Multivalent vaccine compositions and uses thereof

Abstract

Compositions and methods are described for inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC), thereby providing immune protection against diseases associated with ExPEC. In particular, compositions are described that include conjugates of E. coli polysaccharide antigens O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75, and further include O153 or O21, or both O153 and O21, covalently linked to a carrier protein, for 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 compositions comprising Escherichia coli (E. coli) O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75 antigenic polysaccharides, and further comprising O153 or O21, or both O153 and O21 antigenic polysaccharides. The present invention further relates to the use of such compositions to induce an immune response against E. coli for the prevention or treatment of E. coli infection, particularly for preventing extraintestinal pathogenic E. coli (ExPEC) disease.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 383,841, filed November 15, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference to an electronically submitted sequence listing The contents of the electronic sequence listing (004852_206WO1.xml, size: 30,323 bytes, created date: November 13, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0004] Extraintestinal pathogenic Escherichia coli (E. coli) (ExPEC) typically reside harmlessly in the human intestine alongside commensal E. coli strains. However, ExPEC strains possess virulence factors that allow them to colonize and infect sites outside the gastrointestinal tract, causing a variety of severe invasive diseases and resulting in significant morbidity, mortality, and costs annually. ExPEC strains are the most common cause of urinary tract infections (UTIs). ExPEC strains are also responsible for surgical site infections and neonatal meningitis, are associated with abdominal and pelvic infections and nosocomial pneumonia, and are occasionally involved in other extraintestinal infections, such as osteomyelitis, cellulitis, and wound infections. All of these primary sites of infection can result in ExPEC bacteremia (Russo et al., 2003). Neonates, the elderly, and immunocompromised patients are particularly susceptible to ExPEC infections, including invasive ExPEC disease (IED).

[0005] Bacterial resistance to antibiotics is a major concern in the fight against bacterial infections, and multidrug-resistant (MDR) Escherichia coli (E. coli) strains are becoming increasingly prevalent.

[0006] O-antigen serotypes are based on the chemical structure of the O polysaccharide antigen, the outer membrane portion of the lipopolysaccharide (LPS) of Gram-negative bacteria. Although over 180 serologically unique Escherichia coli (E. coli) O antigens have been described (Stenutz et al., FEMS Microbial Rev. 2006;30:382-403), most ExPEC isolates are classified into fewer than 20 O-antigen serotypes. Full-length E. coli O antigens typically consist of approximately 10 to 25 repeating sugar units attached to a highly conserved LPS core structure, with each component synthesized separately by enzymes primarily encoded by the rfb and rfa gene clusters. After polymerization, the O-antigen polysaccharide backbone is typically modified by the addition of acetyl or glucose residues. These modifications effectively increase serotype diversity by creating antigenically distinct serotypes with a common polysaccharide backbone but different side chains.

[0007] ExPEC infections can be caused by any serotype. Although certain serotypes are overrepresented in ExPEC infections, the surface polysaccharides of 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). Furthermore, certain O antigens can be poorly immunogenic. Furthermore, studies of pneumococcal conjugate vaccines have shown that when multiple serotypes can cause disease, vaccine composition, including the selection of serotypes included and the dosage levels of those serotypes, is important because vaccines against certain serotypes may increase carriage and disease incidence rates for serotypes not included in the vaccine, or may pose similar risks for serotypes included in the vaccine but with weak immune responses (Lipsitch, Emerging Infectious Diseases; 1999, 5:336-345). Ideally, a vaccine should maximize its beneficial effect of preventing disease caused by the serotypes included in the vaccine while minimizing the risk of increased disease due to increased carriage of non-vaccine serotypes.

[0008] Efforts to develop vaccines to prevent ExPEC infection have focused on O-antigen polysaccharide conjugates. A 12-valent O-antigen conjugate vaccine was synthesized by extracting and purifying O-antigen polysaccharide and chemically conjugating it with attenuated Pseudomonas aeruginosa exotoxin A, and safety and immunogenicity were tested in a phase 1 clinical trial (Cross et al., J. Infect. Dis. (1994) v.170, pp.834-40). This vaccine candidate was not approved for clinical use. Recently, a bioconjugation system in Escherichia coli (E. coli) has been developed in which polysaccharide antigens and carrier proteins are both synthesized in vivo and then conjugated in vivo by the activity of the Campylobacter jejuni enzyme oligosaccharyltransferase PglB expressed in E. coli (Wacker et al., Proc. Nat. Acad. Sci. (2006) v. 103, pp. 7088-93). This N-linked protein glycosylation system can transfer a variety of polysaccharides to carrier proteins within the bacterial cell.

[0009] Bioconjugation has been successfully utilized to produce conjugated polysaccharides for a tetravalent E. coli O-antigen candidate vaccine (Poolman and Wacker, J. Infect. Dis. (2016) v.213(1), pp. 6-13). Decavalent and nonavalent vaccine compositions of E. coli O-antigen polysaccharide bioconjugates have previously been described (e.g., WO 2020 / 191082, WO 2022 / 058945). The development of a successful ExPEC vaccine requires coverage of the predominant serotypes, and the presence of additional O-antigen modifications in a subset of ExPEC isolates presents additional challenges in covering isolates that display unmodified and modified LPS. Furthermore, immune responses to vaccine compositions containing O-antigens from multiple serotypes may differ between serotypes. Therefore, there remains a continuing need in the art for vaccines against ExPEC. In particular, there is a need for a surface polysaccharide-based ExPEC vaccine that can be safely and efficiently produced on an industrial scale and that can be used to provide an effective immune response, preferably protection, against ExPEC O153 and / or O21 serotypes, and preferably also against other serotypes circulating among ExPEC disease isolates. Summary of the Invention

[0010] In a first aspect, the present invention relates to a composition comprising Escherichia coli (E. coli) O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75 antigenic polysaccharides, wherein the composition further comprises an O21 antigenic polysaccharide or an O153 antigenic polysaccharide, or an O21 and an O153 antigenic polysaccharide, each of the antigenic polysaccharides independently covalently linked to a carrier protein. In certain embodiments, the O1 antigen is O1A, O4 is glycosylated (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 compositions of the invention comprise a structure as set forth in Table 1 herein.

[0011] In certain embodiments, the compositions of the invention further comprise at least one additional E. coli antigen polysaccharide covalently linked to the carrier protein.

[0012] In certain embodiments, the E. coli O-antigen polysaccharides present in the compositions of the invention consist 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.

[0013] 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), where X can be any amino acid except Pro, preferably a glycosylation consensus sequence having 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.

[0014] In certain embodiments, the E. coli antigen polysaccharide of the composition is covalently linked to the carrier protein by bioconjugation or chemical conjugation. Preferably, the E. coli antigen polysaccharide is covalently linked to the carrier protein by bioconjugation. In certain embodiments, the E. coli antigen polysaccharide is covalently linked to an Asn residue at a glycosylation site in the carrier protein.

[0015] In a second aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a composition described herein.

[0016] In a third aspect, the present invention provides a method of inducing an immune response against Escherichia coli (E. coli), preferably extraintestinal pathogenic E. coli (ExPEC), in a subject, comprising administering to the subject a composition described herein or a pharmaceutical composition described herein. In certain embodiments, the method of inducing an immune response against E. coli limits the severity of or prevents invasive ExPEC disease in the subject, preferably, invasive ExPEC disease includes sepsis and / or bacteremia.

[0017] In a fourth aspect, the present invention provides a recombinant prokaryotic host cell for preparing a bioconjugate of an Escherichia coli (E. coli) O153 antigen polysaccharide covalently linked to a carrier protein, the host cell comprising: a. The nucleotide sequence of the rfb gene cluster of the O153 antigenic polysaccharide; b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser(Thr), where X can be any amino acid except Pro, preferably having SEQ ID NO: 1; and c. A nucleotide sequence encoding the oligosaccharyltransferase PglB A recombinant prokaryotic host cell comprising:

[0018] In certain embodiments, the E. coli O153 antigenic polysaccharide has the 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 wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, preferably from 7 to 25.

[0019] In certain embodiments, PglB in a prokaryotic host cell for preparing a bioconjugate of E. coli O153 comprises the following amino acid mutations relative to wild-type PglB having the amino acid sequence of SEQ ID NO: 4: N311V, K482R, D483H, and A669V. The present invention also includes a method for preparing a bioconjugate of an E. coli O153 antigen polysaccharide covalently linked to a carrier protein, comprising culturing a recombinant prokaryotic host cell described herein to produce the bioconjugate.

[0020] In a fifth aspect, the present invention provides a recombinant prokaryotic host cell for preparing a bioconjugate of an Escherichia coli (E. coli) O21 antigen polysaccharide covalently linked to a carrier protein, the host cell comprising: a. Nucleotide sequence of the rfb gene cluster of the O21 antigen polysaccharide; b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser(Thr), where X can be any amino acid except Pro, preferably having SEQ ID NO: 1; c. a nucleotide sequence encoding the oligosaccharyltransferase PglB, and d. Nucleotide sequence encoding UDP-glucose 4-epimerase A recombinant prokaryotic host cell comprising:

[0021] In certain embodiments, the E. coli O21 antigenic polysaccharide has the formula (O21):

[0022] [ka] wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, and more preferably from 7 to 25.

[0023] In certain embodiments, PglB in a prokaryotic host cell for preparing a bioconjugate of E. coli O21 comprises an amino acid mutation of N311V 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 O21 antigen polysaccharide covalently linked to a carrier protein, comprising culturing a recombinant prokaryotic host cell described herein to produce the bioconjugate. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1. IgG responses induced by O153-EPA bioconjugates. Sprague Dawley rats were immunized three times intramuscularly with formulation buffer or three doses of O153-EPA conjugates (0.04 μg, 0.40 μg, and 4.00 μg O153-EPA). Serum Ab levels were measured by ELISA on days 0, 14, and 42 post-immunization. Individual titers (log EC titers) and GMT ± 95% CI are shown. The dotted line indicates the threshold above which the sample dilution curve is a 4PL fit. [Figure 2] Figure 1. IgG responses induced by O21-EPA bioconjugate. Sprague Dawley rats were immunized three times intramuscularly with formulation buffer or three doses of O21-EPA bioconjugate (0.04 μg, 0.40 μg, 4.00 μg O21-EPA). Serum Ab levels were measured by ELISA on days 0, 14, and 42 post-immunization. Individual titers (log EC50 titers) and GMT ± 95% CI are shown. The dotted line indicates the threshold above which the sample dilution curve is a 4PL fit. [Figure 3]Figure 1. Functionality of antibodies induced by O153-EPA bioconjugate. Sprague Dawley rats were immunized three times intramuscularly with formulation buffer or 4.00 μg / dose of O153-EPA bioconjugate. Antibody-mediated killing of O153 bacteria 42 days post-immunization was measured by OPKA. Individual opsonization index (OI) values ​​and GMT ± 95% CI are shown. ***p<0.001, Wilcoxon rank sum test with Bonferroni correction for multiple comparisons, according to analysis of ELISA results. [Figure 4] Figure 1. Functionality of antibodies induced by O21-EPA bioconjugate. Sprague Dawley rats were immunized three times intramuscularly with formulation buffer or 4.00 μg / dose of O21-EPA bioconjugate. Antibody-mediated killing of O21 bacteria 42 days post-immunization was measured by OPKA. Individual opsonization index (OI) values ​​and GMT ± 95% CI are shown. ***p<0.001, Wilcoxon rank sum test with Bonferroni correction for multiple comparisons, according to analysis of ELISA results. [Figure 5-1] Figure 5. Serum IgG responses induced by ExPEC12V vaccine. Sprague Dawley rats were immunized three times intramuscularly with 4 / 8 μg PS / dose of ExPEC12V (4 μg for all O antigens except O25, which is 8 μg), 0.4 / 0.8 μg PS / dose (0.4 μg for all O antigens except O25, which is 0.8 μg), or formulation buffer. Serum Ab levels specific for 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) were measured by ELISA on days 0, 28, and 42 post-immunization. Individual titers (EC50 titers) and GMT±95% CI are shown. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-1. [Figure 5-4]This is a continuation of Figure 5-1. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition

[0010] Various publications, articles, and patents are cited or described in the background and throughout this specification, each of which is incorporated herein by reference in its entirety. Any discussion of documents, acts, materials, devices, articles and the like which has been included in the present 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 items form part of the prior art with respect to the invention disclosed or claimed.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, specific terms cited herein have the meanings set forth herein. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

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

[0028] The term "about," when used in conjunction with a numerical value, refers to a numerical value within ±10%, such as ±5%, or ±1% of the referenced numerical value.

[0029] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein which equivalents are intended to be encompassed by the present invention.

[0030] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean 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. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," and, when used herein, can also sometimes be replaced with the term "having."

[0031] As used herein, "consisting of" excludes elements, steps, or ingredients not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of aspects or embodiments of the invention, any of the foregoing terms "comprising," "containing," "including," and "having" can be replaced with the terms "consisting of" or "consisting essentially of" to change the scope of the disclosure.

[0032] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are joined by "and / or," the first alternative refers to the applicability of the first element without the second element. The second alternative refers to the applicability of the second element without the first element. The third alternative refers to the applicability of the first and second elements together. Any one of these alternatives is understood to be included within the meaning of, and therefore fulfilling the requirement of, the term "and / or" as used herein. The simultaneous applicability of more than one alternative is also understood to be included within the meaning of, and therefore fulfilling the requirement of, the term "and / or."

[0033] As used herein, the terms "O polysaccharide," "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 sero(sub)type of Gram-negative bacteria. O antigens typically contain two to seven repeating units (RUs) of sugar residues. As used herein, RU is set equal to biological repeating unit (BRU). BRU describes the RU of an O antigen synthesized in vivo. Different serotypes of Escherichia coli (E. coli) express different O antigens. In E. coli, the gene products involved in the biosynthesis of O antigens are encoded by the rfb gene cluster. Reference herein to O-antigen polysaccharides refers to the O-antigen polysaccharide of each E. coli serotype and its existing subserotypes, unless otherwise specified; for example, reference to O1 antigen polysaccharide may refer to the O-antigen polysaccharide of E. coli subserotype O1A, O1A1, O1B, or O1C, while O25 antigen polysaccharide may refer to E. coli O25A or O25B antigen polysaccharide, etc. Many E. coli serotypes and subserotypes and the corresponding structures of the RUs (partial structures, or O units) of their O-antigen polysaccharides are provided in Table 1 of WO 2020 / 039359, which is incorporated herein by reference.

[0034] As used herein, "rfb cluster" and "rfb gene cluster" refer to a gene cluster that encodes the enzymatic machinery capable of synthesizing the O-antigen backbone structure. The term rfb cluster can apply to any O-antigen biosynthetic cluster, and preferably refers to a gene cluster from the genus Escherichia, particularly E. coli.

[0035] As used herein, the term "O1A" refers to the O1A antigen of E. coli (a subserotype of E. coli serotype O1). The term "O2" refers to the O2 antigen of E. coli (E. coli serotype O2). The term "O4" refers to the O4 antigen of E. coli (E. coli serotype O4). The term "O6A" refers to the O6A antigen of E. coli (a subserotype of E. coli serotype O6). The term "O8" refers to the O8 antigen of E. coli (E. coli serotype O8). The term "O15" refers to the O15 antigen of E. coli (E. coli serotype O15). The term "O16" refers to the O16 antigen of E. coli (E. coli serotype O16). The term "O18A" refers to the O18A antigen of E. coli (a subserotype of E. coli serotype O18). The term "O25B" refers to the O25B antigen from E. coli (a subserotype of E. coli serotype O25). The term "O75" refers to the O75 antigen of E. coli (E. coli serotype O75). 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 Escherichia coli (E. coli) (E. coli serotype O4) having a glucose side chain (this term was previously described as "O4Glc+" in WO2020191082 and WO2022 / 058945).

[0036] The structures of several E. coli O-antigen polysaccharides referred to throughout this application are shown in Table 1. A single repeating unit of each E. coli O-antigen polysaccharide is shown.

[0037] [Table 1-1]

[0038] [Table 1-2]

[0039] All monosaccharides described herein have their common meanings as known in the art. Monosaccharides can have the D- or L-configuration. If D or L is not specified, the sugar is understood to have the D-configuration. Monosaccharides are generally referred to by abbreviations commonly known and used in the art. For example, Glc refers to glucose, D-Glc refers to D-glucose, and L-Glc refers to L-glucose. Other common abbreviations for monosaccharides include: Rha for rhamnose, GlcNAc for N-acetylglucosamine, GalNAc for N-acetylgalactosamine, Fuc for fucose, Man for mannose, Man3Me for 3-O-methylmannose, Gal for galactose, FucNAc for N-acetylfucosamine, and Rib for ribose. The suffix "f" refers to furanose, and the suffix "p" refers to pyranose.

[0040] The terms "RU," "repeat unit," and "repeating unit," as used with respect to O antigens, refer to biological repeating units (BRUs) of O antigens synthesized in vivo by cellular machinery (e.g., glycosyltransferases). The number of RUs of an O antigen can vary from serotype to serotype, and in embodiments of the present invention, typically ranges from about 1 to 100 RU, preferably about 1 to 50 RU, 1 to 50 RU, 1 to 40 RU, 1 to 30 RU, 1 to 20 RU, and 1 to 10 RU, more preferably at least 3 RU, at least 4 RU, at least 5 RU, 3 to 50 RU, and preferably 5 to 40 RU, preferably 5 to 30 RU, such as 7 to 25 RU, for example, 10 to 20 RU. However, in some cases, the number of RUs of an O antigen may be 1 to 2. The structure of each O antigen specifically described herein is shown to contain one RU, with the variable "n" specifying the number of RUs. For each O antigen polysaccharide in a bioconjugate of the invention, n is independently an integer between 1 and 100, such as between 1 and 50, 1 and 40, 1 and 30, 1 and 20, 1 and 10, preferably at least 3, more preferably at least 5, such as between 3 and 50, preferably between 5 and 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 between 5 and 30, but optionally can be 1 or 2. In some embodiments, n is independently an integer between about 7 and 25, e.g., between about 10 and 20. This value can vary among the individual O antigen polysaccharides in the composition and is provided herein as an average value. That is, if a bioconjugate is described herein as having n, independently an integer between 5 and 40, the composition will contain a majority of O antigen polysaccharides having between 5 and 40 repeating units, but may also contain O antigen polysaccharides having fewer than 5 repeating units or more than 40 repeating units.

[0041] As used herein, the terms "conjugate" and "glycoconjugate" refer to a sugar or saccharide antigen (e.g., oligosaccharide and polysaccharide)-protein conjugate attached to another chemical species, including, but not limited to, a protein, peptide, lipid, etc. Glycoconjugates can be prepared chemically, for example, by chemical (synthetic) coupling of a protein with a sugar or saccharide antigen. The term glycoconjugate also includes bioconjugates.

[0042] As used herein, in the methods according to embodiments of the present invention, the term "effective amount" in the context of administering an O antigen to a subject refers to an amount of O antigen sufficient to induce a desired immune effect or immune response in the subject. In certain embodiments, "effective amount" refers to an amount of O antigen sufficient to produce immunity in a subject to achieve one or more of the following effects: (i) preventing the occurrence or development of ExPEC infection, preferably invasive ExPEC disease, or symptoms associated therewith; (ii) preventing the recurrence of ExPEC infection, preferably invasive ExPEC disease, or symptoms associated therewith; (iii) preventing, reducing, or ameliorating the severity of ExPEC infection, preferably invasive ExPEC disease, or symptoms associated therewith; (iv) shortening the duration of ExPEC infection, preferably invasive ExPEC disease, or symptoms associated therewith; (v) shortening the duration of ExPEC infection, preferably invasive ExPEC disease, or symptoms associated therewith; (vi) causing regression of ExPEC infection or symptoms associated therewith; (vii) preventing or reducing organ failure associated with ExPEC infection; (viii) reducing the chance or frequency of hospitalization in a subject with an ExPEC infection; (ix) shortening the length of hospitalization in a subject with an ExPEC infection; (x) extending the survival of a subject with an ExPEC infection, preferably invasive ExPEC disease; (xi) eliminating ExPEC infection, preferably invasive ExPEC disease; (xii) inhibiting or reducing ExPEC replication; and / or (xiii) enhancing or improving the prophylactic or therapeutic effect of another therapy.

[0043] The "effective amount" may vary depending on various factors, such as the subject's physical condition, age, weight, and health status; the route of administration, such as oral or parenteral; the target O antigen, other co-administered O antigens, the administration composition, such as adjuvants; and the specific disease for which immunization is desired. When the O antigen is covalently bound to a protein carrier, the effective amount for the O antigen is calculated based solely on the weight of the O antigen polysaccharide portion in the conjugate. All concentrations, amounts, and ratios of conjugates, including bioconjugates described herein, are also calculated solely on the weight of the O antigen polysaccharide portion in the conjugate, regardless of the concentration, amount, or ratio of the conjugated carrier protein, unless otherwise specified. For example, administering 16 μg of a particular bioconjugate means that the administered bioconjugate contains 16 μg of the particular O antigen polysaccharide; the amount of the conjugated carrier protein is not included in this figure. As another example, when a composition is said to contain conjugates of O antigen polysaccharides from serogroups A and B in a 2:1 ratio, this indicates that the concentration or amount of conjugated O antigen polysaccharide A is twice that of conjugated O antigen polysaccharide B, based on the weight of the conjugated O antigen polysaccharide in the composition, not taking into account the weight of the conjugated carrier protein.

[0044] As used herein, the term "invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED)" refers to an acute illness consistent with a systemic bacterial infection, confirmed microbiologically either by the isolation and identification of E. coli from the blood or other normally sterile body sites, or by the isolation and identification of E. coli from the urine of a patient presenting with signs and symptoms of invasive disease (systemic inflammatory response syndrome (SIRS), sepsis, or septic shock) and no other source of infection can be identified. In certain embodiments, IED refers to an acute illness consistent with a systemic bacterial infection, confirmed microbiologically either 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 with life-threatening organ dysfunction due to a dysregulated host response to infection originating from the urinary tract and / or male genital tract and no other source of infection can be identified.

[0045] IEDs include, but are not necessarily limited to, urinary tract infections (UTIs), surgical site infections, abdominal or pelvic infections, pneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, wound infections, pyelonephritis, prostate biopsy-related infections (such as transrectal ultrasound-guided prostate needle biopsy [TRUS-PNB]-related infections), urosepsis, meningitis, peritonitis, cholangitis, soft tissue infections, pyomyositis, septic arthritis, endophthalmitis, suppurative thyroiditis, sinusitis, endocarditis, neutropenic fever, and prostatitis (including but not limited to acute bacterial prostatitis).

[0046] In certain preferred embodiments, the IED comprises sepsis. In certain preferred embodiments, the IED comprises bacteremia. In certain embodiments, the present invention provides a composition according to the present invention for preventing sepsis caused by Escherichia coli (E. coli). In certain embodiments, the present invention provides a composition according to the present invention for preventing bacteremia caused by Escherichia coli (E. coli).

[0047] The term "IED event meeting the criteria for sepsis" refers to an IED case that includes evidence of life-threatening organ dysfunction due to dysregulation of the host response to infection. An IED case meets the criteria for sepsis if there is an acute change in the total Sequential Organ Failure Assessment (SOFA) score of 2 or more points from baseline and is considered secondary to IED. In certain embodiments, the present invention provides a composition described herein for preventing IED that meets the criteria for sepsis. As used herein, the term "urosepsis" refers to sepsis caused by an infection originating from the genitourinary tract and / or male reproductive tract.

[0048] The term "bacteremic IED" refers to an IED case involving the isolation and identification of Escherichia coli (E. coli) from blood. In certain embodiments, the present invention provides a composition according to the present invention for preventing bacteremic IED.

[0049] As used herein, an "immunological response" or "immune response" to an antigen or composition refers to the development in a subject of a humoral and / or cellular immune response to the antigen or antigens present in the composition.

[0050] As used herein, a "composition" comprising two or more E. coli antigenic polysaccharides can be a single pharmaceutical composition comprising two or more E. coli antigenic polysaccharides in the same pharmaceutical composition, or a combination of two or more pharmaceutical compositions comprising two or more E. coli antigenic polysaccharides in separate pharmaceutical compositions. In a preferred embodiment, the composition is a single pharmaceutical composition. In a method for inducing an immune response against E. coli, a "composition" comprising two or more E. coli antigenic polysaccharides can be administered to a subject in need thereof together in a single pharmaceutical composition comprising two or more E. coli antigenic polysaccharides, or can be administered to a subject in combination in separate pharmaceutical compositions. In a preferred embodiment, a single pharmaceutical composition is administered to a subject.

[0051] As used herein, the terms "in combination" or "combination of" in the context of administering two or more O antigens or compositions to a subject does not restrict 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), simultaneously with, or subsequent 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 after) administration of a second composition to a subject. Preferably, the two or more O antigens are administered to the subject substantially simultaneously, e.g., within 5 minutes of each other, more preferably by administering at least two compositions simultaneously, and most preferably by administering a single composition comprising the two or more O antigens.

[0052] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human, that is to be vaccinated or has been vaccinated by the methods or compositions according to the embodiments of the present invention. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, most preferably humans. The terms "subject" and "patient" may be used interchangeably herein.

[0053] The term "percent sequence identity (%)" or "% identity" refers to the number of identical amino acid matches ("hits") between two or more aligned amino acid sequences compared to the number of amino acid residues that make up the entire length of the amino acid sequence. In other words, alignment can be used to determine the percentage of identical amino acid residues (e.g., 90%, 95%, 97%, or 98% identity) between two or more sequences when the sequences are compared and aligned for maximum matching, as measured using a sequence comparison algorithm known in the art, or when aligned manually and visually inspected. Thus, the sequences compared to determine sequence identity may differ due to amino acid substitutions, additions, or deletions. Suitable programs for aligning protein sequences are known to those skilled in the art. The percentage sequence identity of protein sequences can be determined using programs such as, for example, CLUSTALW, Clustal Omega, FASTA or BLAST, e.g., the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

[0054] For example, in the case of amino acid sequences, sequence identity and / or similarity can be determined using standard techniques known in the art, 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.), the BestFit sequence program described in Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by inspection. In certain embodiments, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0055] 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 was obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to the default values.

[0056] A further useful algorithm is Gapped BLAST, as described by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402.

[0057] It is shown herein that vaccination or immunization with a composition comprising Escherichia coli (E. coli) O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75 antigenic polysaccharides, and further comprising O153 or O21, or O153 and O21 antigenic polysaccharides, each independently covalently linked to a carrier protein, induces a strong and robust immune response against all of O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75, as well as O153 and / or O21.

[0058] Thus, in a first aspect, the present invention provides a composition comprising Escherichia coli (E. coli) O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O153 antigenic polysaccharides, each of the antigenic polysaccharides independently covalently linked to a carrier protein.

[0059] In certain embodiments, the concentration of O25 and / or O75 antigenic polysaccharides is increased independently relative to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18 and O153 antigenic polysaccharides.

[0060] In certain embodiments, the weight ratio of the concentration of O25 antigenic polysaccharide to the concentration of each of the O1, O2, O4, O6, O8, O15, O16, O18, O75 and O153 antigenic polysaccharides is from about 1.5:1 to about 2.5:1.

[0061] In certain embodiments, the weight ratio of the concentration of O25 antigenic polysaccharide to the concentration of each of the O1, O2, O4, O6, O8, O15, O16, O18, O75 and O153 antigenic polysaccharides is about 2:1.

[0062] In certain embodiments, the weight ratio of the concentration of O25 and O75 antigenic polysaccharides to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, and O153 antigenic polysaccharides is from about 1.5:1 to about 2.5:1.

[0063] In certain embodiments, the weight ratio of the concentration of O25 and O75 antigenic polysaccharides to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, and O153 antigenic polysaccharides is about 2:1.

[0064] In certain embodiments, the weight ratio of concentrations of E. coli antigenic polysaccharides O1:O2:O4:O6:O8:O15:O16:O18:O153:O25:O75 is 1:1:1:1:1:1:1:1:1:1:2:2.

[0065] In a further aspect, the present invention provides a composition comprising E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75 and O21 antigenic polysaccharides, each of the antigenic polysaccharides independently covalently linked to a carrier protein.

[0066] In certain embodiments, the concentration of O25 and / or O75 antigenic polysaccharides is increased independently relative to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18 and O21 antigenic polysaccharides.

[0067] In certain embodiments, the weight ratio of the concentration of O25 antigenic polysaccharide to the concentration of each of the O1, O2, O4, O6, O8, O15, O16, O18, O75, and O21 antigenic polysaccharides is from about 1.5:1 to about 2.5:1.

[0068] In certain embodiments, the weight ratio of the concentration of O25 antigenic polysaccharide to the concentration of each of the O1, O2, O4, O6, O8, O15, O16, O18, O75 and O21 antigenic polysaccharides is about 2:1.

[0069] In certain embodiments, the weight ratio of the concentration of O25 and O75 antigenic polysaccharides to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, and O21 antigenic polysaccharides is from about 1.5:1 to about 2.5:1.

[0070] In certain embodiments, the weight ratio of the concentration of O25 and O75 antigenic polysaccharides to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, and O21 antigenic polysaccharides is about 2:1.

[0071] In certain embodiments, the weight ratio of concentrations of E. coli antigenic polysaccharides O1:O2:O4:O6:O8:O15:O16:O18:O21:O25:O75 is 1:1:1:1:1:1:1:1:1:1:2:2.

[0072] In yet a further aspect, the present invention provides a composition comprising E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, O153 and O21 antigenic polysaccharides, each of the antigenic polysaccharides independently covalently linked to a carrier protein.

[0073] In certain embodiments, the concentration of O25 and / or O75 antigenic polysaccharides is increased independently relative to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, O153 and O21 antigenic polysaccharides.

[0074] In certain embodiments, the weight ratio of the concentration of O25 antigenic polysaccharide to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, O75, O153, and O21 antigenic polysaccharides is from about 1.5:1 to about 2.5:1.

[0075] In certain embodiments, the weight ratio of the concentration of O25 antigenic polysaccharide to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, O75, O153 and O21 antigenic polysaccharides is about 2:1.

[0076] In certain embodiments, the weight ratio of the concentration of O25 and O75 antigenic polysaccharides to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, O153, and O21 antigenic polysaccharides is from about 1.5:1 to about 2.5:1.

[0077] In certain embodiments, the weight ratio of the concentration of O25 and O75 antigenic polysaccharides to the concentration of each of O1, O2, O4, O6, O8, O15, O16, O18, O153 and O21 antigenic polysaccharides is about 2:1.

[0078] In certain embodiments, the weight ratio of concentrations of E. coli antigenic polysaccharides O1:O2:O4:O6:O8:O15:O16:O18:O21:O153:O25:O75 is 1:1:1:1:1:1:1:1:1:1:1:2:2.

[0079] In certain embodiments described herein, the O1 antigen is O1A, O4 is glycosylated (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 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 of 1 to 100, preferably 3 to 50, such as 5 to 40, preferably 5 to 30, such as 7 to 25, for example 10 to 20.

[0080] 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 between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O1 antigen polysaccharide is part of a chemical or bioconjugate and is coupled 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 obtained by bioconjugation.

[0081] In one embodiment, O2 antigen polysaccharides are 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, where n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O2 antigen polysaccharide is part of a chemical or bioconjugate and is coupled to a carrier protein, such as EPA or CRM. 197In a preferred embodiment, the O2 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably obtained by bioconjugation.

[0082] In one embodiment, the O4 antigen polysaccharide is used in the compositions provided herein. In a specific embodiment, the O4 antigen polysaccharide is a glycosylated O4 antigen polysaccharide, which in a specific embodiment comprises a structure of formula (O4A), as shown in Table 1, wherein n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O4 antigen polysaccharide is part of a chemical or bioconjugate, and is coupled 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 obtained by bioconjugation.

[0083] 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, where n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O6 antigen polysaccharide is part of a chemical or bioconjugate and is coupled 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 obtained by bioconjugation.

[0084] 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 between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O8 antigen polysaccharide is part of a chemical or bioconjugate and is coupled 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 obtained by bioconjugation.

[0085] In one embodiment, an O15 antigenic polysaccharide is used in the compositions provided herein. In a specific embodiment, the O15 antigenic polysaccharide comprises a structure of formula (O15) as shown in Table 1, wherein n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O15 antigenic polysaccharide is part of a chemical or bioconjugate and is coupled 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 obtained by bioconjugation.

[0086] In one embodiment, an O16 antigenic polysaccharide is used in the compositions provided herein. In a specific embodiment, the O16 antigenic polysaccharide comprises a structure of formula (O16) as shown in Table 1, wherein n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O16 antigenic polysaccharide is part of a chemical or bioconjugate and is coupled to a carrier protein, such as EPA or CRM. 197In a preferred embodiment, the O16 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably obtained by bioconjugation.

[0087] In one embodiment, an O18 antigenic polysaccharide is used in the compositions provided herein. In a specific embodiment, the O18 antigenic polysaccharide comprises a structure of formula (O18A) as shown in Table 1, wherein n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O18 antigenic polysaccharide is part of a chemical or bioconjugate and is coupled 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 obtained by bioconjugation.

[0088] In one embodiment, O25 antigen polysaccharides are used in the compositions provided herein. In a specific embodiment, the O25 antigen polysaccharide comprises an O25B antigen polysaccharide, which in a specific embodiment comprises a structure of formula (O25B) as shown in Table 1, where n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O25 antigen polysaccharide is part of a chemical or bioconjugate and is coupled 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 obtained by bioconjugation.

[0089] In one embodiment, O75 antigenic polysaccharides are used in the compositions provided herein. In a specific embodiment, the O75 antigenic polysaccharide comprises an O75 antigenic polysaccharide, which in a specific embodiment comprises a structure of formula (O75), as shown in Table 1, wherein n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O75 antigenic polysaccharide is part of a chemical or bioconjugate, and is coupled 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 obtained by bioconjugation.

[0090] In one embodiment, O153 antigenic polysaccharides are used in the compositions provided herein. In a specific embodiment, the O153 antigenic polysaccharide comprises an O153 antigenic polysaccharide, which in a specific embodiment comprises a structure of formula (O153), as shown in Table 1, wherein n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, for example between 10 and 20. Preferably, the O153 antigenic polysaccharide is part of a chemical or bioconjugate, and is coupled 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 obtained by bioconjugation.

[0091] In one embodiment, O21 antigen polysaccharides are used in the compositions provided herein. In a specific embodiment, the O21 antigen polysaccharide comprises an O21 antigen polysaccharide, which in a specific embodiment comprises a structure of formula (O21) as shown in Table 1, wherein n is an integer between 1 and 100, preferably between 3 and 50, such as between 5 and 40, preferably between 5 and 30, such as between 7 and 25, such as between 10 and 20. Preferably, the O21 antigen polysaccharide is part of a chemical or bioconjugate and is coupled to a carrier protein, such as EPA or CRM.197 In a preferred embodiment, the O21 antigen polysaccharide is covalently linked to an Asn residue in the EPA carrier protein, preferably obtained by bioconjugation.

[0092] In certain embodiments, the compositions described herein further comprise at least one additional E. coli O-antigen polysaccharide covalently linked to the carrier protein.

[0093] 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 compositions do not contain additional E. coli O-antigen polysaccharides.

[0094] 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 compositions do not contain additional E. coli O-antigen polysaccharides.

[0095] 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 compositions do not contain additional E. coli O-antigen polysaccharides.

[0096] In certain embodiments, each carrier protein is selected from the group consisting of detoxified exotoxin A (EPA) of P. aeruginosa, flagellin (FliC) of Escherichia coli, and CRMP. 197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified S. aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli (E. coli) heat-labile enterotoxin, a detoxified mutant of E. coli (E. coli) heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, a detoxified mutant of cholera toxin, E. coli (E. coli) Sat protein, a passenger domain of E. coli (E. coli) Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), P. aeruginosa (PcrV), outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from nontypable Haemophilus influenzae.

[0097] In certain embodiments, the carrier protein is detoxified exotoxin A (EPA) or CRM of Pseudomonas aeruginosa. 197 The amino acid sequence of the CRM197 protein is shown in SEQ ID NO:5.

[0098] For preparation of bioconjugates, the carrier protein is preferably encoded on the host cell, for example, on a plasmid.

[0099] In certain embodiments, the carrier protein is detoxified P. aeruginosa exotoxin A. Various detoxified EPA protein variants have been described in the literature and can be used as carrier proteins. In certain embodiments, the EPA carrier protein contains 1 to 20 glycosylation sites, preferably 1 to 10 glycosylation sites, preferably 2 to 4 glycosylation sites, and more preferably 4 glycosylation sites, each containing the glycosylation consensus sequence Asn-X-Ser(Thr), where X can be any amino acid except Pro, more preferably the amino acid sequence of SEQ ID NO: 1.

[0100] In some embodiments, the EPA carrier protein comprises four glycosylation sites each comprising a glycosylation consensus sequence, e.g., a glycosylation site comprising the glycosylation consensus sequence having SEQ ID NO: 1. As used herein, "EPA-4 carrier protein" and "EPA-4" refer to the detoxified exotoxin A carrier protein of P. aeruginosa, which comprises four glycosylation sites each comprising the glycosylation consensus sequence having SEQ ID NO: 1. An illustrative preferred example of an EPA-4 carrier protein is the EPA carrier protein comprising the amino acid sequence of SEQ ID NO: 2.

[0101] In certain embodiments, the E. coli antigen polysaccharide is covalently linked to the carrier protein by bioconjugation or chemical conjugation. Chemical conjugation includes, for example, reductive amination chemistry (RAC), single-end conjugation, conjugation with a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, cyanylation chemistry (CNBr, CDAP) with or without an ADH spacer, thioether chemistry (maleimide / bromoacetyl linker-based), or EDC-N-hydroxysuccinimide zero linker chemistry. Methods for producing glycoconjugates of E. coli O antigen conjugated to carrier proteins using chemical conjugation to carrier proteins, and compositions containing such glycoconjugates, are also described, for example, in International Publication Nos. 2020 / 039359 and 2022 / 058645, which are incorporated herein by reference.

[0102] Preferably, the term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an O antigen, preferably an Escherichia coli (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, where the host cell machinery links the antigen to the protein (e.g., N-linked), preferably conjugated in vivo within the host cell (see, e.g., the examples herein, and e.g., WO 03 / 074687, WO 2006 / 119987, WO 2015 / 124769, WO 2020 / 191082, WO 2020 / 191088, WO 2022 / 058945). Conjugates prepared using lysates of such cells, i.e., in vitro, but using the same glycosylation machinery (see, e.g., WO 2017 / 117539, U.S. Pat. No. 10,829,795, WO 2020 / 146814), are also considered bioconjugates. Because bioconjugates are prepared by host cell machinery in or in conjunction with host cells, the antigen and protein are covalently linked in the bioconjugate via a glycosidic linkage or bond. Bioconjugates can be prepared in recombinant host cells (or lysates thereof) engineered to express the cellular machinery necessary for synthesis of the O antigen and / or linkage of the O antigen to the target protein. The bioconjugates described herein have advantageous properties compared to chemically prepared glycoconjugates, in which glycans are purified from bacterial cell walls and then chemically conjugated to a carrier protein; for example, bioconjugates require fewer chemicals in their manufacture, are more consistent in the final product produced, and contain less or no free (i.e., not bound to a carrier protein) glycans. Purification of O antigens liberated from lipid A and subsequent chemical conjugation to a carrier protein is a lengthy and laborious process.Furthermore, purification, lipid A detoxification, and chemical conjugation steps can result in epitope loss, antigen heterogeneity, and reduced immunogenicity of the conjugated polysaccharide. Synthesis of glycoconjugates by bioconjugation overcomes these limitations of classical purification and chemical conjugation. Therefore, in typical embodiments, bioconjugates are preferred over chemically produced glycoconjugates. The preparation of bioconjugates for Escherichia coli (E. coli) O1, O2, O6, and O25 antigens is described in detail in WO 2015 / 124769 and WO 2017 / 035181. The preparation of bioconjugates for E. coli O4, particularly O4A, and E. coli O1, O2, O6, O8, O15, O16, O18, O25, and O75 is described in detail in WO 2020 / 191082, the preparation of bioconjugates for E. coli O18 is described in detail in International Application No. PCT / IB2022 / 053013, and the preparation of conjugate compositions having E. coli O75 and other E. coli O antigen conjugates is described in detail in WO 2022 / 058945.

[0103] Suitable host cells for the preparation of bioconjugates are described, for example, in WO 2020 / 191082 and WO 2022 / 058945.

[0104] Thus, in certain embodiments, the present invention provides host cells (eg, prokaryotic host cells) capable of producing bioconjugates of E. coli O153 antigen polysaccharides.

[0105] In certain embodiments, the present invention provides host cells (eg, prokaryotic host cells) capable of producing bioconjugates of E. coli O21 antigen polysaccharides.

[0106] The host cells provided herein are preferably modified (e.g., via genetic engineering) to contain one or more nucleic acids encoding the host cell machinery (e.g., glycosyltransferases) used to produce the E. coli O153 antigen bioconjugates or the E. coli O21 antigen bioconjugates, respectively. In certain embodiments, the prokaryotic cell is a Gram-negative bacterium. Exemplary prokaryotic host cells for use in producing bioconjugates comprising the E. coli O153 or O21 antigen polysaccharides described herein include, but are not limited to, Escherichia spp., Shigella spp., Klebsiella spp., Xhantomonas spp., Salmonella spp., Yersinia spp., Lactococcus spp., Lactobacillus spp., Pseudomonas spp., Corynebacterium spp., Streptomyces spp., Streptococcus spp., Staphylococcus spp., Bacillus spp., and Clostridium spp.

[0107] In certain embodiments, the host cell used to produce the O153 or O21 antigen polysaccharide bioconjugate is Escherichia coli (E. coli). In certain embodiments, the host cell is a K-12 strain of E. coli (as a non-limiting example, E. coli W3110 is a K-12 strain), or a B strain of E. coli (as a non-limiting example, E. coli BL21 is a B strain), or any other well-defined strain of E. coli, e.g., a laboratory strain or a production strain, as opposed to a primary wild-type isolate.

[0108] In certain embodiments, host cells used to produce the O153 antigen polysaccharides and bioconjugates described herein are engineered to contain heterologous nucleic acid, e.g., a heterologous nucleic acid comprising the rfb gene cluster of an O153 antigen serotype, a heterologous nucleic acid encoding one or more carrier proteins and / or glycosyltransferases.

[0109] 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.

[0110] In specific embodiments, 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 the host cells described herein. Such nucleic acids can encode proteins including, but not limited to, oligosaccharyltransferases and / or glycosyltransferases.

[0111] Host cells capable of producing bioconjugates of E. coli O153 antigenic polysaccharide covalently linked to carrier proteins provided herein further comprise the nucleotide sequence of the rfb gene cluster of E. coli O153 antigenic polysaccharide. In certain non-limiting exemplary embodiments, the rfb gene cluster useful for producing E. coli O153 antigenic polysaccharide has the sequence of SEQ ID NO: 3. Another example can be found at GenBank locus KJ755551.

[0112] A host cell capable of producing a bioconjugate of E. coli O21 antigen polysaccharide covalently linked to a carrier protein provided herein as SEQ ID NO:4 further comprises the nucleotide sequence of the rfb gene cluster of the E. coli O21 antigen polysaccharide and a nucleotide sequence encoding a UDP-glucose 4-epimerase. In certain non-limiting exemplary embodiments, the rfb gene cluster useful for producing the E. coli O21 antigen polysaccharide has the sequence of SEQ ID NO:6. Another example can be found at GenBank locus EU694098. In certain non-limiting exemplary embodiments, the nucleotide sequence encoding the UDP-glucose 4-epimerase useful for producing the E. coli O21 antigen polysaccharide has the sequence of SEQ ID NO:7. In certain non-limiting embodiments, the UDP-glucose 4-epimerase enzyme has the amino acid sequence set forth in SEQ ID NO:8.

[0113] Modified nucleic acid sequences encoding the same enzymes as those encoded by the provided rfb cluster sequences or UDP-glucose 4-epimerase nucleotide sequences, 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, and have at least qualitatively the same enzymatic function as the corresponding reference enzyme encoded by the rfb gene cluster or UDP-glucose 4-epimerase (i.e., are 100% identical to the encoded enzyme sequence), can also be used.

[0114] Host cells capable of producing bioconjugates of E. coli O153 or O21 antigen polysaccharides further contain a nucleic acid encoding an oligosaccharyltransferase. The oligosaccharyltransferase transfers lipid-linked oligosaccharides to asparagine residues of nascent polypeptide chains containing an N-glycosylation consensus motif. The nucleic acid encoding the oligosaccharyltransferase can be native to the host cell or can be introduced into the host cell using genetic approaches. In a preferred embodiment, the oligosaccharyltransferase is heterologous to the host cell. Because E. coli does not naturally contain oligosaccharyltransferases, when E. coli is used as a host cell for producing bioconjugates, a heterologous oligosaccharyltransferase is introduced into such host cells, for example, by genetic engineering. In view of the present disclosure, the oligosaccharyltransferase can be obtained from any source known in the art.

[0115] In certain embodiments, alternatives to oligosaccharyltransferases having N-glycosyltransferase activity, such as O-glycosyltransferases, including, but not limited to, PglL, can be used in combination with their own, different, glycosylation consensus sequences in a carrier protein, as described, for example, in WO 2016 / 82597 and WO 2020 / 120569. Thus, other glycosyltransferases, such as O-glycosyltransferases, can also be used as oligosaccharyltransferases according to the present invention.

[0116] In certain preferred embodiments, the oligosaccharyltransferase is derived from Campylobacter. For example, in one embodiment, the oligosaccharyltransferase is derived from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al., 2002, Science 298:1790-1793; see also, e.g., NCBI Gene ID: 3231775, UniProt Accession No. O86154). In another embodiment, the oligosaccharyltransferase is derived from Campylobacter lari (see, e.g., NCBI Gene ID: 7410986).

[0117] In certain embodiments, the oligosaccharyltransferase is a PglB oligosaccharyltransferase from Campylobacter jejuni, including the native (wild-type) protein or any variant thereof, as described in International Patent Applications WO 2016 / 107818 and WO 2016 / 107819. In certain embodiments, the PglB oligosaccharyltransferase comprises SEQ ID NO: 4, or a variant thereof. In certain embodiments, one or more endogenous glycosylation consensus sequences of wild-type PglB have been mutated to avoid PglB autoglycosylation, e.g., SEQ ID NO: 4, including the mutation N534Q. Examples of mutant PglB oligosaccharyltransferases suitable for use in the recombinant host cells provided herein include the PglB oligosaccharyltransferase of SEQ ID NO: 4, which contains at least one mutation selected from the group consisting of N311V, K482R, D483H, A669V, Y77H, S80R, Q287P, and K289R. In a particular embodiment, the mutant PglB oligosaccharyltransferase has SEQ ID NO: 4, which contains the mutation N311V. In a preferred embodiment for the production of a bioconjugate of Escherichia coli (E. coli) O153 antigen polysaccharide covalently linked to a carrier protein, the mutant PglB oligosaccharyltransferase has SEQ ID NO: 4, which contains the mutations N311V, K482R, D483H, and A669V. In a preferred embodiment for the production of a bioconjugate of E. coli O21 antigen polysaccharide covalently linked to a carrier protein, the mutant PglB oligosaccharyltransferase has SEQ ID NO: 4, which contains the mutation N311V.

[0118] Additional 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 further have oligosaccharyl transferase activity, and optionally have one or more particular 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. A preferred PglB enzyme for producing bioconjugates of O antigens from E. coli serotypes O1, O2, O4, O6, O8, O15, O16, O18, O25, and O75 is described in WO 2020 / 191088.

[0119] In certain embodiments, additional modifications can be introduced into the host cells described herein (e.g., using recombinant techniques). For example, host cell nucleic acids (e.g., genes) encoding proteins that form part of a glycosylation pathway that potentially compete or interfere (e.g., compete or interfere with one or more heterologous genes involved in glycosylation that are recombinantly introduced into the host cell) can be deleted or modified in the host cell background (genome) in a manner that renders them inactive / non-functional (i.e., the deleted / modified host cell nucleic acid does not encode a functional protein). In certain embodiments, when a nucleic acid is deleted from the genome of a host cell provided herein, the nucleic acid is replaced with a desired sequence, e.g., a sequence useful for producing an O-antigen polysaccharide or a bioconjugate thereof.

[0120] Exemplary genes or gene clusters that can be deleted in a host cell (and optionally replaced with other desired nucleic acid sequences) include host cell genes or gene clusters involved in glycolipid biosynthesis, such as waaL, the lipid A core biosynthetic cluster (waa), the galactose cluster (gal), the arabinose cluster (ara), the colonic acid cluster (wc), the capsular polysaccharide cluster, the undecaprenol-p biosynthetic genes (e.g., uppS, uppP), the und-P recycling genes, metabolic enzymes involved in nucleotide-activated sugar biosynthesis, the enterobacterial common antigen cluster (eca), the prophage O-antigen modification cluster or region thereof, such as the gtrABS cluster, and the like.

[0121] In specific embodiments, the waaL gene is deleted or functionally inactivated from the genome of a host cell (e.g., a recombinant host cell) provided herein. The terms "waaL" and "waaL gene" refer to an O antigen ligase gene that encodes a membrane-bound enzyme with an active site located in the periplasm. The encoded enzyme transfers undecaprenyl phosphate (UPP)-linked O antigen to the lipid A core to form lipopolysaccharide. Deletion or disruption of the endogenous waaL gene (e.g., a ΔwaaL strain) can inhibit the transfer of O antigen to lipid A and instead promote the transfer of O antigen to another biomolecule, such as a carrier protein.

[0122] In another specific embodiment, one or more of the waaL gene, the gtrA gene, the gtrB gene, the gtrS gene, and the rfb gene cluster are deleted or functionally inactivated from the original genome of a host cell capable of producing a bioconjugate, e.g., an E. coli O153 antigen polysaccharide or an E. coli O21 antigen polysaccharide.

[0123] In certain embodiments, the host cell capable of producing a bioconjugate of the E. coli O153 or O21 antigen polysaccharide further comprises a nucleic acid encoding a carrier protein as described previously herein.

[0124] 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 the glycosylation consensus sequence Asn-X-Ser(Thr), where X can be any amino acid except Pro. Preferably, the Asn residue is present in a glycosylation site comprising the glycosylation consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), where X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 1). The carrier protein can contain 1 to 10 glycosylation sites, preferably 2, 3, or 4 glycosylation sites, and most preferably 4 glycosylation sites, each containing a glycosylation consensus sequence. In certain embodiments, the carrier protein is an EPA-4 carrier protein, for example, an EPA-4 carrier protein comprising the amino acid sequence of SEQ ID NO:2.

[0125] In a further aspect, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and / or excipient and a composition described herein. As used herein, the term "pharmaceutically acceptable" means approved by federal or state regulatory authorities or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. The term "carrier," when used in the context of a pharmaceutically acceptable carrier herein, refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Aqueous solutions of saline, dextrose, and glycerol may also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in textbooks known to those skilled in the art of pharmaceutical formulation.

[0126] In certain embodiments, the compositions described herein (e.g., immunogenic compositions) comprise or are administered in combination with an adjuvant. In some embodiments, the term "adjuvant" refers to a compound that, when administered with or as part of a composition described herein, enhances, potentiates, and / or boosts the immune response to the (bio)conjugate, but does not generate an immune response to the (bio)conjugate when the adjuvant compound is administered alone. Examples of suitable adjuvants include aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum oxide, and the like, including nanoparticle or nanoalum formulations containing 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, U.S. Pat. No. 6,491,919), AS01, AS02, AS03, and AS04 (all GlaxoSmithKline; see, e.g., EP 1126876 for AS04, U.S. Pat. No. 6,491,919). Examples of suitable cyclic nucleotides include, but are not limited to, EP 7357936, and for AS02 see EP 0671948, EP 0761231, and U.S. Pat. No. 5,750,110), MF59 (Novartis), imidazopyridine compounds (see WO 2007 / 109812), imidazoquinoxaline compounds (see WO 2007 / 109813), delta-inulin, STING-activating synthetic cyclic dinucleotides (e.g., U.S. Patent Application Publication No. 20150056224), a combination of lecithin and carbomer homopolymer (e.g., U.S. Pat. No. 6,676,958), and saponins such as QuilA, QS21, Matrix M, Iscom, and Iscomatrix, 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 combined with immunostimulants such as monophosphoryl lipid A. Another adjuvant is CpG. A further example of an adjuvant is liposomes containing immunostimulants such as MPL and QS21, such as in AS01E and AS01B. Another example of an adjuvant is imidazoquinoline (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 (glycopyranosyl lipid adjuvant, a 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 the same as that of GLA), 3D-(6-acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-PHAD, and 3D(6A)PHAD are synthetic lipid A variants; see, e.g., avantilipids.com / divisions / adjuvants, which also provides the structures of these molecules), E6020 (CAS No. 287180-63-6), ONO4007, OM-174, and the like.

[0127] In certain preferred embodiments, the compositions described herein do not include an adjuvant other than the bioconjugate and / or are not administered in combination with an adjuvant other than the bioconjugate (should the bioconjugate contain any intrinsic adjuvant properties, in these embodiments, these would be ignored and no exogenous adjuvant would be added).

[0128] Those skilled in the art will recognize that various formulations can be used in the compositions of the present invention. In one embodiment, the compositions of the present invention comprise a (bio)conjugate described herein in Tris-buffered saline (TBS) pH 7.4 (e.g., containing 25 mM, 137 mM, and 2.7 mM Tris, NaCl, and KCl, respectively). In another embodiment, the compositions of the present invention comprise a (bio)conjugate described herein in about 10 mM KH2PO4 / Na2HPO4 buffer, about 5% (w / v) sorbitol, about 10 mM methionine, and about 0.02% (w / v) polysorbate 80, at a pH of about 7.0. In other embodiments, the compositions of the invention comprise a (bio)conjugate described herein in about 10 mM KH2PO4 / Na2HPO4 buffer at about pH 7.0, about 8% (w / v) sucrose, about 1 mM EDTA, and about 0.02% (w / v) polysorbate 80. (See, e.g., WO 2018 / 077853 for suitable buffers for bioconjugates of Escherichia coli (E. coli) O antigen covalently bound to an EPA carrier protein.) In other embodiments, the compositions of the invention comprise a (bio)conjugate described herein in about 5 mM succinic acid / 0.9% NaCl, pH 6.0.

[0129] Typically, the compositions of the invention can be prepared by first obtaining individual glycoconjugates for each of the E. coli O antigen polysaccharides described herein by independently covalently linking these O antigen polysaccharides to a carrier protein, e.g., by chemical or bioconjugation, and then mixing the individual glycoconjugates in the amounts and ratios described herein to obtain the compositions described in the invention. Accordingly, a further aspect of the invention is to provide a method for preparing the compositions described in the invention, which method comprises providing each of the required O antigen conjugates (e.g., by obtaining or manufacturing them, e.g., in the form of a drug substance) and mixing them in the desired ratios and / or amounts to obtain a composition described in the invention (e.g., polyvalent E. coli, particularly ExPEC, vaccine composition, sometimes referred to as a drug product).

[0130] Accordingly, in one aspect, the invention also provides a method for preparing a composition of the invention by producing a bioconjugate of an E. coli O153 antigenic polysaccharide covalently linked to a carrier protein, the method comprising culturing a recombinant prokaryotic host cell of the invention, as described herein, to produce an E. coli O153 bioconjugate of the invention, and mixing the bioconjugate with a further glycoconjugate, preferably a bioconjugate (e.g., a bioconjugate in which each of the E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, and / or O21 antigenic polysaccharides is independently covalently linked to a carrier protein), to obtain a composition according to the invention. In one aspect, the invention also provides a method for preparing a composition of the invention by producing a bioconjugate of an E. coli O21 antigenic polysaccharide covalently linked to a carrier protein, the method comprising culturing a recombinant prokaryotic host cell of the invention, as described herein, to produce an E. coli O21 bioconjugate of the invention, and mixing the bioconjugate with an additional glycoconjugate, preferably a bioconjugate (e.g., a bioconjugate in which each of the E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25, O75, and / or O153 antigenic polysaccharides is independently covalently linked to a carrier protein), to obtain a composition of the invention.

[0131] 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 of inducing an immune response in a subject described herein result in vaccination of the subject against infection or resulting disease by an E. coli strain of the serotype whose O antigen is present in the composition.

[0132] Thus, in a further aspect, the present invention provides a method for inducing an immune response against Escherichia coli (E. coli), preferably extraintestinal pathogenic E. coli (ExPEC), in a subject, comprising administering to the subject a composition as described herein or a pharmaceutical composition as described herein.

[0133] In certain aspects, provided herein are compositions described herein for use in methods of inducing an immune response against Escherichia coli (E. coli), preferably ExPEC, in a subject. In certain aspects, provided herein is use of a composition described herein in the manufacture of a medicament for inducing an immune response against Escherichia coli (E. coli), preferably ExPEC, in a subject.

[0134] In certain embodiments, the subject is a human. In some embodiments, the subject is a human who has or is at risk of having an ExPEC infection or invasive ExPEC disease. In one embodiment, the subject has an Escherichia coli (E. coli) (e.g., ExPEC) infection at the time of administration. In a preferred embodiment, the subject does not have an Escherichia coli (E. coli) (e.g., ExPEC) infection or invasive ExPEC disease at the time of administration.

[0135] In certain embodiments according to the methods of the present invention, the immune response limits the severity of or prevents invasive ExPEC disease in the subject, preferably, the invasive ExPEC disease includes sepsis and / or bacteremia.

[0136] In certain embodiments, the immune response induced in a subject after administration of a composition described herein is effective in preventing or alleviating symptoms resulting from ExPEC infection, preferably in at least 30%, more preferably at least 40%, e.g., at least 50% of subjects to which the composition is administered. Symptoms of ExPEC infection may vary depending on the nature of the infection and may include, but are not limited to, dysuria, increased urinary frequency or urgency, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea (e.g., in subjects with ExPEC urinary tract infection); high fever, headache, stiff neck, nausea, vomiting, convulsions, drowsiness, and / or photosensitivity (e.g., in subjects with ExPEC meningitis); fever, increased heart rate, increased respiratory rate, decreased urine output, decreased platelet count, abdominal pain, dyspnea, and / or abnormal cardiac function (e.g., in subjects with ExPEC sepsis). [Example]

[0137] The following examples of the present invention are presented to further illustrate the nature of the present invention, but it should be understood that the following examples do not limit the present invention, the scope of which is determined by the appended claims.

[0138] [Example 1] Production of bioconjugates of Escherichia coli (E. coli) O153 antigen polysaccharide linked to EPA carrier protein O153 is a serotype of Escherichia coli (E. coli) observed in more than 1% of ExPEC blood culture isolates from patient populations in various regions of the world, with a relatively high prevalence of more than 8% in isolates from South America (Weerdenburg et al., 2022, Clin Infect Dis., doi: 10.1093 / cid / ciac421). Therefore, it would be useful to include in a vaccine composition an antigen capable of protecting against the E. coli O153 serotype, for example, in the form of a conjugate of an 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.

[0139] For the rfb gene cluster, an E. coli O153 blood isolate was selected as the O153 donor (collected in the Netherlands and obtained from Utrecht University Medical Center). The parent strain used was E. coli K12 strain W3110. This strain is available, for example, from the E. coli Genetic Stock Center (Yale University, New Haven, CT, USA, product number CGSC#4474). The genome sequence has been published (PMID: 16738553).

[0140] The parent strain, E. coli K12 strain W3110, was modified by replacing the chromosomal rfb cluster of E. coli W3110 with the rfb cluster of a clinical isolate, E. coli O153 (rfb cluster having SEQ ID NO: 3), as previously described (see, e.g., WO 2020 / 191082). Additionally, the O-antigen ligase waaL was deleted to prevent transfer of O-antigen to lipid A, thereby facilitating transfer of O-antigen to carrier protein. The chromosomal gtrABS gene was disrupted by homologous recombination to prevent the addition of branched glucose to the O153 antigen.

[0141] To confirm the production of O153 antigen, the engineered strain was cultured overnight at 37°C in antibiotic-free LB medium. After harvesting, the cell pellet was resuspended in Laemmli buffer and the proteins were digested with proteinase K. LPS / LLO were separated by SDS-PAGE, and O153 antigen was detected by Western blotting using O153-specific antiserum. The structure of the O153 antigen was confirmed by MALDI-MS / MSMS.

[0142] Plasmids encoding the gene for genetically attenuated Pseudomonas aeruginosa exotoxin (EPA) with two glycosylation sites (EPA-2) or the 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. N311V,K482R,D483H,A669V It was determined that a plasmid encoding PglB (which has the amino acid sequence set forth in SEQ ID NO:4 and is a mutant of PglB with amino acid substitutions N311V, K482R, D483H, and A669V) gave the best results among the mutants tested, which was an unexpected result. Therefore, a plasmid encoding this best PglB mutant was selected and introduced into engineered host cells to generate a production strain for producing a bioconjugate of E. coli O153 antigen polysaccharide linked to the EPA carrier protein.

[0143] To confirm the production of the O153 bioconjugate, the producer strain was grown at 37°C in TB medium supplemented with phosphate buffer, MgCl2, and antibiotics. When the optical density at 600 nm reached approximately 4.0, IPTG and arabinose were added to induce bioconjugate production. After overnight growth, cells were harvested and the bioconjugate was released by osmotic shock. The O153 bioconjugate was separated by SDS-PAGE and detected by Western blotting using O153- and EPA-specific antisera.

[0144] Bioreactor production and purification of the bioconjugates was performed using known methods (see, e.g., WO 2020 / 191082, WO 2022 / 214620).

[0145] [Example 2] Production of bioconjugates of Escherichia coli (E. coli) O21 antigen polysaccharide linked to EPA carrier protein O21 is a serotype of Escherichia coli (E. coli) that has been observed in more than 1% of ExPEC blood culture strains isolated from patient populations in North America (Weerdenburg et al., 2022, Clin Infect Dis., doi: 10.1093 / cid / ciac421). Therefore, it would be useful to include in a vaccine composition an antigen capable of protecting against the E. coli O21 serotype, for example, in the form of a conjugate of an O antigen linked to a carrier protein. This example describes the production of a bioconjugate of the E. coli O21 antigen polysaccharide linked to EPA as a carrier protein.

[0146] An E. coli O21 blood isolate (collected in the Netherlands and obtained from Utrecht University Medical Center) was selected as the O21 donor and examined for the rfb gene cluster and gne gene (encoding UDP-glucose 4-epimerase). The parent strain used was E. coli K12 strain W3110. This strain is available, for example, from the E. coli Genetic Stock Center (Yale University, New Haven, CT, USA, product number CGSC#4474). The genome sequence has been published (PMID: 16738553).

[0147] The parent strain, E. coli K12 strain W3110, was modified by replacing the chromosomal rfb cluster of E. coli W3110 with the rfb cluster of a clinical isolate, E. coli O21 (rfb cluster having SEQ ID NO: 6), as described previously (see, e.g., WO 2020 / 191082), and by introducing the gne gene encoding UDP-glucose 4-epimerase (having SEQ ID NO: 8) in the form of a nucleotide sequence (SEQ ID NO: 7). Additionally, the O-antigen ligase waaL was deleted to prevent transfer of the O-antigen to lipid A, thereby facilitating 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.

[0148] To confirm the production of O21 antigen, the engineered strain was cultured overnight at 37°C in antibiotic-free LB medium. After harvesting, the cell pellet was resuspended in Laemmli buffer and the proteins were digested with proteinase K. LPS / LLO were separated by SDS-PAGE, and O21 antigen was detected by Western blotting using O21-specific antiserum. The structure of the O21 antigen was confirmed by MALDI-MS / MSMS.

[0149] Plasmids encoding the gene for a genetically attenuated Pseudomonas aeruginosa exotoxin with two glycosylation sites (EPA-2) or a gene for a genetically attenuated Pseudomonas aeruginosa exotoxin with four glycosylation sites (EPA-4) were introduced. Several plasmids encoding the oligosaccharyltransferase Campylobacter jejuni PglB were tested in separate experiments. N311V It was determined that a plasmid encoding PglB (which is a mutant of PglB having the amino acid sequence set forth in SEQ ID NO:4 and the amino acid substitution N311V) gave the best results among the mutants tested, confirming that the optimal PglB for bioconjugate preparation varies in unpredictable ways depending on the E. coli serotype (see previous examples and WO 2020 / 191088). Thus, this optimal PglB mutant was introduced into engineered host cells to generate a production strain for producing bioconjugates of E. coli O21 antigen polysaccharide linked to the EPA carrier protein.

[0150] To confirm the production of the O21 bioconjugate, the producer strain was grown at 37°C in TB medium supplemented with phosphate buffer, MgCl2, and antibiotics. When the optical density at 600 nm reached approximately 4.0, IPTG and arabinose were added to induce bioconjugate production. After overnight growth, 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 O21- and EPA-specific antisera.

[0151] Bioreactor production and purification of the bioconjugates was performed using known methods (see, e.g., WO 2020 / 191082, WO 2022 / 214620).

[0152] [Example 3] Immunogenicity of O153 bioconjugates Experimental design Female Sprague Dawley rats (5–6 weeks old) were immunized intramuscularly with 0.04, 0.40, or 4.00 mg of O153-EPA bioconjugate on days 0, 14, and 28. Control groups received formulation buffer alone. Blood samples were collected pre-immunization (day 0) and post-immunization (days 14 and 42). Blood was processed, and serum samples were stored at -20°C. Total IgG Ab responses in serum were measured by ELISA (as described above). Briefly, ELISA plates were coated with O153-LPS, blocked with skim milk, and incubated with rat serum samples. The plates were then incubated with HRP-conjugated goat anti-rat IgG, developed with TMB substrate, and OD450 was measured. EC50 titers, defined as the 50% effective concentration, were calculated based on duplicate 12-step titration curves plotted using a 4PL nonlinear regression model.

[0153] result Immunization with 0.04 μg, 0.40 μg, and 4.00 μg per dose of O153-EPA bioconjugate significantly elevated IgG antibody levels on day 42 compared to formulation buffer (Figure 1). A significant increase in Ab titers was also observed on day 14 in rats immunized with 0.04 μg of conjugate per dose compared to formulation buffer (Figure 1). Furthermore, Ab levels induced by 0.04 μg, 0.40 μg, and 4.00 μg of conjugate were significantly increased on day 42 compared to those detected on days 0 and 14 post-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 administration of 0.04 μg or 4.00 μg of O153-EPA conjugate can induce a significant increase in IgG titers (Figure 1). The significant increase in IgG titers between days 14 and 42 at all three concentrations of bioconjugate indicates that the O153-EPA conjugate can boost antibody responses. The highest Ab titers were detected in the group of animals immunized with the 0.04 μg / dose, followed by the 0.4 μg / dose. A dose-crossover analysis confirmed these findings, suggesting that the lowest dose of O153-EPA conjugate resulted in better Ab responses.

[0154] This example demonstrates that bioconjugates in which the E. coli O153 antigen polysaccharide is covalently linked to a carrier protein are immunogenic.

[0155] [Example 4] Immunogenicity of O21 bioconjugates Experimental design Female Sprague Dawley rats (5–6 weeks old) were immunized intramuscularly with 0.04, 0.40, or 4.00 μg of the O21-EPA bioconjugate on days 0, 14, and 28. A control group received formulation buffer alone. Blood samples were collected pre-immunization (day 0) and post-immunization (days 14 and 42). Blood was processed, and serum samples were stored at -20°C. Total IgG Ab responses in serum were measured by ELISA (as described above). Briefly, ELISA plates were coated with O21-LPS, blocked with skim milk, and incubated with rat serum samples. The plates were then incubated with HRP-conjugated goat anti-rat IgG, developed with TMB substrate, and OD450 was measured. EC50 titers, defined as the 50% effective concentration, were calculated based on duplicate 12-step titration curves plotted using a 4PL nonlinear regression model.

[0156] result Immunization with 0.04 μg, 0.40 μg, and 4.00 μg per dose of O21-EPA bioconjugate significantly elevated IgG antibody levels on day 42 compared to formulation buffer (Figure 2). Furthermore, Ab levels induced by the 0.04 μg, 0.40 μg, and 4.00 μg conjugates were significantly increased on day 42 compared to baseline (day 0) and day 14 post-immunization (Figure 2). The significant increase in IgG titers between days 14 and 42 at all three bioconjugate concentrations indicates that the O21-EPA conjugate can boost antibody responses. A significant decrease in Ab levels was observed on day 42 compared to day 0 in the group administered formulation buffer (Figure 2), although many samples in this group did not show 4PL fitting. The highest titers were detected following immunization with the 0.04 μg / dose. The highest Ab titers were detected in the group of animals immunized with 0.04 μg / dose, and a dose-crossover analysis confirmed this finding, suggesting that the lowest dose of O21-EPA conjugate elicited better Ab responses.

[0157] This example demonstrates that bioconjugates in which the E. coli O21 antigen polysaccharide is covalently linked to a carrier protein are immunogenic.

[0158] [Example 5] Antibody-mediated opsonophagocytic killing induced by O153-EPA or O21-EPA bioconjugates Experimental design The functional activity of serum antibodies was measured by an opsonophagocytic killing assay (OPKA; protocol previously described). Briefly, serum samples were heat-inactivated at 56°C for 30 min and diluted 1:10 (starting dilution), followed by eight three-fold serial dilutions. All serum samples were tested in duplicate. Serum was incubated with bacteria (approximately 103 CFU) at room temperature for 30 min, followed by the addition of complement and HL60 cells and incubation at 37°C for 1 h. The final reaction mixture was spotted onto agar plates and incubated at 33°C for 15–16 h. The number of bacterial colonies on the plates was counted, and the opsonization index (OI), defined as the serum dilution that killed 50% of the bacteria, was calculated using the Opsoiter3 Excel-based program (Department of Microbiology, University of Alabama, Birmingham). Due to the complexity of the assay, antibody functionality was evaluated only in samples from rats immunized on day 42 with formulation buffer or 4.00 μg / dose of O153-EPA / O21-EPA conjugates, respectively.

[0159] result A significant increase in E. coli OIs was observed in serum samples from animals immunized with the O153-EPA conjugate (Figure 3) and the O21-EPA conjugate (Figure 4) compared to the formulation buffer. These results indicate that the O153- and O21-induced antibodies induced by administration of the O153-EPA and O21-EPA conjugates, respectively, are functional and can mediate E. coli killing.

[0160] [Example 6] Production of a composition containing 12 E. coli O-antigen conjugates The preparation of bioconjugates for E. coli O1 (O1A), O2, O4 (O4A), O6 (O6A), O8, O15, O16, O18, O25 (O25B), and O75 is described in detail in WO 2020 / 191082, the preparation of bioconjugates for E. coli O18 is described in detail in International Application No. PCT / IB2022 / 053013, and the preparation of conjugate compositions having E. coli O75 and other E. coli O-antigen conjugates is described in detail in WO 2022 / 058945. Production of E. coli O153 and E. coli O21 bioconjugates was performed as described herein. The 12 conjugates were mixed to provide the immunogenic composition described in Table 2.

[0161] [Table 2]

[0162] A composition comprising 12 bioconjugates is referred to herein as "ExPEC12V."

[0163] [Example 7] Induction of immune responses by ExPEC12V compositions Experimental design Female Sprague-Dawley rats (5-6 weeks old) were immunized intramuscularly with the ExPEC12V vaccine on days 0, 14, and 28. Animals were divided into three different treatment groups: Group 1: 4.0 μg of each polysaccharide (PS) / dose (except for O25B, which received 8.0 μg PS / dose); Group 2: 0.4 μg of each PS / dose (except for O25B, which received 0.8 μg PS / dose); and Group 3: a control group that received formulation buffer. Blood samples were collected pre-immunization (day 0) and post-immunization (days 28 and 42). Serum total IgG levels 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. The plates were then incubated with HRP-conjugated goat anti-rat IgG, developed with TMB substrate, and OD450 was measured. EC50 titers, defined as the 50% effective concentration, were calculated based on duplicate 12-step titration curves plotted using a 4PL nonlinear regression model.

[0164] result Immunization with 0.4 / 0.8 μg PS / dose of ExPEC12V resulted in significantly higher IgG antibody titers on day 42 compared to formulation buffer. Furthermore, the Ab levels induced by 0.4 / 0.8 μg PS / dose of ExPEC12V were significantly increased on day 42 compared to levels detected at baseline (day 0) for all conjugates tested (Figure 5). For many conjugates, except for O1A, O21, and O25B, Ab levels were significantly elevated already on day 28 compared to day 0. Furthermore, for all conjugates with 0.4 / 0.8 μg PS / dose of ExPEC12V, a significant increase in Ab levels on day 42 compared to day 28 was observed, indicating that a booster dose of ExPEC12V administered on day 28 can increase Ab responses (Figure 5).

[0165] Comparing the groups receiving 0.4 / 0.8 μg PS / dose or 4 / 8 μg PS / dose, immunization with 0.4 / 0.8 μg PS / dose induced the highest Ab responses to O2, O4, and O153 conjugates compared with 4.0 / 8.0 μg PS / dose.

[0166] This example demonstrates that E. coli O21 and O153 conjugates are also immunogenic in multivalent vaccine compositions containing several other bioconjugates of E. coli O antigens representing different serotypes, and that other bioconjugates previously shown to be immunogenic in 10-valent compositions (see, e.g., WO 2020 / 191082) remain immunogenic when E. coli O21 and O153 bioconjugates are added to generate a 12-valent composition.

[0167] [Example 8] Ab-mediated opsonophagocytic killing induced by ExPEC12V vaccine Experimental design The functional activity of serum Abs was measured by monoplex opsonophagocytic killing assay (OPKA) for O1A, O2, O4, O8, O15, O16, O18, O21, O75, and O153, and by multiplex 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 min and diluted 1:10 (starting dilution), followed by eight three-fold serial dilutions. The serum was incubated with bacteria for 30 min at room temperature. Subsequently, complement and HL60 cells were added and incubated at 37°C for 1 h. The final reaction mixture was spotted onto agar plates and incubated at 33°C for 15–16 h. Plates containing different types of antibiotics were used to select for specific antibiotic-resistant strains in the reaction mixture. The number of bacterial colonies on the plates was counted, and the opsonization index (OI), defined as the serum dilution that killed 50% of the bacteria, was calculated using the Opsoiter3 Excel-based program (Department of Microbiology, University of Alabama, Birmingham). Due to the complexity of the assay, Ab functionality was evaluated only in samples taken 42 days after immunization.

[0168] result Serum Ab functional activity was measured by OPKA for each serotype included in the ExPEC12V vaccine. Immunization with 0.4 / 0.8 μg / PS / dose of ExPEC12V resulted in significantly higher opsonophagocytic titers against Escherichia coli (E. coli) strains O4, O8, O15, O18, O25B, and O153 on day 42 compared with the formulation buffer (Table 3). For O1A, bacterial kill was observed in only 2 of 15 animals. For O2, bacterial kill was observed in 5 of 15 animals. For O21, bacterial kill was observed in 4 of 15 animals administered 0.4 / 0.8 μg / PS / dose of ExPEC12V. For O6A, sera from 13 of 15 animals mediated killing, whereas sera from only 7 of 15 animals in the buffer-treated group mediated killing; 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, but killing was also observed in the buffer-treated group, indicating no significant difference in opsonophagocytic titers for these serotypes. Immunization with 4 / 8 μg / PS / dose of ExPEC12V resulted in significantly higher opsonophagocytic titers on day 42 only against Escherichia coli O4 and O15 strains compared with the formulation buffer. For O8, deaths were observed in only 6 of 15 animals, for O21, deaths were observed in 3 of 15 animals, for O25B, deaths were observed in 4 of 15 animals administered 4 / 8 μg / PS / dose of ExPEC12V, for O6A, deaths were observed in 7 of 15 animals, and for O16 and O75, deaths were observed in 15 of 15 animals immunized with the 4 / 8 μg / PS / dose of ExPEC12V vaccine, but similar numbers of animals were detected in the buffer group.

[0169] A summary of the GMT values ​​of the opsonization index measured by OPKA for each serotype contained in the ExPEC12V vaccine is shown in Table 3.

[0170] [Table 3]

[0171] Overall, the ExPEC12V composition was able to induce functional antibodies against several serotypes for which the O antigen was present in the composition; however, for some serotypes, significant induction of functional antibodies was not observed; this is not uncommon in studies such as this conducted in rats and is usually a result of high background levels of functional antibodies against the respective serotypes. This is related to the assay and the E. coli strain used in the assay, rather than indicating that functional antibodies against such serotypes are not induced by the composition; functional antibodies would usually be detectable when tested in other animals and / or humans.

[0172] array SEQ ID NO: 1 (optimized glycosylation consensus sequence) Asp(Glu)-X-Asn-Z-Ser(Thr), where X and Z are independently selected from any amino acid except Pro.

[0173] SEQ ID NO: 2 (EPA carrier protein (EPA-4) containing four glycosylation consensus sequences)

[0174] [ka]

[0175] SEQ ID NO: 3 rfb cluster E. coli O153

[0176] [ka]

[0177] [ka]

[0178] [ka]

[0179] [ka]

[0180] SEQ ID NO: 4 (Example PglB sequence ("wild type"))

[0181] [ka]

[0182] SEQ ID NO: 5 (Example of sequence of CRM197)

[0183] [ka]

[0184] SEQ ID NO: 6 rfb cluster E. coli O21

[0185] [ka]

[0186] [ka]

[0187] [ka]

[0188] SEQ ID NO: 7 (nucleotide sequence encoding UDP-glucose 4-epimerase)

[0189] [ka]

[0190] SEQ ID NO: 8 (Amino acid sequence of UDP-glucose 4-epimerase)

[0191] [ka]

Claims

1. 1. A composition comprising Escherichia coli (E. coli) O1, O2, O4, O6, O8, O15, O16, O18, O25 and O75 antigenic polysaccharides, said composition further comprising an O21 antigenic polysaccharide or an O153 antigenic polysaccharide, or an O21 and an O153 antigenic polysaccharide, each of said antigenic polysaccharides independently covalently linked to a carrier protein.

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

3. (i) The E. coli O1 antigen polysaccharide has the formula (O1A): 【Chemistry 1】 The structure of (ii) the E. coli O2 antigen polysaccharide has the formula (O2): 【Chemistry 2】 The structure of (iii) the E. coli O4 antigen polysaccharide has the formula (O4A): 【Transformation 3】 The structure of (iv) The E. coli O6 antigenic polysaccharide has the formula (O6A): 【Chemistry 4】 The structure of (v) The E. coli O8 antigenic polysaccharide has the formula (O8): α-DManpp3Mem(1→−3)-β-D-Manpp-(1→ )-α-､-Manp-(1→2)-α--Manp-(1→) ｎ The structure of (vi) The E. coli O15 antigenic polysaccharide has the formula (O15): [→2) β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→] ｎ The structure of (vii) The E. coli O16 antigenic polysaccharide has the formula (O16): 【Transformation 5】 The structure of (viii) The E. coli O18 antigenic polysaccharide has the formula (O18A): 【Transformation 6】 The structure of (ix) The E. coli O25 antigen polysaccharide has the formula (O25B): 【Transformation 7】 The structure of (x) The E. coli O75 antigenic polysaccharide has the formula (O75): 【Transformation 8】 The structure of (xi) The E. coli O153 antigenic polysaccharide has the formula (O153): [→2) β-D-Rib-(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 antigenic polysaccharide has the formula (O21): 【Chemistry 9】 The structure of 3. The composition of claim 1 or 2, wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, more preferably from 7 to 25.

4. 4. The composition of claim 1, further comprising at least one additional E. coli antigen polysaccharide covalently linked to the carrier protein.

5. the E. coli O-antigen polysaccharide present in the composition is (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 The composition of any one of claims 1 to 4, consisting of:

6. The carrier protein is detoxified exotoxin A (EPA) or CRM of Pseudomonas aeruginosa. １９７ 6. The composition of claim 1, wherein

7. The composition of claim 1 , wherein the carrier protein is EPA.

8. 8. The composition of any one of claims 1 to 7, wherein the carrier protein comprises 1 to 20 glycosylation consensus sequences having the amino acid sequence Asn-X-Ser(Thr), where X can be any amino acid except Pro.

9. 9. The composition of claim 1, wherein each carrier protein comprises the amino acid sequence of SEQ ID NO:

2.

10. 10. The composition of claim 1, wherein the E. coli antigen polysaccharide is covalently linked to the carrier protein by bioconjugation or by chemical conjugation.

11. 11. The composition of claim 1, wherein the E. coli antigen polysaccharide is covalently linked to the carrier protein by bioconjugation.

12. 12. The composition of claim 1, wherein the E. coli antigen polysaccharide is covalently linked to an Asn residue at a glycosylation site in the carrier protein.

13. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the composition of any one of claims 1 to 12.

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

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

16. 1. A recombinant prokaryotic host cell for preparing a bioconjugate of an E. coli O153 antigen polysaccharide covalently linked to a carrier protein, comprising: a. the nucleotide sequence of the rfb gene cluster of the O153 antigenic polysaccharide; b. a nucleotide sequence encoding said carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser(Thr), where X can be any amino acid except Pro, preferably having SEQ ID NO: 1; and c. A nucleotide sequence encoding the oligosaccharyltransferase PglB; Including, The E. coli O153 antigenic polysaccharide has the formula (O153): [→2) β-D-Rib-(1→4)-β-D-Galp-(1→4)-α-D-GlcpNAc-(1→4)-β-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, more preferably from 7 to 25; Recombinant prokaryotic host cells.

17. 1. A recombinant prokaryotic host cell for preparing a bioconjugate of an E. coli O21 antigen polysaccharide covalently linked to a carrier protein, comprising: a. the nucleotide sequence of the rfb gene cluster of the O21 antigenic polysaccharide; b. a nucleotide sequence encoding said carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having the sequence Asn-X-Ser(Thr), where X can be any amino acid except Pro, preferably having SEQ ID NO: 1; and c. A nucleotide sequence encoding the oligosaccharyltransferase PglB; d. a nucleotide sequence encoding a UDP-glucose 4-epimerase; Including, The E. coli O21 antigenic polysaccharide has the formula (O21): 【Chemistry 10】 wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, more preferably from 7 to 25; Recombinant prokaryotic host cells.

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

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

4.

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

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

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

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

24. (i) The E. coli O1 antigen polysaccharide has the formula (O1A): 【Chemistry 11】 The structure of (ii) the E. coli O2 antigen polysaccharide has the formula (O2): 【Chemistry 12】 The structure of (iii) the E. coli O4 antigen polysaccharide has the formula (O4A): 【Chemistry 13】 The structure of (iv) The E. coli O6 antigenic polysaccharide has the formula (O6A): 【Chemistry 14】 The structure of (v) The E. coli O8 antigenic polysaccharide has the formula (O8): α-DManp3Mem(1→−3)-β-D-Manpp-(1→ )-α-､-Manp-(1→2)-α--Manp-(1→) ｎ The structure of (vi) The E. coli O15 antigenic polysaccharide has the formula (O15): [→2) β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→] ｎ The structure of (vii) The E. coli O16 antigenic polysaccharide has the formula (O16): 【Chemistry 15】 The structure of (viii) The E. coli O18 antigenic polysaccharide has the formula (O18A): 【Chemistry 16】 The structure of (ix) The E. coli O25 antigen polysaccharide has the formula (O25B): 【Chemistry 17】 The structure of (x) The E. coli O75 antigenic polysaccharide has the formula (O75): [Chemistry 18] The structure of (xi) The E. coli O153 antigenic polysaccharide has the formula (O153): [→2) β-D-Rib-(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 antigenic polysaccharide has the formula (O21): 【Chemistry 19】 The structure of 24. The composition of claim 22 or 23, wherein each n is independently an integer from 1 to 40, preferably from 5 to 30, more preferably from 7 to 25.

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 the carrier protein.

26. the E. coli O-antigen polysaccharide present in the composition is (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 26. The composition of any one of claims 22 to 25, consisting of:

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

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

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

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

2.

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

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

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

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

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

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