Multivalent vaccine compositions and uses thereof

Abstract

Compositions and methods for inducing an immune response against parenteral pathogenic Escherichia coli (ExPEC) to provide immune protection from a disease associated with ExPEC are described. In particular, compositions and methods are described for preventing invasive ExPEC disease using conjugates of E. coli polysaccharide antigen O75 covalently bound to a carrier protein.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 17, 2021, application number 202180049038.6, and invention name “Multivalent vaccine composition and its use”. Background Art

[0002] Extraintestinal pathogenic Escherichia coli (ExPEC) are normally harmless inhabitants of the human intestine along with commensal E. coli strains. However, ExPEC strains may possess virulence factors for colonization and infection of sites outside the gastrointestinal tract, leading to a variety of serious invasive diseases, which result in significant morbidity, mortality, and costs each year (see, e.g., Johnson et al., J Lab Clin Med. 2002; 139(3): 155-162; Kohler et al., Int J Med Microbiol. 2011; 301(8): 642-647; Foxman, Am J Med. 2002; 113 Suppl 1A: 5S-13S; and Russo et al., Microbes Infect. 2003; 5(5): 449-456). ExPEC strains are the most common cause of urinary tract infections (UTIs). They are also contributors to surgical site infections and neonatal meningitis (Johnson et al., 2002; Russo et al., 2003), are associated with abdominal and pelvic infections and hospital-acquired pneumonia, and are occasionally involved in other extraintestinal infections such as osteomyelitis, cellulitis, and wound infections. All of these major infection sites can lead to ExPEC bacteremia (Russo et al., 2003). Neonates, the elderly, and immunocompromised patients are particularly susceptible to ExPEC infections, including invasive ExPEC disease (IED).

[0003] Bacterial resistance to antibiotics is a major problem in the fight against bacterial infections, and multidrug-resistant (MDR) Escherichia coli strains are becoming increasingly common (see, e.g., Schito et al., 2009, Int. J. Antimicrob. Agents 34(5):407-413; and Pitout et al., 2012, Expert Rev. Anti. Infect. Ther. 10(10):1165-1176). The emergence and rapid global spread of ExPEC sequence type 131 (ST131) is considered a major driver of increasing drug resistance, including multidrug resistance (Johnson et al., Antimicrob Agents Chemother. 2010;54(1):546-550; Rogers et al., J Antimicrob Chemother. 2011;66(1):1-14). This clone is present in 12.5% ​​to 30% of all ExPEC clinical isolates, predominantly exhibiting serotype O25B:H4, and displays high levels of fluoroquinolone resistance, which is often accompanied by trimethoprim / sulfamethoxazole resistance and extended-spectrum β-lactamases that confer resistance to cephalosporins (Rogers et al., 2011, and Banerjee et al., Antimicrob Agents Chemother. 2014;58(9):4997-5004).

[0004] O antigen serotypes are based on the chemical structure of the O polysaccharide antigen, which is the outer membrane portion of the lipopolysaccharide (LPS) in Gram-negative bacteria. More than 180 serologically unique E. coli O antigens have been reported (Stenutz et al., FEMS Microbial Rev. [FEMS Microbiology Review] 2006; 30: 382-403), although the vast majority of ExPEC isolates are classified into less than 20 O antigen serotypes. The full-length E. coli O antigen is typically composed of approximately 10 to 25 repeating sugar units attached to a highly conserved LPS core structure, each component being independently synthesized by enzymes encoded primarily in the rfb and rfa gene clusters. After O antigen polymerization, the O antigen polysaccharide backbone can typically be modified by adding acetyl or glucose residues. These modifications effectively increase the diversity of serotypes by producing antigenically distinct serotypes with a common polysaccharide backbone but different side branches. Genes encoding O-antigen modification enzymes are typically located outside the rfb cluster on the chromosome and, in some cases, are present in lysogenic phages.

[0005] ExPEC infections can be caused by any serotype. Although certain serotypes are overrepresented in ExPEC infections, surface polysaccharides from ExPEC isolates still exhibit considerable antigenic diversity, making the development of surface polysaccharide-based ExPEC vaccines extremely challenging (Russo et al., Vaccine. 2007; 25: 3859-3870). Moreover, some O antigens may be poorly immunogenic. Furthermore, based on studies of pneumococcal conjugate vaccines, when multiple serotypes can cause disease, vaccine composition (e.g., the selection of serotypes to be included in the vaccine and the dose levels of the included serotypes) may be crucial, as the use of vaccines targeting certain serotypes may potentially increase carriage and disease caused by serotypes not included in the vaccine, or even increase carriage and disease caused by serotypes that are included in the vaccine but are not as effective in immunizing against them (Lipsitch, Emerging Infectious Diseases; 1999, 5: 336-345). Ideally, a vaccine should maximize its beneficial effect in preventing disease caused by the serotypes included in the vaccine, while minimizing the risk of new disease arising from increased carriage of non-vaccine serotypes.

[0006] Efforts to develop vaccines to prevent ExPEC infections have focused on O-antigen polysaccharide conjugates. A 12-valent O-antigen conjugate vaccine was synthesized by extracting and purifying the O-antigen polysaccharide and chemically conjugating it to detoxified Pseudomonas aeruginosa exotoxin A, and its safety and immunogenicity were tested in a Phase 1 clinical study (Cross et al., J. Infect. Dis. [Journal of Infectious Diseases] (1994) Vol. 170, pp. 834-40). This candidate vaccine has never been approved for clinical use. Recently, a bioconjugation system has been developed in Escherichia coli, in which both the polysaccharide antigen and the carrier protein are synthesized in vivo and subsequently conjugated in vivo by the activity of the oligosaccharyltransferase PglB (a Campylobacter jejuni enzyme expressed in E. coli) (Wacker et al., Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] (2006) Vol. 103, pp. 7088-93). This N-linked protein glycosylation system enables the transfer of a variety of glycans to carrier proteins, allowing for the purification of the conjugates using straightforward methods.

[0007] Bioconjugation has been successfully used to produce conjugated polysaccharides for a tetravalent O antigen vaccine candidate against Escherichia coli (Poolman and Wacker, J. Infect. Dis. (2016) Vol. 213(1), pp. 6-13). However, the development of a successful ExPEC vaccine requires coverage of the major serotypes, and the presence of further O antigen modifications in a subset of ExPEC isolates presents a further challenge in terms of coverage of isolates exhibiting both unmodified and modified LPS. Furthermore, the immune response to a vaccine composition comprising O antigens from multiple serotypes may vary depending on the serotype. Therefore, there is a continuing need in the art for vaccines against ExPEC. In particular, there is a need for an ExPEC vaccine based on surface polysaccharides that can be used to provide effective immune protection against the ExPEC O75 serotype and other serotypes prevalent in ExPEC. Summary of the Invention

[0008] It was surprisingly found that E. coli O75 antigen appeared to be less immunogenic than other E. coli O antigens (e.g., O1, O2, and O6) when tested as conjugates of O antigens each covalently bound to a carrier protein in a composition having the same concentration of such other O antigens as in clinical trials. Vaccination with a composition containing a conjugate of E. coli O75 antigen and a conjugate of one or more additional E. coli O antigens at appropriate doses and ratios provided an enhanced immune response against the ExPEC O75 serotype and one or more additional ExPEC O-serotypes.

[0009] Thus, in a first general aspect, provided herein is a composition comprising E. coli 01, 02, 04, 015, 016, 018, 025, 075, and 06 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration of each of the O75 and O25 antigenic polysaccharides is independently increased relative to the concentration of each of the O1, O2, O4, O15, O16, O18, and O6 antigenic polysaccharides.

[0010] In certain embodiments, the concentration weight ratio of the 075 antigen polysaccharide to each of the 01, 02, 04, 015, 016, 018, and 06 antigen polysaccharides, independently, is about 1.5:1 to about 2.5:1.

[0011] In certain embodiments, the concentration weight ratio of the 075 antigen polysaccharide to each of the 01, 02, 04, 015, 016, 018, and 06 antigen polysaccharides, independently, is about 2:1.

[0012] In a second general aspect, provided herein is a composition comprising E. coli O1, O2, O4, O15, O16, O18, O25, O75, and O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration weight ratio of the O75 antigenic polysaccharide to the O1, O2, and / or O6 antigenic polysaccharides is from about 1.5:1 to about 4:1. Preferably, the concentration weight ratio of the O75 antigenic polysaccharide to the O1, O2, and / or O6 antigenic polysaccharides is about 2:1.

[0013] In certain embodiments, the concentration weight ratio of the O75 antigen polysaccharide to the O4, O15, O16 and / or O18 antigen polysaccharide is about 1.5: 1 to about 4: 1. Preferably, the concentration weight ratio of the O75 antigen polysaccharide to the O4, O15, O16 and / or O18 antigen polysaccharide is about 2:1.

[0014] In a third general aspect, provided herein is a composition comprising E. coli O1, O2, O4, O15, O16, O18, O25, O75, and O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration of each of the O75 and O25 antigenic polysaccharides is independently increased relative to the concentration of each of the O1, O2, O4, O15, O16, O18, and O6 antigenic polysaccharides, wherein the weighted ratio of the concentration of the O75 antigenic polysaccharide to the O1, O2, and / or O6 antigenic polysaccharides is from about 1.5:1 to about 4:1. Preferably, the weighted ratio of the concentration of the O75 antigenic polysaccharide to the O1, O2, and / or O6 antigenic polysaccharides is about 2:1.

[0015] Certain embodiments of the first, second and third general aspects are now described.

[0016] In certain embodiments, the concentration weight ratio of the O75 antigen polysaccharide to the O25 antigen polysaccharide in the composition is about 1:1.

[0017] In certain embodiments, the concentration weight ratio of E. coli antigen polysaccharide O1:O2:O4:O6:O15:O16:O18:O25:O75 is 1:1:1:1:1:1:1:1:2:2.

[0018] In certain embodiments, the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B, wherein:

[0019] (i) Escherichia coli O1 antigen polysaccharide comprises the structure of formula (O1A) shown in Table 1,

[0020] (ii) E. coli O2 antigen polysaccharide comprises the structure of formula (O2) shown in Table 1:

[0021] (iii) E. coli O4 antigen polysaccharide comprises the structure of the formula (O4-Glc+) shown in Table 1,

[0022] (iv) E. coli O6 antigen polysaccharide comprises the structure of formula (O6A) shown in Table 1,

[0023] (v) Escherichia coli O15 antigen polysaccharide comprises the structure of formula (O15) shown in Table 1,

[0024] (vi) Escherichia coli O16 antigen polysaccharide comprises the structure of formula (O16) shown in Table 1,

[0025] (vii) the Escherichia coli O18 antigen polysaccharide comprises the structure of formula (O18A) shown in Table 1,

[0026] (viii) the E. coli O25 antigen polysaccharide comprises the structure of formula (O25B) shown in Table 1, and

[0027] (ix) Escherichia coli O75 antigen polysaccharide comprises the structure of formula (O75) shown in Table 1,

[0028] wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

[0029] In certain embodiments, the concentration of the O75 antigen polysaccharide is about 8 to about 64 μg / mL, preferably about 8 to about 50 μg / mL, preferably about 12 to about 40 μg / mL, preferably about 16 to about 32 μg / mL, preferably about 28 to about 36 μg / mL, preferably about 32 μg / mL.

[0030] In certain embodiments, the E. coli O-antigen polysaccharide present in the composition consists of O1, O2, O4, O15, O16, O18, O25, O75, and O6.

[0031] In certain embodiments, the composition further comprises at least one additional E. coli antigenic polysaccharide covalently linked to a carrier protein, preferably wherein the at least one additional E. coli antigenic polysaccharide comprises an O8 antigenic polysaccharide having the formula (O8) shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

[0032] In certain embodiments, the carrier protein is Pseudomonas aeruginosa detoxified exotoxin A (EPA) or CRM 197, preferably EPA. Preferably, the carrier protein comprises 1 to 20, such as 1 to 10 or 2 to 4, glycosylation consensus sequences having the amino acid sequence of SEQ ID NO: 1, for example, a consensus sequence having the amino acid sequence of SEQ ID NO: 2. More preferably, the carrier protein comprises four glycosylation consensus sequences. Most preferably, each carrier protein is EPA comprising the amino acid sequence of SEQ ID NO: 3.

[0033] In certain embodiments, the E. coli antigen polysaccharide is covalently linked to the carrier protein by bioconjugation or by chemical conjugation, preferably the E. coli antigen polysaccharide is covalently linked to the carrier protein by bioconjugation, preferably the polysaccharide is covalently linked to an Asn residue in a glycosylation site in the carrier protein.

[0034] In another aspect, there is provided a method of inducing an immune response against E. coli, preferably extraintestinal pathogenic E. coli (ExPEC), in a subject, the method comprising administering to the subject a composition of the invention.

[0035] In another aspect, a method of inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject is provided, the method comprising administering to the subject an effective amount of each of Escherichia coli O1, O2, O4, O15, O16, O18, O25, O75 and O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, wherein the effective amount of each of the O75 and O25 antigenic polysaccharides is independently increased relative to the effective amount of each of the O1, O2, O4, O15, O16, O18 and O6 antigenic polysaccharides.

[0036] In certain embodiments of the methods according to the invention, an effective amount of the O75 antigen polysaccharide is administered in a weight ratio of about 1.5:1 to about 2.5:1, preferably about 2:1, to each of the O1, O2, O4, O15, O16, O18, and O6 antigen polysaccharides, independently.

[0037] In certain embodiments of the methods according to the present invention, an effective amount of O75 antigen polysaccharide is administered in a weight ratio of about 1.5:1 to about 4:1, preferably about 2:1, independently to O1, O2 and / or O6 antigen polysaccharides, and further preferably, an effective amount of O75 antigen polysaccharide is administered in a weight ratio of about 1:1 to O25 antigen polysaccharide.

[0038] In another aspect, a method of inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject is provided, the method comprising administering to the subject an effective amount of each of Escherichia coli O1, O2, O4, O15, O16, O18, O25, O75, and O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and an effective amount of the O75 antigenic polysaccharide is administered at a weight ratio of 1.5:1 to about 4:1 to the O1, O2, and / or O6 antigenic polysaccharide, independently. The effective amount of the O75 antigenic polysaccharide may preferably be administered at a weight ratio of about 1:1 to the O25 antigenic polysaccharide. Preferably, the effective amount of the O75 antigenic polysaccharide is administered at a weight ratio of about 2:1 to the O1, O2, and / or O6 antigenic polysaccharide, independently.

[0039] Examples of any method aspects of the invention are provided below.

[0040] In certain embodiments of the methods according to the invention, the immune response limits the severity of or prevents invasive ExPEC disease in the subject, preferably wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

[0041] In certain embodiments of the methods according to the present invention, the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B. Preferably, the O1A, O2, glucosylated O4, O6A, O15, O16, O18A, O25B, and O75 antigen polysaccharides comprise the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B), and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20, and wherein the weight ratio of the O75 antigen polysaccharide to the O6 antigen polysaccharide is from about 1.5:1 to about 4:1, more preferably about 2:1.

[0042] In certain embodiments of the methods according to the present invention, the E. coli O-antigen polysaccharide administered to the subject consists of 01, 02, 04, 015, 016, 018, 025, 075, and 06.

[0043] In certain embodiments of the methods according to the present invention, the E. coli O-antigen polysaccharide administered to the subject further comprises 1 to 15 additional E. coli O-antigen polysaccharides, each independently covalently linked to a carrier protein.

[0044] In certain embodiments of the methods according to the present invention, the subject is a human having or at risk of having an E. coli (preferably ExPEC) infection, preferably an invasive ExPEC disease.

[0045] In certain embodiments of the methods according to the present invention, 8-16 μg, preferably about 16 μg, of the O75 antigen polysaccharide is administered for each administration.

[0046] In certain embodiments of the methods according to the present invention, the effective amount of E. coli antigen polysaccharide O1:O2:O4:O6:O15:O16:O18:O25:O75 administered is administered in a weight ratio of 1:1:1:1:1:1:1:1:2:2. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings.It is to be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0048] Figure 1 Figure 1 shows the antibody response induced by the ExPEC10V vaccine in New Zealand white rabbits. Animals received three intramuscular ExPEC10V immunizations or saline, administered 2 weeks apart. The ExPEC10V vaccine was administered at three different concentrations (Group 1: high dose, Group 2: medium dose, and Group 3: low dose, Table 11), and the control group received only saline (Group 4, 0.9% (w / v) sodium chloride solution). Antibody levels were measured by ELISA on day 0 (before vaccination) and days 14, 27, and 42 (after vaccination). Individual titers (EC50 titers) and geometric mean titers (GMT) ± 95% CI are shown. Multiple comparisons were performed using the Wilcoxon rank sum test with Bonferroni correction. Animals vaccinated with ExPEC10V (Groups 1, 2, and 3) were compared with saline controls (Group 4). *p≤0.05, **p≤0.01; ***p≤0.001; ****p≤0.0001. LOD: limit of detection.

[0049] Figure 2 Figure 5 Antibody responses induced by ExPEC10V are shown. New Zealand white rabbits received three intramuscular immunizations of ExPEC10V (105.6 μg total polysaccharide) or 0.9% w / v sodium chloride solution (control). IgG titers were determined by ELISA on day 1 (before immunization, n = 20 / group), day 31 (after immunization, n = 20 / group), and day 50 (after immunization, n = 10 / group). The insets show the individual titers and geometric mean ± 95% confidence intervals for each group. The differences in IgG titers between ExPEC10V and control groups were analyzed using the Tobit model with a likelihood ratio test. P values ​​≤ 0.05 were considered significant. *P ≤ 0.05, ****P ≤ 0.0001.

[0050] Figure 3A-3BShown is the overall study design for the Phase 1 / 2a clinical trial in humans using the ExPEC10V vaccine. Figure 3A Showing the overall study design for Cohort 1, Figure 3B Shows the overall study design for Cohort 2. See Example 7 for details.

[0051] Figure 4 The percentage of participants in the BAC1001 clinical trial whose serum O antigen-specific antibody titers increased at least 2-fold on day 15 after vaccination compared to baseline (day 1) measurements is shown (see Example 7 for details). O antigen-specific serum antibody titers were measured by an ECL-based immunoassay.

[0052] Figure 5 The percentage of participants in the BAC1001 clinical trial who showed at least a 2-fold increase in serum opsonophagocytic antibody titers at day 15 post-vaccination compared to baseline (day 1) measurements (see Example 7 for details). Serum opsonophagocytic antibody levels were measured by MOPA.

[0053] Figure 6 Shown are serum antibody levels (total IgG) induced by different ExPEC vaccine compositions in New Zealand white rabbits. See Example 8 for details. Each panel shows individual titers (EC50 titers) and geometric mean titers (GMT) ± 95% confidence intervals. Comparisons between vaccine groups and saline controls were statistically evaluated using the Tobit model with a likelihood ratio test and Bonferroni correction for multiple comparisons. p values ​​≤ 0.05 were considered statistically significant. ****p ≤ 0.0001. DETAILED DESCRIPTION

[0054] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like included in this specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0055] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms cited herein have the meanings as set forth in the specification. It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0056] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0057] When used in conjunction with a number, the term "about" refers to any number that is within ±10% (eg, ±5 or ±1%) of the cited number.

[0058] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the present invention.

[0059] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or any other group of integers or steps. When used herein, the term "comprising" may be replaced by the terms "containing" or "including" or, at times, by the term "having" when used herein.

[0060] As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of one aspect or embodiment of the present invention, any of the aforementioned terms "comprising," "containing," "including," and "having" may be replaced by the terms "consisting of" or "consisting essentially of" to alter the scope of the present disclosure.

[0061] As used herein, the connecting term "and / or" between multiple referenced elements should be understood to cover both individual and combined options. For example, when two elements are connected by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any of these options should be understood to fall within this meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one of these options should also be understood to fall within this meaning and therefore meet the requirements of the term "and / or".

[0062] As used herein, the terms "O polysaccharide," "O-antigen," "O-antigen," "O-antigen polysaccharide," "O polysaccharide antigen," and the abbreviation "OPS" all refer to the O antigen of Gram-negative bacteria, which is a component of lipopolysaccharide (LPS) and is specific for each serotype or serotype (subtype) of Gram-negative bacteria. O antigens typically comprise repeating units (RU) of two to seven sugar residues. As used herein, RU is set to be equal to the biological repeat unit (BRU). BRU describes the RU of O antigen as it is synthesized in vivo. Different serotypes of Escherichia coli express different O antigens. In E. coli, the gene products involved in O antigen biogenesis are encoded by the rfb gene cluster. Unless otherwise indicated, whenever an O antigen polysaccharide is mentioned herein, it may refer to the O antigen polysaccharide of each E. coli serotype and any serosubtype thereof, for example, when an O1 antigen polysaccharide is mentioned, it may be an O antigen polysaccharide of E. coli serosubtype O1A, O1A1, O1B or O1C, while an O25 antigen polysaccharide may refer to an O25A or O25B antigen polysaccharide, etc. Table 1 of WO 2020 / 039359 (incorporated herein by reference) provides the corresponding structures of a number of E. coli serotypes and serosubtypes and their O antigen polysaccharides (RUs or O units).

[0063] As used herein, "rfb cluster" and "rfb gene cluster" refer to a gene cluster encoding the enzymatic machinery capable of synthesizing the O antigen backbone structure. The term rfb cluster can apply to any O antigen biosynthetic cluster, and preferably refers to a gene cluster from Escherichia, particularly Escherichia coli.

[0064] As used herein, the term "O1A" refers to the O1A antigen of Escherichia coli (a serotype of Escherichia coli serotype O1). The term "O2" refers to the O2 antigen of Escherichia coli (a serotype of Escherichia coli serotype O2). The term "O4" refers to the O4 antigen of Escherichia coli (a serotype of Escherichia coli serotype O4). The term "O6A" refers to the O6A antigen of Escherichia coli (a serotype of Escherichia coli serotype O6). The term "O8" refers to the O8 antigen of Escherichia coli (a serotype of Escherichia coli serotype O8). The term "O15" refers to the O15 antigen of Escherichia coli (a serotype of Escherichia coli serotype O15). The term "O16" refers to the O16 antigen of Escherichia coli (a serotype of Escherichia coli serotype O16). The term "O18A" refers to the O18A antigen of Escherichia coli (a serotype of Escherichia coli serotype O18). The term "O25B" refers to the O25B antigen from E. coli (a serosubtype of E. coli serotype O25). The term "O75" refers to the O75 antigen of E. coli (E. coli serotype O75). As used herein, the terms "glucosylated O4," "glucose-branched O4," "O4 Glc+," and "Glc+O4" O antigen refer to E. coli O4 O antigens (E. coli serotype O4) with glucose side branches, while "non-glucosylated O4," "O4 Glc-," and "Glc-O4" refer to O antigens without glucose side branches.

[0065] The structures of several E. coli O antigen polysaccharides mentioned in this application are shown in Table 1 below. A single repeating unit of each E. coli O antigen polysaccharide is shown.

[0066] Table 1: Structure of Escherichia coli O-antigen polysaccharide

[0067]

[0068]

[0069] 1 Each n is independently an integer from 1 to 100, such as 1-50, 1-40, 1-30, 1-20 and 1-10, 3-50, 3-40, 5-30, such as at least 5, such as 5-40, such as 7-30, such as 7 to 25, such as 10 to 20, but in some cases can be 1-2.

[0070] All monosaccharides described herein have their common meanings as known in the art. Monosaccharides can have a D or L configuration. If D or L is not specified, the sugar is understood to have a D configuration. Monosaccharides are typically represented by abbreviations known and used in the art. For example, Glc refers to glucose; D-Glc refers to D-glucose; and L-Glc refers to L-glucose. Other common abbreviations for monosaccharides include: Rha, rhamnose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose; Man, mannose; Man3Me, 3-O-methyl-mannose; Gal, galactose; FucNAc, N-acetylfucosamine; and Rib, ribose. The suffix "f" refers to furanose, and the suffix "p" refers to pyranose.

[0071] The terms "RU," "repeat unit," and "repeating unit" used with respect to O antigen refer to the biological repeat unit (BRU) of the O antigen as it is synthesized in vivo by cellular machinery (e.g., glycosyltransferases). The number of RUs of the O antigen can vary depending on the serotype and, in embodiments of the present invention, typically ranges from about 1-100 RUs, preferably about 1 to 50 RUs, such as 1-50 RUs, 1-40 RUs, 1-30 RUs, 1-20 RUs, and 1-10 RUs, and more preferably at least 3 RUs, at least 4 RUs, at least 5 RUs, such as 3-50 RUs, preferably 5-40 RUs, preferably 5-30 RUs, such as 7-25 RUs, such as 10-20 RUs. However, in some cases, the number of RUs of the O antigen may be 1-2. The structure of each O antigen specifically described herein is shown as containing one RU, with the variable "n" representing the number of RUs. In each O antigen polysaccharide in the bioconjugate of the present invention, n is independently an integer from 1 to 100, such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, preferably at least 3, more preferably at least 5, such as 3 to 50, preferably 5 to 40 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40), more preferably 5 to 30, but in some cases can be 1 to 2. In some embodiments, n is independently an integer from about 7 to 25, such as about 10 to 20. These values ​​may vary between individual O-antigen polysaccharides in the composition and are provided herein as average values, i.e., if a bioconjugate is described herein as having n independently being an integer from 5 to 40, the composition contains a majority of O-antigen polysaccharides having 5 to 40 repeating units, but may also contain some O-antigen polysaccharides having fewer than 5 or more than 40 repeating units.

[0072] As used herein, the terms "conjugate" and "glycoconjugate" refer to a sugar or sugar antigen (e.g., oligosaccharides and polysaccharides)-protein conjugate that is linked to another chemical substance, including but not limited to proteins, peptides, lipids, etc. Glycoconjugates can be prepared chemically, for example, by chemical (synthetic) linkage of a protein and a sugar or sugar antigen. The term glycoconjugate also includes bioconjugates.

[0073] As used herein, in the context of administering an O antigen to a subject in a method according to embodiments of the present invention, the term "effective amount" refers to an amount of an O antigen sufficient to induce a desired immune effect or immune response in the subject. In certain embodiments, an "effective amount" refers to an amount of an O antigen sufficient to produce immunity in a subject to achieve one or more of the following effects in the subject: (i) prevent the development or onset of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (ii) prevent the recurrence of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (iii) prevent, reduce, or ameliorate the severity of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (iv) reduce the duration of an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (v) prevent an ExPEC infection, preferably an invasive ExPEC disease, or symptoms associated therewith; (x) increase the survival of subjects with ExPEC infection, preferably invasive ExPEC disease; (xi) eliminate ExPEC infection, preferably invasive ExPEC disease; (xii) inhibit or reduce ExPEC replication; and / or (xii) enhance or improve one or more prophylactic or therapeutic effects of another therapy.

[0074] The "effective amount" can vary depending on a variety of factors, such as the subject's physical condition, age, weight, health status, etc.; the route of administration, such as oral or parenteral; the composition administered, such as the target O antigen, other co-administered O antigens, adjuvants, etc.; and the specific disease for which immunity is desired. When the O antigen is covalently bound to a protein carrier, the effective amount of the O antigen is calculated based solely on the O antigen polysaccharide portion of the conjugate. All concentrations, amounts, and ratios of the conjugates (including bioconjugates) described herein are also calculated solely based on the weight of the O antigen polysaccharide portion of the conjugate, regardless of the concentration, amount, or ratio of any conjugated carrier protein, unless otherwise specified. For example, administering 16 μg of a particular bioconjugate means that the administered bioconjugate contains 16 μg of the particular O antigen polysaccharide, and the amount of the conjugated carrier protein is not included in this number. As another example, if a composition is said to comprise a conjugate of O antigen polysaccharides from serotypes A and B in a ratio of 2:1, this means that in the composition, based on the weight of the conjugated O antigen polysaccharides, without taking into account the weight of the conjugated carrier protein, the concentration or amount of conjugated O antigen polysaccharide A is twice the concentration or amount of conjugated O antigen polysaccharide B.

[0075] As used herein, the term "invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED)" is an acute illness concurrent with a systemic bacterial infection that is microbiologically confirmed by isolation and identification of E. coli from the blood or other normally sterile body sites, or by isolation and identification of E. coli from the urine of a patient with signs and symptoms of an invasive disease (systemic inflammatory response syndrome (SIRS), sepsis, or septic shock) and no other identifiable source of infection. In certain embodiments, IED is an acute illness concurrent with a systemic bacterial infection that is microbiologically confirmed by (i) isolation and identification of E. coli from the blood or other normally sterile body sites, or by (ii) 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 reproductive organs and no other identifiable source of infection.

[0076] IEDs may include, but are not necessarily limited to, urinary tract infection (UTI), surgical site infection, abdominal or pelvic infection, pneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, wound infection, pyelonephritis, prostate biopsy-associated infection (e.g., transrectal ultrasound-guided prostate needle biopsy [TRUS-PNB]-associated infection), urosepsis, meningitis, peritonitis, cholangitis, soft tissue infection, pyomyositis, septic arthritis, endophthalmitis, suppurative thyroiditis, sinusitis, endocarditis, neutropenic fever, and prostatitis (including but not limited to acute bacterial prostatitis [ABP]).

[0077] In certain preferred embodiments, the IED includes sepsis. In certain preferred embodiments, the IED includes bacteremia. In certain embodiments, the present invention provides a composition according to the present invention for preventing sepsis caused by Escherichia coli. In certain embodiments, the present invention provides a composition according to the present invention for preventing bacteremia caused by Escherichia coli.

[0078] The term "IED event meeting sepsis criteria" refers to an IED case that includes evidence of life-threatening organ dysfunction due to a dysregulated host response to infection. If there is an abrupt change of 2 points or greater in the Sequential Organ Failure Assessment (SOFA) total score from baseline, the IED case meets sepsis criteria and is considered secondary to an IED. In certain embodiments, the present invention provides a composition according to the present invention for preventing IED meeting sepsis criteria. As used herein, the term "urosepsis" is sepsis caused by infection originating from the genitourinary tract and / or male reproductive organs.

[0079] The term "bacteremic IED" is a case of IED that involves the isolation and identification of E. coli from blood. In certain embodiments, the present invention provides a composition according to the present invention for use in preventing bacteremic IED.

[0080] 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 an antigen present in the composition.

[0081] As used herein, a "composition" comprising more than one E. coli antigenic polysaccharide can be a single pharmaceutical composition comprising more than one E. coli antigenic polysaccharide in the same pharmaceutical composition, or a combination of more than one pharmaceutical compositions comprising more than one E. coli antigenic polysaccharide in separate pharmaceutical compositions. In a preferred embodiment, the composition is a single pharmaceutical composition. In a method of inducing an immune response to E. coli, a "composition" comprising more than one E. coli antigenic polysaccharide can be administered to a subject in need as a single pharmaceutical composition comprising more than one E. coli antigenic polysaccharide, or can be administered to a subject as a combination of separate pharmaceutical compositions. In a preferred embodiment, a single pharmaceutical composition is administered to a subject.

[0082] As used herein, in the context of administering two or more O antigens or compositions to a subject, the terms "combination" or "combination of..." do not limit the order in which the O antigens or compositions are administered to the subject. For example, a first composition can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second composition to the subject. Preferably, the two or more O antigens are administered to the subject substantially simultaneously, e.g., within five minutes of each other, and more preferably the two or more O antigens are administered simultaneously by administering at least two compositions simultaneously, most preferably by administering a single composition comprising the two or more O antigens.

[0083] As used herein, "subject" means any animal, preferably a mammal, and most preferably a human, that will or has been vaccinated using the methods or compositions described in the embodiments of the present invention. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like, most preferably humans. The terms "subject" and "patient" are used interchangeably herein.

[0084] The term "percent (%) sequence identity" or "% identity" describes the number of matches ("hits") of identical amino acids between two or more aligned amino acid sequences compared to the number of amino acid residues that make up the total length of the amino acid sequences. In other words, using an alignment, for two or more sequences, when the sequences are compared and aligned for maximum correspondence (as measured using a sequence comparison algorithm known in the art), or when manually aligned and visually inspected, the percentage of identical amino acid residues (e.g., 90%, 95%, 97% or 98% identity) can be determined. The sequences compared to determine sequence identity can thus differ by one or more substitutions, one or more additions or one or more deletions of an amino acid. Suitable programs for aligning protein sequences are known to those skilled in the art. The percentage sequence identity of a protein sequence can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, for example using the NCBI BLAST algorithm (Altschul SF et al. (1997), Nucleic Acids Res. 25: 3389-3402).

[0085] For example, for amino acid sequences, sequence identity and / or similarity can be determined by using standard techniques known in the art, or by inspection, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, the Genetics Computer Group, and the like). Group, 575 Science Drive, Madison, Wis.); the best fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395 (preferably using default settings). In certain embodiments, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and join penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss Company.

[0086] Another example of a useful algorithm is the BLAST algorithm, which is described in: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, which is available from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values.

[0087] Another useful algorithm is Gapped BLAST reported by Altschul et al., 1993, Nucl. Acids Res. 25: 3389-3402.

[0088] Escherichia coli O antigen

[0089] In the present invention, it was surprisingly found that, in a multivalent vaccine composition, at the same polysaccharide concentration, the E. coli O75 antigen conjugated to a carrier protein appeared to be less immunogenic than other E. coli O antigens (e.g., O1A, O2, O4, O6A, O15, O16, and O18A) conjugated to a carrier protein. This discovery led to further studies on the dose of E. coli O75 antigen and the dose ratio of various E. coli O antigens in multivalent vaccines, thereby developing multivalent vaccines and immunization methods based on E. coli O antigens to enhance immune responses against the O75 serotype and other ExPEC serotypes.

[0090] Embodiments of the present invention relate to compositions and methods involving E. coli O75 antigen polysaccharide and one or more additional E. coli O antigen polysaccharides. Preferably, the one or more additional O antigens are commonly found in clinical isolates of E. coli. Examples of such E. coli antigens that can be used in the present invention include, but are not limited to, E. coli O1, O2, O4, O6, O8, O15, O16, O18, and O25 antigens. As desired, the composition can include more than one additional E. coli O antigen, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen additional E. coli O antigens, to provide immune protection against multiple E. coli serotypes in addition to the E. coli O75 serotype. In some embodiments, the additional E. coli O antigen is selected from the group consisting of E. coli O1, O2, O4, O6, O15, O16, O18, and O25 antigens. In preferred embodiments, the additional E. coli O antigen is selected from the group consisting of O1A, O2, glucosylated O4, O6A, O15, O16, O18A, and O25B. More preferably, the composition comprises all of the O1A, O2, glucosylated O4, O6A, O15, O16, O18A, and O25B E. coli O antigens. In a specific embodiment, the E. coli O antigen comprises the structure shown in Table 1, wherein n is an integer from 1 to 100. In some embodiments, n is an integer from 3 to 50, such as 5 to 40, preferably 5 to 30, such as 7 to 25, and for example 10 to 20.

[0091] In one embodiment, the composition of the present invention comprises Escherichia coli O75 and O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration ratio of O75 antigenic polysaccharide to O6 antigenic polysaccharide is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, more preferably about 2:1, for example, about 1:5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1. In some embodiments, the composition further comprises one or more, preferably all, Escherichia coli O1, O2, O4, O15, O16, O18, and O25 antigen polysaccharides, wherein each antigen polysaccharide is independently covalently linked to a carrier protein, preferably, the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B.

[0092] The E. coli O antigens useful in the present invention can be produced by methods known in the art in light of the present disclosure. For example, they can be produced by cells, preferably recombinant cells optimized for O antigen biosynthesis. See, for example, WO 2006 / 119987, WO 2009 / 104074, International Patent Application No. PCT / EP2015 / 053739, and Ihssen et al., 2010, Microbial Cell Factories 9, 61 for disclosures regarding nucleic acids, proteins, host cells, production methods, etc. for E. coli O antigen biosynthesis, the disclosures of which are incorporated herein by reference in their entirety.

[0093] Carrier protein

[0094] According to an embodiment of the present invention, each E. coli O antigen is covalently bound to a carrier protein, preferably via a glycosidic linkage. In view of the present disclosure, any carrier protein known in the art may be used. Suitable carrier proteins include, but are not limited to, detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM 197 , Escherichia coli flagellin (FliC), maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clot factor A, clot factor B, Escherichia coli heat-labile enterotoxin, detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae hemolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae. Bioconjugation to a variety of different carrier proteins containing the desired consensus glycosylation sequence has been described, indicating that a variety of proteins can be glycosylated using this technology (see, for example, WO 06 / 119987, WO 2015 / 124769, WO 2015 / 158403, WO 2015 / 82571, WO 2017 / 216286 and WO 2017 / 67964, which together show a variety of carrier proteins that have been successfully used for bioconjugation).

[0095] In certain embodiments, the carrier proteins are modified, for example, in a manner that renders the protein less toxic and / or more susceptible to glycosylation. In a particular embodiment, the carrier proteins used herein are modified so as to maximize the number of glycosylation sites in the carrier proteins, in such a manner that, for example, the concentration of the protein to be administered in an immunogenic composition (particularly in the form of a bioconjugate thereof) is lower.

[0096] Thus, in certain embodiments, the carrier proteins described herein are modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycosylation sites than the glycosylation sites typically associated with the carrier protein (e.g., relative to the number of glycosylation sites associated with the carrier protein in its native / natural, i.e., "wild-type" state). The introduction of glycosylation sites into the carrier protein can be achieved by inserting a glycosylation consensus sequence into any position in the primary structure of the protein, for example, by adding new amino acids to the primary structure of the protein so as to add glycosylation sites in whole or in part, or by mutating existing amino acids in the protein to generate glycosylation sites. One of ordinary skill in the art will recognize that the amino acid sequence of a protein can be easily modified using methods known in the art (e.g., recombinant methods comprising modifying the nucleic acid sequence encoding the protein). In specific embodiments, a glycosylation consensus sequence is introduced into a specific region of the carrier protein, such as a surface structure of the protein, the N or C terminus of the protein, and / or a loop stabilized by a disulfide bond at the bottom of the protein. In some embodiments, the glycosylation consensus sequence can be extended by adding lysine residues to achieve more efficient glycosylation.

[0097] Illustrative examples of glycosylation consensus sequences that can be inserted or generated in a carrier protein include Asn-X-Ser(Thr) (SEQ ID NO: 1), wherein X can be any amino acid except Pro; and preferably Asp(Glu)-X-Asn-Z-Ser(Thr) (SEQ ID NO: 2), wherein X and Z are independently selected from any amino acid except Pro.

[0098] In some embodiments, the E. coli O-antigen polysaccharide is covalently linked to an asparagine (Asn) residue in the carrier protein (e.g., N-linked), wherein the Asn residue is present in a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, more preferably having SEQ ID NO: 2. Typically, the carrier protein comprises 1-10 glycosylation sites, preferably 2-4 glycosylation sites, most preferably 4 glycosylation sites, e.g., 1-10, preferably 2-4, and more preferably 4 glycosylation sites, each glycosylation site comprising a glycosylation consensus sequence having the amino acid sequence of SEQ ID NO: 1, more preferably the amino acid sequence of SEQ ID NO: 2.

[0099] In a specific embodiment, the carrier protein is Pseudomonas aeruginosa detoxified exotoxin A. For EPA, various detoxified protein variants have been described in the literature and can be used as carrier proteins. For example, according to Lukac et al., 1988, Infect Immun [infection and immunity], 56: 3095-3098 and Ho et al., 2006, Hum Vaccin [human vaccines], 2: 89-98, detoxification can be achieved by mutating and deleting the catalytic essential residues L552V and ΔE553. As used herein, "EPA" refers to Pseudomonas aeruginosa detoxified exotoxin A. In those embodiments where the carrier protein is EPA, the Escherichia coli antigenic polysaccharide can be covalently linked to an Asn residue in a glycosylation site comprising a glycosylation consensus sequence with SEQ ID NO: 1, and preferably covalently linked to an Asn residue in a glycosylation site comprising a glycosylation consensus sequence with SEQ ID NO: 2. Preferably, the EPA carrier protein comprises 1-10 glycosylation sites, preferably 2-4 glycosylation sites, most preferably 4 glycosylation sites, for example 1-10, preferably 2-4, and more preferably 4 glycosylation sites, each glycosylation site comprising a glycosylation consensus sequence having the amino acid sequence of SEQ ID NO: 1, more preferably the amino acid sequence of SEQ ID NO: 2.

[0100] In some embodiments, the EPA carrier protein comprises four glycosylation sites, each of which comprises a glycosylation consensus sequence, such as a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 2. As used herein, "EPA-4 carrier protein" and "EPA-4" refer to a Pseudomonas aeruginosa detoxified exotoxin A carrier protein comprising four glycosylation sites, each of which comprises a glycosylation consensus sequence having SEQ ID NO: 2. An exemplary preferred example of an EPA-4 carrier protein is an EPA carrier protein comprising the amino acid sequence of SEQ ID NO: 3.

[0101] In certain embodiments, the EPA carrier protein can be produced with a signal sequence (e.g., the signal peptide of E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), etc.) that targets the carrier protein to the periplasmic space of the host cell in which it is expressed. The EPA carrier protein can also be modified with a "tag," i.e., an amino acid sequence that allows for isolation and / or identification of the carrier protein.

[0102] EPA carrier proteins useful in the present invention can be produced by methods known in the art in view of the present disclosure. See, for example, Ihssen et al., 2010, Microbial Cell Factories 9, 61 and the relevant disclosures in WO 2006 / 119987, WO 2009 / 104074 and WO 2015 / 124769, the disclosures of which are incorporated herein by reference in their entirety.

[0103] In other embodiments, the carrier protein is CRM 197 .CRM 197 The protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from diphtheria toxin. CRM 197 Non-toxigenic phage β197 tox- The non-toxigenic phage β197 is produced by Corynebacterium diphtheriae infection. tox- Produced by nitrosoguanidine mutagenesis of toxigenic coryneform bacteriophage beta (Uchida et al. (1971) Nature New Biology 233:8-11). CRM 197 The protein has the same molecular weight as diphtheria toxin, but differs from diphtheria toxin by a single base change in the structural gene (guanine to adenine). This single base change results in an amino acid substitution in the mature protein (glycine to glutamic acid) and eliminates the toxicity of diphtheria toxin. CRM 197 Protein is a safe and effective T cell-dependent carrier for sugars. 197 The amino acid sequence of the protein is shown in SEQ ID NO: 20. 197 Further details of its production can be found in, for example, U.S. Patent No. 5,614,382. In one embodiment, the O antigen polysaccharide of the present invention is conjugated to a CRM 197 Protein or CRM 197 In one embodiment, the O antigen polysaccharide of the present invention is conjugated to the CRM obtained by expressing recombinant E. coli. 197In one embodiment, the O antigen polysaccharide of the present invention is independently conjugated to the CRM 197 In one embodiment, the O antigen polysaccharides of the present invention are each independently conjugated to a CRM 197 A chain.

[0104] Conjugate

[0105] The term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and a sugar or sugar antigen (e.g., oligosaccharides and polysaccharides) produced in a host cell background, preferably a bacterial host cell (e.g., an E. coli host cell), wherein the host cell machinery links the antigen to the protein (e.g., N-linked). Preferably, the term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an O antigen, preferably an E. coli O antigen (e.g., O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, O75, etc.), produced in a host cell background, wherein the host cell machinery links the antigen to the protein (e.g., N-linked). Because bioconjugates are produced in host cells by host cell machinery, the antigen and protein are covalently linked via glycosidic linkages or bonds in the bioconjugate. Bioconjugates can be produced in recombinant host cells that are engineered to express the cellular machinery required to synthesize the O antigen and / or link the O antigen to the target protein. As described herein, bioconjugates have advantageous properties over chemically prepared glycoconjugates (in which polysaccharides are purified from bacterial cell walls and subsequently chemically coupled to a carrier protein), for example, bioconjugates require fewer chemicals in their manufacture and are more consistent in the final product produced, and contain less or no free (i.e., not bound to the carrier protein) polysaccharides. Purification of lipid A-free O antigens and subsequent chemical conjugation to a carrier protein is a lengthy and laborious process. Furthermore, the purification, lipid A detoxification, and chemical conjugation processes can result in epitope loss, antigenic heterogeneity, and reduced immunogenicity of the conjugated polysaccharide. Synthesizing glycoconjugates by bioconjugation can overcome these limitations of classical purification and chemical conjugation. Therefore, in typical embodiments, bioconjugates are preferred over chemically produced glycoconjugates.

[0106] In certain embodiments, the host cell can produce E. coli O antigen and EPA carrier protein, and the O antigen is covalently bound to the EPA carrier protein to form a bioconjugate useful in the present invention. See, for example, Ihssen et al., 2010, Microbial Cell Factories 9, 61, and the relevant disclosures in WO 2006 / 119987, WO 2009 / 104074, and WO 2015 / 124769, the disclosures of which are incorporated herein by reference in their entirety.

[0107] In a specific embodiment, the carrier protein is N-linked to an E. coli O antigen that can be used in the present invention. For example, the E. coli O antigen is linked to an Asn residue in the glycosylation sequence of the carrier protein, such as Asn-X-Ser (Thr) (SEQ ID NO: 2), where X can be any amino acid except Pro, preferably Asp (Glu) -X-Asn-Z-Ser (Thr) (SEQ ID NO: 3), where X and Z are independently selected from any natural amino acid except Pro.

[0108] Alternatively, glycoconjugates can be prepared by chemical synthesis, i.e., prepared outside the host cell (in vitro). For example, the E. coli O antigens described herein (e.g., O75 antigen) can be conjugated to a carrier protein using methods known to those skilled in the art, including by using activated reactive groups in polysaccharides / oligosaccharides and protein carriers. See, for example, Pawlowski et al., 2000, Vaccine [Vaccine], 18: 1873-1885; and Robbins et al., 2009, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 106: 7974-7978, the disclosures of which are incorporated herein by reference. Such methods include extracting antigenic polysaccharides / oligosaccharides from host cells, purifying these polysaccharides / oligosaccharides, chemically activating these polysaccharides / oligosaccharides, and conjugating these polysaccharides / oligosaccharides to a carrier protein. WO 2020 / 039359 also describes methods for preparing glycoconjugates of E. coli O antigen conjugated to a carrier protein using chemical conjugation to a carrier protein, as well as compositions comprising such glycoconjugates.

[0109] For example, conjugates can be prepared using CDAP chemistry. In these examples, the polysaccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then linked directly or via a spacer (linker) group to a carrier protein (e.g., EPA or CRM). 197) is coupled to an amino group on the spacer. For example, the spacer can be cystamine or cysteamine to give a thiolated polysaccharide that can be coupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido] propionate (SBAP)). Preferably, the cyanate ester (optionally prepared by CDAP chemistry) is coupled to hexamethylenediamine or adipic acid dihydrazide (ADH), and the amino-derivatized sugar is conjugated to the carrier protein (e.g., EPA or CRM) via the carboxyl groups on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. 197 ). Such conjugates are described, for example, in WO 93 / 15760, WO 95 / 08348 and WO 96 / 129094.

[0110] Other suitable conjugation techniques use carbodiimides, hydrazides, active esters, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Various conjugation techniques are described in WO 98 / 42721. Conjugation may involve a carbonyl linker, which can be formed by reacting the free hydroxyl groups of the sugar with CDI (see Bethell et al. (1979) J. Biol. Chem. 254: 2572-2574; Hearn et al. (1981) J. Chromatogr. 218: 509-518), followed by reaction with the protein to form a carbamate linkage. This may involve reduction of the anomeric end of the primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

[0111] In other embodiments, the conjugate is prepared using reductive amination. Reductive amination involves two steps: (1) oxidation of the polysaccharide, and (2) reduction of the activated polysaccharide and carrier protein to form the conjugate. Prior to oxidation, the polysaccharide is optionally hydrolyzed. Mechanical or chemical hydrolysis can be employed. Chemical hydrolysis can be performed using acetic acid.

[0112] The oxidation step may involve a reaction with a periodate. For the purposes of the present invention, the term "periodate" includes both periodate and periodic acid; the term also includes metaperiodate (IO4 -) and orthoperiodate (IO6 5- ) and includes various periodates (e.g., sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably sodium periodate (NaIO4). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably periodic acid.

[0113] In one embodiment, the oxidant is a stable nitroxyl or nitrooxy group compound, such as a piperidine-N-oxy or pyrrolidine-N-oxy compound, which selectively oxidizes primary hydroxyl groups in the presence of an oxidant (as described in WO 2014 / 097099). In the reaction, the actual oxidant is an N-oxoammonium salt in the catalytic cycle. On the one hand, the stable nitroxyl or nitrooxy group compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. On the one hand, the stable nitroxyl or nitrooxy group compound has a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. On the one hand, the stable nitroxyl group compound is TEMPO or a derivative thereof. On the one hand, the oxidant is a molecule with an N-halogenated moiety. In one aspect, the oxidant is selected from the group consisting of: N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidant is N-chlorosuccinimide.

[0114] Optionally, the oxidation reaction is quenched by adding a quencher selected from a vicinal diol, a 1,2-amino alcohol, an amino acid, glutathione, a sulfite, a bisulfate, a dithionite, a metabisulfite, a thiosulfate, a phosphite, a hypophosphite or a phosphorous acid (such as glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid).

[0115] After the polysaccharide oxidation step, the polysaccharide is said to be activated and is referred to hereinafter as the "activated polysaccharide". The activated polysaccharide and the carrier protein can be lyophilized (freeze-dried) separately (dispersed lyophilization) or together (co-lyophilization). In one embodiment, the activated polysaccharide and the carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and the carrier protein are lyophilized separately.

[0116] In one embodiment, lyophilization is performed in the presence of a non-reducing sugar. Possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinitol.

[0117] The second step of the conjugation process is to reduce the activated polysaccharide and carrier protein using a reducing agent to form the conjugate (so-called reductive amination). Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride, sodium triacetoxyborohydride or sodium borohydride or zinc borohydride in the presence of Bronsted or Lewis acids), amine boranes (such as pyridine borane, 2-methylpyridine borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB)) or borohydride exchange resin. In one embodiment, the reducing agent is sodium cyanoborohydride.

[0118] In one embodiment, the reduction reaction is carried out in an aqueous solvent (e.g., selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine or HEPB, pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5); in another embodiment, the reaction is carried out in an aprotic solvent. In one embodiment, the reduction reaction is carried out in DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. DMSO or DMF solvents can be used to reconstitute lyophilized activated polysaccharides and carrier proteins.

[0119] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4).

[0120] In other embodiments, the conjugates are prepared using eTEC conjugation as described in WO 2014 / 027302. The glycoconjugates comprise a saccharide covalently conjugated to a carrier protein via one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer via a carbamate linkage, and wherein the carrier protein is covalently conjugated to the eTEC spacer via an amide linkage. The eTEC-linked glycoconjugates of the present invention can be represented by the general formula (I):

[0121]

[0122] The atoms that make up the eTEC spacer are contained in the central box.

[0123] The eTEC spacer comprises seven linear atoms (i.e., —C(O)NH(CH2)2SCH2C(O)—) and provides a stable thioether bond and an amide bond between the sugar and the carrier protein. The synthesis of eTEC-linked glycoconjugates involves the reaction of an activated hydroxyl group of the sugar with an amino group of a thioalkylamine reagent (e.g., cystamine or cysteamine or a salt thereof) to form a carbamate linkage with the sugar to provide a thiolated sugar. The generation of one or more free sulfhydryl groups is achieved by reaction with a reducing agent to provide an activated thiolated sugar. The reaction of the free sulfhydryl groups of the activated thiolated sugar with an activated carrier protein having one or more α-haloacetamide groups on an amine-containing residue generates a thioether bond to form a conjugate, wherein the carrier protein is attached to the eTEC spacer via an amide bond.

[0124] The conjugates described herein can be purified by any method known in the art for purifying proteins, for example, by chromatography (e.g., ion exchange chromatography, anion exchange chromatography, affinity chromatography, and size fractionation column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. See, for example, Saraswat et al., 2013, Biomed. Res. Int. [International Biomedical Research] ID# 312709 (pp. 1-18); see also the methods described in WO 2009 / 104074. The actual conditions used to purify a particular conjugate will depend in part on the synthesis strategy (e.g., synthetic production versus recombinant production) and factors such as the net charge, hydrophobicity, and / or hydrophilicity of the bioconjugate, and will be apparent to those skilled in the art.

[0125] host cells

[0126] Described herein are host cells, e.g., prokaryotic host cells, capable of producing E. coli O antigens and bioconjugates comprising such E. coli O antigens. Preferably, the host cells described herein are modified (e.g., by genetic engineering) to contain one or more nucleic acids encoding host cell machinery (e.g., glycosyltransferases) for producing E. coli O antigen polysaccharides and / or bioconjugates thereof.

[0127] Any host cell known to those skilled in the art can be used to produce the E. coli O-antigen polysaccharide (e.g., E. coli O75 polysaccharide) and the bioconjugates comprising the E. coli O-antigen polysaccharide (e.g., bioconjugates of E. coli O75 antigen polysaccharide) described herein, including archaea, prokaryotic host cells, and eukaryotic host cells. In a preferred embodiment, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells that can be used to produce the E. coli O-antigen polysaccharides described herein and bioconjugates comprising the E. coli O-antigen polysaccharides described herein include, but are not limited to, Escherichia species, Shigella species, Klebsiella species, Xanthomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species.

[0128] In a specific embodiment, the host cell used to produce the E. coli O-antigen polysaccharide described herein and the bioconjugates comprising the E. coli O-antigen polysaccharide described herein is a prokaryotic host cell, preferably E. coli.

[0129] In certain embodiments, host cells for producing the E. coli O antigen polysaccharides and bioconjugates described herein are engineered to contain heterologous nucleic acids, e.g., heterologous nucleic acids comprising the rfb gene cluster of the desired O antigen serotype, heterologous nucleic acids encoding one or more carrier proteins and / or glycosyltransferases. In a specific embodiment, heterologous rfb genes and / or heterologous nucleic acids encoding proteins involved in glycosylation pathways (e.g., prokaryotic and / or eukaryotic glycosylation pathways) can be introduced into host cells described herein. Such nucleic acids can encode proteins including, but not limited to, oligosaccharyltransferases and / or glycosyltransferases.

[0130] Described herein are sequences of various genes and gene clusters encoding glycosyltransferases that can be used to prepare recombinant host cells that can, for example, be used to prepare E. coli O-antigen polysaccharides and bioconjugates thereof. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, a protein having a particular amino acid sequence can be encoded by a variety of different nucleic acids. Thus, those skilled in the art will appreciate that the nucleic acids provided herein can be altered in such a way that their sequence differs from that provided herein without affecting the amino acid sequence of the protein encoded by the nucleic acid.

[0131] Described herein are host cells (e.g., recombinant host cells) for producing bioconjugates of E. coli O75 antigen polysaccharide, O1A antigen polysaccharide, O2 antigen polysaccharide, glucosylated or non-glucosylated O4 antigen polysaccharide, O6A antigen polysaccharide, O8 antigen polysaccharide, O15 antigen polysaccharide, O16 antigen polysaccharide, O18A antigen polysaccharide, or O25B antigen polysaccharide. The host cells described herein contain a nucleic acid encoding an enzyme (e.g., a glycosyltransferase) capable of producing an E. coli O antigen polysaccharide. The host cells described herein may naturally express a nucleic acid capable of producing the desired O antigen, or the host cells may be caused to express such a nucleic acid. In certain embodiments, the nucleic acid is heterologous to the host cell and is introduced into the host cell using genetic methods known in the art. For example, nucleic acids can be introduced into host cells by genetic manipulation (e.g., expression of the gene cluster on one or more plasmids or integration into the host cell genome) (see, e.g., WO 2014 / 037585, WO 2014 / 057109, WO 2015 / 052344).

[0132] Also described herein are host cells (e.g., recombinant host cells) capable of producing bioconjugates of E. coli O1A, O2, glucosylated or non-glucosylated O4, O6A, O8, O15, O16, O18A, O25B, or O75 antigen polysaccharides covalently linked to a carrier protein. Such host cells (e.g., recombinant host cells) contain nucleotide sequences of an rfb gene cluster specific for an O antigen polysaccharide. The rfb gene cluster can be isolated from a wild-type E. coli strain and combined with nucleic acids encoding an oligosaccharyltransferase (e.g., PglB) and a carrier protein (e.g., EPA) within a host cell to obtain a recombinant host cell that produces the desired E. coli O antigen or its bioconjugate. For example, recombinant methods can be used to engineer such host cells to include one or more plasmids, using, for example, the bioconjugation techniques described in WO 2015 / 124769, WO 2014 / 037585, WO 2009 / 104074, and WO2009 / 089396, wherein the one or more plasmids include an rfb gene cluster, an oligosaccharyl transferase (e.g., PglB), and a carrier protein (e.g., EPA). Preferably, the host cell includes the rfb gene cluster integrated into its genome. In certain embodiments, nucleic acids encoding oligosaccharyl transferases and carrier proteins, where applicable, gtrS genes, are also integrated into the genome of the host cell. In certain embodiments, heterologous or homologous gtrA and gtrB genes are also integrated into the genome of the host cell.

[0133] Preparation of bioconjugates of O1A, O2, O6A and O25B antigens is described in detail in WO 2015 / 124769 and WO 2017 / 035181. Exemplary gene clusters (rfb loci) for each E. coli O antigen are described in Iguchi A et al., DNA Research, 2014, 1-7 and DebRoy C et al., PLoS One. 2016, 11(1):e0147434; revision: PLoS One. 2016, 11(4):e0154551. The nucleic acid sequences of the rfb clusters and the amino acid sequences of the proteins encoded therein can also be found in public databases such as GenBank. Exemplary sequences of rfb clusters in production strains that can be used for bioconjugates with polysaccharide antigens of the serotypes disclosed herein are also provided in SEQ ID NOs: 9 and 11-19. Thus, for each desired bioconjugate described above, the corresponding rfb cluster can be introduced into a host cell to obtain a host cell having the specific rfb cluster for the desired O antigen and containing nucleic acids encoding an oligosaccharyltransferase and a carrier protein. For the reasons noted above, the host cell is preferably a recombinant host cell, preferably derived from a relatively well-characterized strain, such as a laboratory or production strain of E. coli, such as E. coli K12 or E. coli BL21. Preferably, the rfb cluster is heterologous to the host cell, for example, introduced into a precursor cell of the host cell and preferably integrated into its genome. Preferably, if the original rfb gene cluster is present in the precursor cell, it has been replaced by the rfb gene cluster for the desired O antigen in the host cell, so as to enable production of the bioconjugate with the desired O antigen. Preferably, the oligosaccharyltransferase is heterologous to the host cell, and in certain embodiments, the nucleic acid encoding such an oligosaccharyltransferase is integrated into the host cell genome.

[0134] Any host cell described herein (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell) comprises a nucleic acid encoding an additional enzyme active in N-glycosylation of proteins, for example, a host cell described herein may further comprise a nucleic acid encoding an oligosaccharyltransferase or a nucleic acid encoding other glycosyltransferases.

[0135] The host cells described herein typically contain a nucleic acid encoding an oligosaccharyl transferase. Such oligosaccharyl transferases transfer lipid-linked oligosaccharides to asparagine residues of nascent polypeptide chains containing N-glycosylation consensus motifs. The nucleic acid encoding the oligosaccharyl transferase can be native to the host cell or can be introduced into the host cell using genetic methods. In a preferred embodiment, the oligosaccharyl transferase is heterologous to the host cell. Escherichia coli does not naturally contain oligosaccharyl transferases, so if Escherichia coli is used as a host cell to produce bioconjugates, a heterologous oligosaccharyl transferase is contained in such a host cell, for example, after introduction by genetic engineering. In view of the present disclosure, the oligosaccharyl transferase can be from any source known in the art.

[0136] In certain embodiments, alternatives to oligosaccharyltransferases having N-glycosyltransferase activity, such as O-glycosyltransferases, such as, for example, PglL, as described, for example, in WO 2016 / 82597 and WO 2020 / 120569, can be used in combination with different glycosylation consensus sequences in the carrier protein itself. Other glycosyltransferases (such as O-glycosyltransferases) can therefore also be used as oligosaccharyltransferases according to the present invention.

[0137] In certain preferred embodiments, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter. For example, in one embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al., 2002, Science 298:1790-1793; see also, e.g., NCBI gene ID: 3231775, UniProt accession number 086154). In another embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter lari (see, e.g., NCBI gene ID: 7410986).

[0138] In a specific embodiment, the oligosaccharyltransferase is a PglB oligosaccharyltransferase from Campylobacter jejuni, including the native (wild-type) protein or any variant thereof, such as those described in WO 2016 / 107818 and WO 2016 / 107819. PglB can transfer lipid-linked oligosaccharides to asparagine residues in the consensus sequences SEQ ID NO: 1 and SEQ ID NO: 2. In a specific embodiment, the PglB oligosaccharyltransferase comprises SEQ ID NO: 6 or a variant thereof. In certain embodiments, one or more endogenous glycosylation consensus sequences in wild-type PglB have been mutated to prevent PglB autoglycosylation, such as SEQ ID NO: 6 comprising the mutation N534Q. Examples of variant PglB oligosaccharyltransferases suitable for use in recombinant host cells provided herein include PglB oligosaccharyltransferases having SEQ ID NO: 6 comprising at least one mutation selected from the group consisting of N311V, K482R, D483H, A669V, Y77H, S80R, Q287P, and K289R. In a specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising the mutation N311V. In another specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising the mutations Y77H and N311V. In another specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising the mutations N311V, K482R, D483H, and A669V. In another specific embodiment, a variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising mutations Y77H, S80R, Q287P, K289R, and N311V. Certain PglB oligosaccharyltransferase variants were discovered and described in PCT / US20 / 23415, filed March 18, 2020, to surprisingly improve yields in the production of bioconjugates of E. coli O antigens of specific serotypes. Improved or optimal PglB variants for a given E. coli O antigen are unpredictable. Therefore, the present invention also discloses, in certain aspects, methods for producing bioconjugates of specific E. coli O antigens using specific PglB variants as oligosaccharyltransferases.Other variants of PglB that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 6 and still have oligosaccharyltransferase activity, preferably with one or more specific amino acids at the specified positions disclosed in the combinations herein (e.g., 77Y, 80S, 287Q, 289K, 311N, 482K, 483D, 669A; or 311V; or 311V, 482R, 483H, 669V; or 77H, 80R, 287P, 289R, 311V; or 77H, 311V; etc.) can also be used to generate bioconjugates.

[0139] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli O75 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni having an amino acid sequence of SEQ ID NO: 6 or preferably SEQ ID NO: 6 comprising the mutation N311V.

[0140] In other specific embodiments, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli O1A, O6A or O15 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni having an amino acid sequence of SEQ ID NO: 6 or preferably comprising mutations N311V, K482R, D483H and A669V.

[0141] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni having an amino acid sequence of SEQ ID NO: 6 or preferably SEQ ID NO: 6 comprising the mutation N311V, or more preferably SEQ ID NO: 6 comprising the mutations Y77H and N311V. Preferably, the recombinant host cell for producing a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein further comprises a sequence encoding a glucosyltransferase GtrS, and a nucleotide sequence encoding a translocase GtrA and a glycosyltransferase GtrB having an amino acid sequence of SEQ ID NO: 7 and 8, respectively (or variants thereof containing amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 7 and 8, respectively), wherein the glucosyltransferase GtrS has an amino acid sequence of SEQ ID NO: 4 (or variants thereof having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 7 and 8, respectively). coli O4 antigen polysaccharide) and is capable of modifying E. coli O4 antigen polysaccharide by adding glucose to produce a glucosylated E. coli O4 antigen polysaccharide, wherein the translocase is capable of translocating the glucose linked to the bacterial terpene alcohol and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol. To produce a bioconjugate of the non-glucosylated E. coli O4 antigen polysaccharide covalently linked to a carrier protein, the cell does not need to encode such sequences of the GtrS, GtrA, and GtrB proteins. PCT / US20 / 23404, filed March 18, 2020, which is incorporated herein by reference in its entirety, describes the use of a novel GtrS specific for glucosylation of the E. coli O4 antigen polysaccharide to produce a bioconjugate of the glucosylated E. coli O4 antigen polysaccharide covalently linked to a carrier protein.

[0142] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli O16 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni having an amino acid sequence of SEQ ID NO: 6 or preferably comprising mutations Y77H, S80R, Q287P, K289R, and N311V.

[0143] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli O8, O18A, O25B or O2 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni having an amino acid sequence of SEQ ID NO: 6, preferably wherein SEQ ID NO: 6 does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669.

[0144] In some embodiments, any of the host cells described herein comprises a nucleic acid encoding a carrier protein (eg, a protein produced by the host cell glycosylation machinery to which one or more O-antigen polysaccharides can be attached to form a bioconjugate). In view of the present disclosure, the host cell may comprise a nucleic acid encoding any carrier protein known to those skilled in the art, including but not limited to, Pseudomonas aeruginosa detoxified exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clot factor A, clot factor B, Escherichia coli heat-labile enterotoxin, a detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, a detoxified variant of cholera toxin, Escherichia coli Sat protein, a passenger domain of Escherichia coli Sat protein, pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

[0145] In a preferred embodiment, the host cell further comprises a nucleic acid encoding Pseudomonas aeruginosa detoxified exotoxin A (EPA). Preferably, the EPA carrier protein comprises 1-10 glycosylation sites, preferably 2-4 glycosylation sites, most preferably 4 glycosylation sites, for example 1-10, preferably 2-4, and more preferably 4 glycosylation sites, each glycosylation site comprising a glycosylation consensus sequence having an amino acid sequence of SEQ ID NO: 1, more preferably having an amino acid sequence of SEQ ID NO: 2. In a specific embodiment, the host cell further comprises a nucleic acid encoding an EPA-4 carrier protein comprising SEQ ID NO: 3.

[0146] In certain embodiments, the carrier protein used in the production of bioconjugates by the host cells described herein comprises a "tag," i.e., a sequence of amino acids that allows separation and / or identification of the carrier protein. For example, adding a tag to a carrier protein can be used to purify the protein, thereby purifying a conjugate vaccine comprising the tagged carrier protein. Exemplary tags that can be used herein include, but are not limited to, histidine (HIS) tags (e.g., hexahistidine tags or 6XHis tags), FLAG tags, and HA tags. In certain embodiments, the tags used herein are removable, once they are no longer needed, e.g., after the protein has been purified, e.g., by chemical reagents or enzymatic removal. In other embodiments, the carrier protein does not comprise a tag.

[0147] In certain embodiments, the carrier proteins described herein comprise a signal sequence that targets the carrier protein to the periplasmic space of the host cell in which it is expressed. In a specific embodiment, the signal sequence is from E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), Erwinia carotovorans pectate lyase (PelB), FlgI, NikA, or Bacillus species endoxylanase (XynA), thermolabile E. coli enterotoxin LTIIb, Bacillus endoxylanase XynA, or E. coli flagellin (FlgI). In one embodiment, the signal sequence comprises SEQ ID NO: 10. After the protein is translocated to the periplasm, the signal sequence can be cleaved and therefore may no longer be present in the final carrier protein of the bioconjugate.

[0148] In certain embodiments, additional modifications can be introduced (e.g., using recombinant techniques) into the host cells described herein. For example, host cell nucleic acids (e.g., genes) encoding proteins that form part of a glycosylation pathway that may compete with or interfere with one or more heterologous genes involved in glycosylation introduced recombinantly into the host cell can be deleted or modified in the host cell background (genome) in a manner that renders them inactive / dysfunctional (i.e., the deleted / modified host cell nucleic acids do not encode functional proteins). In certain embodiments, when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced with desired sequences (e.g., sequences that can be used to produce O-antigen polysaccharides or bioconjugates thereof).

[0149] Exemplary genes or gene clusters that can be deleted in the host cell (and in some cases, replaced by other desired nucleic acid sequences) include genes or gene clusters of the host cell involved in glycolipid biosynthesis, such as waaL (see, e.g., Feldman et al., 2005, PNAS USA 102: 3016-3021), lipid A core biosynthesis cluster (waa), galactose cluster (gal), arabinose cluster (ara), colic acid cluster (wc), capsular polysaccharide cluster, undecanol-p biosynthesis genes (e.g., uppS, uppP), und-P recycling genes, metabolic enzymes involved in nucleotide activated sugar biosynthesis, enterobacterial common antigen cluster (eca), and prophage O antigen modification cluster (e.g., gtrABS cluster or region thereof). In a specific embodiment, the host cells described herein are modified so that they do not produce any O antigen polysaccharide other than the desired O antigen polysaccharide (e.g., glucosylated O4 antigen polysaccharide).

[0150] In a specific embodiment, the waaL gene is deleted from the genome of a host cell (e.g., a recombinant host cell) provided herein or its function is inactivated. The terms "waaL" and "waaL gene" refer to an O-antigen ligase gene encoding a membrane-bound enzyme whose active site is located in the periplasm. The encoded enzyme transfers the O-antigen bound to undecaprenylphosphate (UPP) to the lipid A core to form lipopolysaccharide. Deletion or disruption of the endogenous waaL gene (e.g., a ΔwaaL strain) disrupts the transfer of the O-antigen to lipid A and can instead enhance the transfer of the O-antigen to another biomolecule (e.g., a carrier protein).

[0151] In another specific embodiment, one or more of the waaL gene, gtrA gene, gtrB gene, gtrS gene, and rfb gene cluster are deleted from the original genome of the prokaryotic host cell provided herein or their functions are inactivated.

[0152] In one embodiment, the host cell used herein is E. coli, which produces a bioconjugate of a glucosylated O4 antigen polysaccharide, wherein the waaL gene is deleted from the host cell genome or its function is inactivated, and a gtrS gene specific for the E. coli O4 antigen polysaccharide is inserted. In certain embodiments of the strain producing the glucosylated O4 O antigen bioconjugate, the gtrS gene encoding a glucosyltransferase having at least 80% sequence identity to SEQ ID NO:4 is inserted into the position of the gtrS gene of the parent strain, replacing the gtrS gene in the parent strain with the gene responsible for glucosylation of the O4 antigen. An example of such a parent strain is E. coli K-12 strain W3110. The gtrA and gtrB genes can be homologous to the parent strain, or alternatively, one or both of these genes can be heterologous to the parent strain. Typically, unlike the gtrS gene, these gtrA and gtrB genes are not specific for the O antigen structure.

[0153] This paper also describes a method for preparing a recombinant host cell. The recombinant host cells produced by the methods described herein can be used to produce bioconjugates of E. coli O antigens. These methods include introducing one or more recombinant nucleic acid molecules into cells to produce recombinant host cells. Typically, the recombinant nucleic acid molecules are heterologous. In view of the present disclosure, any method known in the art can be used to introduce recombinant nucleic acid molecules into host cells. Any method known to those of ordinary skill in the art, such as electroporation, chemical transformation, heat shock, natural transformation, phage transduction and conjugation, can be used to introduce recombinant nucleic acid into host cells as described herein. In a specific embodiment, a plasmid is used to introduce recombinant nucleic acid into a host cell as described herein. For example, heterologous nucleic acid can be expressed in a host cell by a plasmid (e.g., an expression vector). In another specific embodiment, a method for inserting a genome is used to introduce a heterologous nucleic acid into a host cell as described herein, as described in, for example, WO 2014 / 037585, WO 2014 / 057109 or WO 2015 / 052344.

[0154] In molecular biology, the Escherichia coli strains routinely used as tools and model organisms can, for example, be used as the parents of host cells in certain embodiments. Non-limiting examples include Escherichia coli K12 strains (for example, such as W1485, W2637, W3110, MG1655, DH1, DH5α, DH10, etc.), B strains (for example, BL-21, REL606, etc.), C strains or W strains. In a specific embodiment, the host strain is derived from parent strain W3110. For example, the strain can be obtained from the Escherichia coli Genetic Resource Collection Center (E.coli Genetic Stock Center) of Yale University. For more information about Escherichia coli, see for example Ecoliwiki.net.

[0155] In some embodiments, the host cells described herein can be used to produce bioconjugates comprising an E. coli O antigen polysaccharide covalently linked to a carrier protein. Methods for producing such bioconjugates using host cells are known in the art. See, for example, WO 2003 / 074687 and WO 2006 / 119987. Such methods include culturing any recombinant host cell described herein under conditions that produce a bioconjugate. In view of the present disclosure, any method known in the art can be used to isolate, separate and / or purify the bioconjugate from the recombinant host cell. For example, the bioconjugate can be purified by any method known in the art for purifying proteins, for example, by chromatography (e.g., ion exchange chromatography, anion exchange chromatography, affinity chromatography, and size fractionation column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. See, for example, the methods described in WO 2009 / 104074. In addition, the bioconjugate can be fused to a heterologous polypeptide sequence to facilitate purification. The actual conditions used to purify a particular bioconjugate will depend in part on factors such as the net charge, hydrophobicity, and / or hydrophilicity of the bioconjugate and will be apparent to those skilled in the art. For example, WO 2015 / 124769 and WO 2017 / 035181 describe the preparation of bioconjugates of O1A, O2, O6A, and O25B, as well as vaccine compositions comprising these bioconjugates.

[0156] Also provided are bioconjugates produced by the methods described herein, ie, using the recombinant host cells described herein.

[0157] In some embodiments, a method for preparing a bioconjugate of an E. coli O-antigen polysaccharide covalently linked to a carrier protein comprises: (i) providing a recombinant host cell comprising: (a) a nucleotide sequence of the rfb gene cluster of the O-antigen polysaccharide; (b) a nucleotide sequence encoding a carrier protein, preferably EPA, comprising at least one glycosylation site, the at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2, and more preferably, the carrier protein comprising four glycosylation sites, each glycosylation site comprising a consensus sequence having SEQ ID NO: 2; and (c) a nucleotide sequence encoding an oligosaccharyltransferase, such as PglB oligosaccharyltransferase or a variant thereof.

[0158] In certain embodiments, the E. coli O-antigen polysaccharide is covalently bound to the carrier protein at a specific polysaccharide to protein ratio (w / w) by weight. The ratio of the amount of O-antigen polysaccharide covalently bound to the carrier protein by weight is referred to as the "polysaccharide / protein ratio" or "polysaccharide / protein ratio" or "PS / protein ratio". In some embodiments, the O-antigen polysaccharide is covalently bound to the carrier protein at a polysaccharide to protein (w / w) ratio of about 1:20 to 20:1, preferably 1:10 to 10:1, more preferably 1:3 to 3:1. In certain non-limiting embodiments of the bioconjugates described herein, the polysaccharide / protein ratio is about 0.1 to 0.5, such as 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5. In such embodiments, the weight ratio of O-antigen polysaccharide to protein is about 1:10 to 1:2, such as 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, or 1:2, depending on the specific O-antigen serotype. In certain embodiments, the polysaccharide / protein ratio is about 0.15 to about 0.45. Generally, a higher polysaccharide / protein ratio of O-antigen polysaccharide to carrier protein is preferred because, in some cases, large amounts of carrier protein may cause immunological interference. In addition, a higher polysaccharide / protein ratio will help to obtain sufficient O-antigen polysaccharide for administration in the form of a bioconjugate while keeping the amount of carrier protein relatively low, which is particularly beneficial for multivalent compositions that cover multiple serotypes, for example, compositions comprising bioconjugates from at least 4 different O-antigens, at least 5 different O-antigens, at least 6 different O-antigens, at least 7 different O-antigens, at least 8 different O-antigens, at least 9 different O-antigens, at least 10 different O-antigens, etc.

[0159] The glycan / protein ratio of the conjugates according to the present invention can be determined by determining the amount of protein and the amount of glycans. The amount of protein can be determined by measuring the UV absorbance at 280 nm (A280). The amount of glycans can be determined based on ion chromatography pulsed amperometric detection (IC-PAD) of the sugars in the repeating unit (e.g., Man for O8 in Table 1 and GlcNAc for the other glycans in Table 1), after which the total amount of glycans can be calculated using the structural information of the repeating unit (e.g., the molar mass of the repeating unit of O1A is 845 Da, and one mole of this repeating unit contains one mole of GlcNAc, so when the amount of GlcNAc has been determined by IC-PAD, the total amount of glycans can be calculated).

[0160] In some embodiments, the bioconjugates of the E. coli O25B antigen polysaccharide covalently linked to a carrier protein as described herein have a degree of acetylation of the L-Rh saccharide at position 2. The degree of O-acetylation of the O25B antigen polysaccharide in the (bio)conjugate is preferably at least 30%, preferably at least 50%, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0161] Similarly, the degree of O-acetylation of the E. coli O16 antigenic polysaccharide in the (bio)conjugate is preferably at least 30%, preferably at least 50%, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0162] In certain embodiments, a method for preparing a bioconjugate of an O-antigen polysaccharide comprises providing a recombinant host cell comprising a nucleic acid sequence encoding a specific oligosaccharyltransferase, particularly a PglB oligosaccharyltransferase or a variant thereof, depending on the O-antigen polysaccharide bioconjugate to be produced. The specific oligosaccharyltransferase variant may affect the yield of the bioconjugate produced by the host cell. Typically, because yield affects the cost of producing a particular bioconjugate, higher yields are preferred, which is particularly important for multivalent compositions comprising several different bioconjugates.

[0163] In a specific embodiment, when the O antigen is the O75 antigen polysaccharide, the PglB oligosaccharyltransferase comprises an amino acid mutation N311V, wherein the amino acid mutation is relative to the wild-type PglB having the amino acid sequence of SEQ ID NO:6.

[0164] In another specific embodiment, when the O antigen is an O1A, O6A or O15 antigen polysaccharide, the PglB oligosaccharyltransferase comprises amino acid mutations N311V, K482R, D483H and A669V, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

[0165] In another specific embodiment, when the O antigen is a glucosylated O4 antigen polysaccharide, the PglB oligosaccharyltransferase comprises the amino acid mutation N311V, or the amino acid mutations Y77H and N311V, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

[0166] In another specific embodiment, when the O antigen is an O16 antigen polysaccharide, the PglB oligosaccharyltransferase comprises amino acid mutations Y77H, S80R, Q287P, K289R, and N311V, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

[0167] In another specific embodiment, when the O antigen is O8, O18A, O25B or O2 antigen polysaccharide, the PglB oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6, wherein SEQ ID NO: 6 does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669. In certain embodiments thereof, the PglB oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6.

[0168] In some embodiments, according to the O antigen / PglB oligosaccharyltransferase pairing noted above, the O antigen polysaccharide bioconjugate produced by recombinant host cells encoding the oligosaccharyltransferase preferably has one or more of the preferred properties described herein, such as the glycan / protein ratio and / or the percentage of polyglycosylated carrier protein.

[0169] Compositions and combinations

[0170] Provided herein are compositions and combinations comprising E. coli O75 and O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and optionally further comprising one or more, preferably all, E. coli O1, O2, O4, O15, O16, O18, O25 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein. The combination of O antigenic polysaccharides or conjugates (e.g., bioconjugates) can comprise a variety of compositions, but it is preferred that the combination of O antigenic polysaccharides or conjugates (e.g., bioconjugates) is present in the same composition.

[0171] The compositions and combinations described herein can be used to treat and prevent infection of subjects (e.g., human subjects) with E. coli, preferably to prevent invasive ExPEC disease. In some embodiments, the composition is an immunogenic composition. As used herein, an "immunogenic composition" refers to a composition that can elicit an immune response in a host or subject to which the composition is administered. The compositions and immunogenic compositions may further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the U.S. Pharmacopoeia, or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. As used herein, in the context of a pharmaceutically acceptable carrier, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with the pharmaceutical composition. Saline solutions and aqueous dextrose solutions, as well as glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0172] In one embodiment, the composition of the invention comprises a (bio)conjugate as described herein in Tris-buffered saline (TBS) pH 7.4 (e.g., containing, for example, 25 mM, 137 mM, and 2.7 mM Tris, NaCl, and KCl, respectively). In other embodiments, the composition of the invention comprises a (bio)conjugate as described herein in about 10 mM KH2PO4 / Na2HPO4 buffer (pH about 7.0), about 5% (w / v) sorbitol, about 10 mM methionine, and about 0.02% (w / v) polysorbate 80. In other embodiments, the compositions of the invention comprise a (bio)conjugate as described herein in about 10 mM KH2PO4 / Na2HPO4 buffer (pH about 7.0), about 8% (w / v) sucrose, about 1 mM EDTA, and about 0.02% (w / v) polysorbate 80 (for suitable buffers for bioconjugates of E. coli O antigen covalently bound to an EPA carrier protein, see, e.g., WO 2018 / 077853). In other embodiments, the compositions of the invention comprise a (bio)conjugate as described herein in about 5 mM succinate / 0.9% NaCl (pH 6.0).

[0173] The compositions provided herein (e.g., pharmaceutical compositions and / or immunogenic compositions) are multivalent compositions, such as bivalent, trivalent, tetravalent, etc. compositions. For example, a multivalent composition comprises more than one antigen (e.g., E. coli O antigen), saccharide conjugate, or bioconjugate thereof. In specific embodiments, a multivalent composition provided herein comprises a bioconjugate of an E. coli O75 antigen polysaccharide and a bioconjugate of an E. coli O6A antigen polysaccharide. In some embodiments, a multivalent composition provided herein comprises at least one additional antigen or bioconjugate.

[0174] Typically, the composition of the present invention can be prepared by first obtaining individual glycoconjugates of each E. coli O-antigen polysaccharide as described herein by independently covalently linking the O-antigen polysaccharide to a carrier protein (e.g., by chemical conjugation or bioconjugation), and then mixing these individual glycoconjugates in the amounts and ratios as described herein to obtain the composition according to the present invention.

[0175] In one embodiment, the composition (eg, pharmaceutical composition and / or immunogenic composition) is a multivalent composition comprising an E. coli O75 antigenic polysaccharide covalently linked to a carrier protein as described herein and at least one additional antigen.

[0176] In some embodiments, the additional antigen is an antigenic saccharide or polysaccharide, more preferably an E. coli O antigen polysaccharide, such as an E. coli O antigen of one or more serotypes 01, 02, 04, 06, 08, 015, 016, 018, and 025, and serosubtypes thereof, preferably each individually conjugated to a carrier protein, wherein the carrier protein for each serotype may be the same or may be different between some or all serotypes. Preferably, the multivalent composition comprising a (bio)conjugate of an E. coli O75 polysaccharide further comprises an E. coli O antigen of each of serotypes 01, 02, 04, 06, 015, 016, 018, and 025, or serosubtypes thereof. In some embodiments, each additional E. coli O antigen polysaccharide is a glycoconjugate, meaning that the E. coli O antigen polysaccharide is covalently linked to another chemical substance, such as a protein, peptide, lipid, etc., most preferably a carrier protein, for example, by chemical or enzymatic methods. In a preferred embodiment, each additional E. coli O antigen polysaccharide is a bioconjugate, wherein the O antigen polysaccharide is enzymatically covalently linked to, for example, a carrier protein via a glycosidic bond by host cell machinery. In certain embodiments, the multivalent composition further comprises an E. coli O antigen of the serotype O8, preferably in the form of a glycoconjugate, preferably a bioconjugate. In certain embodiments, the compositions provided herein may comprise 1-20 additional glycoconjugates, more preferably bioconjugates of E. coli O antigen polysaccharides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional glycoconjugates or preferably bioconjugates of E. coli O antigen polysaccharides. Other antigens (such as peptides, proteins or lipid antigens, etc.) may be included in the compositions provided herein.

[0177] In some embodiments, the composition (e.g., a pharmaceutical composition and / or an immunogenic composition) comprises a (bio)conjugate of an Escherichia coli O75 antigenic polysaccharide and a (bio)conjugate of an Escherichia coli O6 antigenic polysaccharide, and at least one additional antigenic polysaccharide selected from the group consisting of: an Escherichia coli O1 antigenic polysaccharide, an Escherichia coli O2 antigenic polysaccharide, an Escherichia coli glucosylated O4 antigenic polysaccharide, an Escherichia coli O8 antigenic polysaccharide, an Escherichia coli O15 antigenic polysaccharide, an Escherichia coli O16 antigenic polysaccharide, an Escherichia coli O18 antigenic polysaccharide and an Escherichia coli O25 antigenic polysaccharide, preferably at least an Escherichia coli O1 antigenic polysaccharide, an Escherichia coli O2 antigenic polysaccharide and an Escherichia coli O25 antigenic polysaccharide, more preferably at least an Escherichia coli O1 antigenic polysaccharide, an Escherichia coli O2 antigenic polysaccharide, an Escherichia coli O25 antigenic polysaccharide, Preferably, the O1 antigen polysaccharide is an O1A antigen polysaccharide, the O6 antigen polysaccharide is an O6A antigen polysaccharide, the O4 antigen polysaccharide is a glucosylated O4 antigen polysaccharide, the O18 antigen polysaccharide is an O18A antigen polysaccharide, and the O25 antigen polysaccharide is an O25B antigen polysaccharide. Preferably, each additional O antigen polysaccharide is covalently linked to a carrier protein, and more preferably, each additional O antigen polysaccharide is a bioconjugate.

[0178] In some embodiments, the composition comprises E. coli O75 and at least one of E. coli O1, O2 or O6 antigenic polysaccharides, preferably at least one of E. coli O1 or O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration ratio of O75 antigenic polysaccharide to O1, O2 and / or O6 antigenic polysaccharide is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, more preferably about 2:1, for example, about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1. In a preferred embodiment, the composition further comprises an E. coli O25 antigen polysaccharide, preferably an E. coli O25B antigen polysaccharide, covalently linked to a carrier protein, and wherein the concentration ratio of the O25 antigen polysaccharide to the O1, O2 and / or O6 antigen polysaccharide is from about 1.5:1 to about 4:1, preferably about 2:1. In such embodiments, the concentration ratio of the O25 antigen polysaccharide to the O75 antigen polysaccharide is preferably from about 2:1 to about 1:1, preferably about 1.5:1, more preferably about 1:1. In certain embodiments thereof, the concentration ratio of the O25 antigen polysaccharide to the O2 antigen polysaccharide is from about 4:1 to about 2:1. In certain embodiments, the composition comprises E. coli O75, O1, O2, O6, and O25 antigenic polysaccharides, preferably wherein the O1, O6, and O25 antigenic polysaccharides are O1A, O6A, and O25B polysaccharides, respectively, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration ratio of the antigenic polysaccharides O75:O1:O2:O6:O25 is approximately 2:1:1:1:2. In certain embodiments, the O antigen is covalently linked to the carrier protein by bioconjugation, for example, via an N-linkage to an Asn residue in the carrier protein. In certain embodiments, the concentration of the O75 antigenic polysaccharide in the composition is about 8-64 μg / mL, preferably about 16-32 μg / mL, preferably about 28-36 μg / mL, for example, about 32 μg / mL. In certain embodiments, the composition further comprises one or more, preferably all, E. coli O4, O15, O16, and O18 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, preferably wherein O4 is glucosylated and the O18 antigen is O18A. In certain embodiments, the composition further comprises an E. coli O8 antigenic polysaccharide covalently linked to a carrier protein. In certain embodiments, the concentration ratio of the antigenic polysaccharides O75:O4:O15:O16:O18:O8 (when each of these is present in the composition) is approximately 2:1:1:1:1:1:1. In further embodiments, the composition may comprise one or more other E. coli O antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein.

[0179] In one embodiment, the O1 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with the O75 antigen polysaccharide or its bioconjugate). In a specific embodiment, the O1 antigen polysaccharide comprises the structure of formula (O1A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O1 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O1 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, such as about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0180] In one embodiment, the O2 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with an O75 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O2 antigen polysaccharide comprises a structure of formula (O2) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O2 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O2 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, such as about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0181] In one embodiment, the O4 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with the O75 antigen polysaccharide or its bioconjugate). In a specific embodiment, the O4 antigen polysaccharide is a glucosylated O4 antigen polysaccharide, and in a specific embodiment, it comprises a structure of the formula (O4-Glc+) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O4 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O4 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, such as about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0182] In one embodiment, the O6 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with the O75 antigen polysaccharide or its bioconjugate). In a specific embodiment, the O6 antigen polysaccharide comprises a structure of formula (O6A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O6 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O6 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, such as about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0183] In one embodiment, the O8 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with the O75 antigen polysaccharide or its bioconjugate). In a specific embodiment, the O8 antigen polysaccharide comprises a structure of formula (O8) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O8 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O8 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, such as about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0184] In one embodiment, the O15 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with an O75 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O15 antigen polysaccharide comprises a structure of formula (O15) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O15 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O15 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, for example, about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0185] In one embodiment, the O16 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with an O75 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O16 antigen polysaccharide comprises a structure of formula (O16) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O16 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197In certain embodiments, the weight ratio between the O75:O16 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, such as about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0186] In one embodiment, the O18 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with an O75 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O18 antigen polysaccharide comprises a structure of formula (O18A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O18 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O18 antigen polysaccharides in the composition and / or administered to the subject is between about 1.2:1 and 8:1, preferably between about 1.5:1 and 4:1, more preferably about 2:1, for example, about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1.

[0187] In one embodiment, the O25 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with an O75 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O25 antigen polysaccharide comprises an O25B antigen polysaccharide, and in a specific embodiment, it comprises a structure of formula (O25B) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, the O25 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein (e.g., EPA or CRM). 197 In certain embodiments, the weight ratio between the O75:O25 antigen polysaccharides in the composition and / or administered to the subject is between about 1:4 and 1:0.5, preferably between about 1:2 and 1:1, more preferably about 1:1, such as about 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1.0, 1:0.9, 1:0.8, 1:0.7, 1:0.6 or 1:0.5.

[0188] In another embodiment, the composition (e.g., pharmaceutical composition and / or immunogenic composition) comprises at least E. coli O75, O1, O2, O4, O6 and O25 antigen polysaccharides conjugated to a carrier protein, preferably comprising a carrier protein (e.g., EPA or CRM). 197 ) covalently linked 075, 01A, 02, glucosylated 04, 06A, and 025B antigen polysaccharides (i.e., at least a hexavalent composition). In one embodiment, the weight ratio of the 075:01:02:04:06:025 antigen polysaccharides in the composition and / or administered to a subject is about 2:1:1:1:1:2.

[0189] In a preferred embodiment, the composition (e.g., pharmaceutical composition and / or immunogenic composition) comprises at least E. coli O1, O2, O4, O6, O15, O16, O18, O25 and O75 antigen polysaccharides conjugated to a carrier protein, preferably comprising a carrier protein (e.g., EPA or CRM). 197 ) covalently linked 01A, 02, glucosylated 04, 06A, 015, 016, 018A, 025B, and 075 antigen polysaccharides (i.e., a composition with at least 9 valencies). In one embodiment, the weight ratio of the 075:01:02:04:06:015:016:018:025 antigen polysaccharides in the composition and / or administered to a subject is about 2:1:1:1:1:1:1:1:2.

[0190] In another preferred embodiment, the composition (e.g., pharmaceutical composition and / or immunogenic composition) comprises at least E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25 and O75 antigen polysaccharides conjugated to a carrier protein, preferably comprising a carrier protein (e.g., EPA or CRM) 197 ) covalently linked 01A, 02, glucosylated 04, 06A, 08, 015, 016, 018A, 025B, and 075 antigen polysaccharides (i.e., at least a 10-valent composition). In one embodiment, the weight ratio of the 075:01:02:04:06:08:015:016:018:025 antigen polysaccharides in the composition and / or administered to a subject is about 2:1:1:1:1:1:1:1:1:2.

[0191] Also contemplated herein are compositions that optionally further comprise additional O antigens from other E. coli serotypes (e.g., in isolated form or as part of a glycoconjugate or bioconjugate). In some embodiments, the composition (e.g., pharmaceutical composition and / or immunogenic composition) comprises at least a 6-valent, 9-valent, or 10-valent composition as described above, and further comprises 1 to 15 additional E. coli antigenic polysaccharides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional E. coli antigenic polysaccharides, preferably in combination with a carrier protein (e.g., EPA or CRM). 197 ) is a (bio)conjugate of an antigenic polysaccharide covalently linked to a 6-valent, 9-valent or 10-valent composition as described above. In certain embodiments, the 6-valent, 9-valent or 10-valent composition as described above does not contain additional E. coli antigenic polysaccharides or their conjugates, that is, such compositions include 6, 9 or 10 but not more E. coli antigenic polysaccharides, each of which is preferably in the form of a conjugate, preferably a bioconjugate. Therefore, one embodiment is to provide a composition comprising 9 conjugates, in particular E. coli O1, O2, O4, O6, O15, O16, O18, O25 and O75 antigenic polysaccharides conjugated to a carrier protein, preferably to a carrier protein (e.g., EPA or CRM). 197 ) covalently linked to the O1A, O2, glucosylated O4, O6A, O15, O16, O18A, O25B and O75 antigen polysaccharides (i.e., a 9-valent composition), and the composition does not contain an additional conjugate of the E. coli O antigen polysaccharide covalently linked to a carrier protein. Therefore, another embodiment provides a composition comprising 10 conjugates, in particular, E. coli O1, O2, O4, O6, O8, O15, O16, O18, O25 and O75 antigen polysaccharides conjugated to a carrier protein, preferably to a carrier protein (e.g., EPA or CRM). 197 ) covalently linked to the O1A, O2, glucosylated O4, O6A, O8, O15, O16, O18A, O25B, and O75 antigen polysaccharides (i.e., a 10-valent composition), and the composition does not contain an additional conjugate of the E. coli O antigen polysaccharide covalently linked to a carrier protein.

[0192] In some preferred embodiments, each of the additional E. coli O1, O2, O4, O6, O15, O16, O18, and / or O25 antigen polysaccharides is covalently linked to a carrier protein. The O antigen polysaccharide can be linked to the carrier protein by chemical or other synthetic methods, or the O antigen polysaccharide can be part of a bioconjugate, and is preferably part of a bioconjugate. Preferably, each serotype of O antigen covered by the composition is individually coupled to a carrier protein, i.e., each glycoconjugate or bioconjugate covering a particular serotype can be produced separately before being mixed into the composition according to the present invention. The carrier protein can preferably be the same for each glycoconjugate or bioconjugate in the composition; alternatively, the carrier protein can be different for some or all of the E. coli O antigens, for example, O antigens of different serotypes can be conjugated to different carrier proteins. In view of the present disclosure, any carrier protein known in the art can be used. Suitable carrier proteins include, but are not limited to, Pseudomonas aeruginosa detoxified exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxified hemolysin A, clot factor A, clot factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae. Preferably, the carrier protein is EPA or CRM197. 197 .

[0193] In some embodiments, each of the additional E. coli 01(A), 02, (glucosylated) 04, 06(A), 015, 016, 018(A) and / or 025(B) 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) (SEQ ID NO: 1), wherein X can be any amino acid except Pro, preferably wherein the Asn residue is present in a glycosylation site comprising the glycosylation consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr) (SEQ ID NO: 2), wherein X and Z are independently selected from any amino acid except Pro. The carrier protein may comprise 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, and most preferably 4 glycosylation sites, each glycosylation site comprising a glycosylation consensus sequence. In a specific embodiment, the carrier protein is an EPA-4 carrier protein, such as an EPA-4 carrier protein comprising the amino acid sequence of SEQ ID NO:3.

[0194] In a specific embodiment, provided herein are compositions (eg, pharmaceutical compositions and / or immunogenic compositions) comprising: (i) a polypeptide comprising SEQ ID NO:3 of Pseudomonas aeruginosa detoxified exotoxin A carrier protein (EPA-4 carrier protein) covalently linked to Escherichia coli O75 antigen polysaccharide, wherein the Escherichia coli O75 antigen polysaccharide comprises the structure of formula (O75); (ii) a bioconjugate of Escherichia coli O1A antigen polysaccharide covalently linked to EPA-4 carrier protein, wherein the Escherichia coli O1A antigen polysaccharide comprises the structure of formula (O1A); (iii) a bioconjugate of Escherichia coli O2 antigen polysaccharide covalently linked to EPA-4 carrier protein, wherein the Escherichia coli O2 antigen polysaccharide comprises the structure of formula (O2); (iv) a bioconjugate of Escherichia coli O6A antigen polysaccharide covalently linked to EPA-4 carrier protein, wherein the Escherichia coli O6A antigen polysaccharide comprises the structure of formula (O6A); (v) a bioconjugate of Escherichia coli O15 antigen polysaccharide covalently linked to EPA-4 carrier protein, wherein the Escherichia coli O15 antigen polysaccharide comprises the structure of formula (O15); (vi) a bioconjugate of Escherichia coli O15 antigen polysaccharide covalently linked to EPA-4 carrier protein, wherein the Escherichia coli O15 antigen polysaccharide comprises the structure of formula (O15); coli O16 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O16 antigen polysaccharide comprises a structure of formula (O16); (vii) a bioconjugate of an E. coli O18A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O18A antigen polysaccharide comprises a structure of formula (O18A); (viii) a bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O25B antigen polysaccharide comprises a structure of formula (O18A); The antigen polysaccharide comprises a structure of formula (O25B); and (ix) a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli glucosylated O4 antigen polysaccharide comprises a structure of formula (O4-Glc+), wherein the individual formulas are provided in Table 1, and for each formula, n is independently an integer from 1 to 100, such as 1 to 50, preferably 3 to 50, such as 5 to 40, preferably 5-30, such as 7 to 25, such as 10 to 20.

[0195] In a specific embodiment, the composition (e.g., a pharmaceutical composition and / or an immunogenic composition) further comprises: (x) a bioconjugate of an E. coli O8 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O8 antigen polysaccharide comprises a structure of formula (O8) as shown in Table 1, wherein for the structure, n is an integer from 1 to 100, e.g., 1 to 50, preferably 3 to 50, e.g., 5 to 40, preferably 5-30, e.g., 7 to 25, e.g., 10 to 20.

[0196] In some embodiments, the compositions provided herein comprise a conjugate of an E. coli O75 antigen polysaccharide and a conjugate of at least an E. coli O6 antigen polysaccharide, preferably an O6A antigen polysaccharide, wherein the conjugate of the E. coli O75 antigen polysaccharide is present in the composition at a concentration that is about 1.2 to 8 times higher, for example, about 2 to 4 times higher, such as 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 4, 5, 6, 7 or 8 times higher than the concentration of the O6 antigen polysaccharide present in the composition (all concentrations are based on the weight of the O antigen polysaccharide). Preferably, the concentration of E. coli O75 antigenic polysaccharide present in the composition is about 1.5 to 2.5 times the concentration of O6 antigenic polysaccharide in the composition, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 times.

[0197] In particular embodiments, the composition comprises a bioconjugate of E. coli O1A, O2, glucosylated O4, O6A, O15, O16, O18, O25B, and O75 antigen polysaccharides, wherein the bioconjugate of O1A:O2:glucosylated O4:O6A:O15:O16:O18:O25B:O75 is present in a ratio of 1:1:1:1:1:1:1:1:2:2, 1:1:1:1:1:1:1:1:2:1.5, 2:1:1:2:1:1:1:1:4:3, 2:1:1:2:1:1:1:1:4:4, 2:1:2:2:2:2:2:4:4, preferably about 1:1:1:1:1:1:1:1:2:2 (by weight of O antigen polysaccharide).

[0198] In particular embodiments, the composition comprises a bioconjugate of E. coli O1A, O2, glucosylated O4, O6A, O8, O15, O16, O18A, O25B, and O75 antigen polysaccharides, wherein the bioconjugate of O1A:O2:glucosylated O4:O6A:O8:O15:O16:O18A:O25B:O75 is present in a ratio of 1:1:1:1:1:1:1:1:1:2:2, 2:1:2:2:2:2:2:2:4:4, 2:1:1:2:1:1:1:1:1:4:3, or 2:1:1:2:1:1:1:1:4:4, preferably about 1:1:1:1:1:1:1:1:1:2:2 (by weight of O antigen polysaccharide).

[0199] In some embodiments, the compositions provided herein comprise a bioconjugate of an E. coli O75 antigenic polysaccharide and a bioconjugate of at least an E. coli O6 antigenic polysaccharide, wherein the bioconjugate of the E. coli O75 antigenic polysaccharide is present in the composition at a concentration of about 8 to about 50 μg / mL, preferably about 12 to about 40 μg / mL, more preferably about 16 to about 32 μg / mL, such as 16, 18, 20, 22, 24, 26, 28, 30 or 32 μg / mL, preferably about 32 μg / mL. The concentration of the bioconjugate of the E. coli O75 antigenic polysaccharide is preferably about 1.2 to about 8 times higher than the concentration of the O6 bioconjugate present in the composition, for example, about 2 to 4 times higher, such as 1.5, 2, 3, 4, 5, 6, 7 or 8 times higher.

[0200] In a specific embodiment, a pharmaceutical composition is provided, comprising:

[0201] (i) a bioconjugate of an E. coli O1A antigen polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0202] (ii) a bioconjugate of an E. coli O2 antigen polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0203] (iii) a bioconjugate of an E. coli O4 antigen polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0204] (iv) a bioconjugate of an E. coli O6A antigen polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0205] (v) a bioconjugate of an E. coli O15 antigenic polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0206] (vi) a bioconjugate of an E. coli O16 antigenic polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0207] (vii) a bioconjugate of an E. coli O18A antigenic polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0208] (viii) a bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to an EPA-4 carrier protein at a polysaccharide concentration of about 28 to 36, preferably about 32 μg / mL; and

[0209] (ix) a bioconjugate of an E. coli O75 antigenic polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 28 to 36, preferably about 32 μg / mL;

[0210] wherein the EPA-4 carrier protein comprises the amino acid sequence of SEQ ID NO: 3, and

[0211] The O1A, O2, O4, O6A, O15, O16, O18A, O25B and O75 antigen polysaccharides respectively comprise the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, more preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

[0212] In certain embodiments, the composition further comprises:

[0213] (x) a bioconjugate of an E. coli O75 antigenic polysaccharide covalently linked to an EPA-4 carrier protein, the polysaccharide concentration being about 12 to 20, preferably about 16 μg / mL;

[0214] The O8 antigen polysaccharide comprises the structure of formula (O8) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as 5 to 40, more preferably from 5 to 30, such as 7 to 25, such as 10 to 20.

[0215] In one embodiment, a composition is provided that comprises a bioconjugate of an E. coli O75 antigen polysaccharide covalently linked to a carrier protein, wherein the concentration of the O75 antigen polysaccharide is at least about 32 μg / mL.

[0216] In one embodiment, a composition is provided, comprising:

[0217] (i) a bioconjugate of an E. coli O1A antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O1A antigen polysaccharide comprises a structure of formula (O1A);

[0218] (ii) a bioconjugate of an E. coli O2 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O2 antigen polysaccharide comprises a structure of formula (O2);

[0219] (iii) a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide of E. coli comprises a structure of the formula (O4-Glc+);

[0220] (iv) a bioconjugate of an E. coli O6A antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O6A antigen polysaccharide comprises a structure of formula (O6A);

[0221] (v) a bioconjugate of an E. coli O15 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O15 antigen polysaccharide comprises a structure of formula (O15);

[0222] (vi) a bioconjugate of an E. coli O16 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O16 antigen polysaccharide comprises a structure of formula (O16);

[0223] (vii) a bioconjugate of an E. coli O18A antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O18A antigen polysaccharide comprises a structure of formula (O18A);

[0224] (viii) a bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O25B antigen polysaccharide comprises a structure of formula (O25B); and

[0225] (ix) a bioconjugate of an E. coli O75 antigen polysaccharide covalently linked to a carrier protein, wherein the E. coli O75 antigen polysaccharide comprises a structure of formula (O75);

[0226] The structures of formulae (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) are shown in Table 1, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, more preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20,

[0227] The E. coli antigenic polysaccharide present in the composition is composed of O1A, O2, O4-Glc+, O6A, O15, O16, O18A, O25B and O75. Preferably, the carrier protein is Pseudomonas aeruginosa detoxified exotoxin A (EPA-4), more preferably, EPA-4 comprises the amino acid sequence of SEQ ID NO: 3. Preferably, the concentration ratio of the O75 antigenic polysaccharide to the O1A, O2 and / or O6A antigenic polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, more preferably about 2:1. Preferably, the E. coli antigenic polysaccharide present in the composition is composed of O1A, O2, O4-Glc+, O6A, O15, O16, O18A, O25B and O75 in a ratio of 1:1:1:1:1:1:1:2:2.

[0228] In one general aspect, provided herein is a composition comprising E. coli O75 and O6 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration ratio of the O75 antigenic polysaccharide to the O6 antigenic polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, preferably from about 1.5:1 to about 2.5:1, and more preferably about 2:1.

[0229] In certain embodiments, the composition further comprises one or more, preferably all, E. coli O1, O2, O4, O15, O16, O18, O25 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein.

[0230] Preferably, the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B.

[0231] In certain embodiments,

[0232] The Escherichia coli O1 antigen polysaccharide comprises the structure of formula (O1A) shown in Table 1,

[0233] The Escherichia coli O2 antigen polysaccharide comprises the structure of formula (O2) shown in Table 1,

[0234] The E. coli O4 antigen polysaccharide comprises the structure of the formula (O4-Glc+) shown in Table 1,

[0235] The Escherichia coli O6 antigen polysaccharide comprises the structure of formula (O6A) shown in Table 1,

[0236] The Escherichia coli O15 antigen polysaccharide comprises the structure of formula (O15) shown in Table 1,

[0237] The Escherichia coli O16 antigen polysaccharide comprises the structure of formula (O16) shown in Table 1,

[0238] The Escherichia coli O18 antigen polysaccharide comprises the structure of formula (O18A) shown in Table 1,

[0239] The E. coli O25 antigen polysaccharide comprises the structure of formula (O25B) shown in Table 1, and

[0240] The Escherichia coli O75 antigen polysaccharide comprises the structure of formula (O75) shown in Table 1,

[0241] wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

[0242] In certain embodiments, the weight ratio of E. coli antigen polysaccharide O1:O2:O4:O6:O15:O16:O18:O25:O75 is 1:1:1:1:1:1:1:1:2:2.

[0243] In certain embodiments, the concentration of the O75 antigen polysaccharide is about 8 to about 50 μg / mL, preferably 12 to 40 μg / mL, such as 16-32 μg / mL, preferably about 32 μg / mL.

[0244] In certain embodiments, the composition further comprises at least one additional E. coli antigenic polysaccharide covalently linked to a carrier protein.

[0245] In a particular embodiment, the at least one additional E. coli antigenic polysaccharide comprises an O8 antigenic polysaccharide having the formula (O8) shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as 5 to 40, preferably from 5 to 30, such as 7 to 25, such as 10 to 20.

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

[0247] In a specific embodiment, the carrier protein is Pseudomonas aeruginosa detoxified exotoxin A (EPA) or CRM197.

[0248] In certain embodiments, the carrier protein comprises 1 to 20, such as 1 to 10 or 2 to 4, glycosylation consensus sequences having the amino acid sequence of SEQ ID NO: 1, preferably a consensus sequence having the amino acid sequence of SEQ ID NO: 2, and most preferably, the carrier protein comprises four glycosylation consensus sequences.

[0249] In a specific embodiment, each carrier protein is EPA comprising the amino acid sequence of SEQ ID NO:3.

[0250] In certain embodiments, the E. coli antigenic polysaccharide is covalently linked to the carrier protein by bioconjugation or by chemical conjugation. Chemical conjugation can include, for example, reductive amination chemistry (RAC), single-end conjugation, conjugation with a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, cyanidation chemistry (CNBr, CDAP) with or without an ADH spacer, thioether chemistry (based on maleimide / bromoacetyl linkers), or EDC-N-hydroxysuccinimide zero linker chemistry.

[0251] In certain embodiments, the E. coli antigenic polysaccharide is covalently linked to a carrier protein by bioconjugation, preferably the polysaccharide is covalently linked to an Asn residue in a glycosylation site in the carrier protein.

[0252] In another aspect, provided herein is a composition comprising: a bioconjugate of an E. coli O1A antigenic polysaccharide covalently linked to a Pseudomonas aeruginosa detoxified exotoxin A (EPA-4) carrier protein, wherein the E. coli O1A antigenic polysaccharide comprises a structure of formula (O1A);

[0253] A bioconjugate of an E. coli O2 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O2 antigen polysaccharide comprises a structure of formula (O2);

[0254] A bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to an EPA-4 carrier protein, wherein the glucosylated O4 antigen polysaccharide of E. coli comprises a structure of the formula (O4-Glc+);

[0255] A bioconjugate of an E. coli O6A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O6A antigen polysaccharide comprises a structure of formula (O6A);

[0256] A bioconjugate of an E. coli O15 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O15 antigen polysaccharide comprises a structure of formula (O15);

[0257] A bioconjugate of an E. coli O16 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O16 antigen polysaccharide comprises a structure of formula (O16);

[0258] A bioconjugate of an E. coli O18A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O18A antigen polysaccharide comprises a structure of formula (O18A);

[0259] A bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O25B antigen polysaccharide comprises a structure of formula (O25B); and

[0260] A bioconjugate of an E. coli O75 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O75 antigen polysaccharide comprises a structure of formula (O75);

[0261] wherein the EPA-4 comprises the amino acid sequence of SEQ ID NO: 3,

[0262] The structures of formulae (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) are shown in Table 1, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, more preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20, and

[0263] The ratio of O75 antigen polysaccharide to O6A antigen polysaccharide is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, and more preferably about 2:1.

[0264] In certain embodiments, the composition further comprises 1 to 15 additional E. coli antigenic polysaccharides, each antigenic polysaccharide independently covalently linked to a carrier protein.

[0265] In certain embodiments, the composition comprises a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide of E. coli comprises a structure of the formula (O4-Glc+) shown in Table 1, wherein n is an integer from 3 to 50, such as 5 to 40, preferably 5 to 30, such as 7 to 25, such as 10 to 20, wherein the bioconjugate of the glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein has been produced in an E. coli cell comprising:

[0266] (i) nucleotide sequence of the rfb gene cluster of Escherichia coli O4 antigen polysaccharide;

[0267] (ii) a nucleotide sequence encoding a glucosyltransferase having at least 80%, preferably at least 90%, and preferably at least 95% sequence identity to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying E. coli O4 antigen polysaccharide to produce an E. coli glucosylated O4 antigen polysaccharide;

[0268] (iii) a nucleotide sequence encoding a translocase and a glycosyltransferase having at least 80%, preferably at least 90%, preferably at least 95% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating a glucose to which a bacterial terpene alcohol is attached, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0269] (iv) a nucleotide sequence encoding a carrier protein; and

[0270] (v) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glucosylated O4 antigen polysaccharide of E. coli to a carrier protein, preferably wherein the oligosaccharyltransferase is PglB from Campylobacter jejuni.

[0271] In certain embodiments, the compositions described herein further comprise a preservative, such as the mercury derivative thimerosal. In a specific embodiment, the pharmaceutical compositions described herein comprise 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.

[0272] In certain embodiments, the compositions described herein (e.g., immunogenic compositions) comprise an adjuvant, or are administered in combination with an adjuvant. The adjuvant for administration in combination with the compositions described herein can be administered prior to (e.g., within 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, 10 minutes), simultaneously with, or after (e.g., within 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, 10 minutes) administration of the composition. In some embodiments, the term "adjuvant" refers to a compound that, when combined with or administered as part of a composition described herein, increases, enhances, and / or promotes an immune response to a bioconjugate, but does not produce an immune response to the bioconjugate when the adjuvant compound is administered alone. In some embodiments, the adjuvant produces an immune response to the bioconjugate peptide without producing allergic or other adverse reactions. Adjuvants can enhance the immune response by several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. In certain preferred embodiments, the compositions described herein do not comprise an adjuvant other than the bioconjugate, and / or the compositions described herein are not administered in combination with an adjuvant other than the bioconjugate (if the bioconjugate would contain some inherent adjuvant properties, these would be disregarded and, in these embodiments, no exogenous adjuvant would be added).

[0273] Examples of suitable adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles or nanoalum formulations comprising alum), calcium phosphate, monophosphoryl lipid A (MPL) or 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (see, e.g., GB 2220211, EP 0971739, EP 1194166, US6491919), AS01, AS02, AS03, and AS04 (all from GlaxoSmithKline; for AS04, see, e.g., EP 1126876, US 7357936; for AS02, see, e.g., EP 0671948, EP 0761231, US5750110), MF59 (Novartis), imidazopyridine compounds (see WO 200802064). 2007 / 109812), imidazoquinoxaline compounds (see WO 2007 / 109813), delta-inulin, STING-activating synthetic cyclic dinucleotides (e.g., US20150056224), combinations of lecithin and carbomer homopolymers (e.g., US 6676958), and saponins such as QuilA and QS21 (see, e.g., Zhu D and W Tuo, 2016, Nat Prod Chem Res 3:e113), Matrix M, Iscoms, Iscomatrix, etc., optionally in combination with QS7 (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (Powell and Newman, eds., Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540)). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally in combination with immunostimulants such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. [New England Journal of Medicine] 336, 86-91 (1997)). Another adjuvant is CpG. Other examples of adjuvants are liposomes containing immunostimulants such as MPL and QS21, as in AS01E and AS01B (e.g., US2011 / 0206758). Other examples of adjuvants are imidazoquinolines (such as imiquimod and R848). See, for example, Reed G et al., 2013, Nature Med [Natural Medicine], 19: 1597-1608. In certain embodiments, the adjuvant contains a toll-like receptor 4 (TLR4) agonist.TLR4 agonists are well known in the art, see, for example, Ireton GC and SG Reed, 2013, Expert Rev Vaccines [Vaccine Expert Review] 12:793-807. In certain embodiments, the adjuvant comprises a TLR4 agonist comprising lipid A or an analog or derivative thereof, such as MPL, 3D-MPL, RC529 (e.g., EP1385541), PET-lipid A, GLA (pyranosyl lipid adjuvant, a synthetic disaccharide glycolipid; e.g., US20100310602, US8722064), SLA (e.g., Carter D et al., 2016, Clin Transl Immunology [Clinical and Translational Immunology] 5:e108 (doi:10.1038 / cti.2016.63), which describes a structure-function approach for optimizing TLR4 ligands for human vaccines), PHAD (a phosphorylated hexaacyl disaccharide), 3D-PHAD (whose structure is identical to that of GLA), 3D-(6-acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-PHAD, and 3D(6A)PHAD are synthetic lipid A variants, see, for example, avantilipids.com / divisions / adjuvants, which also provides the structures of these molecules), E6020 (CAS No. 287180-63-6), ONO4007, OM-174, etc.

[0274] In certain embodiments, the compositions described herein do not comprise an adjuvant, or are not administered in combination with an adjuvant.

[0275] In certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration. In a specific embodiment, the pharmaceutical composition can be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal, or pulmonary administration. In certain embodiments, the compositions described herein are administered by intramuscular injection.

[0276] In certain embodiments, the compositions described herein additionally comprise one or more buffers, such as Tris-buffered saline, phosphate buffer, or sucrose phosphate glutamate buffer.

[0277] In certain embodiments, the compositions described herein additionally comprise one or more salts, such as Tris-hydrochloride, sodium chloride, calcium chloride, potassium chloride, sodium phosphate, monosodium glutamate, or an aluminum salt (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts).

[0278] The compositions described herein can be included in a container, pack, or dispenser together with instructions for administration.

[0279] The compositions described herein can be stored prior to use, for example, the compositions can be stored frozen (e.g., at about -20°C or at about -70°C); stored under refrigerated conditions (e.g., at about 4°C); or stored at room temperature.

[0280] In one aspect, the present invention also provides methods for preparing the compositions according to the invention, which methods comprise providing each of the desired O-antigen conjugates (e.g., by obtaining or manufacturing these, e.g., in the form of bulk drugs) and mixing them in the desired ratios and / or amounts to obtain the composition of the invention (e.g., a multivalent E. coli, in particular ExPEC vaccine composition, sometimes also referred to as a drug product).

[0281] Methods of inducing an immunological response

[0282] The bioconjugates and compositions provided herein can be used to induce antibodies against E. coli O antigens in a subject, or to vaccinate a subject against E. coli. The methods described herein for inducing an immune response in a subject result in the subject being vaccinated against infection with an ExPEC strain whose O antigen is present in one or more compositions. When O antigen subtypes are used, the methods of the present invention can also induce an immune response to another O antigen subtype with similar antigenicity. An example is cross-reactivity between serosubtypes. For example, immunization with the O25B antigen induces antibodies that recognize O-LPS of serotypes O25B and O25A, and immunization with glucosylated O4 induces antibodies that recognize O-LPS of glucosylated O4 and non-glucosylated O4 serotypes. In other examples, cross-immunization can also be performed against other serotypes that have different O antigens but still have some similarity in antigenic structure (e.g., some sera cross-react in serotyping studies).

[0283] In some embodiments, the subject is a human. In some embodiments, the subject is a human having or at risk of having an ExPEC infection or invasive ExPEC disease.

[0284] In some embodiments, the invasive ExPEC disease comprises sepsis. In some embodiments, the invasive ExPEC disease comprises bacteremia. In some embodiments, the invasive ExPEC disease comprises one or more of: urinary tract infection, surgical site infection, abdominal or pelvic infection, pneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, wound infection, pyelonephritis, meningitis, peritonitis, cholangitis, soft tissue infection, pyomyositis, septic arthritis, endophthalmitis, suppurative thyroiditis, sinusitis, endocarditis, and prostatitis.

[0285] In certain embodiments, the immune response induced in a subject following administration of a composition described herein limits the severity of or prevents invasive ExPEC disease in the subject. In one embodiment, the subject has an E. coli (e.g., ExPEC) infection at the time of administration. In a preferred embodiment, the subject does not have an E. coli (e.g., ExPEC) infection at the time of administration.

[0286] In certain embodiments, the immune response induced in a subject following administration of a composition described herein is preferably effective in preventing or alleviating symptoms caused by an ExPEC infection in at least 30%, more preferably at least 40%, such as at least 50% of the subjects to whom the composition is administered. Symptoms of an ExPEC infection may vary depending on the nature of the infection and may include, but are not limited to: difficulty urinating, increased frequency or urgency of urination, pyuria, hematuria, back pain, pelvic pain, pain during urination, fever, chills, and / or nausea (e.g., in subjects with a urinary tract infection caused by an ExPEC); high fever, headache, stiff neck, nausea, vomiting, seizures, lethargy, and / or light sensitivity (e.g., in subjects with meningitis caused by an ExPEC); fever, increased heart rate, increased respiratory rate, decreased urine output, decreased platelet count, abdominal pain, difficulty breathing, and / or abnormal heart function (e.g., in subjects with sepsis caused by an ExPEC).

[0287] In certain embodiments, the immune response induced in a subject following administration of a composition described herein is effective to reduce the likelihood of hospitalization of a subject with an ExPEC infection. In some embodiments, the immune response induced in a subject following administration of a composition described herein is effective to reduce the duration of hospitalization of a subject with an ExPEC infection.

[0288] In certain embodiments, vaccination with the compositions of the present invention is to prevent invasive ExPEC disease (IED), such as urosepsis, bacteremia, sepsis, etc. In certain embodiments, vaccination is to prevent or reduce the occurrence or severity of urinary tract infections. In certain embodiments, the IED may be hospital-acquired, such as in patients undergoing genitourinary and / or abdominal surgery. In certain embodiments, the IED may be healthcare-related, such as in patients receiving healthcare for another condition, such as via a central line, catheter, etc., such as in a hospital, ambulatory surgical center, end-stage renal disease facility, long-term care facility, etc. In certain embodiments, the IED may be community-acquired, such as in patients who have not recently been exposed to healthcare risks.

[0289] In some embodiments, methods of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject comprise administering to the subject a composition described herein.

[0290] In some embodiments, a method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject comprises administering to the subject a first effective amount of an E. coli O75 antigenic polysaccharide and a second effective amount of an E. coli O6 antigenic polysaccharide, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, for example, about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, more preferably about 2:1. In some preferred embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O6 antigenic polysaccharide covalently linked to the carrier protein are administered as a composition. In some alternative embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O6 antigenic polysaccharide covalently linked to the carrier protein are administered as a combination of separate compositions.

[0291] In some embodiments, the method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject further comprises administering to the subject one or more, preferably all, of the E. coli O1, O2, O4, O15, O16, O18, and O25 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein. Preferably, the O1 antigen is O1A, the O4 antigen is a glucosylated O4 antigenic polysaccharide, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B. Preferably, the O1, O2, O4, O6, O15, O16, O18, O25 and O75 antigens have the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. According to the present invention, the ratio of the O75 antigen polysaccharide to the O6 antigen polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, more preferably about 2:1. In some embodiments, the method further comprises administering to the subject 1 to 15 additional E. coli antigen polysaccharides, each antigen polysaccharide independently covalently linked to a carrier protein. In certain embodiments, such additional E. coli antigens include O8 antigen polysaccharides, which preferably have the structure of formula (O8) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, each E. coli antigen polysaccharide covalently linked to a carrier protein is a bioconjugate. In some embodiments, these bioconjugates are administered as a composition. In some embodiments, these bioconjugates are administered as a combination of two or more separate compositions. It is preferred to limit the number of separate administrations, and thus it is preferred to use a single composition comprising most or all of the antigens.

[0292] In one embodiment is a method of administering a composition for inducing an immune response to Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), wherein the composition comprises a bioconjugate of an E. coli O75 antigenic polysaccharide covalently linked to a carrier protein, and wherein the concentration of the administered O75 antigenic polysaccharide is at least 16 μg / dose.

[0293] In some embodiments, a method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject comprises administering to the subject a first effective amount of an E. coli O75 antigenic polysaccharide and a second effective amount of an E. coli O1 antigenic polysaccharide, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is between about 1.2:1 and about 8:1, preferably between about 1.5:1 and about 4:1, for example, about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, more preferably about 2:1. In some preferred embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O1 antigenic polysaccharide covalently linked to the carrier protein are administered as a composition. In some alternative embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O1 antigenic polysaccharide covalently linked to the carrier protein are administered as a combination of compositions. In some embodiments, the method further comprises administering to the subject one or more, preferably all, of the E. coli O2, O4, O6, O15, O16, O18, and O25 antigen polysaccharides, wherein each antigen polysaccharide is independently covalently linked to a carrier protein. Preferably, the O1 antigen is O1A, the O4 antigen is a glucosylated O4 antigen polysaccharide, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B. Preferably, the O1, O2, O4, O6, O15, O16, O18, O25 and O75 antigens have the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. According to the present invention, the ratio of the O75 antigen polysaccharide to the O1 antigen polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, more preferably about 2:1. In some embodiments, the method further comprises administering to the subject 1 to 15 additional E. coli antigen polysaccharides, each antigen polysaccharide independently covalently linked to a carrier protein. In certain embodiments, such additional E. coli antigens include O8 antigen polysaccharides, which preferably have the structure of formula (O8) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, each E. coli antigen polysaccharide covalently linked to a carrier protein is a bioconjugate. In some embodiments, these bioconjugates are administered as a composition. In some embodiments, these bioconjugates are administered as a combination of two or more separate compositions. It is preferred to limit the number of separate administrations, and thus it is preferred to use a single composition comprising most or all of the antigens.

[0294] In some embodiments, a method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject comprises administering to the subject a first effective amount of an E. coli O75 antigenic polysaccharide and a second effective amount of an E. coli O2 antigenic polysaccharide, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, for example, about 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, more preferably about 2:1. In some preferred embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O2 antigenic polysaccharide covalently linked to the carrier protein are administered as a composition. In some alternative embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O2 antigenic polysaccharide covalently linked to the carrier protein are administered as a combination of compositions. In some embodiments, the method further comprises administering to the subject one or more, preferably all, of the E. coli O1, O4, O6, O15, O16, O18, and O25 antigen polysaccharides, wherein each antigen polysaccharide is independently covalently linked to a carrier protein. Preferably, the O1 antigen is O1A, the O4 antigen is a glucosylated O4 antigen polysaccharide, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B. Preferably, the O1, O2, O4, O6, O15, O16, O18, O25 and O75 antigens have the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. According to the present invention, the ratio of the O75 antigen polysaccharide to the O2 antigen polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, more preferably about 2:1. In some embodiments, the method further comprises administering to the subject 1 to 15 additional E. coli antigen polysaccharides, each antigen polysaccharide independently covalently linked to a carrier protein. Preferably, each E. coli antigen polysaccharide covalently linked to a carrier protein is a bioconjugate. In some embodiments, the bioconjugates are administered as a single composition. In some embodiments, the bioconjugates are administered as a combination of two or more separate compositions. It is preferred to limit the number of separate administrations, so a single composition containing most or all of the antigen is preferably used.

[0295] In some embodiments, a method of inducing an immune response against extraintestinal pathogenic Escherichia coli (ExPEC) in a subject comprises administering to the subject a first effective amount of an E. coli O75 antigenic polysaccharide and a second effective amount of an E. coli O25 antigenic polysaccharide, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is about 1:4 and about 1:0.5, preferably about 1:2 and 1:1, more preferably about 1:1, for example, about 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1.0, 1:0.9, 1:0.8, 1:0.7, 1:0.6 or 1:0.5. In some preferred embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O25 antigenic polysaccharide covalently linked to the carrier protein are administered as a composition. In some alternative embodiments, the O75 antigenic polysaccharide covalently linked to the carrier protein and the O25 antigenic polysaccharide covalently linked to the carrier protein are administered as a combination of compositions. In some embodiments, the method further comprises administering to the subject one or more, preferably all, of the E. coli O1, O2, O4, O6, O15, O16, and O18 antigen polysaccharides, wherein each antigen polysaccharide is independently covalently linked to a carrier protein. Preferably, the O1 antigen is O1A, the O4 antigen is a glucosylated O4 antigen polysaccharide, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B. Preferably, the O1, O2, O4, O6, O15, O16, O18, O25 and O75 antigens have the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. According to the present invention, the ratio of the O75 antigen polysaccharide to the O25 antigen polysaccharide is from about 1:4 to about 1:0.5, preferably between about 1:2 and 1:1, more preferably about 1:1. In some embodiments, the method further comprises administering to the subject 1 to 15 additional E. coli antigen polysaccharides, each antigen polysaccharide independently covalently linked to a carrier protein. In certain embodiments, such additional E. coli antigens include O8 antigen polysaccharides, which preferably have the structure of formula (O8) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20. Preferably, each E. coli antigen polysaccharide covalently linked to a carrier protein is a bioconjugate. In some embodiments, these bioconjugates are administered as a composition. In some embodiments, these bioconjugates are administered as a combination of two or more separate compositions. It is preferred to limit the number of separate administrations, and thus it is preferred to use a single composition comprising most or all of the antigens.

[0296] In certain embodiments, the compositions, combinations and bioconjugates described herein can be administered to a subject to induce an immune response comprising the production of antibodies, preferably antibodies with opsonophagocytic activity. Such antibodies can be isolated using techniques known to those skilled in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.).

[0297] The ability of the bioconjugates and compositions described herein to generate an immune response in a subject can be assessed using any method known to those skilled in the art or described herein. In some embodiments, the ability of a bioconjugate to generate an immune response in a subject can be assessed by immunizing a subject (e.g., mouse, rat, rabbit, or monkey) or group of subjects with a bioconjugate described herein and immunizing another subject (e.g., mouse, rat, rabbit, or monkey) or group of subjects with a control (e.g., PBS). The subject or group of subjects can then be challenged with ExPECs, and the ability of the ExPECs to cause disease (e.g., UTI, bacteremia, or other disease) in the subject or group of subjects can be determined. One skilled in the art will recognize that if the subject or group of subjects immunized with the control develops the disease after challenge with the ExPECs, but the subject or group of subjects immunized with one or more bioconjugates described herein or compositions thereof develops less disease or no disease, then the bioconjugate is capable of generating an immune response in the subject. The ability of one or more bioconjugates described herein, or compositions thereof, to induce antisera that cross-react with the O antigen from ExPEC can be tested, for example, by an immunoassay such as an ELISA (see, e.g., Van den Dobbelsteen et al., 2016, Vaccine 34:4152-4160) or an ECL-based immunoassay.

[0298] For example, the ability of the bioconjugates described herein to generate an immune response in a subject can be assessed using a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPK assay, or OPKA), which represent established and accepted methods that have been used to obtain approval for vaccines based on saccharide conjugates. Such assays are well known in the art and, briefly, comprise the steps of generating and isolating antibodies against a target of interest (e.g., an O-antigen polysaccharide, e.g., an E. coli O75 antigen polysaccharide) by administering an antibody-eliciting compound to a subject (e.g., a mouse, rat, rabbit, or monkey). Subsequently, the bactericidal ability of the antibody can be assessed by, for example, culturing the bacterium in question (e.g., E. coli of the relevant serotype) in the presence of the antibody and complement and, depending on the assay, neutrophils, and determining the ability of the antibody to mediate killing and / or neutralization of the bacteria, e.g., using standard microbiological methods. For examples of OPK assays for E. coli bioconjugate vaccines, see, for example, Abbanat et al., 2017, Clin. Vaccine Immunol. 24:e00123-17. OPK assays can be performed in a single or multiplex format, with multiplex formats (e.g., testing multiple serotypes simultaneously) typically being preferred. Multiplex OPK assays are sometimes referred to herein as "MOPAs."

[0299] In certain embodiments, wherein the compositions provided herein comprise a bioconjugate of an E. coli O75 antigenic polysaccharide and at least a bioconjugate of an E. coli O6A antigenic polysaccharide, the effective amount of the E. coli O75 antigenic polysaccharide is about 1.2 to 8 times greater than the concentration of any other bioconjugate present in the composition, such as about 2 to 4 times greater, such as 1.5, 2, 3, 4, 5, 6, 7, or 8 times greater. In such embodiments, the effective amount of the E. coli O75 antigenic polysaccharide is, for example, about 5 to 18 μg per administration, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 μg per administration. Preferably, about 8-16 μg of the O75 antigenic polysaccharide is administered per administration.

[0300] In certain embodiments, a subject is administered a composition described herein in combination with one or more other therapies (e.g., antibacterial or immunomodulatory therapies). The one or more other therapies may be beneficial in treating or preventing an ExPEC infection, or may improve symptoms or conditions associated with an ExPEC infection. In some embodiments, the one or more other therapies are analgesics or antipyretics. In certain embodiments, the therapies are administered less than 5 minutes apart to less than 1 week apart. Any antibacterial agent known to those skilled in the art (e.g., antibiotics) may be used in combination with the compositions described herein.

[0301] In certain embodiments, a bioconjugate or composition according to the present invention is administered once to a subject. In certain embodiments, a bioconjugate or composition according to the present invention is administered to a subject more than once, for example, in a prime-boost regimen. In certain embodiments, the time between administrations is at least two weeks, at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least five years, at least ten years, or at least fifteen years. In humans, a desired immune response can typically be generated by a single administration of a bioconjugate or composition according to the present invention. In certain embodiments, repeated administration is provided, for example, after ten years.

[0302] The compositions provided herein can be used to induce antibodies against E. coli O antigens in a subject, and to vaccinate a subject against E. coli, particularly extraintestinal pathogenic E. coli (ExPEC). As used herein, "subject" means any animal, preferably a mammal, to which a bioconjugate or composition provided herein will be administered or has been administered. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) (such as monkeys or apes), humans, etc. In certain embodiments, the subject is a human. The human subject can be of any age. In certain embodiments, the subject is a person aged about two months to about 18 years old, for example, 1 to 18 years old. In certain embodiments, the subject is a person of at least 18 years old. In certain embodiments, the subject is a person aged 15 to 50 years old, for example, 18 to 45 years old, for example, 20 to 40 years old. In certain embodiments, the subject is a man. In certain embodiments, the subject is a woman. In certain embodiments, the subject is immunocompromised. In certain embodiments, the subject is a human who is at least 50 years old, at least 55 years old, at least 60 years old, or at least 65 years old. In certain embodiments, the subject is a human who is no more than 100 years old, no more than 95 years old, no more than 90 years old, no more than 85 years old, no more than 80 years old, or no more than 75 years old. In certain embodiments, the subject is a human who is at least 60 years old and no more than 85 years old. In certain embodiments, the subject is a human who is in stable health. In certain embodiments, the subject is an adult who is at least 60 years old and no more than 85 years old and in stable health. In certain embodiments, the subject is a human who has a history of urinary tract infection (UTI, i.e., a bacterial infection in the urethra, bladder, ureters, and / or kidneys; in some embodiments, this includes pyelonephritis), i.e., has had at least one episode of UTI in his or her lifetime. In certain embodiments, the subject is a human who has had a history of UTI in the past 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year. In certain embodiments, the subject is a human who has had a history of UTI in the past two years. In certain embodiments, the subject is a human subject with a history of recurrent UTIs, i.e., at least two UTIs within six months or at least three UTIs within a year. In certain embodiments, the subject is a human subject with a history of recurrent UTIs within the past two years. In certain embodiments, the subject is a human 60 years of age or older in a stable health state. In certain embodiments, the subject is a human 60 years of age or older with a history of UTIs within the past two years. In certain embodiments, the subject is a human at least 60 years of age and less than 75 years of age with a history of UTIs within the past two years. In certain embodiments, the subject is a human 75 years of age or older with a history of UTIs within the past two years.In certain embodiments, the subject is a patient scheduled for elective genitourinary surgery and / or abdominal surgery, such as transrectal ultrasound-guided prostate biopsy (TRUS-PNB). In certain embodiments, the subject is a man with a history of prostatitis (including but not limited to acute bacterial prostatitis (ABP), i.e., a bacterial infection of the prostate), i.e., at least one episode of prostatitis in his lifetime (e.g., in the last ten, nine, eight, seven, six, five, four, three, two, or one year).

[0303] In a preferred embodiment, the immune response induced by the composition or the method of inducing an immune response according to the present invention comprises antibodies with opsonophagocytic activity. Compositions comprising an E. coli O antigen polysaccharide covalently linked to a carrier protein (i.e., glycoconjugates of E. coli O antigen) have been shown to induce functional antibodies of this type in humans, and such antibodies have been shown to mediate bacterial killing in vivo and, via this mechanism, to protect against E. coli infection.

[0304] In another aspect, provided herein are methods of inducing an immune response against E. coli, preferably extraintestinal pathogenic E. coli (ExPEC), in a subject, comprising administering to the subject a composition as described herein.

[0305] In another aspect, provided herein is a method of vaccinating a subject against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), comprising administering a composition as described herein to the subject. In certain aspects, provided herein is a composition as described herein for inducing antibodies against Escherichia coli, preferably ExPEC. In certain aspects, provided herein is a composition as described herein for vaccinating against Escherichia coli, preferably ExPEC. In certain aspects, provided herein is use of a composition as described herein for the manufacture of a medicament for inducing antibodies against Escherichia coli, preferably ExPEC, in a subject. In certain aspects, provided herein is use of a composition as described herein for the manufacture of a medicament for vaccinating a subject against Escherichia coli, preferably ExPEC.

[0306] In certain aspects, provided herein are compositions as described herein for use in a method of inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject. In certain aspects, provided herein are uses of compositions as described herein in the manufacture of a medicament for inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject.

[0307] In another aspect, provided herein is a method for inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject, the method comprising administering to the subject a first effective amount of an E. coli O75 antigenic polysaccharide and a second effective amount of an E. coli O6 antigenic polysaccharide, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, and more preferably about 2:1.

[0308] In certain embodiments, the immune response limits the severity of or prevents aggressive ExPEC disease in the subject.

[0309] In certain embodiments, invasive ExPEC disease comprises sepsis and / or bacteremia.

[0310] In certain embodiments, the method further comprises administering to the subject one or more, preferably all, of the E. coli O1, O2, O4, O15, O16, O18, and O25 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein.

[0311] Preferably, the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B,

[0312] More preferably, the O1A, O2, glucosylated O4, O6A, O15, O16, O18A, O25B and O75 antigenic polysaccharides each comprise the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20,

[0313] And wherein the ratio of O75 antigen polysaccharide to O6 antigen polysaccharide is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, more preferably about 2:1.

[0314] In certain embodiments, the method further comprises administering to the subject 1 to 15 additional E. coli antigenic polysaccharides, each antigenic polysaccharide independently covalently linked to a carrier protein.

[0315] In a specific embodiment, each carrier protein comprises the amino acid sequence of SEQ ID NO:3.

[0316] In certain embodiments, the subject is a human having or at risk of developing an E. coli (preferably ExPEC) infection, preferably an invasive ExPEC disease.

[0317] In certain embodiments, about 8-16 μg, preferably about 16 μg, of the O75 antigen polysaccharide is administered for each administration.

[0318] In certain embodiments, the weight ratio of E. coli antigen polysaccharide O1:O2:O4:O6:O15:O16:O18:O25:O75 administered is 1:1:1:1:1:1:1:2:2.

[0319] In certain embodiments, the glucosylated O4 is a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide of E. coli comprises a structure of the formula (O4-Glc+) shown in Table 1, wherein n is an integer from 3 to 50, such as 5 to 40, preferably 5 to 30, such as 7 to 25, such as 10 to 20,

[0320] wherein a bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein has been produced in E. coli cells comprising:

[0321] (i) nucleotide sequence of the rfb gene cluster of Escherichia coli O4 antigen polysaccharide;

[0322] (ii) a nucleotide sequence encoding a glucosyltransferase having at least 80%, preferably at least 90%, and preferably at least 95% sequence identity to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying E. coli O4 antigen polysaccharide to produce an E. coli glucosylated O4 antigen polysaccharide;

[0323] (iii) a nucleotide sequence encoding a translocase and a glycosyltransferase having at least 80%, preferably at least 90%, preferably at least 95% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating a glucose to which a bacterial terpene alcohol is attached, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0324] (iv) a nucleotide sequence encoding a carrier protein; and

[0325] (v) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glucosylated O4 antigen polysaccharide of E. coli to a carrier protein, preferably wherein the oligosaccharyltransferase is PglB from Campylobacter jejuni.

[0326] In one aspect, the present invention also provides a composition comprising E. coli O1, O2, O4, O6, O15, O16, O18, O25 and O75 antigen polysaccharides (each having the structure indicated above for each antigen), wherein each antigen polysaccharide is independently covalently linked to a carrier protein, and the composition does not comprise other E. coli O antigen polysaccharides covalently linked to the carrier protein (i.e., a 9-valent composition). In certain embodiments of this aspect, the carrier protein is EPA. In other embodiments of this aspect, the carrier protein is CRM. 197 In certain embodiments of this aspect, the covalent linkage is the result of chemical conjugation. In other embodiments, the covalent linkage is the result of biological conjugation. In certain embodiments of this aspect, the carrier protein is a CRM 197 and the covalent linkage is the result of chemical conjugation. In other embodiments of this aspect, the carrier protein is EPA and the covalent linkage is the result of biological conjugation. In preferred embodiments of this aspect, the composition comprises an increased amount of E. coli O75 antigenic polysaccharide compared to the O1, O2 or O6 antigenic polysaccharide, preferably compared to each of the O1, O2 and O6 antigenic polysaccharides, preferably an increase of about 1.5-4 times, preferably an increase of about two times.

[0327] Example

[0328] Embodiment 1 is a composition comprising Escherichia coli O75 antigenic polysaccharide and at least one additional Escherichia coli O antigenic polysaccharide selected from the group consisting of O1, O2, O4, O6, O15, O16 and O18, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the concentration ratio of O75 antigenic polysaccharide to the additional O antigenic polysaccharide is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, preferably about 1.5:1 to about 2.5:1, and more preferably about 2:1.

[0329] Embodiment 1a is the composition of embodiment 1, wherein the at least one additional O-antigen polysaccharide is O1, preferably O1A.

[0330] Embodiment 1a1 is the composition of embodiment 1a, wherein the concentration ratio of O75 antigen polysaccharide to O1 antigen polysaccharide is 2:1.

[0331] Embodiment 1b is the composition of embodiment 1, wherein the at least one additional O-antigen polysaccharide is O2.

[0332] Example 1b1 is the composition of Example 1b, wherein the concentration ratio of O75 antigen polysaccharide to O1 antigen polysaccharide is 2:1.

[0333] Embodiment 1c is the composition of embodiment 1, wherein the at least one additional O-antigen polysaccharide is O4, preferably glucosylated O4.

[0334] Example 1c1 is the composition of Example 1c, wherein the concentration ratio of O75 antigen polysaccharide to O4 antigen polysaccharide is 2:1.

[0335] Embodiment 1d is the composition of embodiment 1, wherein the at least one additional O-antigen polysaccharide is O6, preferably O6A.

[0336] Example 1d1 is a composition as described in Example 1d, wherein the concentration ratio of O75 antigen polysaccharide to O6 antigen polysaccharide is 2:1.

[0337] Embodiment 1e is the composition of embodiment 1, wherein the at least one additional O-antigen polysaccharide is O15.

[0338] Embodiment 1e1 is the composition of embodiment 1e, wherein the concentration ratio of O75 antigen polysaccharide to O15 antigen polysaccharide is 2:1.

[0339] Embodiment 1f is the composition of embodiment 1, wherein the at least one additional O-antigen polysaccharide is O16.

[0340] Example 1f1 is a composition as described in Example 1f, wherein the concentration ratio of O75 antigen polysaccharide to O16 antigen polysaccharide is 2:1.

[0341] Embodiment 1g is the composition of embodiment 1, wherein the at least one additional O-antigen polysaccharide is O18, preferably O18A.

[0342] Example 1g1 is the composition of Example 1g, wherein the concentration ratio of O75 antigen polysaccharide to O18 antigen polysaccharide is 2:1.

[0343] Embodiment 1h is the composition of embodiment 1, further comprising an O25 antigen polysaccharide independently covalently linked to a carrier protein, preferably the O25 antigen is O25B.

[0344] Example 1h1 is the composition of Example 1h, wherein the concentration ratio of O75 antigen polysaccharide to O25 antigen polysaccharide is 1:1.

[0345] Embodiment 2 is a composition as described in embodiment 1, wherein the composition comprises Escherichia coli O75 antigenic polysaccharide and two or more, preferably all, Escherichia coli O1, O2, O4, O6, O15, O16, O18, and O25 antigenic polysaccharides, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, preferably the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B.

[0346] Embodiment 3 is the composition as described in embodiment 2, wherein

[0347] (i) The E. coli O1 antigen polysaccharide comprises the structure of formula (O1A):

[0348]

[0349] (ii) The E. coli O2 antigen polysaccharide comprises the structure of formula (O2):

[0350]

[0351] (iii) The E. coli O4 antigen polysaccharide comprises a structure of the formula (O4-Glc+):

[0352]

[0353] (iv) The E. coli O6 antigen polysaccharide comprises a structure of formula (O6A):

[0354]

[0355] (v) The E. coli O15 antigen polysaccharide comprises the structure of formula (O15):

[0356] [→2)-β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→]n,

[0357] (vi) The E. coli O16 antigen polysaccharide comprises the structure of formula (O16):

[0358]

[0359] (vii) The E. coli O18 antigen polysaccharide comprises the structure of formula (O18A):

[0360]

[0361] (viii) The E. coli O25 antigen polysaccharide comprises the structure of formula (O25B):

[0362] and

[0363] (ix) The E. coli O75 antigen polysaccharide comprises the structure of formula (O75):

[0364]

[0365] wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

[0366] Embodiment 4 is a composition as described in embodiment 2 or 3, wherein the weight ratio of these Escherichia coli antigen polysaccharides O1:O2:O4:O6:O15:O16:O18:O25:O75 is 1:1:1:1:1:1:1:1:2:2.

[0367] Embodiment 5 is a composition as described in any of embodiments 1-4, wherein the concentration of the O75 antigen polysaccharide is about 8 to about 50 μg / mL, preferably 12 to 40 μg / mL, such as 16-32 μg / mL, preferably about 26-38 μg / mL, preferably about 32 μg / mL.

[0368] Embodiment 6 is the composition of any one of embodiments 1-5, further comprising at least one additional E. coli antigenic polysaccharide covalently linked to a carrier protein.

[0369] Embodiment 7 is the composition of embodiment 6, wherein the another additional E. coli antigenic polysaccharide comprises an O8 antigenic polysaccharide having formula (O8):

[0370] α-D-Manp3Me-(1→[3)-β-D-Manp-(1→2)-α-D-Manp-(1→2)-α-D-Manp-(1→]n,

[0371] wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

[0372] Embodiment 8 is a composition as described in any one of embodiments 1-7, wherein each carrier protein is independently selected from the group consisting of: Pseudomonas aeruginosa detoxified exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM 197 , maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clot factor A, clot factor B, Escherichia coli heat-labile enterotoxin, detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from non-typeable Haemophilus influenzae.

[0373] Embodiment 9 is a composition as described in embodiment 8, wherein the carrier protein is Pseudomonas aeruginosa detoxified exotoxin A (EPA) or CRM 197 .

[0374] Embodiment 10 is the composition of any one of embodiments 1-9, wherein the carrier protein comprises 1 to 20, such as 1 to 10 or 2 to 4, glycosylation consensus sequences having the amino acid sequence of SEQ ID NO: 1, such as a consensus sequence having the amino acid sequence of SEQ ID NO: 2, most preferably, the carrier protein comprises four glycosylation consensus sequences.

[0375] Embodiment 11 is the composition of any one of embodiments 1-10, wherein each carrier protein is EPA comprising the amino acid sequence of SEQ ID NO: 3.

[0376] Embodiment 12 is a composition as described in any of embodiments 1-11, wherein the E. coli antigenic polysaccharides are covalently linked to the carrier protein by bioconjugation or chemical conjugation, such as reductive amination chemistry (RAC), single-end conjugation, or conjugation with a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

[0377] Embodiment 13 is the composition of embodiment 12, wherein the E. coli antigenic polysaccharides are covalently linked to a carrier protein by bioconjugation, preferably the polysaccharide is covalently linked to an Asn residue in a glycosylation site in the carrier protein.

[0378] Example 14 is a composition comprising:

[0379] (i) a bioconjugate of an Escherichia coli O1A antigenic polysaccharide covalently linked to a Pseudomonas aeruginosa detoxified exotoxin A (EPA-4) carrier protein, wherein the Escherichia coli O1A antigenic polysaccharide comprises a structure of formula (O1A);

[0380] (ii) a bioconjugate of an E. coli O2 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O2 antigen polysaccharide comprises a structure of formula (O2);

[0381] (iii) a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to an EPA-4 carrier protein, wherein the glucosylated O4 antigen polysaccharide of E. coli comprises a structure of the formula (O4-Glc+);

[0382] (iv) a bioconjugate of an E. coli O6A antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O6A antigen polysaccharide comprises a structure of formula (O6A);

[0383] (v) a bioconjugate of an E. coli O15 antigenic polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O15 antigenic polysaccharide comprises a structure of formula (O15);

[0384] (vi) a bioconjugate of an E. coli O16 antigenic polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O16 antigenic polysaccharide comprises a structure of formula (O16);

[0385] (vii) a bioconjugate of an E. coli O18A antigenic polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O18A antigenic polysaccharide comprises a structure of formula (O18A);

[0386] (viii) a bioconjugate of an E. coli O25B antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O25B antigen polysaccharide comprises a structure of formula (O25B); and

[0387] (ix) a bioconjugate of an E. coli O75 antigen polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O75 antigen polysaccharide comprises a structure of formula (O75);

[0388] wherein the EPA-4 comprises the amino acid sequence of SEQ ID NO: 3,

[0389] The structures of formulae (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) are shown in Table 1, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, more preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20, and

[0390] The ratio of O75 antigen polysaccharide to O6A antigen polysaccharide is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, and more preferably about 2:1.

[0391] Embodiment 15 is the composition of embodiment 14, further comprising 1 to 15 additional E. coli antigenic polysaccharides, each antigenic polysaccharide independently covalently linked to a carrier protein.

[0392] Embodiment 16 is a composition as described in any one of embodiments 2-15, wherein the glucosylated O4 is a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide of E. coli comprises a structure of the formula (O4-Glc+) shown in Table 1, wherein n is an integer from 3 to 50, such as 5 to 40, preferably 5 to 30, such as 7 to 25, such as 10 to 20,

[0393] wherein a bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein has been produced in E. coli cells comprising:

[0394] (i) the nucleotide sequence of the rfb gene cluster of the Escherichia coli O4 antigen polysaccharide;

[0395] (ii) a nucleotide sequence encoding a glucosyltransferase having at least 80%, preferably at least 90%, and preferably at least 95% sequence identity to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying E. coli O4 antigen polysaccharide to produce E. coli glucosylated O4 antigen polysaccharide;

[0396] (iii) a nucleotide sequence encoding a translocase and a glycosyltransferase having at least 80%, preferably at least 90%, preferably at least 95% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating a glucose to which a bacterial terpene alcohol is attached, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0397] (iv) a nucleotide sequence encoding the carrier protein; and

[0398] (v) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glucosylated O4 antigen polysaccharide of E. coli to a carrier protein, preferably wherein the oligosaccharyltransferase is PglB from Campylobacter jejuni.

[0399] Example 17 is a method of inducing an immune response against Escherichia coli (preferably extraintestinal pathogenic Escherichia coli, ExPEC) in a subject, the method comprising administering to the subject the composition of any one of Examples 1-16.

[0400] Embodiment 17a is the method of embodiment 17, wherein the subject is in need of the immune response.

[0401] Example 18 is a method of inducing an immune response against Escherichia coli (preferably extraintestinal pathogenic Escherichia coli, ExPEC) in a subject, the method comprising administering to the subject a first effective amount of an E. coli O75 antigenic polysaccharide and a second effective amount of an E. coli O6 antigenic polysaccharide, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, and more preferably about 2:1.

[0402] Embodiment 18a is the method of embodiment 18, wherein the subject is in need of the immune response.

[0403] Embodiment 19 is the method of any one of embodiments 17-18a, wherein the immune response limits the severity of or prevents aggressive ExPEC disease in the subject.

[0404] Embodiment 20 is the method of embodiment 19, wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

[0405] Embodiment 20a is the method of embodiment 20, wherein the invasive ExPEC disease comprises sepsis.

[0406] Embodiment 20b is the method of embodiment 20, wherein the invasive ExPEC disease comprises bacteremia.

[0407] Embodiment 21 is the method of any one of embodiments 18-20, further comprising administering to the subject one or more, preferably all, of the O1, O2, O4, O15, O16, O18, and O25 antigen polysaccharides, wherein each antigen polysaccharide is independently covalently linked to a carrier protein,

[0408] Preferably, the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B,

[0409] More preferably, each of the O1A, O2, glucosylated O4, O6A, O15, O16, O18A, O25B and O75 antigen polysaccharides comprises the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20, and wherein the ratio of the O75 antigen polysaccharide to the O6 antigen polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, more preferably about 2:1.

[0410] Embodiment 22 is the method of any one of embodiments 18-21, further comprising administering to the subject 1 to 15 additional E. coli antigenic polysaccharides, each antigenic polysaccharide independently covalently linked to a carrier protein.

[0411] Embodiment 23 is the method of any one of embodiments 18-22, wherein each carrier protein comprises the amino acid sequence of SEQ ID NO: 3.

[0412] Embodiment 24 is the method of any one of embodiments 17-23, wherein the subject is a human having or at risk of E. coli (preferably ExPEC) infection or invasive ExPEC disease.

[0413] Embodiment 25 is the method of any one of embodiments 17-24, wherein about 8-16 μg, preferably about 16 μg, of the O75 antigen polysaccharide is administered for each administration.

[0414] Embodiment 26 is the method of any one of embodiments 21-25, wherein the weight ratio of the administered E. coli antigen polysaccharide O1:O2:O4:O6:O15:O16:O18:O25:O75 is 1:1:1:1:1:1:1:1:2:2.

[0415] Embodiment 27 is a method as described in any one of embodiments 21-26, wherein the glucosylated O4 is a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide of E. coli comprises a structure of the formula (O4-Glc+) shown in Table 1, wherein n is an integer from 3 to 50, such as 5 to 40, preferably 5 to 30, such as 7 to 25, such as 10 to 20,

[0416] wherein a bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein has been produced in E. coli cells comprising:

[0417] (i) the nucleotide sequence of the rfb gene cluster of the Escherichia coli O4 antigen polysaccharide;

[0418] (ii) a nucleotide sequence encoding a glucosyltransferase having at least 80%, preferably at least 90%, and preferably at least 95% sequence identity to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying E. coli O4 antigen polysaccharide to produce E. coli glucosylated O4 antigen polysaccharide;

[0419] (iii) a nucleotide sequence encoding a translocase and a glycosyltransferase having at least 80%, preferably at least 90%, preferably at least 95% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating a glucose to which a bacterial terpene alcohol is attached, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0420] (iv) a nucleotide sequence encoding the carrier protein; and

[0421] (v) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glucosylated O4 antigen polysaccharide of E. coli to a carrier protein, preferably wherein the oligosaccharyltransferase is PglB from Campylobacter jejuni.

[0422] Embodiment 27 is the composition of any one of embodiments 1-16, for use in a method of inducing an immune response against Escherichia coli (preferably extraintestinal pathogenic Escherichia coli, ExPEC) in a subject.

[0423] Embodiment 28 is use of the composition of any one of embodiments 1-16 in the manufacture of a medicament for inducing an immune response against Escherichia coli (preferably extraintestinal pathogenic Escherichia coli, ExPEC) in a subject.

[0424] Example 29 is a combination of a first effective amount of an Escherichia coli O75 antigenic polysaccharide and a second effective amount of an Escherichia coli O6 antigenic polysaccharide for use in a method of inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, and more preferably about 2:1.

[0425] Embodiment 29a is the combination of embodiment 29, wherein the first effective amount and the second effective amount are in the same composition.

[0426] Embodiment 29b is the combination of embodiment 29, wherein the first effective amount and the second effective amount are in separate compositions.

[0427] Embodiment 29c is the combination of any one of embodiments 29-29b, wherein the subject is in need of said immune response.

[0428] Embodiment 30 is the combination of any one of embodiments 29-29c, wherein the immune response limits the severity of or prevents aggressive ExPEC disease in the subject.

[0429] Embodiment 31 is the combination of embodiment 30, wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

[0430] Embodiment 32 is a combination as described in any one of embodiments 29-31, which further comprises an effective amount of one or more, preferably all, E. coli O1, O2, O4, O15, O16, O18, O25 antigen polysaccharides, wherein each antigen polysaccharide is independently covalently linked to a carrier protein, preferably the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B, preferably O1A, O2, glucosylated O4, O6A, O15, O16, O18A, O25B and O25A. Each of the 75 antigen polysaccharides comprises the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as 5 to 40, preferably from 5 to 30, such as 7 to 25, such as 10 to 20, and wherein the ratio of O75 antigen polysaccharide to O6 antigen polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, and more preferably about 2:1.

[0431] Embodiment 33 is the combination of any one of embodiments 29-32, further comprising an effective amount of 1 to 15 additional E. coli antigenic polysaccharides, each antigenic polysaccharide independently covalently linked to a carrier protein.

[0432] Embodiment 34 is the combination of any one of embodiments 29-33, wherein each carrier protein comprises the amino acid sequence of SEQ ID NO: 3.

[0433] Embodiment 35 is the combination of any one of embodiments 29-34, wherein the subject is a human having or at risk of E. coli (preferably ExPEC) infection or invasive ExPEC disease.

[0434] Embodiment 36 is the combination of any one of embodiments 29-35, wherein the effective amount of the O75 antigen polysaccharide is about 8-16 μg, preferably about 16 μg.

[0435] Example 37 is a combination of a first effective amount of an Escherichia coli O75 antigenic polysaccharide and a second effective amount of an Escherichia coli O6 antigenic polysaccharide, in the manufacture of a medicament for inducing an immune response against Escherichia coli, preferably extraintestinal pathogenic Escherichia coli (ExPEC), in a subject, wherein each antigenic polysaccharide is independently covalently linked to a carrier protein, and wherein the ratio of the first effective amount to the second effective amount is about 1.2:1 to about 8:1, preferably about 1.5:1 to about 4:1, and more preferably about 2:1.

[0436] Embodiment 37a is the combination of embodiment 37, wherein the subject is in need of said immune response.

[0437] Embodiment 38 is the combination of embodiment 37 or 37a, wherein the immune response limits the severity of or prevents aggressive ExPEC disease in the subject.

[0438] Embodiment 39 is the combination of embodiment 38, wherein the invasive ExPEC disease comprises sepsis and / or bacteremia.

[0439] Embodiment 40 is a combination as described in any one of embodiments 37-39, which further comprises an effective amount of one or more, preferably all, E. coli O1, O2, O4, O15, O16, O18, O25 antigen polysaccharides, in the manufacture of a medicament, wherein each antigen polysaccharide is independently covalently linked to a carrier protein, preferably the O1 antigen is O1A, O4 is glucosylated, the O6 antigen is O6A, the O18 antigen is O18A, and the O25 antigen is O25B, preferably O1A, O2, O4, O15, O16, O18, O25A, O25B, O1A, O2, O4, O15, O16, O18, O25B, O2A, O2A, O1B, O2B, O2B, O2B, O2B, O2B, O2B, O2B, O1 ... Each of the 5B and O75 antigen polysaccharides comprises the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as 5 to 40, preferably from 5 to 30, such as 7 to 25, such as 10 to 20, and wherein the ratio of O75 antigen polysaccharide to O6 antigen polysaccharide is from about 1.2:1 to about 8:1, preferably from about 1.5:1 to about 4:1, more preferably about 2:1.

[0440] Embodiment 41 is the combination of any one of embodiments 37-40, further comprising, for use in the manufacture of a medicament, an effective amount of 1 to 15 additional E. coli antigenic polysaccharides, each antigenic polysaccharide independently covalently linked to a carrier protein.

[0441] Embodiment 42 is the combination of any one of embodiments 37-41, wherein each carrier protein comprises the amino acid sequence of SEQ ID NO: 3.

[0442] Embodiment 43 is the combination of any one of embodiments 37-42, wherein the subject is a human having or at risk of E. coli (preferably ExPEC) infection or invasive ExPEC disease.

[0443] Embodiment 44 is the combination of any one of embodiments 37-43, wherein the effective amount of one dose of O75 antigen polysaccharide for administration to a subject is about 8-16 μg, preferably about 16 μg.

[0444] Examples

[0445] Example 1: Epidemiological data of Escherichia coli infection

[0446] A global surveillance study was conducted to determine the distribution of O serotypes of Escherichia coli causing bacteremia. Between 2011 and 2017, over 3200 E. coli bloodstream isolates were collected from hospitalized patients aged 60 years and older in countries across North America, Europe, Asia-Pacific, and South America. The O antigen serotype of each isolate was determined using classical agglutination techniques and sequence-based O genotyping. See Table 2 below.

[0447] Isolated human blood samples were analyzed to determine the identity of the pathogens and their antibiotic resistance patterns. E. coli isolates were obtained from the samples after analysis. E. coli identity was verified by MALDI-TOF MS. E. coli isolates were further analyzed using an antiserum-based agglutination assay to determine their O antigen serotype (DebRoy et al. (2011) Animal health research reviews / Conference of Research Workers in Animal Diseases 12, 169-185). Isolates that could not be typed by agglutination were further analyzed by whole genome sequencing, followed by O genotyping based on the O serotype-specific wzy and wzx gene sequences.

[0448] Table 2. Distribution of the most common bacteremia-associated Escherichia coli O serotypes based on O serotyping by agglutination plus O genotyping for isolates that could not be typed by agglutination, from a collection of 3217 blood isolates collected worldwide between 2011 and 2017. The subjects were hospitalized in the following countries: United States, Canada, Argentina, Brazil, United Kingdom, Germany, Spain, Italy, Netherlands, France, Japan, Thailand, South Korea, and Australia.

[0449] O-serotype Popularity n (%) O25 737(22.9％) O2 268(8.3％) O6 261(8.1％) O1 255(7.9％) O75 145(4.5％) O15 110(3.4％) O8 104(3.2％) O16 103(3.2％) O4 96(3.0％) O18 91(2.8％)

[0450] Stratification of the global collection of bacteremia-associated E. coli by geographic location revealed that the prevalence of the top 10 O serotypes was independent of location, indicating that these O serotypes are the predominant O serotypes globally associated with bacteremia-causing E. coli.

[0451] In a global collection of bacteremia-associated multidrug-resistant E. coli isolates (n=345) (ie, strains resistant to at least three classes of clinically relevant antimicrobial drugs), the prevalence of the top 10 O serotypes was 75.4%.

[0452] Taking all information from epidemiological analyses together, these 10 major O serotypes could cover an estimated 60%–80% of E. coli-associated bacteremic infections, assuming coverage of a subset of nontypeable strains.

[0453] A multivalent vaccine that covers most E. coli serotypes that cause bacteremia would be very useful. Therefore, the O serotypes in Table 2 would be good candidates for a multivalent vaccine based on the O antigen. Such a vaccine can advantageously be prepared using chemical conjugation or bioconjugation techniques.

[0454] Example 2: Production of E. coli O-antigen bioconjugates and bioconjugate products produced therefrom.

[0455] Ten (10) bioconjugates were produced, including E. coli O1A-EPA bioconjugate, O2-EPA bioconjugate, O4-Glc+-EPA bioconjugate (hereinafter also referred to as O4-EPA, i.e., in the following examples, the bioconjugate of the O4 antigen polysaccharide is a variant in which O4 is glucosylated, having the glycan structure (O4-Glc+) shown in Table 1; PCT / US20 / 23404 filed on March 18, 2020 provides details on the use of novel, hitherto unreported and coli O4 antigen polysaccharide by adding glucose to produce E. coli glucosylated O4 antigen, which is incorporated herein by reference), O6A-EPA bioconjugates, O8-EPA bioconjugates, O15-EPA bioconjugates, O16-EPA bioconjugates, O18A-EPA bioconjugates, O25B-EPA bioconjugates, and O75-EPA bioconjugates. The structures of the glycans in these conjugates can be found in the formulas in Table 1. The composition containing these 10 bioconjugates is referred to herein as 'ExPEC10V'. The composition containing the O1A-EPA, O2-EPA, O6A-EPA, and O25B-EPA bioconjugates is referred to as 'ExPEC4V' (and has been previously described, for example, in WO 2015 / 124769 and WO 2017 / 035181). The production of these ten bioconjugates and exemplary production strains of these bioconjugates are described in detail in PCT / US20 / 23404, filed on March 18, 2020, which is incorporated herein by reference.

[0456] Escherichia coli W3110 parent strain

[0457] The non-pathogenic E. coli K12 strain W3110 was used as the parent strain for the construction of all ten production strains. E. coli K12 strain W3110 was obtained from the Coli Genetic Stock Center (Yale University, New Haven, Connecticut (CT), USA, product number CGSC #4474). Its related genotype (E. coli W3110, F-, λ-, IN (rrnD-rrnE) 1, rph-1) has been previously described, and its genome sequence has been previously published (Hayashi K et al., 2006, Mol. Syst. Biol. 2006.0007 (doi: 10.1038 / msb4100049)). The E. coli W3110 strain was genetically modified to enable it to produce each of these E. coli O antigen bioconjugates (Table 3).

[0458] Bioconjugate production strains

[0459] Both "ExPEC4V" and "ExPEC10V" compositions contain the O2-EPA and O25B-EPA bioconjugates from the same production strain. The "ExPEC4V" composition contains the O1A-EPA bioconjugate from either the stGVXN4411 or stLMTB10217 production strain, while the "ExPEC10V" composition contains the O1A-EPA bioconjugate from the stLMTB10217 production strain. The "ExPEC4V" composition contains the O6A-EPA bioconjugate from the stGVXN4112 production strain, while the "ExPEC10V" composition contains the O6A-EPA bioconjugate from the stLMTB10923 production strain. In addition, the "ExPEC10V" composition contains 04-EPA (i.e., (04-Glc+)-EPA), 08-EPA, 015-EPA, 016-EPA, 018A-EPA, and 075-EPA bioconjugates from production strains not used for "ExPEC4V." As shown below, different production strains may differ in the plasmids used to express the EPA carrier protein and / or the oligosaccharyltransferase PglB. An overview of several exemplary production strains is given in Table 3 below.

[0460] Table 3. Overview of genetic engineering of E. coli production strains for O-antigen bioconjugates of ExPEC4V and ExPEC10V vaccine compositions

[0461]

[0462]

[0463] Alternative production strains have also been prepared and used for some bioconjugates, for example to find clones with improved production and / or changes in certain characteristics of the bioconjugate.

[0464] O antigen biosynthesis (rfb) gene cluster

[0465] In all E. coli O antigen production strains, the naturally occurring E. coli W3110 genomic O16::IS5-antigen biosynthesis (rfb) gene cluster was replaced by a selected O antigen-specific biosynthesis cluster encoding a specific serotype O antigen structure (for these O antigen structures, see Table 1) from an E. coli strain of a selected serotype. Ten donor rfb clusters were selected or confirmed after whole genome analysis of E. coli blood isolates. Replacement of the W3110 O16::IS5 rfb gene cluster, which is defective in O antigen biosynthesis, was achieved in a single homologous recombination event. In the case of the O16 and O18 Arfb gene clusters, the donor DNA was recombined via the flanking gnd and rmlCA genes, while the rfb gene clusters of the other strains were recombined via the flanking gnd and galF genes. The sequences of the rfb clusters in the production strains are provided in SEQ ID NOs: 9 and 11-19.

[0466] O antigen ligase (waaL) gene

[0467] All E. coli O-antigen-producing strains carry an artificially introduced deletion in the O-antigen ligase encoded by the waaL gene in the E. coli W3110 genome. In the ΔwaaL strain, O-antigen transfer to lipid A is disrupted, redirecting O-antigen transfer to the carrier protein to increase product yield.

[0468] O antigen glycosylation (gtrABS) gene

[0469] The gtrABS gene, responsible for glycosylation of the O16 O antigen, has been deleted from the E. coli W3110 genome in E. coli O8, O15, O16, O18A, O25B, and O75 production strains. While the gtrA and gtrB genes are highly homologous and interchangeable among different serotypes, the gtrS gene encodes a serotype-specific O antigen glycosyltransferase. In E. coli W3110, GtrS transfers a glucose (Glc) residue to the GlcNAc sugar within the α-L-Rha-(1→3)-d-GlcNAc motif of the E. coli O16 O antigen. No gtrABS gene deletion or replacement has occurred in E. coli O1A, O2, and O6A production strains. These O antigens lack the α-L-Rha-(1→3)-d-GlcNAc motif, the natural substrate for E. coli O16 gtrS. In the E. coli O4 production strain, the W3110 gtrS gene has been replaced by the E. coli O4 gtrS gene to accommodate proper glucosylation of the E. coli O4 O antigen (the coding sequence of the E. coli O4 gtrS gene is provided herein as SEQ ID NO:5, and the amino acid sequence of the E. coli O4 GtrS protein is provided herein as SEQ ID NO:4; see also, e.g., PCT / US20 / 23404 filed March 18, 2020).

[0470] Oligosaccharyltransferase PglB

[0471] All E. coli O-antigen production strains express a variant of the Campylobacter jejuni glycosyltransferase PglB, which can transfer the O-antigen to an amino acid consensus sequence on the carrier protein via N-glycosylation. PglB has broad substrate recognition, but due to low product yields, several production strains expressing PglB variants with altered substrate specificity have been generated, resulting in improved product yields (see, e.g., WO 2016 / 107818 and WO 2016 / 107819). The pglB gene was placed on a plasmid behind an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoter. Table 4 below lists the PglB variants encoded by the plasmids used to produce E. coli O-antigen production strains for the bioconjugates of the ExPEC4V and ExPEC10V compositions described above. Other plasmids with variations in the carrier backbone, antibiotic resistance marker, and / or alternative PglB variants have also been successfully tested for bioconjugate production.

[0472] Table 4. PglB and EPA plasmids used in E. coli O-antigen production strains

[0473]

[0474] 1 SpR, spectinomycin resistance; AmpR, ampicillin resistance; KanR, kanamycin resistance

[0475] As described in more detail, e.g., in PCT / US20 / 23415, filed March 18, 2020, the optimal PglB variant was determined for each bioconjugate of the ten E. coli O antigens in ExPEC10V, which is incorporated herein by reference in its entirety.

[0476] Carrier protein (EPA)

[0477] All E. coli O-antigen production strains express a genetically detoxified Pseudomonas aeruginosa ADP-ribosyltransferase toxoid (EPA) as the carrier protein for the O-antigen. The EPA toxoid differs from wild-type EPA toxin by two residues: Leu552 is changed to Val and Glu553 (in the catalytic domain) is deleted. Deletion of Glu553 has been reported to significantly reduce toxicity. In addition to the detoxification mutations, four (EPA-4) consensus N-glycosylation site motifs were introduced. The epa gene was placed behind an l-arabinose (Ara)-inducible promoter on the plasmid (Table 4). Table 4 is limited to plasmids used in the production strains of the bioconjugates in the "ExPEC4V" and "ExPEC10V" compositions described above. Plasmids with variations in the carrier backbone, antibiotic resistance marker, and / or EPA variants, such as those with different numbers of consensus N-glycosylation site motifs (e.g., EPA-2, which has two such motifs), have also been successfully tested for bioconjugate production.

[0478] Example 3: Optimization of oligosaccharyltransferase to produce bioconjugates of E. coli O antigen.

[0479] Yield optimization of bioconjugate production can be achieved by modifying the Campylobacter jejuni oligosaccharyltransferase PglB, which results in more efficient or higher N-glycosylation of the O-antigen of interest with the EPA carrier protein. This optimization strategy was applied to an E. coli strain used to produce bioconjugates with glucosylated O4 (O4-Glc+) O-antigen polysaccharides, resulting in optimized (O4-Glc+)-specific PglB variants that increased bioconjugate product yield.

[0480] In this method, an O4-Glc+ O-antigen polysaccharide-producing strain containing an EPA expression plasmid was transformed with multiple different PglB expression plasmids, each containing a different amino acid substitution in the PglB protein that altered substrate specificity. The bioconjugate production level and profile of each strain were assessed at the shake flask level in an osmotic shock experiment, and the periplasmic extracts were read using a capillary electrophoresis immunoassay using an O4-Glc+-specific monoclonal antibody.

[0481] One of the tested PglB variants containing the N311V amino acid substitution was found to significantly increase the product yield of the glucosylated O4 bioconjugate (see PCT / US20 / 23415, filed March 18, 2020, which is incorporated herein by reference in its entirety).

[0482] In a further improvement of the N311V PglB variant, the Y77H amino acid substitution further increased the yield of the O4-Glc+ specific product and showed increased levels of di- and tri-glycosylated products compared to the N311V PglB variant, where other improvements were found to be neutral or had a negative impact on product yield (see PCT / US20 / 23415, filed on March 18, 2020, which is incorporated herein by reference in its entirety). Plasmid pLMTB4008 (SpR) encodes an optimized E. coli codon usage, optimized (O4-Glc+) substrate, a PglB variant with mutations Y77H and N311V.

[0483] Compared to the first round of optimized PglB-N311V variant, a PglB variant containing N311V and Y77H amino acid substitutions with optimized substrate specificity for O4-Glc+O antigen polysaccharide relative to wild-type (wt) Campylobacter jejuni glycosyltransferase PglB was found to result in doubled bioconjugate yield.

[0484] Similarly, using screening, the best-yielding PglB variants were also identified for production of E. coli O-antigen bioconjugates for nine other serotypes in the ExPEC10V composition, see, for example, PCT / US20 / 23415, filed March 18, 2020, which is incorporated herein by reference in its entirety.

[0485] For bioconjugates with O1A, O6A or O15 antigenic polysaccharides, PglB with amino acid mutations N311V, K482R, D483H and A669V was found to provide the highest yield.

[0486] For bioconjugates with the 02, 08, 018A or 025B antigenic polysaccharides, wild-type PglB (ie, without amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669) was found to provide the highest yield.

[0487] For the bioconjugate with the O16 antigen polysaccharide, PglB with the amino acid mutations Y77H, S80R, Q287P, K289R and N311V was found to provide the highest yield.

[0488] For the bioconjugate with the O75 antigen polysaccharide, PglB with the amino acid mutation N311V was found to provide the highest yield.

[0489] These results indicate that the optimal PglB variant is different for different O-antigens and that the optimal PglB variant for generating bioconjugates with a given O-antigen polysaccharide is unpredictable.

[0490] Example 4: Quality attributes of bioconjugates of O antigens from 10 E. coli serotypes.

[0491] The O-glycan residues of the target O-antigens are structurally diverse and have variable repeat units. The specificity and affinity of the glycosyltransferase PglB are related to the glycan structure. Therefore, preparing bioconjugates with desired quality attributes (e.g., purity, glycan / protein ratio, etc.) is a challenging and non-trivial task. The correct combination of PglB and EPA carrier protein determines yield and may affect glycosylation efficiency. By optimizing PglB and carrier protein, bioconjugates with desired quality attributes are generated. Maintaining a low threshold of total carrier protein is also important, particularly when one or more O-antigen bioconjugates are combined and administered in a single composition or vaccine, as very large amounts of carrier protein may cause immunological interference. To avoid this phenomenon, conjugates with higher glycan / protein ratios are preferred. Therefore, for the ExPEC10V vaccine, a bioconjugate with a glycosylation ratio at least comparable to that of the previously described ExPEC4V vaccine that has been clinically tested was developed.

[0492] Bioconjugates were produced individually by culturing the respective host cells (Example 2, Table 3) in bioreactors (10 L and / or 200 L volume) and expressing the bioconjugates according to previously described methods. Each drug substance was produced in batches by bacterial fed-batch fermentation to produce biomass containing the expressed bioconjugate of the corresponding polysaccharide serotype. The cells were cultured and induced with IPTG and arabinose. The bioconjugates were isolated from the periplasm of the cells in the bioreactor cultures by osmotic shock and chromatographic purification. This process was performed for each of the 10 bioconjugates.

[0493] The E. coli O antigen bioconjugates thus prepared are the drug substances (DS) for ExPEC10V and ExPEC4V, which exhibit comparable critical quality attributes: (1) process-related purity (measured by RP-HPLC) greater than 95%, (2) polysaccharide / protein ratios ranging from approximately 0.1 to 0.5, with the majority being between 0.15 and 0.45, and (3) bacterial endotoxins (Ph. Eur. 2.2.3) less than 0.5 EU / μg polysaccharide. The average length of individual polysaccharide chains is typically between approximately 10 and 20 repeat units (measured using high-resolution SDS-PAGE).

[0494] The structures of the polysaccharide repeating units were confirmed (by NMR and MS / MS on the conjugates (intact or trypsin-digested)) to be the structures shown in Table 1 for each formula corresponding to the serotype, and all ten bioconjugates were DS of the ExPEC10V composition described above.

[0495] The ExPEC10V drug product (DP) comprises a mixture of the ten monovalent DSs described above.

[0496] Example 5: Toxicology of ExPEC10V vaccine.

[0497] A single-dose pilot toxicity and local tolerance study (non-GLP) was conducted in female NZW rabbits using ExPEC10V. One group (n=2) received an intramuscular (IM) injection (on day 0) of a control (normal saline), while a second group (n=4) received an IM injection of 105.6 μg total polysaccharide (PS) / dose (9.6:9.6:9.6:9.6:9.6:9.6:9.6:9.6:19.2:9.6 μg PS / dose for ExPEC10V serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively) using a dose volume of 0.6 mL (176 μg PS / mL). Necropsy was performed on day 2.

[0498] No deaths were observed. In addition, no vaccine-related effects were recorded in clinical observations (including injection site effects using the Draize score), body weight, food consumption, and body temperature. Histopathologically, no vaccine-related changes were observed at the administration site or draining (iliac) lymph nodes. A small increase in the formation of germinal centers in the spleen was observed in one of the four treated animals (day 2), which is considered to be a normal immunological response to the injected vaccine. Overall, a single IM dose of ExPEC10V was well tolerated in female rabbits.

[0499] Example 6: Immunogenicity of ExPEC10V mixed formulation in rabbits.

[0500] The ExPEC4V vaccine (a bioconjugate comprising E. coli serotypes O1A, O2, O6A, and O25B) has previously been shown to be immunogenic against these four serotypes in rats, rabbits, and humans (see, e.g., WO 2015 / 124769; WO 2017 / 035181; Huttner et al., 2017, Lancet Infect Dis, http: / / dx.doi.org / 10.1016 / S1473-3099(17)30108-1; RW Frenck Jr et al., 2019, Lancet Infect Dis 19(6):631-640, http: / / dx.doi.org / 10.1016 / S1473-3099(18)30803-X). The immunogenicity of bioconjugates of E. coli serotypes O4-Glu+, O8, O15, O16, O18A, and O75 (all with EPA-2 as a carrier protein) when administered alone (monovalently) to rats demonstrated that each of these bioconjugates was also immunogenic, as ELISA data indicated that each of these bioconjugates could elicit high levels of E. coli O antigen-specific antibodies (not shown).

[0501] The immunogenicity of a 10-valent vaccine containing a mixture of the 10 bioconjugates described above was also tested. New Zealand White (NZW) rabbits (female, 12-16 weeks old) received three intramuscular immunizations of ExPEC10V or saline, administered 2 weeks apart (Table 5; administered on days 0, 14, and 27). The 10 polysaccharides used as part of the ExPEC10V vaccine in these experiments were conjugated to the carrier protein EPA (EPA-4), which contains four glycosylation sites. The vaccine was formulated at three different doses: Group 1 ('high dose'): 8 μg / dose of O1A, O2, O6A, O4, O8, O15, O16, O18, and O75, and 16 μg / dose of O25B; Group 2 ('medium dose'): 4 μg / dose of O2, O4, O8, O15, O16, O18, and O75, 8 μg / dose of O1A and O6A, and 16 μg / dose of O25B; Group 3 ('low dose'): 0.4 μg / dose of O2, O4, O8, O15, O16, O18, and O75, 0.8 μg / dose of O1A and O6A, and 1.6 μg / dose of O25B. Animals from the control group (Group 4) received only saline (0.9% (w / v) sodium chloride solution) (Table 5).

[0502] Antibody responses were assessed at day 0 (before immunization) and 14, 27, and 42 after immunization. Serum antibody levels induced by each bioconjugate and carrier protein EPA included in the vaccine were measured by ELISA (total IgG) using a specific type of LPS as a coating material. Antibody titers were reported as EC50 values ​​based on a 12-step titration curve replicate drawn in a 4-parameter logistic nonlinear regression model, corresponding to half the maximal effective concentration. Functional activity was determined by OPK.

[0503] Table 5. Description of experimental groups.

[0504]

[0505]

[0506] The results are shown in Figure 1 and are summarized in Table 6.

[0507] Table 6. Summary of E. coli O antigen-specific antibody responses induced by ExPEC10V in NZW rabbits.

[0508]

[0509]

[0510]

[0511] Serotype-specific antibody responses where the p-value was statistically significant are indicated with an asterisk. Serotype-specific antibody responses where the p-value was not statistically significant are designated as ns. Multiple comparisons were performed using the Wilcoxon rank sum test with Bonferroni correction. Animals vaccinated with ExPEC10V (Groups 1, 2, and 3) were compared with saline controls (Group 4). *p ≤ 0.05, **p ≤ 0.01. # P values ​​were statistically significant after excluding abnormal animals from the control group (sensitivity analysis).

[0512] The high dose of ExPEC10V (Group 1) induced significantly higher IgG antibody levels at all time points studied (days 14, 27, and 42 after immunization) compared to saline controls in O1A, O2, O4, O6A, O16, O18A, and O25B ( Figure 2 , Table 6). On days 27 and 42 after immunization, significantly higher antibody titers were observed in response to the O8 and O75 conjugates compared to the saline control ( Figure 1 , Table 6).

[0513] Compared with saline controls in O1A, O2, O4, O6A, O16, and O25B, the medium dose of ExPEC10V (Group 2) and the low dose (Group 3) induced significantly higher antibody levels at all time points studied (days 14, 27, and 42 after immunization). Figure 1 , Table 6). Significantly higher antibody titers induced by the O8, O18A, and O75 conjugates compared to saline controls were observed on days 27 and 42 post-immunization, indicating that boosting doses in rabbits increased responses to these O serotypes ( Figure 1 , Table 6).

[0514] For the O15 conjugate, sensitivity analysis excluding outlier animals from the control group showed that all three doses of ExPEC10V vaccine induced significantly increased antibody responses compared with saline controls at days 14, 27, and 42 after immunization ( Figure 1 , Table 6).

[0515] At all time points studied (days 14, 27, and 42), the carrier protein EPA induced antibodies that were significantly higher than the EPA antibody titers in the saline-treated (control) group for the three doses tested (high, medium, and low). Figure 1 ).

[0516] Dose comparisons (not shown) showed that high doses of ExPEC10V induced significantly higher antibody responses compared to low doses of most tested conjugates (O1A, O2, O4, O6A, O15, O16, O18A, and O25B) at day 14 post-vaccination. A medium dose of ExPEC10V also induced significantly higher antibody responses compared to low doses of O1A, O2, O4, O18A, O25B, and O75. For the O8 conjugate, all three formulations of ExPEC10V induced similar levels of antibodies at day 14 post-vaccination.

[0517] Compared with high and medium doses of ExPEC10V for the O1A, O2, O4, O16, O25B, and O75 conjugates, low doses of ExPEC10V induced significantly greater antibody responses at day 42 post-vaccination (after the prime dose and two booster doses). These findings are consistent with other experience with conjugate vaccines, for example, no clear relationship between dose and magnitude of antibody response to prime vaccination was observed in infants vaccinated with pneumococcal conjugate vaccines (Poolman JT et al. Expert Rev Vaccines. 2013, 12(12):1379-94).

[0518] For the O6A, O8, and O15 conjugates, there were no significant differences among the three doses of ExPEC10V tested at day 42 post-vaccination. For the O18A conjugate, the high dose of ExPEC10V induced significantly higher antibody responses compared to the medium dose at day 42 post-vaccination.

[0519] For the carrier protein (EPA), high and medium doses of ExPEC10V induced significantly higher antibody responses compared to the low dose on day 14 after vaccination. The high dose of the vaccine also induced significantly higher antibody responses compared to the low dose on day 42 after vaccination.

[0520] In conclusion, three formulations of ExPEC10V (high, medium, and low) were immunogenic in rabbits when administered via intramuscular injection on days 0, 14, and 27.

[0521] To date, the vaccine has been shown to induce functional antibodies in rabbits capable of killing E. coli strains of serotypes O1A, O2, O4, O6A, O15, O16, and O25B.

[0522] In another experiment, as part of a toxicology study, a GMP batch of ExPEC10V vaccine (for production, see Example 4 above) was prepared and injected into NZW rabbits (Table 7). In this study, NZW rabbits (male and female) received three intramuscular injections (0.6 mL) of ExPEC10V vaccine (days 1, 15, and 29), with a control group receiving 0.9% (w / v) sodium chloride solution (normal saline). Each dose of vaccine contained 9.6 μg polysaccharide (PS) for serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, and O75, and 19.2 μg PS for serotype O25B, corresponding to 105.6 μg total PS (176 μg total PS / mL) and 382.8 μg total EPA (638 μg EPA / mL). IgG titers to O antigen and carrier protein (EPA) were determined from samples collected during the pre-treatment period (day 1) and on days 31 and 50 after immunization.

[0523] Compared with the control group that received saline only, significantly increased antibody responses against all O antigens and the carrier protein EPA were observed in the group that received ExPEC10V at days 31 and 50 after vaccination ( Figure 2 , Table 8). For the O1A serotype, significantly higher antibody responses were also observed on day 1 (baseline) when vaccinated animals were compared to controls. These results suggest that some animals were pre-exposed to E. coli or had antibodies that cross-reacted with O1A-LPS.

[0524] Table 7. ExPEC10V doses used in experimental groups and NZW rabbits.

[0525]

[0526] *Each dose (0.6 mL dosing volume) contains 9.6:9.6:9.6:9.6:9.6:9.6:9.6:9.6:9.6:19.2:9.6 μg polysaccharide (PS) (176 μg total PS / mL) for serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively. Each dose contains 382.8 μg EPA protein (638 μg EPA / mL).

[0527] Table 8. Immunogenicity of ExPEC10V in NZW rabbits as part of toxicology studies.

[0528]

[0529] Antibody responses induced by ExPEC10V. Serotypes for which a significant increase in antibody responses was observed in the vaccinated group compared with controls are indicated with asterisks. Tobit model using a likelihood ratio test. ****P ≤ 0.0001.

[0530] Example 7: Phase 1 / 2a trial of ExPEC10V vaccine in humans.

[0531] Currently, there is no available vaccine to prevent IED. The serotypes that comprise the ExPEC10V vaccine (O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75) were selected to address invasive disease caused by the most clinically relevant ExPEC strains, which also represent the majority of ExPEC isolates (including ST131) that cause antimicrobial IED. The selected serotypes represent the ten prevalent ExPEC O serotypes that cause bloodstream infections in the elderly population and are responsible for approximately 70% of bloodstream infections caused by ExPEC.

[0532] Because the conjugate vaccine's mechanism of action in preventing invasive disease is not expected to be affected by antibiotic resistance mechanisms, the ExPEC10V vaccine is believed to provide protection against IED caused by both drug-resistant and drug-susceptible O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75 serotypes.

[0533] There is previous clinical experience with ExPEC4V, an earlier vaccine candidate that contains a subset of the four E. coli O-antigen conjugates (O1A, O2, O6A, and O25B) also found in ExPEC10V. Based on results from four completed clinical studies (two Phase 1 studies and two Phase 2 studies), ExPEC4V was well tolerated by study participants, and no vaccine-related safety signals were observed at doses up to 16 μg polysaccharide (PS) / serotype (O1A, O2, O6A, and O25B). Most adverse events (AEs) were Grade 1 and 2, with few Grade 3 AEs reported. Late-onset, induced local AEs (AEs that began after day 5 of vaccination) were primarily observed at higher doses of ExPEC4V. In each study, the ExPEC4V vaccine demonstrated immunogenicity, as demonstrated by a dose-dependent immune response and an increase in O-antigen-specific immunoglobulin G (IgG) titers, as measured by enzyme-linked immunosorbent assay (ELISA). Functional activity of the antibodies was demonstrated using an optimized opsonophagocytic killing assay (OPKA) using ExPEC4V. Co-analysis of ELISA and OPKA test results demonstrated correlation between these assay responses (Pearson correlation coefficients ≥0.61 and ≥0.48 at day 30 and day 360, respectively, in a Phase 2 clinical trial [Study 4V-BAC2001]), confirming the use of the ELISA as a primary measure of ExPEC4V antibody titers and predicting functional antibody activity. Analysis of immunogenicity data demonstrated persistence of the immune response up to three years after vaccination with ExPEC4V. It was also observed that serum from humans vaccinated with ExPEC4V and containing high titers of serotype-specific opsonophagocytic antibodies mediated in vivo protection when passively transferred into mice subsequently challenged intraperitoneally with E. coli strains of serotypes O25B or O2, as evidenced by significant reductions in E. coli colony counts (CFU) in the blood, spleen, and liver (data not shown). Thus, ExPEC4V-specific opsonophagocytic human antibodies mediated bacterial killing in vivo, which is consistent with other conjugate vaccines in which the proposed mechanism of protection is through the induction of opsonophagocytic antibodies that mediate bacterial killing.

[0534] ExPEC10V includes a total of ten serotypes and increases the coverage of bloodstream infections caused by ExPEC in adults aged 60 years and older from approximately 50% (ExPEC4V) to approximately 70%. Based on the clinical experience with ExPEC4V and the preclinical data for ExPEC10V discussed in the examples above, it is expected that administration of ExPEC10V in humans will also induce immune responses to E. coli serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75. Each O-antigen polysaccharide (PS) is individually bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) from Pseudomonas aeruginosa, the production of which has been described above. The O4 PS is a glucosylated form with the structure of the formula (O4-Glc+) in Table 1.

[0535] A randomized, observer-blinded, first-in-human Phase 1 / 2a study was conducted in humans aged 60 to 85 years in stable health to evaluate the safety, reactogenicity, and immunogenicity of three different doses of the ExPEC10V vaccine (Study 10V-BAC1001). The study design included two cohorts: a total of 824 participants were enrolled in the study, 404 participants aged ≥60 to ≤85 years in stable health in Cohort 1 (100 participants / ExPEC10V dose), and approximately 420 additional participants aged ≥60 years in stable health with a history of UTI within the past 5 years in Cohort 2 (280 in the ExPEC10V group and 140 in the placebo group). (Initially, 600 participants were planned for Cohort 2, but after some protocol modifications, this number was reduced to approximately 420 during the study.)

[0536] Purpose and destination

[0537] Cohort 1-1 / 2a Observer-blind Period and Open-label Long-term Follow-up Period (N=404):

[0538]

[0539]

[0540]

[0541] Cohort 2 - Double-blind period and double-blind long-term follow-up period (N=420):

[0542]

[0543]

[0544]

[0545] Overall design

[0546] This was a randomized, multicenter, interventional study including two groups.

[0547] For Cohort 1, the study used an observer-blind, active-controlled design and included a total of 404 adult participants aged ≥60 to ≤85 years, in stable health, with or without a history of UTI. The Cohort 1 study design consisted of three periods: a screening period of up to 28 days, an observer-blind 181-day follow-up period (vaccination on Day 1), and an open-label LTFU period from Day 182 to 3 years after vaccination (Day 1096). Figure 3A Only participants from the selected dose group of ExPEC10V (approximately 100 participants) and those from the Prevnar 13 group progressed to the LTFU stage. The endpoint for Cohort 1 was the last participant's 3-year visit (Day 1096).

[0548] For Cohort 2, the study used a double-blind, placebo-controlled design and included a total of approximately 420 adult participants aged ≥60 years, in stable health, with a history of UTI within the past 5 years. Enrollment began after completion of the Phase 1 / 2a primary analysis and dose selection of ExPEC10V from Cohort 1. The Cohort 2 study design consisted of three periods: a screening period of up to 28 days, a double-blind 181-day follow-up period (vaccination on Day 1), and a double-blind LTFU period (vaccination from Day 182 until 3 years after vaccination (Day 1096)). Figure 3B All participants in Cohort 2 progressed to the LTFU stage. The study endpoint was the 3-year visit (Day 1096) for the last participant in Cohort 2.

[0549] Group 1: 1 period

[0550] In Phase 1 of Cohort 1, a total of 84 participants were enrolled in a staggered approach according to a stepwise dose escalation schedule, and safety assessments were performed appropriately before moving from one step to the next. The study commissioned an internal Data Review Committee (DRC) to review physical examination data (baseline and target), baseline demographics, and safety data 14 days after vaccination (including provoked local and systemic AEs, unprovoked AEs, SAEs, clinical laboratory data, and vital signs) for these 84 Phase 1 participants. During this study period, participants were enrolled and randomized into six steps:

[0551] Step 1: Four sentinel participants were enrolled and randomized; two participants were in the ExPEC10V low-dose group (Table 11) and one participant each was in the ExPEC4V group and the Prevnar 13 group.

[0552] Step 2: Twenty-four participants were enrolled and randomized; 18 participants were in the ExPEC10V low-dose group (Table 11), and three participants each were in the ExPEC4V group and the Prevnar 13 group.

[0553] Step 3: Four sentinel participants were enrolled and randomized; two participants were in the ExPEC10V medium-dose group (Table 11) and one participant each was in the ExPEC4V group and the Prevnar 13 group.

[0554] Step 4: Twenty-four participants were enrolled and randomized; 18 participants were in the ExPEC10V medium-dose group (Table 11) and three participants each were in the ExPEC4V group and the Prevnar 13 group.

[0555] Step 5: Four sentinel participants were enrolled and randomized; two participants were in the ExPEC10V high-dose group (Table 11) and one participant each was in the ExPEC4V group and the Prevnar 13 group.

[0556] Step 6: Twenty-four participants were enrolled and randomized; 18 participants were in the ExPEC10V high-dose group (Table 11), and three participants each were in the ExPEC4V group and the Prevnar 13 group.

[0557] All participants received a single intramuscular (IM) injection of ExPEC10V (1 of 3 doses), ExPEC4V, or Prevnar 13 on Day 1, depending on the assigned study vaccination group. Twenty-four hours after vaccination, four sentinel participants in each of Steps 1, 3, and 5 were contacted by telephone to collect safety information. The principal investigator (PI), study physician (SRP), and sponsor medical manager (SML) reviewed blinded safety data 24 hours after vaccination for the four sentinel participants in each group. Randomization of additional participants to the next step was not permitted until the completion of the sentinel safety assessment on Day 2.

[0558] In the absence of any clinically significant findings, an additional 24 participants (for steps 2, 4, and 6) were enrolled and randomized to one of three study vaccination groups (Table 11) to receive a single IM injection of ExPEC10V (1 of 3 doses), ExPEC4V, or Prevnar 13 on day 1.

[0559] After vaccination of an additional 24 participants at each dose level (low dose in step 2, medium dose in step 4, and high dose in step 6), the DRC reviewed the 14-day post-vaccination safety data for all 28 (4+24) participants at each dose level before entering the next dose level or Phase 2a.

[0560] Group 1: Phase 2a

[0561] Based on acceptable safety and reactogenicity as determined by the DRC after reviewing the safety data for the initial 84 participants 14 days after vaccination (in the absence of any safety issues or any events that met the specific study suspension rules), the remaining 320 participants from Cohort 1 were randomized and dosed in Phase 2a of the study. These additional 320 participants were enrolled and randomly assigned in parallel in a 2:2:2:1:1 ratio to one of the five study vaccination groups to receive a single IM injection of ExPEC10V (1 of 3 doses), ExPEC4V, or Prevnar 13 on Day 1 (Table 11). In addition to the 14-day safety review of the initial 84 participants, the DRC also evaluated the safety data of Cohort 1 during the study and reviewed any events that met the specific study vaccination suspension rules or any other safety issues that may have occurred.

[0562] For Cohort 1, the primary analysis occurred when all participants completed the Day 30 visit (Visit 4) or discontinued early. The final analysis occurred when all participants completed the Day 181 visit or discontinued early. For participants entering the open-label long-term follow-up (LTFU) period (selected dose groups of ExPEC10V and Prevnar 13), annual follow-up analyses included safety and immunogenicity data (multiplex ECL-based immunoassay and MOPA) collected at visits up to Year 1 (Day 366), Year 2 (Day 731), and Year 3 (Day 1096) post-vaccination.

[0563] Group 2

[0564] In Cohort 2, the safety, reactogenicity, and immunogenicity of the selected dose of ExPEC10V (a high dose was selected based on the results of the primary analysis of Cohort 1) were evaluated in participants aged ≥60 years, in stable health, and with a history of UTI within the past 5 years. For Cohort 2, the study used a double-blind, placebo-controlled design, and a total of approximately 420 participants were enrolled and randomized in a 2:1 ratio in parallel (approximately 280 participants in the ExPEC10V group and approximately 140 participants in the placebo group).

[0565] All participants received a single IM injection of the selected dose of ExPEC10V or placebo on Day 1 according to the assigned study vaccination group (Table 12).

[0566] For Cohort 2, the primary analysis included safety and immunogenicity data and occurred when all participants completed the Day 30 visit (Visit 4) or discontinued early. The final analysis occurred when all participants completed the Day 181 visit or discontinued early. For all participants, annual follow-up analyses included safety and immunogenicity data (multiplex ECL-based immunoassay and MOPA) collected at visits up to Year 1 (Day 366), Year 2 (Day 731) (not applicable for MOPA), and Year 3 (Day 1096) post-vaccination.

[0567] Fecal sample analysis was performed in a selected subset of participants to assess the impact of ExPEC10V on the prevalence of pathogens (e.g., Clostridium difficile) and ExPEC10V serotypes in the gut microbiota using metagenomics.

[0568] Number of participants

[0569] A total of approximately 824 participants were included in the study; 404 participants were in cohort 1 and approximately 420 participants were in cohort 2.

[0570] Intervention group: Description of the intervention

[0571] ExPEC10V: An E. coli bioconjugate vaccine in phosphate-buffered saline containing the O antigens (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to the EPA carrier protein. One of three doses of ExPEC10V was administered as a single 0.5 mL IM (deltoid muscle) injection on day 1.

[0572] ExPEC4V: An E. coli bioconjugate vaccine in saline buffer containing the O antigen PS of ExPEC serotypes O1A, O2, O6A, and O25B, respectively, bioconjugated to the EPA carrier protein (4:4:4:8 μg PS / ExPEC serotype). Administer a single 0.5 mL IM (deltoid muscle) injection of ExPEC4V on day 1.

[0573] Prevnar 13: A sterile suspension of saccharides of the capsular antigens of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, respectively, linked to the non-toxic diphtheria CRM197 protein. Administer a single 0.5 mL IM (deltoid muscle) injection on day 1, supplied in a single-dose prefilled syringe.

[0574] Placebo: Normal saline. Placebo was administered as a single 0.5 mL IM (deltoid muscle) injection on Day 1.

[0575] The ExPEC study intervention materials are described in Table 9.

[0576] Table 9. BAC1001MV ExPEC Investigational Vaccines

[0577]

[0578] EPA = genetically detoxified form of exotoxin A from Pseudomonas aeruginosa; PS = polysaccharide

[0579] ExPEC4V consists of O-antigen polysaccharides (PS) of ExPEC serotypes O1A, O2, O6A, and O25B, respectively, bioconjugated to the EPA carrier protein.

[0580] ExPEC10V consists of O-antigen polysaccharides (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to the EPA carrier protein.

[0581] Dosage is based on PS only. EPA (μg) is a measured value.

[0582] ExPEC10V consists of 10 monovalent drug substances (DSs). For this clinical study, two different concentrations (medium and high) of drug product (DP) were produced (Table 10). A third (low) concentration was obtained clinically by diluting the high concentration 1:1 with dilution buffer, which was the same as the formulation buffer. Each DP was formulated in sodium / potassium phosphate buffer (0.02% [w / w] polysorbate 80, 5% [w / w] sorbitol, 10 mM methionine) at pH 7.0.

[0583] Table 10: Composition of the ExPEC10V vaccine used in the Phase 1 / 2a clinical study

[0584]

[0585] EPA = genetically detoxified Pseudomonas aeruginosa exotoxin A, used as a carrier protein

[0586] a The active ingredient is a biosynthetic conjugate consisting of PS antigen and carrier protein (EPA); the dosage is calculated based on the PS portion only.

[0587] b The "low concentration" is the safety assessment obtained by diluting the "high concentration" 1:1 with dilution buffer in clinical practice

[0588] Key safety assessments included provoked local and systemic AEs, unprovoked AEs, SAEs, physical examinations, vital sign measurements, and clinical laboratory tests.

[0589] Immunogenicity assessment

[0590] Key immunogenicity assessments of the collected sera included evaluation of vaccine-elicited serotype-specific total IgG antibody levels to ExPEC10V and ExPEC4V (as measured by a multiplex ECL-based immunoassay), and serotype-specific functional antibodies to ExPEC10V and ExPEC4V (as measured by a multiplexed opsonophagocytic killing assay (OPKA), or MOPA). Immunogenicity assessment of pneumococcal antibody titers elicited by Prevnar 13 was not performed.

[0591] Serum antibody levels induced by ExPEC10V were measured using a multiplex electrochemiluminescence (ECL)-based immunoassay. The assay incorporates high-binding carbon electrodes into a multi-well 96-well microplate format coated with different E. coli O-LPS antigens or the carrier protein EPA. A secondary antibody (anti-human IgG) labeled with a SULFO tag was used to detect the levels of antigen-specific antibodies present in the serum samples. The SULFO tag emits light in the presence of electrical stimulation at an intensity that increases proportionally to the amount of bound IgG antibodies. The assay complies with the recommendations of the International Conference on Harmonisation (ICH).

[0592] The levels of functional antibodies induced by ExPEC10V were measured by a multiplex opsonophagocytosis assay (MOPA). Briefly, heat-inactivated serum samples were serially diluted and incubated with different E. coli strains specifically resistant to different types of antibiotics. After this, human complement and phagocytes (HL60) were added to the reaction, and after a second incubation period, aliquots of the reaction mixture were transferred to different PVDF hydrophilic membrane filter plates containing medium supplemented with a specific antibiotic that selectively allows the growth of strains resistant to that specific antibiotic. After overnight growth, colony-forming units (CFU) were counted to determine the number of surviving bacteria. This assay complies with ICH recommendations.

[0593] The following immunogenicity measures were evaluated and listed for all immunogenicity time points for the study vaccine groups for ExPEC10V serotype antibodies as measured by multiplex ECL-based immunoassay and MOPA, and for EPA measured by multiplex ECL-based immunoassay only:

[0594] - Proportion of participants with ≥2-fold and ≥4-fold increases in serum antibody titers from Day 1 (pre-vaccination)

[0595] -Geometric mean titer (GMT)

[0596] -GMR: fold change from baseline, calculated from post-baseline / baseline values.

[0597] For the LTFU period, a descriptive summary of immunogenicity is provided for each serotype.

[0598] Late-stage dose selection considered all available indications at the time of the primary analysis of Cohort 1 (Day 30 results).

[0599] Table 11: Group 1: Vaccination schedule

[0600]

[0601] *ExPEC10V consists of the O-antigen polysaccharide (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.

[0602] **ExPEC4V consists of the O-antigen polysaccharide (PS) of ExPEC serotypes O1A, O2, O6A, and O25B, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.

[0603] ***Prevnar 13, Pneumococcal 13-valent Conjugate Vaccine (Diphtheria CRM197 Protein), is a sterile suspension of saccharides of the capsular antigens of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, respectively, linked to the avirulent diphtheria CRM197 protein.

[0604] Table 12: Group 2: Vaccination schedule

[0605]

[0606] a ExPEC10V consists of the O-antigen polysaccharide (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) from Pseudomonas aeruginosa.

[0607] Participants in Cohort 2 of the study were randomized in a 2:1 ratio (ExPEC10V:placebo). The dose of ExPEC10V used in Cohort 2 was based on the results of the primary analysis (Day 30) of Cohort 1 and was the higher dose used in Cohort 1.

[0608] state

[0609] Enrollment and vaccination of Cohort 1 of the above study have been completed. No major safety issues were identified, and the ExPEC10V vaccine had an acceptable safety profile.

[0610] Immunogenicity analysis was performed on Cohort 1 clinical samples, and the results are described below.

[0611] Cohort 2 vaccination was initiated with a high dose of ExPEC10V, and this portion of the study is ongoing.

[0612] Cohort 1 Safety and Immunogenicity Results

[0613] The results presented here apply to the BAC1001 clinical study (Cohort 1). There were 416 participants in the full analysis set. Each participant was randomly assigned to 1 of 3 single intramuscular doses of ExPEC10V or 1 of 2 active control doses of ExPEC4V or Prevnar; 104 received low-dose ExPEC10V, 102 received medium-dose ExPEC10V, 104 received high-dose ExPEC10V, 52 received ExPEC4V (4:4:4:8 μg O1A, O2, O6A, and O25B polysaccharides / dose), and 54 received Prevnar. A total of 413 participants completed the Day 30 visit. 392 (94.2%) participants were included in the per-protocol immunogenicity analysis at Day 15.

[0614] Security

[0615] Overall, all ExPEC10V doses were well tolerated. There was a trend toward increased reactogenicity with higher doses, but with the exception of erythema, swelling, and nausea, the reactogenicity of ExPEC10V at higher doses was lower or comparable to that of Prevnar. More delayed local reactogenicity was observed in the ExPEC10V group compared with Prevnar, with injection site erythema and swelling reported as late events in >90% of participants experiencing these local events. However, the reactogenicity profile of ExPEC10V was acceptable compared to other licensed vaccines used in the elderly population.

[0616] Immunogenicity

[0617] Vaccine-induced immune responses were assessed at baseline (day 1 before vaccination) and day 15 after vaccination using a multiplex electrochemiluminescence (ECL)-based immunoassay that measures serotype-specific serum antibody (total immunoglobulin G [IgG]) levels and a multiplex opsonophagocytic killing assay (MOPA) that measures antibody-mediated opsonophagocytic killing of bacteria.

[0618] Antigen-specific serum antibody levels (ECL)

[0619] For all tested ExPEC10V doses and all vaccine-associated serotypes, the levels of O-antigen-specific serum antibodies (total IgG) increased significantly on day 15 after vaccination. It was observed that at least 80% of subjects who received the high-dose ExPEC10V vaccine showed a two-fold or greater increase in O-antigen-specific antibody titers against eight of the 10 serotypes (including O25B, O6A, O2, O1A, O4, O15, O16, and O18A) on day 15 after vaccination. For the ExPEC10V medium-dose group, at least 80% of subjects showed a two-fold or greater increase in O-antigen-specific antibody titers against five of the 10 serotypes (including O25B, O2, O1A, O4, and O15) on day 15 after vaccination; and for the ExPEC10V low-dose group, this was also the case for four of the 10 serotypes (including O25B, O2, O15, and O16). Figure 4 , Table 13). Small changes in GMT from day 1 to day 15 were observed in the groups of subjects who received Prevnar or ExPEC4V (for non-ExPEC4V serotypes) ( Figure 4 , Table 13).

[0620] For the ECL assay, all subjects were analyzed and included in all immunogenicity subsets. Subjects with serious violations were excluded from the per-protocol analysis based on protocol exclusion criteria. The geometric mean titer (GMT), GMT FI (geometric mean titer fold increase) and the percentage of participants with at least a 2-fold and 4-fold increase from baseline in the ECL-based immunoassay are summarized in Table 13 below.

[0621] Table 13. ECL data of BAC1001 group 1

[0622] Serotype O1A

[0623]

[0624]

[0625] Serotype O2

[0626]

[0627] Serotype O4

[0628]

[0629] Serotype O6A

[0630]

[0631]

[0632] Serotype O8

[0633]

[0634] Serotype O15

[0635]

[0636]

[0637] Serotype O16

[0638]

[0639] Serotype O18A

[0640]

[0641]

[0642] Serotype O25B

[0643]

[0644] Serotype O75

[0645]

[0646] Group 1: Output of preliminary analysis

[0647] 1 =GM ​​from baseline (Day 1) FI

[0648] 2 = Percentage of participants who doubled their score from baseline (Day 1)

[0649] CI = confidence interval; GM = geometric mean, GMT = geometric mean titer; FI = fold increase. The 95% CIs for GMT and GM FI are based on a t distribution.

[0650] The 95% CIs for the percentages of participants with 2-fold and 4-fold increases were obtained using the Clopper-Pearson method.

[0651] From the data ( Figure 4 ) It can be seen that the total IgG response to the O75 polysaccharide antigen was lower (e.g., a lower percentage of subjects with at least a two-fold increase on Day 15 compared to Day 1) compared to the other antigens administered at the same amount, which is a surprising result. The data also showed that increasing the amount of O75 polysaccharide antigen in the composition (i.e., 8 μg at the high dose versus 4 μg at the low and medium doses) increased the induced immune response to the O75 polysaccharide antigen.

[0652] Serum antibody functionality (MOPA)

[0653] Partial data were included in the Cohort 1 MOPA analysis. Table 14 shows the number of participants included in the MOPAGMT analysis at Day 15. Although partial data were used to assess the functionality of the antibody responses induced by ExPEC10V, the conclusions of the analysis did not change when the complete data set became available.

[0654] Table 14. Number of participants included in the MOPAGMT analysis

[0655]

[0656] The levels of opsonophagocytic antibodies measured by MOPA were generally consistent with the levels of O-antigen-specific antibodies (total IgG) measured by ECL-based immunoassay. In all dose groups tested, increased levels of functional antibodies were observed at day 15 against nine of the ten ExPEC10V serotypes ( Figure 5 ).

[0657] For serotype O8, a qualified MOPA assay failed to demonstrate a vaccine-mediated increase in functional antibodies at day 15 after ExPEC10V vaccination on day 1, compared to other ExPEC10V serotypes ( Figure 5 ), although an increase in total IgG titers was observed for serotype O8 ( Figure 4). Studies were initiated to distinguish whether this qualified assay was unable to detect functional O8 antibodies. Subsequent experiments using the improved MOPA assay and day 1 and day 15 sera successfully showed a 2-fold or greater increase in functional antibodies on day 15 (data not shown), indicating that the ExPEC10V vaccine did in fact also induce functional antibodies to serotype O8. For clinical development, the improved O8 MOPA would have required requalification of the assay and retesting of clinical samples, which would have been time consuming, so the decision was made to initially proceed with the development of a vaccine composition that did not include the O8 polysaccharide antigen. However, it became clear from subsequent experiments that the O8 bioconjugate in the 10-valent ExPEC vaccine composition did induce functional antibodies to the E. coli serotype O8, and therefore this bioconjugate is likely to be successfully added to the nine-valent vaccine composition described in the examples below.

[0658] The highest levels of opsonophagocytic antibodies were observed for serotype O2; 95% of subjects who received the high dose of the vaccine showed a 2-fold or greater increase in anti-O2 opsonophagocytic antibody titers on day 15 after vaccination. Excluding the O8 serotype, for which limited levels of functional antibodies were detected as discussed above, the lowest levels of opsonophagocytic antibodies were observed for serotype O75; 36% of subjects who received the high dose of the vaccine showed a 2-fold or greater increase in anti-O75 opsonophagocytic antibody titers on day 15 after vaccination. Thus, the MOPA data for the O75 serotype are consistent with the ECL data for this serotype described above, confirming that the conjugate of the E. coli O75 antigen polysaccharide is relatively less immunogenic than several other conjugates at the same concentration in the composition (e.g., E. coli O6, O1, or O2 conjugates). In addition, minimal changes in GMT from day 1 to day 15 were observed in the groups of subjects who received Prevnar or ExPEC4V (for non-ExPEC4V serotypes) Figure 5 ), which is in line with expectations for serotypes not present in the respective vaccine compositions.

[0659] For each of the 10 serotypes, the geometric mean titer (GMT), geometric mean fold increase (GMR FI), and the percentage of participants with at least a 2-fold and 4-fold increase from baseline in MOPA are summarized in Table 15.

[0660] Table 15. MOPA data of BAC1001 group 1

[0661] Serotype O1A

[0662]

[0663]

[0664] Serotype O2

[0665]

[0666] Serotype O4

[0667]

[0668]

[0669] Serotype O6A

[0670]

[0671] Serotype O8

[0672]

[0673]

[0674] Serotype O15

[0675]

[0676] Serotype O16

[0677]

[0678] Serotype O18A

[0679]

[0680]

[0681] Serotype O25B

[0682]

[0683] Serotype O75

[0684]

[0685]

[0686] Group 1: Output of preliminary analysis

[0687] 1 =GM ​​from baseline (Day 1) FI

[0688] 2 = Percentage of participants who doubled their score from baseline (Day 1)

[0689] CI = confidence interval; GM = geometric mean, GMT = geometric mean titer; FI = fold increase

[0690] The 95% CIs for GMT and GM FI are based on t distributions.

[0691] The 95% CIs for the percentages of participants with 2-fold and 4-fold increases were obtained using the Clopper-Pearson method.

[0692] Dose selection

[0693] An immunogenicity dose selection algorithm based on the mean log10 fold increase from baseline to day 15 for each dose group, taking into account two analysis sets (PPI and FAS) and two assays (ECL and MOPA), determined the ExPEC10V high dose for cohort 2 of the trial.

[0694] For serotypes O2, O4, O15, O16, and O18A, in both assays, the high-dose groups had higher GMTs, GMRs, and the percentages of participants with a 2-fold and 4-fold increase at day 15 compared to the other two ExPEC10V doses. For serotype O25B, the medium-dose group generally had higher GMTs, GMRs, and the percentages of participants with a 2-fold and 4-fold increase across the three ExPEC10V dose groups; for this serotype, the O antigen dose was the same for both the medium and high ExPEC10V dose groups (16 μg). For both serotypes O1A and O6A, results were mixed between the high and medium doses. In serotype O75, there was a measurable functional antibody response to the ExPEC10V dose. As discussed above, no measurable functional antibody response was detected in the qualified assay for serotype O8. In non-ExPEC4V serotypes, lower functional antibody responses were observed in the two active controls; this antibody response was from pre-existing antibodies rather than a vaccine-induced response. Based on these results, a high dose of ExPEC10V was selected for cohort 2 of the BAC1001 clinical trial.

[0695] in conclusion

[0696] The ExPEC10V vaccine induced robust antibody responses, with increases in O-antigen-specific serum antibodies observed against all vaccine-relevant serotypes at day 15 post-vaccination compared to baseline titers (day 1). Functional antibodies induced by ExPEC10V that mediated opsonophagocytic killing of E. coli were detected against all vaccine-relevant serotypes except serotype O8 in the qualified MOPA. For serotype O8, functional antibody responses were measured in subsequent studies using a modified MOPA. Notably, responses to the 4 μg and 8 μg doses of O75 were weaker than those to the other serotypes at the same dose, a surprising and unexpected finding of the study. In order to improve the immune response against E. coli serotype O75 produced by the multivalent saccharide conjugate composition, in one aspect, the present invention increases the dose of serotype O75 antigenic polysaccharide, for example, to about 1.2 to 8 times the dose of some other serotypes, for example, to about 1.5 to 4 times, for example, about 1.5 to 2.5 times, for example about 2 times, of the dose of some other serotypes (such as O6 or O1), for example, to about 16 μg, which is higher than the dose of, for example, the three most relevant and prevalent serotypes O1A, O2 and O6A, and is similar to the dose of serotype O25B antigenic polysaccharide. It is noteworthy that in early experiments (see, for example, the above discussion in this example and Examples 5 and 6, i.e., before the clinical data for the ExPEC10V vaccine disclosed herein were obtained), the dose of the O75 antigen polysaccharide was at most as high as the dose of the O1A, O2, and O6A antigen polysaccharides, or in fact, in some test groups, even two-fold lower than the O1A and O6A antigen polysaccharides, and even two to four-fold lower than the O25B antigen polysaccharide. In contrast, in certain aspects of the present invention, the dose of O75 was actually increased compared to the dose of O1, O2, and / or O6.

[0697] Cohort 2 Safety and Immunogenicity Results

[0698] In cohort 2 of the study, a total of approximately 420 participants aged ≥60 years, in stable health, with a history of UTI within the past 5 years were enrolled and randomized in a 2:1 ratio to parallel groups (the subset for which the full results were analyzed included 278 participants in the ExPEC10V (high-dose) group and 138 participants in the placebo group).

[0699] Security

[0700] Overall, the ExPEC10V vaccine was well tolerated. The reactogenicity profile was generally comparable to that observed in Cohort 1 using the medium and high doses of ExPEC10V. The most common locally induced AE was pain / tenderness, and the most frequently reported systemic AEs were myalgia, headache, and fatigue. Injection site erythema and swelling were the most common late-onset events (time to first onset >5 days post-vaccination). The reactogenicity profile of ExPEC10V was acceptable compared to other licensed vaccines used in the elderly population.

[0701] Immunogenicity

[0702] Vaccine-induced antibody responses were assessed at baseline (day 1 before vaccination) and on days 15 and 30 after vaccination using a multiplex ECL-based immunoassay measuring serotype-specific serum antibody (total IgG) levels.

[0703] Antigen-specific serum antibody levels (ECL, days 1, 15, and 30)

[0704] The ExPEC10V vaccine was immunogenic against all serotypes based on increases in GMT, GMT FI, and the percentage of participants with at least a 2-fold increase in titer from baseline from day 1 to day 15. Changes in all three measures from day 15 to day 30 were minimal.

[0705] The highest antibody responses were observed against serotypes O2, O4, O15, and O16, with at least 80% of participants vaccinated with ExPEC10V showing a two-fold or greater increase in antibody responses on days 15 and 30 post-vaccination. For serotypes O1A, O6A, and O25B, at least 70% of participants showed a two-fold or greater increase in antibody responses, and for serotypes O8 and O18A, at least 60% of participants showed a two-fold or greater increase in antibody responses. Lower antibody responses were observed against serotype O75, however, an increase of two-fold or greater in antigen-specific antibody responses was observed in at least 50% of participants. Small changes in GMT from day 1 to day 15 and to day 30 were observed in the group of subjects who received placebo (Table 16).

[0706] The geometric mean titer (GMT), GMT FI (geometric mean titer fold increase), and percentage of participants with at least a 2-fold and 4-fold increase from baseline in the ECL-based immunoassay are summarized in Table 16 below.

[0707] Table 16. ECL data for BAC1001 cohort 2 (days 1, 15, and 30) Serotype O1A

[0708]

[0709]

[0710] Serotype O2

[0711]

[0712]

[0713] Serotype O4

[0714]

[0715] Serotype O6A

[0716]

[0717] Serotype O8

[0718]

[0719]

[0720] Serotype O15

[0721]

[0722]

[0723] Serotype O16

[0724]

[0725]

[0726] Serotype O18A

[0727]

[0728] Serotype O25B

[0729]

[0730] Serotype O75

[0731]

[0732]

[0733] Group 2: Output of preliminary analysis

[0734] 1 = GM from baseline (Day 1) FI

[0735] 2 = Percentage of participants who doubled from baseline (Day 1)

[0736] CI = confidence interval; GM = geometric mean, GMT = geometric mean titer; FI = fold increase

[0737] The 95% CIs for GMT and GM FI are based on t distributions.

[0738] The 95% CIs for the percentages of participants with 2-fold and 4-fold increases were obtained using the Clopper-Pearson method.

[0739] The magnitude of responses at day 15 (GMT) was similar between Cohorts 1 and 2 for all serotypes.

[0740] Example 8: Novel ExPEC compositions and immunogenicity in rabbits.

[0741] A new study was designed that included two adapted compositions of ExPEC vaccines (ExPEC9V a and b, Table 17) (also referred to herein as adapted formulations). These adapted formulations removed serotype O8 polysaccharide and increased the polysaccharide content of E. coli serotype O75 (ExPEC9V b) due to the low immunogenicity of these antigens observed during analysis of cohort 1 from the Ph1 / 2a clinical trial. As a control to validate the earlier experiments described above, the ExPEC10V composition was also evaluated. A saline control (unvaccinated group) was also included.

[0742] The overall goal of this study was to assess whether the changes in the vaccine composition compared to the aforementioned ExPEC10V (removal of the O8 polysaccharide and, in particular, an increase in the dose of the O75 antigen polysaccharide) had any effect on the immune response induced by the vaccine. In particular, the aim was to observe whether increasing the amount of the conjugate of the E. coli O75 antigen polysaccharide, compared to, for example, the conjugate of the E. coli O6 antigen polysaccharide in a multivalent conjugate vaccine composition, indeed selectively enhances the immune response against serotype O75 E. coli, thereby resulting in a more uniform increase in the immune response across the various constituent E. coli serotypes. To this end, the antibody responses induced by different formulations of the ExPEC vaccine were evaluated in an established immunogenic preclinical model using New Zealand White (NZW) rabbits.

[0743] Experimental design

[0744] New Zealand white rabbits (NZW, 13 weeks old at the start of the study) received three intramuscular immunizations of different formulations of the ExPEC vaccine (500 μL / injection) or saline, administered 2 weeks apart (Table 17). The study included four different groups (Table 18): Group 1 (ExPEC10V) received 8 μg / dose of O1A, O2, O6A, O4, O8, O15, O16, O18, and O75 and 16 μg / dose of O25B (i.e., 0.5 mL of a composition containing 16 μg / mL of each of the O1A, O2, O6A, O4-Glc+, O8, O15, O16, O18A, and O75 antigen polysaccharides and 32 μg / mL of the O25B antigen polysaccharide, each bioconjugated to an EPA carrier protein, see Examples above); Group 2 (ExPEC9V) received 8 μg / dose of O1A, O2, O6A, O4-Glc+, O8, O15, O16, O18A, and O75 antigen polysaccharides and 16 μg / mL of the O25B antigen polysaccharide, each bioconjugated to an EPA carrier protein, see Examples above); a) received 8 μg / dose of O1A, O2, O6A, O4, O15, O16, O18A, and O75 and 16 μg / dose of O25B (i.e., 0.5 mL of a composition comprising 16 μg / mL of each of the O1A, O2, O6A, O4-Glc+, O15, O16, O18A, and O75 antigenic polysaccharides and 32 μg / mL of the O25B antigenic polysaccharide, each bioconjugated to an EPA carrier protein, see Examples above); Group 3 (ExPEC9V b) received 8 μg / dose of O1A, O2, O6A, O4, O15, O16, and O18A and 16 μg / dose of O75 and O25B (i.e., 0.5 mL of a composition comprising 16 μg / mL of each of the O1A, O2, O6A, O4-Glc+, O15, O16, and O18A antigenic polysaccharides and 32 μg / mL of the O25B and O75 antigenic polysaccharides, each bioconjugated to an EPA carrier protein, see Examples above).

[0745] The buffer used for the complex contains 6.19mM KH2PO4, 3.81mM Na2HPO4, 5% sorbitol (w / w), 10mM methionine, 0.02% PS80 (w / w), pH 7.0. Animals from the control group (Group 4) received normal saline (0.9% (w / v) sodium chloride solution). Each experimental group contained 10 animals. Serum antibody levels (total IgG) measured by ELISA were assessed before immunization (Day 0) and after immunization (Days 14, 28, and 42).

[0746] Table 17. Different multivalent ExPEC vaccine compositions.

[0747] Serotype ExPEC10V ExPEC9V a ExPEC9V b O1A 8 8 8 O2 8 8 8 O4 8 8 8 O6A 8 8 8 O8 8 0 0 O15 8 8 8 O16 8 8 8 O18A 8 8 8 O25B 16 16 16 O75 8 8 16 Total PS (μg) 88 80 88

[0748] Table 18. Experimental groups.

[0749] Group deal with Dosing day Number of animals 1 ExPEC10V 0，14，28 10 2 ExPEC9V a (1:1 ratio of O75:O6) 0，14，28 10 3 ExPEC9V b (2:1 ratio of O75:O6) 0，14，28 10 4 Normal saline 0，14，28 10

[0750] Monitor for possible injection site reactions using the Draize scoring system before each dose, approximately 6 hours after each dosing day, and daily after injection for 7 days. If injection site effects are observed, continue daily observation until the score returns to 0.

[0751] Weigh the animals individually (in grams) before treatment, once daily after each dose (for 3 days), and weekly thereafter. Observe the animals at least once daily for any impairment of health or adverse clinical effects. On the dosing day, examine the animals for their response to treatment before dosing and approximately 6 hours after treatment (simultaneously with the injection site inspection). Monitor body temperature using a rectal digital thermometer and record it before dosing, 6 hours after each dose, and 24 hours after each dose. When the temperature exceeds the normal range of 38°C-40°C for rabbits, perform additional measurements daily until the temperature returns to within these values.

[0752] Animals received three intramuscular immunizations of ExPEC10V, ExPEC9V a, ExPEC9V b or saline, administered 2 weeks apart. Antibody levels were measured by ELISA on day 0 (before vaccination) and days 14, 28, and 42 (after vaccination).

[0753] Serum antibody levels induced by each O antigen included in the ExPEC vaccine composition were analyzed by ELISA using Gen 5 software. OD at 450 nm was analyzed in a 4-parameter (4PL) nonlinear regression model. The half-maximal effective concentration (EC50) was calculated for each individual sample based on replicate 12-step titration curves. Sample results are expressed as EC50 titers.

[0754] Evaluation of antigen-specific serum antibody responses in NZW rabbits showed that all ExPEC vaccine compositions tested (ExPEC10V, ExPEC9Va, and ExPEC9Vb) induced significantly increased antibody responses against all vaccine antigens at days 14, 28, and 42 post-vaccination compared to saline controls ( Figure 6 The second dose of all ExPEC vaccine compositions tested boosted antibody responses, as demonstrated by significant increases in antigen-specific antibody responses from days 14 to 28 post-vaccination. For some antigens and compositions, a further boosting effect of a third dose of ExPEC vaccine was evident.

[0755] The geometric mean antibody titer (GMT) of O75 antigen increased with the increase of O75 polysaccharide concentration ( Figure 6Compared to ExPEC9V a and ExPEC10V compositions containing only 8 μg of O75 polysaccharide, ExPEC9V b compositions containing increasing concentrations of polysaccharide O75 (16 μg) induced higher O75 GMTs and fold-increases in GMTs at days 14, 28, and 42 post-vaccination (Table 19).

[0756] Table 19. Evaluation of O75 antibody responses to different ExPEC vaccine compositions.

[0757]

[0758] na: not applicable; GMT: geometric mean antibody titer; CI: confidence interval; GMT is the fold increase compared to day 0 (pre-vaccination baseline).

[0759] Evaluation of body weight, temperature, and injection site reactions indicated that all ExPEC vaccine compositions tested were well tolerated by the animals (data not shown).

[0760] In summary, all ExPEC vaccine compositions tested were immunogenic in NZW rabbits, with significant increases in antigen-specific serum antibody responses observed at 14, 28, and 42 days post-vaccination compared to saline controls. All ExPEC vaccine compositions tested enhanced polysaccharide-specific antibody responses. Furthermore, all ExPEC vaccine compositions tested were well tolerated by the animals, with no vaccine-related adverse events observed. Importantly, ExPEC9Vb compositions containing increased concentrations of the O75 polysaccharide induced higher O75 antibody responses (GMT and fold increase in GMT) compared to compositions containing lower concentrations of the O75 polysaccharide (ExPEC9Va and ExPEC10V). Thus, the increased dose of O75 in the ExPEC9Vb compositions enhanced immune responses against the O75 serotype, resulting in a more uniform increase in immune responses across serotypes in the ExPEC9Vb compositions.

[0761] Example 9. Human Phase 3 efficacy trial using a novel ExPEC9V composition.

[0762] A human efficacy study was conducted using a 9-valent ExPEC vaccine composition with increasing concentrations of the O75 antigen polysaccharide. The study is titled: "A randomized, double-blind, placebo-controlled, multicenter, phase 3 study to evaluate the efficacy, safety, and immunogenicity of vaccination with ExPEC9V in preventing invasive extraintestinal pathogenic Escherichia coli disease in adults aged 60 years and older with a history of urinary tract infection within the past 2 years," and is referred to herein as the 'BAC3001' study.

[0763] ExPEC9V is a 9-valent vaccine candidate being developed for the prevention of invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED) in adults aged 60 years and older. ExPEC9V consists of the O-antigen polysaccharides (PS) of ExPEC serotypes O1A, O2, O4, O6A, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) from Pseudomonas aeruginosa, as described in detail in the previous Examples. The ExPEC9V vaccine covers nine designated E. coli serotypes, which are the most prevalent O serotypes of ExPEC and cause approximately 65% ​​of all IED.

[0764] Purpose and destination

[0765]

[0766]

[0767]

[0768]

[0769]

[0770] Overall design

[0771] This is a randomized, double-blind, placebo-controlled, parallel-group, multicenter, interventional Phase 3 study that will be conducted in approximately 18,556 medically stable adults aged ≥60 years with a history of UTI within the past 2 years.

[0772] All participants were enrolled and randomly assigned in a 1:1 ratio to receive ExPEC9V or placebo in parallel and received the study vaccine (0.5 mL intramuscularly (deltoid muscle)) on day 1. The final analysis of the first primary endpoint (microbiological confirmation from blood, other sterile sites, or urine) was performed when 72 vaccine serotype IED events were observed during the study, or at the latest when the last participant had been followed for 36 months after vaccination. When the first primary endpoint showed statistical significance, the second primary endpoint (microbiological confirmation from blood or other sterile sites) was tested in a hierarchical manner. For the final analysis of the second primary endpoint (microbiological confirmation from blood or other sterile sites), 53 vaccine serotype IED events according to the second primary endpoint had to be observed. The ExPEC9V vaccine composition comprises 16 μg / mL of each of the O1A, O2, O6A, O4-Glc+, O15, O16, and O18A antigenic polysaccharides and 32 μg / mL of the O25B and O75 antigenic polysaccharides, each bioconjugated to an EPA carrier protein, see the examples above (e.g., this is the same composition used in rabbit immunization group 3 of Example 8 above; the excipients are the same as those described for the ExPEC10V vaccine used in the BAC1001 study of Example 7). The placebo was 0.9% w / v sodium chloride.

[0773] Safety Assessment

[0774] Key safety assessments included SAEs, physical examinations, and vital signs. In addition, provoked local and systemic AEs and unprovoked AEs were recorded for participants in the "safety subset" (i.e., approximately 4,000 participants from selected study sites who gave informed consent before randomization for additional assessments).

[0775] Immunogenicity assessment

[0776] To assess immunogenicity, levels of IgG antibodies elicited by the vaccine against each of the nine vaccine O serotypes and the carrier protein EPA were measured by a multiplex ECL-based immunoassay, and serotype-specific functional antibodies were measured by MOPA. Immunogenicity analysis was perf...

Claims

1. A composition comprising any one of the following i)-viii): i) Escherichia coli O75 antigen polysaccharide and Escherichia coli O1 antigen polysaccharide; ii) Escherichia coli O75 antigen polysaccharide and Escherichia coli O2 antigen polysaccharide; iii) Escherichia coli O75 antigenic polysaccharide and Escherichia coli O4 antigenic polysaccharide; iv) Escherichia coli O75 antigen polysaccharide and Escherichia coli O6 antigen polysaccharide; v) Escherichia coli O75 antigenic polysaccharide and Escherichia coli O15 antigenic polysaccharide; vi) Escherichia coli O75 antigenic polysaccharide and Escherichia coli O16 antigenic polysaccharide; vii) Escherichia coli O75 antigenic polysaccharide and Escherichia coli O18 antigenic polysaccharide; viii) Escherichia coli O75 antigenic polysaccharide and Escherichia coli O25 antigenic polysaccharide; Each antigenic polysaccharide is independently covalently linked to a carrier protein.

2. The composition of claim 1, wherein i) the concentration ratio of O75 antigen polysaccharide to O1 antigen polysaccharide is 2:1; ii) the concentration ratio of O75 antigen polysaccharide to O2 antigen polysaccharide is 2:1; iii) the concentration ratio of O75 antigen polysaccharide to O4 antigen polysaccharide is 2:1; iv) the concentration ratio of O75 antigen polysaccharide to O6 antigen polysaccharide is 2:1; v) the concentration ratio of O75 antigen polysaccharide to O15 antigen polysaccharide is 2:1; vi) the concentration ratio of O75 antigen polysaccharide to O16 antigen polysaccharide is 2:1; vii) the concentration ratio of O75 antigen polysaccharide to O18 antigen polysaccharide is 2:1; and / or viii) The concentration ratio of O75 antigen polysaccharide to O25 antigen polysaccharide is 1:

1.

3. The composition of claim 1 or 2, wherein the O1 antigen is O1A, the O4 is glycosylated, the O6 antigen is O6A, the O18 antigen is O18A, and / or the O25 antigen is O25B.

4. The composition of any one of claims 1 to 3, wherein: (i) The E. coli O1 antigen polysaccharide comprises a structure of formula (O1A): (ii) the E. coli O2 antigen polysaccharide comprises a structure of formula (O2): (iii) the E. coli O4 antigen polysaccharide comprises a structure of formula (O4-Glc+): (iv) the E. coli O6 antigen polysaccharide comprises a structure of formula (O6A): (v) The E. coli O15 antigen polysaccharide comprises a structure of formula (O15): [→2)-β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→]n, (vi) The E. coli O16 antigen polysaccharide comprises a structure of formula (O16): (vii) The E. coli O18 antigen polysaccharide comprises the structure of formula (O18A): (viii) the E. coli O25 antigen polysaccharide comprises the structure of formula (O25B): and (ix) The E. coli O75 antigen polysaccharide comprises the structure of formula (O75): wherein each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

5. The composition of any one of claims 1-3, wherein the concentration of the O75 antigen polysaccharide is about 8 to about 50 μg / mL, preferably about 12 to about 40 μg / mL, such as about 16 to about 32 μg / mL, preferably about 28 to about 36 μg / mL, preferably about 32 μg / mL.

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

7. The composition of claim 6, wherein the at least one additional E. coli antigenic polysaccharide comprises an O8 antigenic polysaccharide having formula (O8): α-D-Member3Me-(1→[3)-β-D-Member-(1→2)-α-D-Member-(1→2)-α-D-Member-(1→] n , wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, preferably from 5 to 30, such as from 7 to 25, such as from 10 to 20.

8. The composition of any one of claims 1-3, wherein each carrier protein is independently selected from the group consisting of: Pseudomonas aeruginosa detoxified exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM 197 , maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, agglutination factor A, agglutination factor B, Escherichia coli heat-labile enterotoxin, a detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, a detoxified variant of cholera toxin, Escherichia coli Sat protein, the passenger domain of Escherichia coli Sat protein, pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from non-typeable Haemophilus influenzae.

9. A host cell (eg, a recombinant host cell) for producing a conjugate of an E. coli antigenic polysaccharide covalently linked to a carrier protein, wherein the host cell comprises a nucleic acid encoding an enzyme (eg, a glycosyltransferase) capable of producing: i) Escherichia coli O75 antigenic polysaccharide, ii) Escherichia coli O1A antigenic polysaccharide, iii) Escherichia coli O2 antigenic polysaccharide, iv) glycosylated or non-glucosylated Escherichia coli O4 antigenic polysaccharide, v) Escherichia coli O6A antigenic polysaccharide, vi) Escherichia coli O8 antigenic polysaccharide, vii) Escherichia coli O15 antigenic polysaccharide, viiii) Escherichia coli O16 antigenic polysaccharide, ix) Escherichia coli O18A antigenic polysaccharide, or x) Escherichia coli O25B antigenic polysaccharide.

10. The host cell of claim 9, wherein the host cell naturally expresses a nucleic acid capable of producing an O antigen of interest, or the host cell is caused to express such a nucleic acid.

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