121] In a specific embodiment, the carrier protein is a detoxified exotoxin A of Pseudomonas aeruginosa. 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 comprises 1-20 glycosylation sites, preferably 1-10 glycosylation sites, preferably 2-4, and more preferably 4 glycosylation sites, each glycosylation site comprising a glycosylation consensus sequence having the amino acid sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro, and more preferably the amino acid sequence of SEQ ID NO: 1.

[0122] In some embodiments, the EPA carrier protein comprises four glycosylation sites, each of which comprises a glycosylation consensus sequence, such as a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1. As used herein, "EPA-4 carrier protein" and "EPA-4" refer to a detoxified exotoxin A carrier protein of Pseudomonas aeruginosa comprising four glycosylation sites, each of which comprises a glycosylation consensus sequence having SEQ ID NO: 1. An exemplary preferred example of an EPA-4 carrier protein is an EPA carrier protein comprising the amino acid sequence of SEQ ID NO: 2.

[0123] In certain embodiments, the E. coli antigen polysaccharide is covalently linked to a carrier protein by bioconjugation or by chemical conjugation. Chemical conjugation can, for example, include reductive amination chemistry (RAC), single-end conjugation, conjugation with (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, cyanide chemistry (CNBr, CDAP), thioether chemistry (based on maleimide / bromoacetyl linker) or EDC-N-hydroxysuccinimide zero linker chemistry with or without ADH spacer. Methods for preparing glycoconjugates of the E. coli O-antigens conjugated to a carrier protein using chemical conjugation with a carrier protein, and compositions comprising such glycoconjugates, are also described in, for example, WO2020 / 039359 and WO2022 / 058645, which are incorporated herein by reference.

[0124] Preferably, the term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an O-antigen, preferably an E. coli O-antigen (e.g., O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O21, O153, etc.), prepared in a host cell background, wherein the host cell machinery links the antigen to the protein (e.g., N-linked), preferably within the host cell endosome (see, e.g., the examples herein and, e.g., WO03 / 074687, WO2006 / 119987, WO2015 / 124769, WO2020 / 191082, WO2020 / 191088, WO2022 / 058945). Lysates of such cells, i.e., conjugates prepared in vitro but using the same glycosylation mechanism (see, e.g., WO2017 / 117539, US10,829,795, WO2020 / 146814) are also considered to be bioconjugates. Because bioconjugates are prepared in host cells or with host cells by host cell mechanisms, antigens and proteins are covalently linked via glycosidic linkages or bonds in bioconjugates. Bioconjugates can be prepared in recombinant host cells (or their lysates), which are engineered to express synthetic O-antigens and / or the cellular mechanisms required for connecting O-antigens to target proteins. Bioconjugates as described herein have advantageous properties over chemically prepared glycoconjugates (wherein polysaccharides are purified from bacterial cell walls and subsequently chemically coupled to carrier proteins), for example, bioconjugates require fewer chemicals in manufacturing and are more consistent in terms of the final product produced, and contain less or no free (i.e., not bound to carrier proteins) polysaccharides. Purification of lipid A-free O-antigens and subsequent chemical conjugation to carrier proteins is a lengthy and laborious process. Furthermore, the purification, lipid A detoxification, and chemical conjugation processes can result in epitope loss, antigenic heterogeneity, and reduced immunogenicity of the conjugated polysaccharide. Synthesizing glycoconjugates by bioconjugation can overcome these limitations of classical purification and chemical conjugation. Therefore, in typical embodiments, bioconjugates are preferred over chemically produced glycoconjugates. The preparation of bioconjugates of O1, O2, O6, and O25 antigens is described in detail in WO2015 / 124769 and WO2017 / 035181. The preparation of bioconjugates of E. coli O4, in particular O4A, and bioconjugates of E. coli O1, O2, O6, O8, O15, O16, O18, O25 and O75 is described in detail in WO2020 / 191082, the preparation of bioconjugates of E. coli O18 is described in detail in PCT / IB2022 / 053013, and the preparation of conjugate compositions with E. coli O75 and other E. coli O-antigen conjugates is described in detail in WO2022 / 058945.

[0125] Host cells suitable for preparing bioconjugates are described, for example, in WO2020 / 191082 and WO2022 / 058945.

[0126] Thus, in certain embodiments, the present invention provides host cells (eg, prokaryotic host cells) capable of producing bioconjugates of E. coli O153 antigenic polysaccharides.

[0127] In certain embodiments, the present invention provides host cells (eg, prokaryotic host cells) capable of producing bioconjugates of E. coli 021 antigenic polysaccharides.

[0128] The host cells provided herein are preferably modified to contain (e.g., by genetic engineering) one or more nucleic acids encoding host cell machinery (e.g., glycosyltransferases) for producing an E. coli O153 antigen bioconjugate or producing an E. coli O21 antigen bioconjugate, respectively. In certain embodiments, the prokaryotic cell is a Gram-negative bacterium. Exemplary prokaryotic host cells for producing bioconjugates comprising the E. coli 0153 or 021 antigenic polysaccharides described herein include, but are not limited to, Escherichia species, Shigella species, Klebsiella species, Xanthomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species.

[0129] In certain embodiments, the host cell used to produce the O153 or O21 antigen polysaccharide bioconjugate is E. coli. In certain embodiments, the host cell is E. coli K-12 (as a non-limiting example, E. coli strain W3110 is a K-12 strain), or E. coli B strain (as a non-limiting example, E. coli strain BL21 is a B strain), or any other well-defined strain of E. coli, such as a laboratory strain or production strain, as opposed to a primary wild-type isolate.

[0130] In certain embodiments, the host cells used to produce the O153 antigen polysaccharides and bioconjugates described herein are engineered to contain heterologous nucleic acids, e.g., heterologous nucleic acids comprising the rfb gene cluster of an O153 antigen serotype, heterologous nucleic acids encoding one or more carrier proteins and / or glycosyltransferases.

[0131] In certain embodiments, host cells used to produce the O21 antigen polysaccharides and bioconjugates described herein are engineered to contain heterologous nucleic acid, e.g., heterologous nucleic acid comprising the rfb gene cluster of an O21 antigen serotype, heterologous nucleic acid encoding one or more carrier proteins and / or glycosyltransferases.

[0132] In a specific embodiment, heterologous rfb genes and / or heterologous nucleic acids encoding proteins involved in glycosylation pathways (e.g., prokaryotic and / or eukaryotic glycosylation pathways) can be introduced into host cells as described herein. Such nucleic acids can encode proteins including, but not limited to, oligosaccharyl transferases and / or glycosyl transferases.

[0133] The host cell capable of producing the bioconjugate of the E. coli O153 antigen polysaccharide covalently linked to the carrier protein provided herein further comprises the nucleotide sequence of the rfb gene cluster of the E. coli O153 antigen polysaccharide. In certain non-limiting exemplary embodiments, the rfb gene cluster that can be used to produce the E. coli O153 antigen polysaccharide has the sequence of SEQ ID NO: 3. Another example can be found in GenBank locus KJ755551.

[0134] The host cell capable of producing the bioconjugate of the E. coli O21 antigen polysaccharide covalently linked to the carrier protein of SEQ ID NO: 4 provided herein further comprises the nucleotide sequence of the rfb gene cluster of the E. coli O21 antigen polysaccharide and the nucleotide sequence encoding UDP-glucose 4-epimerase. In certain non-limiting exemplary embodiments, the rfb gene cluster that can be used to produce the E. coli O21 antigen polysaccharide has the sequence of SEQ ID NO: 6. Another example can be found in GenBank locus EU694098. In certain non-limiting exemplary embodiments, the nucleotide sequence encoding the UDP-glucose 4-epimerase that can be used to produce the E. coli O21 antigen polysaccharide has the sequence of SEQ ID NO: 7. In certain non-limiting embodiments, the UDP-glucose 4-epimerase has the amino acid sequence shown in SEQ ID NO: 8.

[0135] Degenerate nucleic acid sequences encoding the same enzyme as that encoded by the provided rfb cluster sequence or UDP-glucose 4-epimerase nucleotide sequence, or sequences encoding enzymes that are at least 80% identical, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the respective reference enzymes encoded in the rfb gene cluster (i.e., 100% identical to the encoded enzyme sequence) or to UDP-glucose 4-epimerase and have at least qualitatively the same enzymatic function, can also be used.

[0136] The host cell capable of producing a bioconjugate of an E. coli O153 or O21 antigenic polysaccharide further comprises a nucleic acid encoding an oligosaccharyltransferase. The oligosaccharyltransferase transfers lipid-linked oligosaccharides to asparagine residues of nascent polypeptide chains containing a consensus N-glycosylation motif. The nucleic acid encoding the oligosaccharyltransferase may be native to the host cell or may be introduced into the host cell using genetic methods. In a preferred embodiment, the oligosaccharyltransferase is heterologous to the host cell. E. coli does not naturally contain oligosaccharyltransferases, so if E. coli is used as a host cell to produce a bioconjugate, a heterologous oligosaccharyltransferase is contained in such a host cell, for example, after introduction by genetic engineering. In view of the present disclosure, the oligosaccharyltransferase may be from any source known in the art.

[0137] In certain embodiments, alternatives to oligosaccharyltransferases having N-glycosyltransferase activity, such as O-glycosyltransferases, such as, for example, PglL, as described, for example, in WO 2016 / 82597 and WO 2020 / 120569, can be used in combination with their own, different glycosylation consensus sequences in the carrier protein. Other glycosyltransferases, such as O-glycosyltransferases, can therefore also be used as oligosaccharyltransferases according to the present invention.

[0138] In certain preferred embodiments, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter. For example, in one embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al., 2002, Science 298: 1790-1793; see also, e.g., NCBI gene ID: 3231775, UniProt accession number 086154). In another embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter lari (see, e.g., NCBI gene ID: 7410986).

[0139] In certain embodiments, the oligosaccharyltransferase is a PglB oligosaccharyltransferase from Campylobacter jejuni, including native (wild-type) proteins or any variants thereof, such as those described in International Patent Application Publications WO2016 / 107818 and WO2016 / 107819. In a specific embodiment, the PglB oligosaccharyltransferase comprises SEQ ID NO: 4 or a variant thereof. 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: 4 comprising mutation N534Q. Examples of variant PglB oligosaccharyltransferases suitable for use in recombinant host cells provided herein include SEQ ID NO: 4 comprising at least one mutation, wherein the at least one mutation is selected from the group consisting of N311V, K482R, D483H, A669V, Y77H, S80R, Q287P, and K289R. In a specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 4 comprising the mutation N311V. In a preferred embodiment for producing a bioconjugate of an E. coli O153 antigenic polysaccharide covalently coupled to a carrier protein, the variant PglB oligosaccharyltransferase has SEQ ID NO: 4 comprising the mutations N311V, K482R, D483H, and A669V. In a preferred embodiment for producing a bioconjugate of an E. coli O21 antigenic polysaccharide covalently coupled to a carrier protein, the variant PglB oligosaccharyltransferase has SEQ ID NO: 4 comprising the mutation N311V.

[0140] Other variants of PglB that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 4 and still have oligosaccharyltransferase activity, optionally with one or more of the specific amino acids at the positions indicated herein (e.g., 77Y, 80S, 287Q, 289K, 311N, 482K, 483D, 669A; or 311V; or 311V, 482R, 483H, 669V; or 77H, 80R, 287P, 289R, 311V; or 77H, 311V; etc.), can also be used to produce bioconjugates. Preferred PglB enzymes for producing bioconjugates of O-antigens from E. coli serotypes O1, O2, O4, O6, O8, O15, O16, O18, O25 and O75 are described in WO2020 / 191088.

[0141] In certain embodiments, additional modifications can be introduced (e.g., using recombinant techniques) into the host cells described herein. For example, host cell nucleic acids (e.g., genes) encoding proteins that form part of a glycosylation pathway that may compete with or interfere with one or more heterologous genes involved in glycosylation introduced recombinantly into the host cell can be deleted or modified in the host cell background (genome) in a manner that renders them inactive / dysfunctional (i.e., the deleted / modified host cell nucleic acids do not encode functional proteins). In certain embodiments, when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced with desired sequences (e.g., sequences that can be used to produce O-antigen polysaccharides or bioconjugates thereof).

[0142] Exemplary genes or gene clusters that can be deleted in the host cell (and in some cases, replaced by other desired nucleic acid sequences) include genes or gene clusters of the host cell involved in glycolipid biosynthesis, such as waaL, the lipid A core biosynthesis cluster (waa), the galactose cluster (gal), the arabinose cluster (ara), the colonic acid cluster (wc), the capsular polysaccharide cluster, undecaprenol-p biosynthesis genes (e.g., uppS, uppP), und-P recycling genes, metabolic enzymes involved in nucleotide-activated sugar biosynthesis, the enterobacterial common antigen cluster (eca), and the prophage O antigen modification cluster (e.g., the gtrABS cluster or a region thereof).

[0143] In a specific embodiment, the waaL gene is deleted from the genome of the host cell (e.g., a recombinant host cell) provided herein or its function is inactivated. The terms "waaL" and "waaL gene" refer to an O-antigen ligase gene encoding a membrane-bound enzyme whose active site is located in the periplasm. The encoded enzyme transfers the O-antigen bound to undecaprenylphosphate (UPP) to the lipid A core to form lipopolysaccharide. Deletion or disruption of the endogenous waaL gene (e.g., ΔwaaL strain) disrupts the transfer of the O-antigen to lipid A and can instead enhance the transfer of the O-antigen to another biomolecule (e.g., a carrier protein).

[0144] In another specific embodiment, one or more of the waaL gene, gtrA gene, gtrB gene, gtrS gene and rfb gene cluster are deleted or functionally inactivated from the original genome of the host cell capable of producing a bioconjugate (e.g., of E. coli O153 antigen polysaccharide or E. coli O21 antigen polysaccharide).

[0145] In certain embodiments, the host cell capable of producing a bioconjugate of an E. coli 0153 or 021 antigenic polysaccharide further comprises a nucleic acid encoding a carrier protein as previously described herein.

[0146] In certain embodiments, each of the E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O21, and O153 antigenic polysaccharides, particularly when part of a bioconjugate, is covalently linked to an asparagine (Asn) residue in a carrier protein, wherein the Asn residue is present in a glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro. Preferably, the Asn residue is present in a glycosylation site comprising a glycosylation consensus sequence Asp (Glu) -X-Asn-Z-Ser (Thr), wherein X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 1). The carrier protein may comprise 1-10 glycosylation sites, preferably 2, 3, or 4 glycosylation sites, and most preferably 4 glycosylation sites, each glycosylation site comprising a glycosylation consensus sequence. In a specific embodiment, the carrier protein is an EPA-4 carrier protein, such as an EPA-4 carrier protein comprising the amino acid sequence of SEQ ID NO:2.

[0147] On the other hand, the present invention provides a kind of pharmaceutical composition, it comprises pharmaceutically acceptable carrier and / or excipient and composition as described herein.As used herein, term " pharmaceutically acceptable " means to be approved by the regulatory agency of federal or state government or to be listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, more specifically for people.As used herein, in the context of pharmaceutically acceptable carrier, term " carrier " refers to the diluent, adjuvant, excipient or vehicle that is administered together with pharmaceutical composition.Saline solution and dextrose aqueous solution and glycerol solution can also be used as liquid carrier, particularly for injectable solution.Suitable excipient includes starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol etc.The example of suitable pharmaceutical carrier is described in the textbook known to those skilled in the art of pharmaceutical preparation.

[0148] In certain embodiments, the compositions described herein (e.g., immunogenic compositions) comprise an adjuvant, or are administered in combination with an adjuvant. In some embodiments, the term "adjuvant" refers to a compound that, when administered in conjunction with or as part of a composition described herein, augments, enhances, and / or promotes an immune response to a (bio)conjugate, but does not produce an immune response to the (bio)conjugate when the adjuvant compound is administered alone. Examples of suitable adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles or nanoalum formulations comprising alum), 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, US 6491919), AS01, AS02, AS03, and AS04 (all GlaxoSmithKline; see, e.g., EP 1126876, US 735793), 6 for AS04, see EP0671948, EP0761231, US5750110 for AS02), MF59 (Novartis), imidazopyridine compounds (see WO2007 / 109812), imidazoquinoxaline compounds (see WO2007 / 109813), delta-inulin, STING-activating synthetic cyclic dinucleotides (e.g., US20150056224), combinations of lecithin and carbomer homopolymers (e.g., US6676958), and saponins (such as Quil A and QS21), Matrix M, immunostimulatory complexes (Iscoms), Iscomatrix, etc., optionally in combination with QS7. In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally in combination with an immunostimulant (such as monophosphoryl lipid A). Another adjuvant is CpG. Other examples of adjuvants are liposomes containing immunostimulants (such as MPL and QS21 in AS01E and AS01B). Other examples of adjuvants are imidazoquinolines (such as imiquimod and R848. In certain embodiments, the adjuvant contains a Toll-like receptor 4 (TLR4) agonist. TLR4 agonists are well known in the art, see, for example, Ireton GC and.In certain embodiments, the adjuvant comprises a TLR4 agonist comprising lipid A or an analog or derivative thereof, such as MPL, 3D-MPL, RC529, PET-lipid A, GLA (glucopyranosyl lipid adjuvant, synthetic disaccharide glycolipid), SLA (which describes a structure-function approach to optimizing TLR4 ligands for human vaccines), PHAD (phosphorylated hexaacyl disaccharide), 3D-PHAD (whose structure is identical to that of GLA), 3D-(6-acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-PHAD, and 3D(6A)PHAD are synthetic lipid A variants, see, for example, avantilipids.com / divisions / adjuvants, which also provides the structures of these molecules), E6020 (CAS No. 287180-63-6), ONO4007, OM-174, etc.

[0149] In certain preferred embodiments, the compositions described herein do not comprise an adjuvant other than the bioconjugate, and / or the compositions described herein are not administered in combination with an adjuvant other than the bioconjugate (if the bioconjugate would contain some inherent adjuvant properties, these would be disregarded and, in these embodiments, no exogenous adjuvant would be added).

[0150] Those skilled in the art will recognize that various formulations can be used for the compositions of the present invention. In one embodiment, the composition of the present invention comprises a (bio)conjugate as described herein in Tris-buffered saline (TBS) pH 7.4 (e.g., containing, for example, 25 mM, 137 mM, and 2.7 mM Tris, NaCl, and KCl, respectively). In other embodiments, the composition of the present invention comprises a (bio)conjugate as described herein in about 10 mM KH2PO4 / Na2HPO4 buffer (pH about 7.0), about 5% (w / v) sorbitol, about 10 mM methionine, and about 0.02% (w / v) polysorbate 80. In other embodiments, the compositions of the invention comprise a (bio)conjugate as described herein in about 10 mM KH2PO4 / Na2HPO4 buffer (pH about 7.0), about 8% (w / v) sucrose, about 1 mM EDTA, and about 0.02% (w / v) polysorbate 80 (for suitable buffers for bioconjugates of E. coli O-antigen covalently bound to an EPA carrier protein, see, e.g., WO2018 / 077853). In other embodiments, the compositions of the invention comprise a (bio)conjugate as described herein in about 5 mM succinate / 0.9% NaCl (pH 6.0).

[0151] Typically, the composition of the present invention can be prepared by first obtaining a separate glycoconjugate of each E. coli O-antigen polysaccharide as described herein by independently covalently linking the O-antigen polysaccharide to a carrier protein (e.g., by chemical conjugation or biological conjugation), and then mixing these separate glycoconjugates in the amounts and ratios as described herein to obtain the composition according to the present invention. Therefore, in another aspect, the present invention provides a method for preparing a composition according to the present invention, comprising providing each desired O-antigen conjugate (e.g., by obtaining or manufacturing these, e.g., in the form of a bulk drug substance) and mixing them in the desired ratio and / or amount to obtain the composition of the present invention (e.g., a multivalent E. coli, in particular ExPEC vaccine composition, sometimes also referred to as a drug product).

[0152] Therefore, in one aspect, the present invention also provides a method for preparing a composition of the present invention by producing a bioconjugate of an E. coli O153 antigenic polysaccharide covalently linked to a carrier protein, the method comprising culturing the recombinant prokaryotic host cell of the present invention to produce the bioconjugate of E. coli O153 of the present invention, and mixing the bioconjugate with another saccharide conjugate, preferably a bioconjugate (e.g., a bioconjugate of an E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and / or O21 antigenic polysaccharide, each independently covalently linked to a carrier protein) to obtain a composition according to the present invention as described herein. In one aspect, the present invention also provides a method for preparing a composition of the present invention by producing a bioconjugate of an Escherichia coli O21 antigenic polysaccharide covalently linked to a carrier protein, the method comprising culturing the recombinant prokaryotic host cell of the present invention to produce the bioconjugate of the Escherichia coli O21 of the present invention, and mixing the bioconjugate with another saccharide conjugate, preferably a bioconjugate (e.g., a bioconjugate of an Escherichia coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and / or O153 antigenic polysaccharide, each independently covalently linked to a carrier protein) to obtain a composition according to the present invention as described herein.

[0153] The (bio)conjugates and compositions provided herein can be used to induce antibodies against E. coli O-antigens in a subject, or to vaccinate a subject against E. coli. The methods described herein for inducing an immune response in a subject result in vaccination of the subject against infection with, or disease caused by, an E. coli strain from a serotype whose O-antigen is present in one or more compositions.

[0154] Thus, in another aspect, the present invention provides a method of inducing an immune response against E. coli, preferably extraintestinal pathogenic E. coli (ExPEC), in a subject, wherein the method comprises administering to the subject a composition as described herein or a pharmaceutical composition as described herein.

[0155] In certain aspects, provided herein are compositions as described herein for use in a method of inducing an immune response against E. coli, preferably ExPEC, in a subject. In certain aspects, provided herein are uses of compositions as described herein for the preparation of a medicament for inducing an immune response against E. coli, preferably ExPEC, in a subject.

[0156] In certain embodiments, the subject is a human. In some embodiments, the subject is a human having, or at risk for, an ExPEC infection or invasive ExPEC disease. In one embodiment, the subject has an E. coli (e.g., ExPEC) infection at the time of administration. In preferred embodiments, the subject does not have an E. coli (e.g., ExPEC) infection or invasive ExPEC disease at the time of administration.

[0157] In certain embodiments of the methods according to the invention, the immune response limits the severity of or prevents invasive ExPEC disease in the subject, preferably wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

[0158] In certain embodiments, the immune response induced in a subject following administration of a composition described herein is preferably effective in preventing or alleviating symptoms caused by an ExPEC infection in at least 30%, more preferably at least 40%, such as at least 50% of the subjects to whom the composition is administered. Symptoms of an ExPEC infection may vary depending on the nature of the infection and may include, but are not limited to: difficulty urinating, increased frequency or urgency of urination, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea (e.g., in subjects with a urinary tract infection caused by an ExPEC); high fever, headache, stiff neck, nausea, vomiting, seizures, lethargy, and / or light sensitivity (e.g., in subjects with meningitis caused by an ExPEC); fever, increased heart rate, increased respiratory rate, decreased urine output, decreased platelet count, abdominal pain, difficulty breathing, and / or abnormal heart function (e.g., in subjects with sepsis caused by an ExPEC).

[0159] Example

[0160] The following examples of the present invention are intended to further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention, and the scope of the present invention is determined by the appended claims.

[0161] Example 1: Production of bioconjugates of Escherichia coli O153 antigenic polysaccharide linked to EPA carrier protein

[0162] O153 is an E. coli serotype observed in more than 1% of ExPEC blood culture isolates from patient populations in various regions around the world, and there appears to be a relatively high prevalence of more than 8% in such isolates from South America (Weerdenburg et al, 2022, Clin Infect Dis., doi: 10.1093 / cid / ciac421). Therefore, it may be useful to include an antigen that can protect against E. coli O153 serotype in a vaccine composition, for example in the form of a conjugate of the O-antigen linked to a carrier protein. This example describes the production of a bioconjugate of an E. coli O153 antigen polysaccharide linked to EPA as a carrier protein.

[0163] E. coli O153 blood isolate was selected as the O153 donor for the rfb gene cluster (collected in the Netherlands and obtained from Utrecht University Medical Center). E. coli K12 strain W3110 was used as the parent strain. This strain can be obtained, for example, from the E. coli Genetic Stock Center (Yale University, New Haven (CT), USA, product number CGSC#4474). The genomic sequence is publicly available (PMID: 16738553).

[0164] The parental E. coli K12 strain W3110 was modified by replacing the chromosomal rfb cluster of the clinical isolate E. coli O153 strain (rfb cluster having SEQ ID NO: 3) using previously described methods (see, e.g., WO2020 / 191082). Additionally, the O-antigen ligase waaL was deleted to prevent transfer of the O-antigen to lipid A, thereby enhancing transfer of the O-antigen to the carrier protein. The chromosomal gtrABS gene was disrupted by homologous recombination to prevent the addition of branched glucose to the O153 antigen.

[0165] To confirm the production of the O153 antigen, the modified strain was grown overnight in antibiotic-free LB medium at 37°C. After harvesting, the cell pellet was resuspended in Laemmli buffer and the protein was digested with proteinase K. Lipopolysaccharide / lipid-linked oligosaccharide (LPS / LLO) was separated by SDS-PAGE, and the O153 antigen was detected by Western blotting using an O153-specific antiserum. The structure of the O153 antigen was confirmed by MALDI-MS / MS-MS.

[0166] Plasmids encoding either a gene for a genetically detoxified Pseudomonas aeruginosa exotoxin (EPA) with two glycosylation sites (EPA-2) or a gene for EPA with four glycosylation sites (EPA-4; sequence provided as SEQ ID NO: 2) were introduced. Several plasmids encoding the oligosaccharyltransferase Campylobacter jejuni PglB were tested in separate experiments, and the plasmid encoding the oligosaccharyltransferase Campylobacter jejuni PglB was determined. N311V,K482R,D483H,A669V The plasmid encoding the best PglB variant (which is a PglB variant having the amino acid sequence described in SEQ ID NO: 4, having amino acid substitutions N311V, K482R, D483H, and A669V) produced the best results among the tested variants, which was an unexpected result. Therefore, the plasmid encoding this best PglB variant was selected and introduced into the modified host cells to generate a production strain for the production of a bioconjugate of an E. coli O153 antigenic polysaccharide linked to an EPA carrier protein.

[0167] To confirm the production of the O153 bioconjugate, the production strain was grown in TB medium supplemented with phosphate buffer, MgCl2, and antibiotics at 37°C. At an optical density at 600 nm of approximately 4.0, IPTG and arabinose were added to induce bioconjugate production. After overnight growth, the cells were harvested and the bioconjugates were released by osmotic shock. The O153 bioconjugates were separated by SDS-PAGE and detected by Western blotting using antisera specific for O153 and EPA.

[0168] Production and purification of the bioconjugate in a bioreactor is performed using known methods (see, for example, WO2020 / 191082, WO2022 / 214620).

[0169] Example 2: Production of bioconjugates of Escherichia coli O21 antigenic polysaccharide linked to EPA carrier protein

[0170] O21 is an E. coli serotype observed in more than 1% of ExPEC blood culture isolates from North American patient populations (Weerdenburg et al, 2022, Clin Infect Dis., doi: 10.1093 / cid / ciac421). Therefore, it may be useful to include an antigen that can protect against E. coli O21 serotype in a vaccine composition, for example in the form of a conjugate of the O-antigen linked to a carrier protein. This example describes the production of a bioconjugate of an E. coli O21 antigen polysaccharide linked to EPA as a carrier protein.

[0171] E. coli O21 blood isolate was selected as the O21 donor of the rfb gene cluster and the gne gene (encoding UDP-glucose 4-epimerase) (collected in the Netherlands and obtained from Utrecht University Medical Center). E. coli K12 strain W3110 was used as the parent strain. This strain can be obtained, for example, from the E. coli Genetic Stock Center (Yale University, New Haven (CT), USA, product number CGSC#4474). The genomic sequence is disclosed (PMID: 16738553).

[0172] The E. coli W3110 chromosomal rfb cluster was replaced with the rfb cluster of the clinical isolate E. coli O21 strain (rfb cluster with SEQ ID NO: 6) by a previously described method (see, for example, WO2020 / 191082), and the parent E. coli K12 strain W3110 was modified by introducing the gne gene in the form of a nucleotide sequence (SEQ ID NO: 7) encoding UDP-glucose 4-epimerase (with SEQ ID NO: 8). In addition, the O-antigen ligase waaL was deleted to prevent the transfer of the O-antigen to lipid A, thereby enhancing the transfer of the O-antigen to the carrier protein. The chromosomal gtrABS gene was disrupted by homologous recombination to prevent the addition of branched glucose to the O21 antigen.

[0173] To confirm the production of the O21 antigen, the modified strain was grown overnight in antibiotic-free LB medium at 37°C. After harvesting, the cell pellet was resuspended in Laemmli buffer and digested with proteinase K. LPS / LLO were separated by SDS-PAGE, and the O21 antigen was detected by Western blotting using an O21-specific antiserum. The structure of the O21 antigen was confirmed by MALDI-MS / MS-MS.

[0174] Plasmids encoding either a gene for a genetically detoxified Pseudomonas aeruginosa exotoxin with two glycosylation sites (EPA-2) or a gene for a genetically detoxified Pseudomonas aeruginosa exotoxin with four glycosylation sites (EPA-4) were introduced. Several plasmids encoding the oligosaccharyltransferase Campylobacter jejuni PglB were tested in separate experiments, and the plasmid encoding the oligosaccharyltransferase Campylobacter jejuni PglB was identified. N311VThe plasmid (which is a PglB variant having the amino acid sequence described in SEQ ID NO: 4, having the amino acid substitution N311V) produced the best results among the tested variants, confirming that the optimal PglB for preparing bioconjugates varies in an unpredictable manner between E. coli serotypes (see previous examples and WO2020 / 191088). Therefore, this optimal PglB variant was introduced into the modified host cells to generate a production strain for the production of bioconjugates of E. coli O21 antigenic polysaccharide linked to the EPA carrier protein.

[0175] To confirm the production of the O21 bioconjugate, the production strain was grown in TB medium supplemented with phosphate buffer, MgCl2, and antibiotics at 37°C. At an optical density at 600 nm of approximately 4.0, IPTG and arabinose were added to induce bioconjugate production. After overnight growth, the cells were harvested and the bioconjugate was released by osmotic shock. The O21 bioconjugate was separated by SDS-PAGE and detected by Western blotting using antisera specific for O21 and EPA.

[0176] Production and purification of the bioconjugate in a bioreactor is performed using known methods (see, for example, WO2020 / 191082, WO2022 / 214620).

[0177] Example 3: Immunogenicity of O153 bioconjugates

[0178] Experimental design

[0179] Female Sprague Dawley rats (5-6 weeks old) were immunized intramuscularly with 0.04, 0.40 or 4.00 μg O153-EPA bioconjugate on days 0, 14 and 28. The control group received formulation buffer only. Blood samples were drawn before immunization (day 0) and after immunization (days 14 and 42). The blood was processed and serum samples were stored at -20°C. The total IgG Ab response in serum was measured by ELISA (as described above). In brief, ELISA plates were coated with O153-LPS, blocked with skim milk and incubated with rat serum samples. Subsequently, the plates were incubated with HRP-labeled goat anti-rat IgG and the OD450 was measured after development with TMB substrate. The EC50 titer, defined as the half-maximal effective concentration, was calculated based on the repeated 12-step titration curve drawn in the 4PL nonlinear regression model.

[0180] result

[0181] Compared with formulation buffer, immunization with 0.04 μg, 0.40 μg and 4.00 μg O153-EPA bioconjugate per dose induced a significant increase in IgG antibody levels on day 42 ( Figure 1For rats immunized with 0.04 μg of conjugate per dose, a significant increase in Ab titer was also observed on day 14 compared to formulation buffer ( Figure 1 Furthermore, the Ab levels induced by 0.04 μg, 0.40 μg, and 4.00 μg of the conjugate were significantly increased on day 42 compared to those detected on day 0 and day 14 after immunization ( Figure 1 The groups receiving 0.04 μg or 4.00 μg of conjugate per dose also showed a significant increase in antibody (Ab) levels on day 14 compared to day 0, indicating that a single dose of 0.04 μg or 4.00 μg of O153-EPA conjugate was able to induce a significant increase in IgG titers ( Figure 1 ). For all three concentrations of the bioconjugate, a significant increase in IgG titers between days 14 and 42 indicated that the O153-EPA conjugate was able to enhance the antibody response. The highest Ab titers were detected in the group of animals immunized with 0.04 μg / dose followed by 0.4 μg / dose. Across-dose analysis confirmed these findings, indicating that a better Ab response was achieved with the lowest dose of the O153-EPA conjugate.

[0182] This example demonstrates that a bioconjugate in which an E. coli O153 antigenic polysaccharide is covalently linked to a carrier protein is immunogenic.

[0183] Example 4: Immunogenicity of O21 bioconjugates

[0184] Experimental design

[0185] Female Sprague Dawley rats (5-6 weeks old) were immunized intramuscularly with 0.04, 0.40 or 4.00 μg O21-EPA bioconjugate on days 0, 14 and 28. The control group received formulation buffer only. Blood samples were drawn before immunization (day 0) and after immunization (days 14 and 42). The blood was processed and serum samples were stored at -20°C. The total IgG Ab response in serum was measured by ELISA (as described above). In brief, ELISA plates were coated with O21-LPS, blocked with skim milk and incubated with rat serum samples. Subsequently, the plates were incubated with HRP-labeled goat anti-rat IgG and developed with TMB substrate to measure OD450. The EC50 titer, defined as the half-maximal effective concentration, was calculated based on the repeated 12-step titration curve drawn in the 4PL nonlinear regression model.

[0186] result

[0187] Compared with formulation buffer, immunization with 0.04 μg, 0.40 μg and 4.00 μg O21-EPA bioconjugate per dose induced a significant increase in IgG antibody levels on day 42 ( Figure 2Furthermore, the Ab levels induced by 0.04 μg, 0.40 μg, and 4.00 μg of the conjugate were significantly increased on day 42 compared to those detected at baseline (day 0) and day 14 after immunization ( Figure 2 For all three concentrations of bioconjugate, the significant increase in IgG titers between day 14 and day 42 indicated that the O21-EPA conjugate was able to enhance the antibody response. For the group receiving formulation buffer, a significant decrease in Ab levels was observed on day 42 compared to day 0 ( Figure 2 ), however, most samples in this group did not show 4PL fit. The highest titers were detected after immunization with 0.04 μg / dose. The highest Ab titers were detected in the group of animals immunized with 0.04 μg / dose. Analysis across doses confirmed this finding, indicating that a better Ab response was obtained with the lowest dose of the O21-EPA conjugate.

[0188] This example demonstrates that a bioconjugate in which an E. coli O21 antigenic polysaccharide is covalently linked to a carrier protein is immunogenic.

[0189] Example 5: Antibody-mediated opsonophagocytic killing induced by O153-EPA or O21-EPA bioconjugates

[0190] Experimental design

[0191] The functional activity of serum antibodies was measured by opsonophagocytic killing assay (OPKA; protocol as described above). Briefly, serum samples were heat inactivated at 56°C for 30 minutes and diluted 1:10 (starting dilution), followed by 8 steps of 3-fold serial dilution. All serum samples were tested in duplicate. Serum was incubated with bacteria (~103 CFU) at room temperature for 30 minutes, followed by the addition of complement and HL60 cells and incubation at 37°C for 1 hour. The final reaction mixture was spotted onto agar plates and incubated at 33°C for 15-16 hours. The number of bacterial colonies on the plates was counted, and the opsonization index (OI) value, defined as the serum dilution that killed 50% of the bacteria, was calculated using the Opsotiter3 Excel-based program (Department of Microbiology, University of Alabama, Birmingham). Due to the complexity of the assay, only the antibody function of rat samples immunized with formulation buffer or 4.00 μg / dose O153-EPA / O21-EPA conjugate on day 42 was evaluated.

[0192] result

[0193] When compared to formulation buffer, the expression of EPA from animals immunized with O153-EPA conjugate ( Figure 3 ) and animals immunized with O21-EPA conjugate ( Figure 4A significant increase in E. coli OI was observed in serum samples of 1% (95% CI 0.04-0.08) (Table 1). These results indicate that the O153-induced antibodies and O21-induced antibodies induced by administration of O153-EPA and O21-EPA conjugates, respectively, are functional and capable of mediating E. coli killing.

[0194] Example 6: Production of a composition comprising 12 Escherichia coli O-antigen conjugates

[0195] The preparation of bioconjugates of E. coli O1 (O1A), O2, O4 (O4A), O6 (O6A), O8, O15, O16, O18, O25 (O25B), and O75 is described in detail in WO2020 / 191082, the preparation of bioconjugates of E. coli O18 is described in detail in PCT / IB2022 / 053013, and the preparation of conjugate compositions with E. coli O75 and other E. coli O-antigen conjugates is described in detail in WO2022 / 058945. The production of E. coli O153 and E. coli O21 bioconjugates was performed as described herein. The 12 conjugates were mixed together to provide the immunogenic compositions described in Table 2:

[0196] Table 2: 12-valent composition of E. coli bioconjugates

[0197]

[0198] The composition comprising these 12 bioconjugates is referred to herein as 'ExPEC12V'.

[0199] Example 7: Induction of immune responses by ExPEC12V compositions

[0200] Experimental design

[0201] Female Sprague Dawley rats (5-6 weeks old) were immunized intramuscularly with the ExPEC12V vaccine on days 0, 14, and 28. The animals were divided into three different treatment groups: Group 1: 4.0 μg of each polysaccharide (PS) / dose (except O25B, which was given at 8.0 μg PS / dose); Group 2: 0.4 μg of each PS / dose (except O25B, which was given at 0.8 μg PS / dose); Group 3: a control group receiving formulation buffer. Blood samples were drawn before immunization (day 0) and after immunization (days 28 and 42). Total IgG levels in serum were measured by ELISA (according to industry standards). Briefly, ELISA plates were coated with O1A, O2, O4, O6A, O8, O15, O16, O18, O21, O25B, O75, O21, or O153 LPS, blocked with skim milk, and incubated with rat serum samples. Subsequently, the plate was incubated with HRP-labeled goat anti-rat IgG and the OD450 was measured after development with TMB substrate. The EC50 titer, defined as the half-maximal effective concentration, was calculated based on the repeated 12-step titration curves plotted in the 4PL nonlinear regression model.

[0202] result

[0203] Significantly higher IgG antibody titers were observed on day 42 after immunization with 0.4 / 0.8 μg PS / dose of ExPEC12V compared to formulation buffer. In addition, for all tested conjugates, Ab levels induced by 0.4 / 0.8 μg PS / dose of ExPEC12V were significantly increased on day 42 compared to those detected at baseline (day 0) ( FIG. 5 ). For most conjugates, with the exception of O1A, O21, and O25B, Ab levels were already significantly increased on day 28 compared to day 0. In addition, for all conjugates of 0.4 / 0.8 μg PS / dose of ExPEC12V, a significant increase in Ab levels was observed on day 42 compared to day 28, indicating that a booster dose of ExPEC12V administered on day 28 was able to increase the Ab response ( FIG. 5 ).

[0204] Comparison of groups receiving 0.4 / 0.8 μg PS / dose or 4 / 8 μg PS / dose showed that immunization with 0.4 / 0.8 μg PS / dose induced the highest Ab responses to the O2, O4 and O153 conjugates when compared to 4.0 / 8.0 μg PS / dose.

[0205] This example demonstrates that the E. coli O21 and O153 conjugates are also immunogenic in a multivalent vaccine composition comprising several other bioconjugates representing E. coli O-antigens of different serotypes, and shows that other bioconjugates previously shown to be immunogenic in 10-valent compositions (see, e.g., WO2020 / 191082) remain immunogenic when the bioconjugates of E. coli O21 and O153 are added to produce a 12-valent composition.

[0206] Example 8: Ab-mediated opsonophagocytic killing induced by ExPEC12V vaccine

[0207] Experimental design

[0208] The functional activity of serum Abs was measured by single opsonophagocytic killing assay (OPKA) for O1A, O2, O4, O8, O15, O16, O18, O21, O75 and O153 or multiple opsonophagocytic killing assay (MOPA) for O6A and O25B. The OPKA protocol was performed as described in Example 5. For MOPA, serum samples were heat inactivated at 56°C for 30 minutes and diluted 1:10 (starting dilution), followed by 8 steps of 3-fold serial dilutions. The serum was incubated with bacteria at room temperature for 30 minutes. Subsequently, complement and HL60 cells were added and incubated at 37°C for 1 hour. The final reaction mixture was spotted onto agar plates and incubated at 33°C for 15-16 hours, and plates containing different types of antibiotics were used to select for specific antibiotic-resistant strains in the reaction mixture.

[0209] The number of bacterial colonies on the plate was counted and the opsonic index (OI) value, defined as the serum dilution that killed 50% of the bacteria, was calculated using an Excel-based program (Department of Microbiology, University of Alabama, Birmingham). Due to the complexity of the assay, only samples from day 42 post-immunization were used to assess Ab function.

[0210] result

[0211] Functional activity of serum Abs for each serotype included in the ExPEC12V vaccine was measured by OPKA. Significantly higher opsonophagocytic titers were observed against E. coli strains O4, O8, O15, O18, O25B, and O153 on day 42 following immunization with 0.4 / 0.8 μg / PS / dose of ExPEC12V compared to formulation buffer (Table 3). For O1A, bacterial killing was observed in only 2 of 15 animals, for O2, bacterial killing was observed in 5 of 15 animals, and for O21, killing was observed in 4 of 15 animals receiving 0.4 / 0.8 μg / PS / dose of ExPEC12V. For O6A, sera from 13 of 15 animals mediated killing, while in the group receiving buffer, sera from only 7 of 15 animals mediated killing, however, this difference did not reach statistical significance. For O16 and O75, killing was observed in 15 of 15 animals in the group immunized with 0.4 / 0.8 μg / PS / dose of ExPEC12V vaccine, however, since killing was also observed in the buffer group, no significant differences in opsonophagocytic titers could be detected for these serotypes. Significantly higher opsonophagocytic titers were observed only for E. coli strains O4 and O15 on day 42 after immunization with 4 / 8 μg / PS / dose of ExPEC12V compared to formulation buffer. Killing was observed in only 6 of 15 animals for O8, 3 of 15 animals for O21, and 4 of 15 animals for O25B that received 4 / 8 μg / PS / dose of ExPEC12V. For O6A, killing was observed in 7 of 15 animals, and for O16 and O75, killing was observed in 15 of 15 animals in the groups immunized with 4 / 8 μg / PS / dose of ExPEC12V vaccine, however, a similar number of animals were detected with killing in the buffer group.

[0212] A summary of the GMT values of the opsonic index measured by OPKA for each serotype included in the ExPEC12V vaccine is shown in Table 3.

[0213] Table 3: Summary of GMT values of opsonic index (OI) for the indicated serotypes

[0214]

[0215] In summary, the ExPEC12V composition was able to induce functional antibodies against several serotypes whose O-antigens were present in the composition, while for some serotypes no significant induction of functional antibodies was observed, which is not uncommon in such studies conducted in rats and is usually due to high background levels of functional antibodies against the corresponding serotype, i.e., more related to the assay and the E. coli strain used in the assay, rather than an indication that the composition is unable to induce functional antibodies against such serotypes, and functional antibodies are usually detectable when tested in other animals and / or humans.

[0216] sequence

[0217] SEQ ID NO: 1 (optimized glycosylation consensus sequence)

[0218] Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro

[0219] SEQ ID NO: 2 (EPA carrier protein containing 4 glycosylation consensus sequences (EPA-4)

[0220]

[0221] SEQ ID NO: 3 rfb cluster Escherichia coli (E. coli) O153

[0222]

[0223]

[0224]

[0225]

[0226] SEQ ID NO: 4 (Exemplary PglB sequence ('wild type')

[0227]

[0228] SEQ ID NO: 5 (Exemplary CRM197 sequence)

[0229]

[0230] SEQ ID NO: 6rfb cluster Escherichia coli O21

[0231]

[0232]

[0233]

[0234] SEQ ID NO: 7 (nucleotide sequence encoding UDP-glucose 4-epimerase)

[0235]

[0236] SEQ ID NO:8 (UDP-glucose 4-epimerase amino acid sequence)

[0237]

Claims

1. A composition comprising Escherichia coli O1, O2, O4, O6, O8, O15, O16, O18, O25 and O75 antigenic polysaccharides, and wherein the composition further comprises O21 antigenic polysaccharide or O153 antigenic polysaccharide or O21 and O153 antigenic polysaccharides, wherein each of the antigenic polysaccharides is independently covalently linked to a carrier protein.

2. The composition of claim 1, wherein the O1 antigen is O1A, the O4 antigen is glucosylated (O4A), the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B.

3. The composition of claim 1 or 2, wherein (i) The E. coli O1 antigen polysaccharide comprises a structure of formula (O1A): (ii) The E. coli O2 antigen polysaccharide comprises a structure of formula (O2): (iii) the E. coli O4 antigen polysaccharide comprises a structure of formula (O4A): (iv) The E. coli O6 antigen polysaccharide comprises a structure of formula (O6A): (v) The E. coli O8 antigen polysaccharide comprises a structure of formula (O8): α-D-Member3Me-(1→[3)-β-D-Member-(1→2)-α-D-Member-(1→2)-α-D-Member-(1→] n (vi) The E. coli O15 antigen polysaccharide comprises a structure of formula (O15): [→2)-β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→] n (vii) The E. coli O16 antigen polysaccharide comprises the structure of formula (O16): (viii) The E. coli O18 antigen polysaccharide comprises the structure of formula (O18A): (ix) The E. coli O25 antigen polysaccharide comprises a structure of formula (O25B): (x) The E. coli O75 antigen polysaccharide comprises the structure of formula (O75): (xi) The E. coli O153 antigen polysaccharide comprises the structure of formula (O153): [→2)-β-D-Ribf-(1→4)-β-D-Galp-(1→4)-α-D-GlcpNAc-(1→4)-β-D-Galp-(1→3)-α-D-GlcpNAc-(1→]n and (xii) The E. coli O21 antigen polysaccharide comprises the structure of formula (O21): wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, preferably from 7 to 25.

4. The composition of any one of claims 1 to 3, further comprising at least one additional E. coli antigenic polysaccharide covalently linked to a carrier protein.

5. The composition according to any one of the preceding claims, wherein the E. coli O-antigen polysaccharide present in the composition consists of: (i) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O153; (ii) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O21; or (iii) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O153 and O21.

6. The composition of any one of the preceding claims, wherein the carrier protein is detoxified exotoxin A (EPA) or CRM of Pseudomonas aeruginosa 197 .

7. The composition of any preceding claim, wherein the carrier protein is EPA.

8. The composition of any one of the preceding claims, wherein the carrier protein comprises 1 to 20 glycosylation consensus sequences having the amino acid sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro.

9. The composition of any one of the preceding claims, wherein each carrier protein comprises the amino acid sequence of SEQ ID NO:

2.

10. The composition of any one of the preceding claims, wherein the E. coli antigenic polysaccharide is covalently linked to the carrier protein by bioconjugation or by chemical conjugation.

11. The composition of any one of the preceding claims, wherein the E. coli antigenic polysaccharide is covalently linked to the carrier protein by bioconjugation.

12. The composition of any one of the preceding claims, wherein the E. coli antigenic polysaccharide is covalently linked to an Asn residue in a glycosylation site in the carrier protein.

13. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the composition of any one of the preceding claims.

14. A method of inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject, comprising administering to the subject the composition of any one of claims 1 to 12 or the pharmaceutical composition of claim 13.

15. The method of claim 14, wherein the immune response limits the severity of or prevents invasive ExPEC disease in the subject, preferably, wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

16. A recombinant prokaryotic host cell for preparing a bioconjugate of an Escherichia coli O153 antigen polysaccharide covalently linked to a carrier protein, the recombinant prokaryotic host cell comprising: a. The nucleotide sequence of the rfb gene cluster of the O153 antigen polysaccharide; b. a nucleotide sequence encoding the carrier protein, the carrier protein comprising at least one glycosylation site, the glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro, preferably having SEQ ID NO: 1; and c. a nucleotide sequence encoding oligosaccharyltransferase PglB, The E. coli O153 antigen polysaccharide comprises the structure of formula (O153): [→2)-β-D-Rib-(1→4)-β-D-Galp-(1→4)-α-D-GlcpNAc-(1→4)-3-D-Galp-(1→3)-α-D-GlcpNAc-(1→] n wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, preferably from 7 to 25.

17. A recombinant prokaryotic host cell for preparing a bioconjugate of an Escherichia coli O21 antigen polysaccharide covalently linked to a carrier protein, the recombinant prokaryotic host cell comprising: a. The nucleotide sequence of the rfb gene cluster of the O21 antigen polysaccharide; b. a nucleotide sequence encoding the carrier protein, the carrier protein comprising at least one glycosylation site, the glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro, preferably having SEQ ID NO: 1; and c. a nucleotide sequence encoding oligosaccharyltransferase PglB; d. a nucleotide sequence encoding UDP-glucose 4-epimerase, The E. coli O21 antigen polysaccharide comprises the structure of formula (O21): wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, preferably from 7 to 25.

18. The recombinant prokaryotic host cell of claim 16, wherein Relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 4, the PglB comprises amino acid mutations N311V, K482R, D483H, and A669V.

19. The recombinant prokaryotic host cell of claim 17, wherein Relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 4, the PglB comprises the amino acid mutation N311V.

20. A method for preparing a bioconjugate of an E. coli O153 antigenic polysaccharide covalently linked to a carrier protein, the method comprising culturing the recombinant prokaryotic host cell of claim 16 or 18 to produce the bioconjugate.

21. A method for preparing a bioconjugate of an E. coli O21 antigen polysaccharide covalently linked to a carrier protein, the method comprising culturing the recombinant prokaryotic host cell of claim 17 or 19 to produce the bioconjugate.

22. A composition comprising Escherichia coli O1, O2, O4, O6, O8, O15, O16, O18, O25 and O75 antigenic polysaccharides, and wherein the composition further comprises O21 antigenic polysaccharide or O153 antigenic polysaccharide or O21 and O153 antigenic polysaccharides, further comprising a pharmaceutically acceptable excipient, wherein each of the antigenic polysaccharides is independently covalently linked to a carrier protein, for use in a method of inducing an immune response against Escherichia coli, preferably against extraintestinal pathogenic Escherichia coli (ExPEC), in a subject.

23. The composition of claim 22, wherein the O1 antigen is O1A, the O4 is glucosylated (O4A), the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B.

24. The composition of claim 22 or 23, wherein (i) The E. coli O1 antigen polysaccharide comprises the structure of formula (O1A): (ii) The E. coli O2 antigen polysaccharide comprises a structure of formula (O2): (iii) the E. coli O4 antigen polysaccharide comprises a structure of formula (O4A): (iv) The E. coli O6 antigen polysaccharide comprises a structure of formula (O6A): (v) The E. coli O8 antigen polysaccharide comprises a structure of formula (O8): α-D-Member3Me-(1→[3)-β-D-Member-(1→2)-α-D-Member-(1→2)-α-D-Member-(1→] n , (vi) The E. coli O15 antigen polysaccharide comprises a structure of formula (O15): [→2)-β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→] n (vii) the E. coli O16 antigen polysaccharide comprises a structure of formula (O16): (viii) The E. coli O18 antigen polysaccharide comprises the structure of formula (O18A): (ix) The E. coli O25 antigen polysaccharide comprises a structure of formula (O25B): (x) The E. coli O75 antigen polysaccharide comprises the structure of formula (O75): (xi) The E. coli O153 antigen polysaccharide comprises the structure of formula (O153): [→2)-β-D-Ribf-(1→4)-β-D-Galp-(1→4)-α-D-GlcpNAc-(1→4)-β-D-Galp-(1→3)-α-D-GlcpNAc-(1→] n and also (xii) The E. coli O21 antigen polysaccharide comprises the structure of formula (O21): wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, preferably from 7 to 25.

25. The composition of any one of claims 22 to 24, further comprising at least one additional E. coli antigenic polysaccharide covalently linked to a carrier protein.

26. The composition of any one of claims 22 to 25, wherein the E. coli O-antigen polysaccharide present in the composition consists of: (i) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O153; (ii) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O21; or (iii) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O153 and O21.

27. The composition of any one of claims 22 to 26, wherein the carrier protein is detoxified exotoxin A (EPA) or CRM of Pseudomonas aeruginosa 197 .

28. The composition of any one of claims 22-27, wherein the carrier protein is EPA.

29. The composition of any one of claims 22-28, wherein the carrier protein comprises 1 to 20 glycosylation consensus sequences having the amino acid sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro.

30. The composition of any one of claims 22-29, wherein the carrier protein comprises the amino acid sequence of SEQ ID NO:

2.

31. The composition of any one of claims 22-30, wherein the E. coli antigenic polysaccharide is covalently linked to the carrier protein by bioconjugation or by chemical conjugation.

32. The composition of any one of claims 22-31, wherein the E. coli antigenic polysaccharide is covalently linked to the carrier protein by bioconjugation.

33. The composition of any one of claims 22-32, wherein the E. coli antigenic polysaccharide is covalently linked to an Asn residue in a glycosylation site in the carrier protein.

34. The composition of any one of claims 22-33, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

35. The composition of any one of claims 22-34, wherein the immune response limits the severity of or prevents invasive ExPEC disease in the subject, preferably wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

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