E. coli compositions and methods thereof

Abstract

To provide Escherichia coli compositions for alternative treatment to standard therapy for invasive multidrug-resistant (MDR) Escherichia coli and Klebsiella infections.SOLUTION: In one embodiment, the compositions comprise a polypeptide derived from Escherichia coli (E. coli) or a fragment thereof; and modified O-polysaccharide molecules derived from Escherichia coli (E. coli) lipopolysaccharides or conjugates thereof. In a further aspect, the compositions further comprise modified O-polysaccharide molecules derived from Klebsiella pneumoniae or conjugates thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing Reference This application was filed electronically via EFS-Web and contains an electronically submitted sequence listing in .txt format. The .txt file was created on Jan. 28, 2021, and contains a sequence listing entitled "PC72591_PROV2_ST25.txt" having a size of 152KB. The sequence listing contained in this .txt file is a part of the present specification and is incorporated by reference in its entirety.

[0002] Technical Field The present invention relates to Escherichia coli compositions and methods thereof. [Background technology]

[0003] The threat to public health posed by increasing antimicrobial resistance has been described in recent reports published by the WHO and CDC (Thelwall SN et al., Annual Epidemiological Commentary Mandatory MRSA, MSSA and E. coli bacteraemia and C. difficile infection data 2015 / 16 2016; Russo TA et al., Microbes and infection 2003;5:449-56). Priority pathogens described by both agencies include Enterobacteriacea with resistance to third generation cephalosporins, conferred by the production of extended-spectrum beta-lactamases (ESBLs), and to carbapenems, due to the production of carbapenemase enzymes. According to the CDC, Enterobacteriacea expressing ESBLs are a significant threat, while Enterobacteriacea resistant to last-line carbapenem antibiotics are considered an urgent threat. Escherichia coli (ESBL) strains are becoming increasingly widespread, and untreatable infections caused by Klebsiella pneumoniae, which produces both ESBLs and carbapenemases, are becoming increasingly common, especially in developing countries.

[0004] Escherichia coli is responsible for bloodstream infections (70 per 100,000 in the United States) (Marder EP et al. Foodborne pathogens and disease 2014;11:593-5), urinary tract infections (catheter-associated (250,000-525,000 cases per year in the United States) (Al-Hasan MN et al. The Journal of antimicrobial chemotherapy 2009;64:169-7); non-catheter-associated (6-8 million cases per year in the United States) (id.)); surgical site infections (127,500 cases per year in the United States), pneumonia (14,100-23,400 cases per year in the United States) (id.) and severe foodborne illness-associated diarrhea (63,000 cases per year in the United States) (Zowawi HM et al. Nature reviews Urology 2015;12:570-84), are among the most common human bacterial pathogens with clinical findings. They are serologically classified by differences in the structure of the lipopolysaccharide-associated O antigen (>180 known serotypes), the capsular polysaccharide K antigen (>80 serotypes), and the flagellar H antigen (>50 serotypes).

[0005] Urinary tract infections (UTIs) most often manifest as cystitis that may recur repeatedly after resolution in some individuals. If left untreated, they can progress to pyelonephritis and bloodstream infections. Escherichia coli (E. coli) infections are associated with high levels of antibiotic resistance (Rogers BA et al., The Journal of antimicrobial chemotherapy 2011;66:1-14), with many strains being resistant to multiple antibiotics, including last-resort antibiotics such as carbapenems and polymyxins (Nicolas-Chanoine MH et al., Clinical Microbiology Reviews 2014;27:543-74). In particular, O25b serotype multilocus sequence type (MLST) 131 has emerged as a global pandemic clone, causing primarily community-acquired infections and displaying high rates of resistance to extended-spectrum cephalosporins (ESBLs) and fluoroquinolones (Poolman JT et al., The Journal of infectious diseases 2016;213:6-13; Podschun R et al., Clin Microbiol Rev 1998;11:589-603). BSI- and UTI-infecting strains of Escherichia coli (E. coli) are also known as invasive extraintestinal pathogenic E. coli (ExPEC) or uropathogenic E. coli (UPEC). Among ExPEC strains, of the more than 180 identified Escherichia coli (E. coli) O-antigen serotypes, a subset of 10-12 O-serotypes has been reported to account for more than 60% of bacteremia cases (Yinnon AM et al. QJM: monthly journal of the Association of Physicians 1996;89:933-41).

[0006] After E. coli, Klebsiella spp. (including K. pneumoniae and K. oxytoca) are the next most common Gram-negative pathogens associated with invasive infections including UTIs, pneumonia, intra-abdominal infections, and bloodstream infections (BSIs) (Podschun R et al., Clin Microbiol Rev 1998;11:589-603; Anderson DJ et al., PLoS One 2014;9:e91713; Chen L et al., Trends Microbiol 2014;22:686-96; Iredell J et al., Bmj 2016;352:h6420). Klebsiella maintain a high capacity to acquire antibiotic resistance via horizontally transmitted ESBLs and carbapenem resistance-conferring genes (Follador R et al., Microbial Genomics 2016;2:e000073; Schrag SJ, Farley MM, Petit S et al., Epidemiology of Invasive Early-Onset Neonatal Sepsis, 2005-2014, 2016;138:e20162013). Thus, during the last decade, the prevalence of ESBL-resistant Klebsiella producing extended-spectrum β-lactamases (ESBLs) has increased dramatically at a global level. Klebsiella spp. can express up to eight different O types and more than 80 K types. Although there are numerous K antigens associated with virulent Klebsiella strains, just four O antigen serotypes account for >80% of Klebsiella clinical isolates, regardless of specimen collection site (blood, urine, saliva), infection status (invasive vs. noninvasive), or nature of acquisition (community vs. hospital-acquired) (Stoll BJ et al. Pediatrics 2011;127:817-26). Summary of the Invention [Problem to be solved by the invention]

[0007] Increasing rates of invasive multidrug-resistant (MDE) Escherichia coli (E. coli) and Klebsiella (Klebsiella) infections in vulnerable neonatal populations and the elderly highlight the need for vaccine-based approaches as an alternative to antibiotics, the increasingly effective standard of care. [Means for solving the problem]

[0008] To meet these and other needs, the present invention relates to compositions and methods of use for eliciting an immune response against E. coli and Klebsiella pneumoniae serotypes.

[0009] In one embodiment, the invention relates to a polypeptide or fragment thereof derived from FimH; and formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A1, formula O18B, formula O18B1, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25, formula O26, formula O27, formula O28, formula O29, formula O30, formula O31, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, formula O46, formula O47, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula O63, formula O64, formula O65, formula O66, formula O67, formula O68, formula O69, formula O70, formula O71, formula O72, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, 1, Formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O26, Formula O27, Formula O28, Formula O2 9, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, formula O43, formula O44, formula O45, formula O45, formula O45rel, formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O 132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O1 47, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O16 2, a composition comprising a sugar comprising a structure selected from any one of formulas O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

[0010] In one aspect, the composition further comprises at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3 and O5.

[0011] In another embodiment, the composition further comprises a saccharide derived from Klebsiella pneumoniae conjugated to a carrier protein; and a saccharide derived from E. coli conjugated to a carrier protein.

[0012] In another embodiment, the invention provides a composition comprising a polypeptide or fragment thereof derived from FimH; and at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5. In one embodiment, the composition comprises formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18, Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, Formula O18B1, Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O 23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O4 3, Formula O44, Formula O45, Formula O45, Formula O45rel, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O115, formula O 116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O13 2, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147 , formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula and further comprising at least one sugar comprising a structure selected from any one of formulas O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

[0013] In another aspect, a saccharide derived from Klebsiella pneumoniae is conjugated to a carrier protein; and a saccharide derived from E. coli is conjugated to a carrier protein.

[0014] In a further embodiment, the invention relates to at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5; and a saccharide of formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A 1, Formula O18B, Formula O18B1, Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O2 5b, formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O 39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O45, Formula O45rel, Formula O46, Formula O48, Formula O49, Formula O50 , formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O 101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O11 6, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147, Formula O 148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O16 3, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, formula O187.

[0015] In one embodiment, the composition further comprises a polypeptide or fragment thereof derived from FimH. In another embodiment, the E. coli saccharide comprises the formula O8. In another embodiment, the E. coli saccharide comprises the formula O9.

[0016] In another embodiment, the invention provides a method of raising an immune response against Escherichia coli in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of the above embodiments and aspects thereof.

[0017] In a further embodiment, the present invention provides a method of raising an immune response against Klebsiella pneumoniae in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of the above embodiments and aspects thereof.

[0018] In one aspect, the invention relates to a recombinant mammalian cell comprising a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli. In some embodiments, the polynucleotide encodes a polypeptide derived from E. coli flagellar H (fimH) polypeptide or fragment thereof. In some embodiments, the polypeptide or fragment thereof derived from E. coli FimH comprises a phenylalanine residue at the N-terminus of the polypeptide.

[0019] In one aspect, the present invention relates to a method for producing a polypeptide or a fragment thereof from E. coli in a recombinant mammalian cell. The method includes expressing the polypeptide or a fragment thereof by culturing the recombinant mammalian cell under suitable conditions; and collecting the polypeptide or a fragment thereof. In some embodiments, the method further includes purifying the polypeptide or a fragment thereof. In some embodiments, the yield of the polypeptide is at least 0.05 g / L. In some embodiments, the yield of the polypeptide is at least 0.10 g / L.

[0020] In one aspect, the invention relates to a composition comprising a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, or any combination thereof.

[0021] In another aspect, the invention relates to a composition comprising a polypeptide having at least n consecutive amino acids from any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). In some embodiments, the composition further comprises a sugar selected from any one of the formulas in Table 1, preferably Formula O1A, Formula O1B, Formula O2, Formula O6, and Formula O25B, where n is an integer from 1 to 100, preferably 31 to 100. [Brief description of the drawings]

[0022] [Figure 1] 1A-H depict amino acid sequences, including the amino acid sequence of an exemplary polypeptide or fragment thereof derived from E. coli; and the amino acid sequence of an exemplary wzzB sequence. [Diagram 2] 2A-T depict maps of exemplary expression vectors. [Diagram 3] FIG. 3 depicts the expression and purification results. [Figure 4] FIG. 4 depicts the expression and purification results. [Diagram 5] FIG. 5 depicts the expression results. [Figure 6]Figure 6A-C depict pSB02083 and pSB02158 SEC pools and affinities, including yields. [Figure 7] FIG. 7 depicts the results of expression of the pSB2198 FimH dscG Lock mutant construct. [Figure 8] FIG. 8 depicts the results of expression of pSB2307 FimH dscG wild type. [Figure 9] 9A-C depict the structures of O antigens synthesized by the polymerase-dependent pathway that contain four or fewer residues in the backbone. [Figure 10] FIG. 10A depicts the structure of an O antigen synthesized by the polymerase-dependent pathway that contains five or six residues in the backbone; FIG. 10B depicts an O antigen thought to be synthesized by the ABC transporter-dependent pathway. [Figure 11] FIG. 11 depicts computational mutagenesis scanning of Phe1 with other amino acids with aliphatic hydrophobic side chains, such as Ile, Leu and Val, that may stabilize the FimH protein and accommodate mannose binding. [Figure 12] Figures 12A-12B depict the plasmids: pUC replicon plasmid, 500-700 copies per cell, chain length regulator (Figure 12A); and P15a replicon plasmid, 10-12 copies per cell, O-antigen operon (Figure 12B). [Figure 13]Figures 13A-13B depict modulation of O-antigen chain length in serotype O25a and O25b strains by plasmid-based expression of heterologous wzzB and fepE chain length regulators. Genetic complementation of LPS expression in plasmid transformants of wzzB knockout strains O25K5H1 (O25a) and GAR2401 (O25b) is shown. On the left side of Figure 13A, the LPS profile of a plasmid transformant of O25a O25K5HΔwzzB is shown; on the right side, a similar profile of an O25b GAR 2401ΔwzzB transformant is shown. Immunoblots of replicate gels probed with O25-specific serum (Statens Serum Institut) are shown in Figure 13B. Lanes 1-7 correspond to the O25aΔwxxB (knockout) background; lanes 8-15 correspond to the O25b 2401ΔwzzB (knockout) background. [Figure 14] FIG. 14 depicts long O antigen expression mediated by E. coli and Salmonella fepE plasmids in host O25K5H1ΔwzzB. [Figure 15] FIG. 15 depicts Salmonella fepE expression produces long O-antigen LPS in various clinical isolates. [Figure 16] Figures 16A-16B depict plasmid-mediated arabinose-inducible expression of O25b long chain O antigen LPS in an O25b O antigen knockout host strain. Results from SPS PAGE are shown in Figure 16A and results from O25 immunoblots are shown in Figure 16B, where in both Figures 16A and 16B, lane 1 is from clone 1, no arabinose; lane 2 is from clone 1, 0.2% arabinose; lane 3 is from clone 9, no arabinose; lane 4 is from clone 9, 0.2% arabinose; lane 5 is from O55 E. coli LPS standard; and lane 6 is from O111 E. coli LPS standard. [Figure 17] FIG. 17 shows plasmid-mediated arabinose-inducible expression of long-chain O-antigen LPS in a common host strain. [Figure 18] FIG. 18 depicts expression of O25 O-antigen LPS in exploratory bioprocess lines. [Figure 19] Figures 19A-19B depict the SEC profiles and characteristics of short (Figure 19A, strain 1, O25b wt2831) and long O25b O antigens (Figure 19B, strain 2, O25b 2401ΔwzzB / LT2 FepE) purified from GAR2831 and '2401ΔwzzB / fepE strains. [Figure 20] Figures 20A-20B depict vaccination schedules in rabbits: (Figure 20A) Information regarding vaccination schedule for rabbit study 1 VAC-2017-PRL-EC-0723; (Figure 20B) Vaccination schedule for rabbit study 2 VAC-2018-PRL-EC-077. [Figure 21] Figures 21A-21C depict O25b glycoconjugate IgG responses. -●- represents pre-bleed results; -■- bleed 1 (week 6); -▲- bleed 2 (week 8); -◆- bleed 3 (week 12). Figure 21A depicts results from rabbit 1-3 (medium activation); Figure 21B depicts results from rabbit 2-3 (low activation); Figure 21C depicts results from rabbit 3-1 (high activation). [Figure 22]Figures 22A-22F depict IgG responses to O25b long chain O antigen saccharide conjugates, i.e., low activated O25b-CRM197 conjugates (in Figures 22D-F, -●- represents pre-bleed results from rabbit 2-1, -■- represents week 12 antiserum results from rabbit 2-1) and non-conjugated polysaccharides, i.e., free O25b polysaccharides (in Figures 22A-C, -●- represents pre-bleed results from rabbit A-1, -■- represents week 6 antiserum results from rabbit A-1, -▲- represents week 8 antiserum results from rabbit A-1). Note that MFI is plotted on a logarithmic scale to highlight the difference between pre-immune and immune antibodies in the MFI range below 1000. Figure 22A depicts the results for rabbit A-1 (unconjugated poly); Figure 22B depicts the results for rabbit A-3 (unconjugated poly); Figure 22C depicts the results for rabbit A-4 (unconjugated poly); Figure 22D depicts the results for rabbit 2-1 (low activation); Figure 22E depicts the results for rabbit 2-2 (low activation); and Figure 22F depicts the results for rabbit 2-3 (low activation). [Figure 23] Figures 23A-23C depict surface expression of native and long chain O25b O antigens detected using O25b antisera. Figure 23A depicts results where -●- depicts results for O25b 2831 vs. PD3 antisera, -■- represents results for O25b 2831 wt vs. pre-bleed, -▲- represents results for O25b 2831 / fepE vs. PD3 antisera, and -▼- represents results for O25b 2831 / fepE vs. pre-bleed. Figure 23B depicts results where -●- represents results for O25b 2401 vs. PD3 antiserum, -■- represents results for O25b 2401 vs. pre-bleeding, -▲- represents results for O25b 2401 / fepE vs. PD3 antiserum, -▼- represents results for O25b 2401 / fepE vs. pre-bleeding. Figure 23C depicts results where -●- represents results for E. coli K12 vs. PD3 antiserum, -■- represents results for E. coli K12 vs. pre-bleeding. [Figure 24]Figure 24 depicts the generalized structures of the carbohydrate backbones of the outer core oligosaccharides of the five known chemotypes. All glycoses are in the α-anomeric configuration unless otherwise indicated. The genes whose products catalyze the formation of each bond are indicated with dashed arrows. The asterisk indicates the residue of the core oligosaccharide at which attachment of the O antigen occurs. [Diagram 25] FIG. 25 depicts that unconjugated free O25b polysaccharide is not immunogenic (dLIA), where -●- represents the results of antiserum from 4-1 at week 18 (1 week = PD4); -■- represents the results of antiserum from 4-2 at week 18 (1 week = PD4); -▲- represents the results of antiserum from 5-1 at week 18 (1 week = PD4); -▼- represents the results of antiserum from 5-2 at week 18 (1 week = PD4); -*- represents the results of antiserum from 6-1 at week 18 (1 week = PD4); -^- represents the results of antiserum from 6-2 at week 18 (1 week = PD4). [Figure 26] Figures 26A-26C- depict graphs showing specificity of BRC rabbit O25b RAC conjugate immune serum OPA titer. Figure 26A shows OPA titer of rabbit 2-3 with pre-immune serum -●- and post-immune serum week 13 -■-. Figure 26B shows OPA titer of rabbit 1-2 with pre-immune serum -●- and post-immune serum week 19 -■-. Figure 26C shows OPA titer specificity of rabbit 1-2 week 19, where OPA activity of rabbit 1-2 immune serum is blocked by pre-incubation with 100 μg / mL purified unconjugated O25b long chain O antigen polysaccharide. -■- represents rabbit 1-2 immune serum week 19 results, and -▼- represents rabbit 1-2 w / R1 long chain OAg results week 19. [Figure 27]Figure 27A shows an illustration of an exemplary dosing schedule. Figures 27B and 27C show graphs depicting O antigen O25b IgG levels elicited by unconjugated O25b long chain O antigen polysaccharide (Figure 27B, O25b free poly(2 μg)) and induced O25b RAC / DMSO long chain O antigen saccharide conjugate (Figure 27C, O25b-CRM197 RAC long chain(2 μg)), where -...- (dotted line) represents naive CD1 O25b IgG levels. [Figure 28] Figures 28A-28B depict graphs depicting OPA immunogenicity of RAC, eTEC O25b long chain glycoconjugates, and single-end glycoconjugates after dose 2 (Figure 28A) and dose 3 (Figure 28B), where -○- represents single-end short 2 μg results, -●- represents single-end long 2 μg results, -▲- represents RAC / DMSO long 2 μg results, -▼- represents eTEC long 2 μg results, and * represents background control (n=20). †Response rate is % of mice with titers greater than 2x unvaccinated baseline. [Figure 29] Figure 29 depicts a graph showing OPA immunogenicity and altered levels of polysaccharide activation of eTEC chemistry. † Response rate is % of mice with titers greater than 2x unvaccinated baseline. [Diagram 30] Figures 30A-30B show an illustration of an exemplary dosing schedule (Figure 30A); and a graph depicting protection of mice immunized with a dose of E. coli eTEC conjugate from a lethal challenge with an O25b isolate (Figure 30B), where -◇- represents 17% activation of eTEC long chains, -△- represents 10% activation of eTEC long chains, -▽- represents 4% activation of eTEC long chains, -□- represents O25b polysaccharide, and -○- represents unvaccinated controls. [Diagram 31]Figure 31 depicts a schematic diagram showing an exemplary preparation of a single-terminated conjugate, where the conjugation process involves selective activation of 2-keto-3-deoxyoctanoic acid (KDO) with a disulfide amine linker upon unmasking of the thiol functional group. KDO is then conjugated to a bromo-activated CRM197 protein as depicted in Figure 31 (preparation of a single-terminated conjugate). [Diagram 32] 32A-32B depict exemplary process flow diagrams for the activation (FIG. 32A) and conjugation (FIG. 32B) processes used in the preparation of E. coli glycoconjugates to CRM197. [Diagram 33] Figure 33 depicts the structure of the repeating unit (RU) of E. coli and K. pneumoniae polymannan O antigens. Legend: Trimeric E. coli O8 and K. pneumoniae O5 are identical, as are tetrameric E. coli O9A / K. pneumoniae O3a and pentameric E. coli O9 / K. pneumoniae O3. Differences between K. pneumoniae O3 subtypes at the level of biosynthetic enzyme sequences are described in Guachalla LM et al. (Scientific Reports 2017;7:6635). [Diagram 34]Figures 34A-34B depict that E. coli serotype O8 immune sera are bactericidal against invasive K. pneumoniae serotype O5 strains. Legend: Rabbit immune sera elicited by E. coli serotype O8 O antigen CRM197 conjugate were evaluated in bactericidal assays with E. coli O8 strains (Figure 34A) and K. pneumoniae O5 strains (Figure 34B). Strong opsonophagocytosis assay (OPA) activity against E. coli O8 strains was observed after two vaccine doses (week 15), which was not present after preadsorption with unconjugated O8 polysaccharide (O8-OAg) or with matched preimmune sera (week 0). The same rabbit immune serum showed antigen-specific serum bactericidal activity (SBA) against K. pneumoniae O5 strain. BRC-juvenile rabbit complement, hC-IgG / IgM, and human serum were depleted as a complement source. [Diagram 35] Figures 35A-35B depict that E. coli serotype O9 O-antigen immune sera are bactericidal against invasive K. pneumoniae O3 isolates. Legend: Rabbit immune sera elicited with E. coli serotype O9a O-antigen CRM197 conjugate were evaluated in an opsonophagocytosis assay (OPA) with E. coli O9a strain (Figure 35A) and K. pneumoniae O3b strain (Figure 35B). OPA activity against E. coli O9 strains was observed after two vaccine doses (week 15), but was not present after preadsorption with unconjugated O9 polysaccharide (O9-OAg) or with matched preimmune sera (week 0). The same rabbit immune serum also showed potent antigen-specific serum bactericidal activity (SBA) against K. pneumoniae O3b strain. BRC-juvenile rabbit complement, hC-IgG / IgM, was depleted using human serum as a complement source. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Sequence Identifier SEQ ID NO:1 sets forth the amino acid sequence of the wild-type type 1 fimbria D-mannose specific adhesin (E. coli FimH J96). SEQ ID NO:2 sets forth the amino acid sequence of a fragment of FimH corresponding to aa residues 22-300 of SEQ ID NO:1 (mature FimH protein). SEQ ID NO:3 sets forth the amino acid sequence of the FimH lectin domain. SEQ ID NO:4 sets forth the amino acid sequence of the FimH pyrin domain. SEQ ID NO:5 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB02198 - FimH mIgK signal pept / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7 AA linker / FimG A1..K14 / GGHis8 in pcDNA3.1(+)). SEQ ID NO:6 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB02307 - FimH mIgK signal pept / F22..Q300 J96 FimH N28S N91S N249Q / His8 in pcDNA3.1(+)). SEQ ID NO:7 sets forth the amino acid sequence of a fragment of a polypeptide derived from E. coli FimH (pSB02083 FimH lectin domain wild type construct). SEQ ID NO:8 sets forth the amino acid sequence of a fragment of a polypeptide derived from E. coli FimH (pSB02158 FimH lectin domain Lock mutant). SEQ ID NO:9 sets forth the amino acid sequence of a fragment of a polypeptide derived from E. coli FimG (FimG A1..K14). SEQ ID NO:10 sets forth the amino acid sequence of a fragment of a polypeptide derived from E. coli FimC. SEQ ID NO:11 sets forth the amino acid sequence of the 4aa linker. SEQ ID NO: 12 sets forth the amino acid sequence of the 5aa linker. SEQ ID NO: 13 sets forth the amino acid sequence of the 6aa linker. SEQ ID NO: 14 sets forth the amino acid sequence of the 7aa linker. SEQ ID NO: 15 sets forth the amino acid sequence of the 8aa linker. SEQ ID NO: 16 sets forth the amino acid sequence of the 9aa linker. SEQ ID NO:17 sets forth the amino acid sequence of the 10aa linker. SEQ ID NO:18 sets forth the amino acid sequence of the FimH J96 signal sequence. SEQ ID NO: 19 sets forth the amino acid sequence of the signal peptide of SEQ ID NO: 5 (pSB02198 in pcDNA3.1(+) - FimH mIgK signal pept / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7 AA linker / FimG A1..K14 / GGHis8). SEQ ID NO: 20 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH according to SEQ ID NO: 5 (mature protein of pSB02198 in pcDNA3.1(+) - FimH mIgK signal pept / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7 AA linker / FimG A1..K14 / GGHis8). SEQ ID NO:21 sets forth the amino acid sequence of a polypeptide derived from E. coli FimG. SEQ ID NO: 22 sets forth the amino acid sequence of the signal peptide of SEQ ID NO: 6 (pSB02307 - FimH mIgK signal pept / F22..Q300 J96 FimH N28S N91S N249Q / His8 in pcDNA3.1(+). SEQ ID NO: 23 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH according to SEQ ID NO: 6 (mature protein of FimH mIgK signal pept / F22..Q300 J96 FimH N28S N91S N249Q / His8 in pcDNA3.1(+). SEQ ID NO:24 sets forth the amino acid sequence of the E. coli FimH-derived polypeptide according to SEQ ID NO:7 (mature protein of the pSB02083 FimH lectin domain wild-type construct). SEQ ID NO:25 sets forth the amino acid sequence of the His tag. SEQ ID NO: 26 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH according to SEQ ID NO: 8 (mature protein of the pSB02158 FimH lectin domain Lock mutant). SEQ ID NO:27 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (pSB01878). SEQ ID NO:28 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (K12). SEQ ID NO:29 sets forth the amino acid sequence of a polypeptide derived from E. coli FimH (UTI89). SEQ ID NO:30 sets forth the O25b 2401 WzzB amino acid sequence. SEQ ID NO:31 sets forth the O25a:K5:H1 WzzB amino acid sequence. SEQ ID NO:32 sets forth the O25a ETEC ATCC WzzB amino acid sequence. SEQ ID NO:33 sets forth the K12 W3110 WzzB amino acid sequence. SEQ ID NO:34 sets forth the Salmonella LT2 WzzB amino acid sequence. SEQ ID NO:35 sets forth the O25b 2401 FepE amino acid sequence. SEQ ID NO:36 sets forth the O25a:K5:H1 FepE amino acid sequence. SEQ ID NO:37 sets forth the O25a ETEC ATCC FepE amino acid sequence. SEQ ID NO:38 sets forth the O157 FepE amino acid sequence. SEQ ID NO:39 sets forth the Salmonella LT2 FepE amino acid sequence. SEQ ID NO: 40 sets forth the primer sequence of LT2wzzB_S. SEQ ID NO: 41 sets forth the primer sequence of LT2wzzB_AS. SEQ ID NO: 42 sets forth the primer sequence for O25bFepE_S. SEQ ID NO: 43 sets forth the primer sequence for O25bFepE_A. SEQ ID NO: 44 sets forth the primer sequence of wzzB P1_S. SEQ ID NO: 45 sets forth the primer sequence of wzzB P2_AS. SEQ ID NO: 46 sets forth the primer sequence of wzzB P3_S. SEQ ID NO: 47 sets forth the primer sequence of wzzB P4_AS. SEQ ID NO: 48 sets forth the primer sequence for O157 FepE_S. SEQ ID NO: 49 sets forth the primer sequence for O157 FepE_AS. SEQ ID NO: 50 sets forth the primer sequence of pBAD33_adaptor_S. SEQ ID NO: 51 sets forth the primer sequence of pBAD33_adaptor_AS. SEQ ID NO:52 sets forth the primer sequence of JUMPSTART_r. SEQ ID NO:53 sets forth the primer sequence of gnd_f. SEQ ID NO:54 sets forth the amino acid sequence of the mouse IgK signal sequence. SEQ ID NO:55 sets forth the amino acid sequence of the human IgG receptor FcRn large subunit p51 signal peptide. SEQ ID NO:56 sets forth the amino acid sequence of the human IL10 protein signal peptide. SEQ ID NO:57 sets forth the amino acid sequence of the Human respiratory syncytial virus A (A2 strain) fusion glycoprotein F0 signal peptide. SEQ ID NO:58 sets forth the amino acid sequence of the influenza A hemagglutinin signal peptide. SEQ ID NOs:59-101 set forth the amino acid and nucleic acid sequences of nanostructure-associated polypeptides or fragments thereof. SEQ ID NOs: 102-109 set forth the Signal P 4.1 (DTU Bioinformatics) sequences from various species used for signal peptide prediction.

[0024] Detailed Description of the Invention In one embodiment, the invention relates to a polypeptide or fragment thereof derived from FimH; and formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A1, formula O18B, formula O18B1, formula O19, formula O20, formula O21, formula O22, formula O23, formula O24, formula O25, formula O26, formula O27, formula O28, formula O29, formula O30, formula O31, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45, formula O46, formula O47, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula O63, formula O64, formula O65, formula O66, formula O67, formula O68, formula O69, formula O70, formula O71, formula O72, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, 1, Formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O26, Formula O27, Formula O28, Formula O2 9, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, formula O43, formula O44, formula O45, formula O45, formula O45rel, formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, formula O117, formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O 132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O1 47, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O16 2, a composition comprising a sugar comprising a structure selected from any one of formulas O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

[0025] In one embodiment, the composition further comprises at least one saccharide derived from any one of K. pneumoniae type selected from the group consisting of O1, O2, O3 and O5. In another embodiment, the composition further comprises a saccharide derived from Klebsiella pneumoniae type O1. In another embodiment, the composition further comprises a saccharide derived from Klebsiella pneumoniae type O2. In another embodiment, the composition further comprises a saccharide derived from Klebsiella pneumoniae type O3. In another embodiment, the composition further comprises a saccharide derived from Klebsiella pneumoniae type O5. In another embodiment, the composition further comprises a saccharide derived from Klebsiella pneumoniae type O1 and a saccharide derived from Klebsiella pneumoniae type O2.

[0026] In another embodiment, the composition further comprises a saccharide derived from Klebsiella pneumoniae conjugated to a carrier protein; and a saccharide derived from E. coli conjugated to a carrier protein.

[0027] In another embodiment, the composition further comprises a polypeptide derived from Klebsiella pneumoniae.

[0028] In another embodiment, the invention provides a composition comprising a polypeptide or fragment thereof derived from FimH; and at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5. In one aspect, the composition comprises formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18, Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, Formula O18B1, Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O 23A, Formula O24, Formula O25, Formula O25a, Formula O25b, Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O4 3, Formula O44, Formula O45, Formula O45, Formula O45rel, Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O115, formula O 116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O13 2, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147 , formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula and further comprising at least one sugar comprising a structure selected from any one of formulas O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

[0029] In another aspect, a saccharide derived from Klebsiella pneumoniae is conjugated to a carrier protein; and a saccharide derived from E. coli is conjugated to a carrier protein.

[0030] In a further embodiment, the composition further comprises a polypeptide derived from Klebsiella pneumoniae.

[0031] In a further embodiment, the invention relates to at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5; and a saccharide of formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2;K13;K15, formula O6:K54, formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18, formula O18A, formula O18ac, formula O18A 1, Formula O18B, Formula O18B1, Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O23A, Formula O24, Formula O25, Formula O25a, Formula O2 5b, formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O 39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O45, Formula O45rel, Formula O46, Formula O48, Formula O49, Formula O50 , formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73, formula O73, formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O 101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O11 6, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147, Formula O 148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O16 3, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, formula O187.

[0032] In one embodiment, the composition further comprises a polypeptide or fragment thereof derived from FimH. In another embodiment, the E. coli saccharide comprises the formula O8. In another embodiment, the E. coli saccharide comprises the formula O9.

[0033] In a further embodiment, the composition further comprises a polypeptide derived from Klebsiella pneumoniae.

[0034] In one aspect of the above embodiment, the saccharide is covalently attached to a carrier protein. In one aspect, the saccharide further comprises a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety. In another aspect, the carrier protein is CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from P. aeruginosa, maltose binding protein (MBP), detoxified hemolysin A from S. aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae, or Streptococcus pneumoniae. pneumoniae pneumolysin and its detoxified variants, Campylobacter jejuni (C. jejuni) AcrA, Campylobacter jejuni (C. jejuni) native glycoprotein and Streptococcal C5a peptidase (SCP).

[0035] In another embodiment, the invention provides a method of eliciting an immune response against Escherichia coli in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of the above embodiments and aspects thereof. In one aspect, the immune response comprises opsonophagocytic antibodies against E. coli. In another aspect, the immune response protects the mammal against E. coli infection.

[0036] In a further embodiment, the invention provides a method of eliciting an immune response against Klebsiella pneumoniae in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of the above embodiments and aspects thereof. In one aspect, the immune response comprises opsonophagocytic antibodies against Klebsiella pneumoniae. In another aspect, the immune response protects the mammal against Klebsiella pneumoniae infection.

[0037] The present inventors have overcome the challenges of producing polypeptides derived from E. coli adhesin proteins by using mammalian cells for expression. As illustrated in this disclosure and in the Examples section, it has been discovered that mammalian cell expression of recombinant polypeptides consistently achieves higher yields compared to expression of the polypeptides in E. coli. Furthermore, the present inventors have surprisingly identified mutations and expression constructs to stabilize the recombinant polypeptides and fragments thereof in desired conformations.

[0038] Blocking the primary steps of infection, i.e., bacterial binding to host cell receptors and colonization of mucosal surfaces, is important in preventing, treating, and / or reducing the likelihood of bacterial infection. Bacterial binding may involve the interaction of bacterial surface proteins, called adhesins, with host cell receptors. Previous preclinical studies with the FimH adhesin (derived from uropathogenic E. coli) confirmed that antibodies against the adhesin were elicited. Advances in the identification, characterization, and isolation of adhesins are necessary in efforts to prevent infections, from otitis media and dental caries to pneumonia and sepsis.

[0039] To produce adhesin proteins such as FimH and fragments thereof on a commercial scale, it is necessary to identify suitable constructs and suitable hosts capable of expressing the polypeptides and fragments thereof in sufficient amounts over extended periods of time and in a preferred conformation. For example, in some embodiments, the preferred conformation of the recombinant polypeptide is characterized by a low affinity for monomannose (e.g., a K of about 300 μM). d In some embodiments, the preferred conformation exhibits a high affinity for monomannose (e.g., a K of less than 1.2 μM). d ) is shown.

[0040] Adhesin proteins derived from E. coli have been recombinantly expressed in E. coli cells. However, the yield was less than 10 mg / L. Purifying large amounts of fimbria-associated adhesins can be difficult when produced in E. coli. Without being bound by theory or mechanism, it has been suggested that products expressed in E. coli may exhibit a conformation that is not optimal for eliciting an effective immune response in mammals.

[0041] In one aspect, the invention includes a recombinant mammalian cell comprising a polynucleotide sequence encoding a polypeptide or fragment thereof derived from a bacterial adhesin protein.

[0042] In another aspect, the present invention includes a process for producing a polypeptide or fragment thereof in a mammalian cell, comprising: (i) expressing said polypeptide or fragment thereof by culturing mammalian cells under suitable conditions; and (ii) collecting said polypeptide or fragment thereof from the culture. The process may further comprise purifying the polypeptide or fragment thereof. Also disclosed herein is a polypeptide or fragment thereof produced by the process.

[0043] In another aspect, the present invention includes a composition comprising a polypeptide or a fragment thereof as described herein. The composition may comprise a polypeptide or a fragment thereof suitable for in vivo administration. For example, the polypeptide or a fragment thereof in such a composition may have a purity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by mass. The composition may further comprise an adjuvant.

[0044] In further aspects, the invention includes compositions for use in inducing an immune response against E. coli. Use of the compositions described herein for inducing an immune response against E. coli and use of the compositions described herein in the manufacture of a medicament for inducing an immune response against E. coli are also disclosed.

[0045] I. Polypeptides and fragments thereof derived from E. coli In one aspect, disclosed herein is a mammalian cell comprising a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli. The term "derived from" as used herein refers to a polypeptide comprising the amino acid sequence of a FimH polypeptide or FimCH polypeptide complex or fragment thereof as described herein, which has been altered by the introduction of substitutions, deletions or additions of amino acid residues. Preferably, the polypeptide or fragment thereof derived from E. coli comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide or fragment thereof derived from E. coli is identical in full length amino acids to the corresponding wild-type FimH polypeptide or FimCH polypeptide complex or fragment thereof.

[0046] The fragment should comprise at least n contiguous amino acids from the sequence, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more) depending on the particular sequence. Preferably, the fragment comprises an epitope from the sequence. In some embodiments, the fragment comprises an amino acid sequence of at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a polypeptide derived from E. coli.

[0047] In some embodiments, the E. coli derived polypeptide or fragment thereof comprises one or more non-classical amino acids when compared to the corresponding wild-type E. coli FimH polypeptide or fragment.

[0048] In some embodiments, the E. coli derived polypeptide or fragment thereof has a similar or identical function as the corresponding wild-type FimH polypeptide or fragment thereof.

[0049] In a preferred embodiment, a polypeptide or polypeptide complex of the invention, or a fragment thereof, is isolated or purified.

[0050] In some embodiments, a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli is integrated into the genomic DNA of a mammalian cell, such that the E. coli derived polypeptide or fragment thereof is expressed by the mammalian cell when cultured under suitable conditions.

[0051] In a preferred embodiment, the E. coli derived polypeptide or fragment thereof is soluble.

[0052] In some embodiments, the E. coli derived polypeptide or fragment thereof is secreted from a mammalian host cell.

[0053] In some embodiments, the polypeptide or fragment thereof derived from E. coli may include additional amino acid residues, such as an N-terminal or C-terminal extension. Such extensions may include one or more tags, which may facilitate detection (e.g., an epitope tag for detection by monoclonal antibodies) and / or purification (e.g., a polyhistidine tag that allows purification on nickel-chelating resins) of the polypeptide or fragment thereof. In some embodiments, the tag comprises an amino acid sequence selected from any one of SEQ ID NO: 21 and SEQ ID NO: 25. Such affinity purification tags are known in the art. Examples of affinity purification tags include, for example, His tags (e.g., hexahistidine, which can bind to metal ions), maltose binding protein (MBP), which can bind to amylose, glutathione-S-transferase (GST), which can bind to glutathione, FLAG tags, which can bind to anti-flag antibodies, and Strep tags, which can bind to streptavidin or derivatives thereof. In preferred embodiments, the E. coli derived polypeptide or fragment thereof does not include additional amino acid residues such as N-terminal or C-terminal extensions. In some embodiments, the E. coli derived polypeptide or fragment thereof described herein does not include an exogenous tag sequence.

[0054] Although specific strains of E. coli may be mentioned herein, it should be understood that the polypeptides or fragments thereof derived from E. coli are not limited to a specific strain unless specified.

[0055] In some embodiments, the polypeptide or fragment thereof derived from E. coli FimH comprises a phenylalanine residue at the N-terminus of the polypeptide. In some embodiments, the polypeptide or fragment thereof derived from FimH comprises a phenylalanine residue within the first 20 residue positions of the N-terminus. Preferably, the phenylalanine residue is located at position 1 of the polypeptide. For example, in some embodiments, the polypeptide or fragment thereof derived from E. coli FimH does not comprise an additional glycine residue at the N-terminus of the polypeptide or fragment thereof derived from E. coli FimH.

[0056] In some embodiments, the phenylalanine residue at position 1 of wild-type mature E. coli FimH is replaced with an aliphatic hydrophobic amino acid, such as, for example, any one of Ile, Leu, and Val residues.

[0057] In some embodiments, a signal peptide can be used to express a polypeptide or fragment thereof from E. coli. Signal sequences and expression cassettes for producing proteins are known in the art. Generally, leader peptides are 5-30 amino acids long and are typically present at the N-terminus of a newly synthesized polypeptide. Signal peptides generally contain a long stretch of hydrophobic amino acids that have a tendency to form a single alpha-helix. In addition, many signal peptides begin with a short positively charged stretch of amino acids, which may help enforce the correct topology of the polypeptide during translocation. At the end of the signal peptide, there is typically a stretch of amino acids that is recognized and cleaved by a signal peptidase, which can cleave to generate a free signal peptide and a mature protein, either during or after the translocation is complete. In some embodiments, the signal peptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identity to any one of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:22.

[0058] In some embodiments, the polypeptide or fragment thereof derived from E. coli described herein may include a cleavable linker. Such a linker allows the tag to be separated from the purified complex, for example, by the addition of an agent capable of cleaving the linker. Cleavable linkers are known in the art. Such linkers can be cleaved, for example, by irradiation of a photolabile bond or by acid-catalyzed hydrolysis. Another example of a cleavable linker includes a polypeptide linker that includes a protease recognition site and can be cleaved by the addition of a suitable protease enzyme.

[0059] In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a modification compared to the corresponding wild-type E. coli FimH polypeptide or fragment. The modification may comprise a covalent attachment of a molecule to the polypeptide. For example, such modifications may comprise glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, and the like. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a modification compared to the corresponding wild-type E. coli FimH polypeptide or fragment, such as by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In another embodiment, the modification may comprise a covalent attachment of a lipid molecule to the polypeptide. In some embodiments, the polypeptide does not comprise a covalent attachment of a molecule to the polypeptide compared to the corresponding wild-type E. coli FimH polypeptide or fragment.

[0060] For example, proteins and polypeptides produced in cell culture may be glycoproteins that contain covalently linked carbohydrate structures, including oligosaccharide chains. These oligosaccharide chains are attached to the protein via either N- or O-linkages. The oligosaccharide chains may account for a significant portion of the mass of the glycoprotein. Generally, N-linked oligosaccharides are attached to the amino group on the side chain of an asparagine residue within the target consensus sequence of Asn-X-Ser / Thr, where X can be any amino acid except proline. In some embodiments, the glycosylation site comprises an amino acid sequence selected from any one of the following: asparagine-glycine-threonine (NGT), asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS). The polypeptide or fragment thereof derived from E. coli produced in mammalian cells may be glycosylated. Glycosylation may occur at the N-linked glycosylation signal Asn-Xaa-Ser / Thr in the sequence of the polypeptide or fragment thereof derived from E. coli. "N-linked glycosylation" refers to the attachment of a carbohydrate moiety via GIcNAc to an asparagine residue in the polypeptide chain. N-linked carbohydrates include the consensus Man 1-6(Man1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-R core structure, where R represents an asparagine residue in the produced polypeptide or fragment thereof derived from E. coli.

[0061] In some embodiments, the glycosylation site in the polypeptide or fragment thereof derived from E. coli is removed by mutation in the sequence of the polypeptide or fragment thereof derived from E. coli. For example, in some embodiments, the Asn residue of the glycosylation motif (Asn-Xaa-Ser / Thr) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

[0062] In some embodiments, the Ser residue of the glycosylation motif may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and Gln.

[0063] In some embodiments, the Thr residue of the glycosylation motif may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and Gln.

[0064] In some embodiments, glycosylation sites (such as Asn-Xaa-Ser / Thr) in a polypeptide or fragment thereof derived from E. coli are not removed or modified. In some embodiments, a compound that reduces or inhibits glycosylation may be added to the cell culture medium. In such embodiments, the polypeptide or protein contains at least one more non-glycosylated (i.e., aglycosylated) site, i.e., a completely unoccupied glycan site that does not contain an attached carbohydrate moiety, or contains at least one less carbohydrate moiety at the same potential glycosylation site than an otherwise identical polypeptide or protein produced by a cell under identical conditions except in the absence of a glycosylation-inhibiting compound.Such compounds are known in the art and include, but are not limited to, tunicamycin, tunicamycin homologs, streptovirudin, mycospocidin, amphomycin, tsushimycin, antibiotic 24010, antibiotic MM19290, bacitracin, corynetoxin, showdomycin, duimycin, 1-deoxymannonojirimycin, deoxynojirimycin, N-methyl-1-deoxymannojirimycin, brefeldin A, glucose and mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D-(+)-mannose, D-(+) galactose, 2-deoxy-2-fluoro-D- Glucose, 1,4-dideoxy-1,4-imino-D-mannitol (DIM), fluoroglucose, fluoromannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, hydroxymethylglutaryl-CoA reductase inhibitors, 25-hydroxycholesterol, hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, monensin, m-chlorocarbonyl-cyanide phenylhydrazone (CCCP), compactin, dolichyl-phosphoryl^-deoxyglucose, N-acetyl-D-glucosamine, hygoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromoconduritol, conduritol epoxide. epoxide) and conduritol derivatives, glycosylmethyl-p-nitrophenyltriazenes, β-hydroxynorvaline, threo-β-fluoroasparagine, D-(+)-gluconic acid δ-lactone, di(2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl ester of (diphenylmethyl)-phosphate, [2-(diphenylphosphinyloxy)ethyl]trimethylammonium iodide, iodoacetate, 2-deoxy-D-glucose, and fluoroacetate. Those of skill in the art will readily recognize or be able to determine, without undue experimentation, glycosylation inhibitors that can be used in accordance with the methods and compositions of the invention. In such embodiments, glycosylation of a polypeptide or fragment thereof can be controlled without introducing amino acid mutations into the polypeptide or fragment thereof.

[0065] In some embodiments, the level of glycosylation of a polypeptide or fragment thereof produced by a mammalian cell (e.g., the number of glycan sites occupied on the polypeptide or fragment thereof, the size and / or complexity of the glycoform at those sites, etc.) is lower than the level of glycosylation of a polypeptide or fragment thereof produced in the same medium and under otherwise identical conditions but without the inclusion of a glycolysis inhibitor compound, mutation, etc.

[0066] In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not include a site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not include at least one site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not include any site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli includes a site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli includes at most one site for N-linked protein glycosylation. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli includes at most two sites for N-linked protein glycosylation.

[0067] E. coli derived polypeptides or fragments thereof expressed by different cell lines or in transgenic animals may have different glycan site occupancy, glycotype and / or glycosylation patterns compared to each other. In some embodiments, the present invention encompasses E. coli derived polypeptides or fragments thereof, regardless of the glycosylation, glycan occupancy or glycotype of the E. coli derived polypeptides or fragments thereof produced in mammalian cells.

[0068] In some embodiments, the polypeptide or fragment thereof derived from E. coli may be derived from an E. coli FimH polypeptide, where the amino acid residue at position 1 of the polypeptide is phenylalanine, rather than methionine, for example, a polypeptide having the amino acid sequence of SEQ ID NO: 2. Preferably, the polypeptide derived from E. coli FimH comprises a phenylalanine at position 1 of the amino acid sequence of the polypeptide derived from E. coli. In another preferred embodiment, the polypeptide derived from E. coli FimH comprises the amino acid sequence of SEQ ID NO: 3, where the residue at position 1 of the amino acid sequence of the polypeptide derived from E. coli is phenylalanine. In some embodiments, the polypeptide derived from E. coli or fragment thereof may comprise the amino acid sequence of SEQ ID NO: 4, which may be derived from an E. coli FimH polypeptide.

[0069] In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. In some embodiments, the E. coli derived polypeptide or fragment thereof may be derived, for example, from an E. coli FimG polypeptide having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the E. coli derived polypeptide or fragment thereof may be derived, for example, from an E. coli FimC polypeptide having the amino acid sequence of SEQ ID NO: 10.

[0070] A. Polypeptides and fragments thereof derived from E. coli FimH The bacterial fimbrial adhesins FimH and FmlH enable Escherichia coli to exploit distinct urinary tract microenvironments through recognition of specific host cell glycoproteins. FimH binds to mannosylated uroplakin receptors in urothelial cells, while FmlH binds to galactose or N-acetylgalactosamine O-glycans on epithelial surface proteins in the kidney and inflamed bladder. FimH fimbriae also play a role in colonization of the intestine by enterotoxigenic E. coli (ETEC) and multidrug-resistant invasive E. coli through binding to highly mannosylated proteins on the intestinal epithelium.

[0071] Full-length FimH is composed of two domains: an N-terminal lectin domain and a C-terminal pyrin domain, which are connected by a short linker. The lectin domain of FimH contains the carbohydrate recognition domain responsible for binding to mannosylated uroplakin 1a on the urothelial cell surface. The pyrin domain is anchored to the core of the pilus via the donor strand of the subsequent FimG subunit, a process called donor strand complementation.

[0072] The conformation and ligand-binding properties of the lectin domain of FimH are under allosteric control of the pyrin domain of FimH. Under static conditions, the interaction of the two domains of full-length FimH binds the lectin domain to a low affinity mannose (e.g., K)-binding domain characterized by a shallow binding pocket. d Binding to a mannoside ligand induces a conformational change that results in a moderate affinity state, in which the lectin and pyrin domains remain in close contact. However, upon shear stress, the lectin and pyrin domains separate, thereby transitioning to a high affinity state (e.g., K d <1.2 μM).

[0073] Due to the absence of negative allosteric regulation provided by the pyrin domain, the isolated lectin domain of FimH is locked in a high affinity state. Isolated recombinant lectin domains locked in a high affinity state show high stability. However, locking the adhesin in a low binding conformation induces the production of adhesin-inhibiting antibodies. Therefore, there is interest in stabilizing the lectin domain in a low affinity state.

[0074] There is also additional interest in methods to express FimH in high enough yields for product development. An obstacle to developing compositions containing FimH is the low yields achieved with FimH expressed in its native state in the E. coli periplasm. Typical yields reported at lab bench scale are 3-5 mg / L for purified FimCH complex and 4-10 mg / L for FimH(LD), which are below levels deemed scalable for producing clinical trial material. The in vivo conformation of FimH differs from the conformation achieved by purified recombinant forms of the protein. In general, FimH has a native conformation that is determined at least in part by the in vivo interaction of FimH with its periplasmic chaperone protein, called FimC.

[0075] Recombinant production of FimH remains challenging: protein expression and purification are not routine processes.

[0076] In a preferred embodiment, the polypeptide or fragment thereof is derived from E. coli FimH. In some embodiments, the polypeptide or fragment thereof comprises full-length E. coli FimH. Full-length FimH comprises two domains: an N-terminal lectin domain and a C-terminal pyrin domain, which are connected by a short linker. In some embodiments, the full-length E. coli FimH comprises 279 amino acids, including the full-length mature protein of E. coli FimH. In some embodiments, the full-length E. coli FimH comprises 300 amino acids, including the full-length mature protein of E. coli FimH and a signal peptide sequence having a length of 21 amino acids. The primary structure of the 300 amino acid-long wild-type FimH is highly conserved across E. coli strains.

[0077] An exemplary sequence of full-length E. coli FimH is SEQ ID NO:1. The full-length FimH sequence includes a sequence for the lectin domain and a sequence for the pilin domain. The lectin domain of FimH contains the carbohydrate recognition domain responsible for binding to mannosylated uroplakin 1a on the urothelial cell surface. The pilin domain is anchored to the core of the pilus via the donor strand of the subsequent FimG subunit, a process called donor strand complementation.

[0078] Starting from the N-terminus, the names of each domain of full-length FimH and exemplary amino acid sequences in parentheses are as follows: FimH lectin (SEQ ID NO:2) and FimH pilin (SEQ ID NO:3).

[0079] Other suitable polypeptides and fragments thereof derived from E. coli FimH include variants having various degrees of identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29. In certain embodiments, the FimH variant protein (i) forms part of FimH-FimC; (ii) comprises at least one epitope from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29; and / or (iii) is capable of eliciting antibodies in vivo that immunologically cross-react with E. coli FimH.

[0080] In some embodiments, the composition comprises a polypeptide having at least n contiguous amino acids from any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). Preferably, the fragment comprises an epitope from those sequences. In some embodiments, the composition comprises a polypeptide having at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of any one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29.

[0081] In some embodiments, a composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:1. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:2. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:20.In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:23. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:28. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:30.

[0082] Another example of a suitable polypeptide and fragments thereof derived from E. coli FimH as described herein is shown as SEQ ID NO:2, which lacks the wild-type N-terminal signal sequence and corresponds to amino acid residues 22 to 300 of SEQ ID NO:1. Another example of a FimH fragment includes the entire N-terminal signal sequence and the mature protein, such as that set forth in SEQ ID NO:1.

[0083] In some embodiments, the glycosylation site in the polypeptide or fragment thereof derived from E. coli is removed by mutation in the sequence of the polypeptide or fragment thereof derived from E. coli. For example, in some embodiments, the Asn residue at position 7 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by substitution. In some embodiments, the Asn residue at position 7 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

[0084] In some embodiments, the Thr residue at position 10 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by substitution. In some embodiments, the Thr residue at position 7 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and Gln.

[0085] In some embodiments, the Asn residue at position N235 (e.g., according to the numbering of SEQ ID NO:2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the Asn residue at position N228 (e.g., according to the numbering of SEQ ID NO:2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

[0086] In some embodiments, the Asn residue at position 70 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 2) may be mutated, preferably by substitution. In some embodiments, the Asn residue at position 70 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO: 3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

[0087] In some embodiments, the Ser residue at position 72 of the mature E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:2) may be mutated, preferably by substitution. In some embodiments, the Ser residue at position 72 of the lectin domain of the E. coli FimH polypeptide (e.g., according to the numbering of SEQ ID NO:3) may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Asp, Thr, and Gln.

[0088] The term "fragment" as used herein refers to a polypeptide and is defined as any distinct portion of a given polypeptide that is unique or characteristic of that polypeptide. The term as used herein also refers to any distinct portion of a given polypeptide that retains at least a fraction of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity that is retained is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity that is retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity that is retained is at least 95%, 96%, 97%, 98% or 99% of the activity of the full-length polypeptide. In certain embodiments, the fraction of activity that is retained is 100% or more of the activity of the full-length polypeptide. In some embodiments, fragments comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more contiguous amino acids of the full-length polypeptide.

[0089] B. Complex of FimH, FimC, and their fragments In some embodiments, the polypeptide or fragment thereof derived from E. coli FimH is present in a complex with a polypeptide or fragment thereof derived from E. coli FimC. In a preferred embodiment, the polypeptide or fragment thereof derived from E. coli FimH and the polypeptide or fragment thereof derived from E. coli FimC are present in a complex, preferably in a 1:1 ratio in the complex. Without being bound by theory or mechanism, full-length FimH may be stabilized in an active conformation by the periplasmic chaperone FimC, thereby allowing full-length FimH protein to be purified. Thus, in some embodiments, the polypeptide or fragment thereof comprises full-length FimH and full-length FimC.

[0090] In some embodiments, the polypeptide or fragment thereof comprises a fragment of FimH and a fragment of FimC. In some embodiments, the polypeptide or fragment thereof comprises full-length FimH and a fragment of FimC. An exemplary sequence of E. coli FimC is set forth in SEQ ID NO: 10. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a complex-forming fragment of FimH.

[0091] A complexing fragment of FimH may be any part or portion of the FimH protein that retains the ability to form a complex with FimC or a fragment thereof. A suitable complexing fragment of FimH may be obtained or determined by standard assays known in the art, such as co-immunoprecipitation assays, cross-linking, or co-localization by fluorescent staining. SDS-PAGE or Western blots may also be used (e.g., by showing that the FimH fragment and FimC or a fragment thereof are in a complex as evidenced by gel electrophoresis). In certain embodiments, a complexing fragment of FimH (i) forms part of a FimH-FimC complex; (ii) comprises at least one epitope from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29; and / or (iii) is capable of eliciting antibodies that immunologically cross-react with E. coli FimH in vivo.

[0092] In some embodiments, the E. coli derived polypeptide or fragment thereof comprises full length FimH that is not complexed with FimC. In further embodiments, the polypeptide or fragment thereof comprises a fragment of FimH that is not complexed with FimC. In some embodiments, the E. coli derived polypeptide or fragment thereof FimC comprises SEQ ID NO: 10. In some embodiments, the complexes may be expressed from the same plasmid, preferably under the control of separate promoters for each polypeptide or fragment thereof.

[0093] In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH binds to a polypeptide or fragment thereof derived from E. coli FimC that may be engineered into the structure of the polypeptide or fragment thereof derived from E. coli FimH. The portion of the FimC molecule that is bound to FimH in the complex is referred to as the "donor strand," and the mechanism of formation of the native FimH structure using the strand from FimC that is bound to FimH in the FimCH complex is known as "donor strand complementation."

[0094] In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH may be expressed with an appropriate donor strand complemented version of FimH, where the amino acid sequence of FimC that interacts with FimH in the FimCH complex is itself engineered at the C-terminus of FimH to provide a native conformation that does not require the presence of the remainder of the FimC molecule. In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH may be expressed in the form of a complex comprising isolated domains thereof, such as a lectin binding domain and a piling domain, and such domains may be covalently or non-covalently linked to each other. For example, in some embodiments, the binding segment may comprise an amino acid sequence or other oligomeric structure, including a single polymeric structure.

[0095] The methods and compositions of the present invention may include a complex as described herein co-expressed or formed in combination with said polypeptides or fragments thereof derived from E. coli.

[0096] C. Lectin domain, pyrin domain, and their variants The conformation and ligand-binding properties of the lectin domain of FimH may be under allosteric control of the pyrin domain of FimH. Under static conditions, the interaction of the two domains of full-length FimH binds the lectin domain to a low affinity (e.g., K) binding domain characterized by a shallow binding pocket. d Binding to a mannoside ligand can induce a conformational change that results in a moderate affinity state, in which the lectin and pyrin domains remain in close contact. However, upon shear stress, the lectin and pyrin domains separate, thereby transitioning to a high affinity state (e.g., K d <1.2 μM).

[0097] In the absence of negative allosteric regulation provided by the pyrin domain, the isolated lectin domain of FimH is in a high affinity state (e.g., K d <1.2 μM). Isolated recombinant lectin domains locked in a high affinity state. However, adhesins can be locked in low affinity conformations (e.g., K d Locking the lectin domain at a low affinity (≈300 μM) induces the production of adhesin-inhibiting antibodies, so it is of interest to stabilize the lectin domain in a low affinity state.

[0098] In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a lectin domain of E. coli FimH. Exemplary sequences of the lectin domain include any one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:26. In some embodiments, the lectin domain of E. coli FimH comprises a cysteine ​​substitution. In a preferred embodiment, the lectin domain of E. coli FimH comprises a cysteine ​​substitution within the first 50 amino acid residues of the lectin domain. In some embodiments, the lectin domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine ​​substitutions. Preferably, the lectin domain comprises two cysteine ​​substitutions. See, for example, pSB02158 and pSB02198.

[0099] Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to the sequence set forth in SEQ ID NO:3, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 3. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises the pyrin domain of E. coli FimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants having various degrees of identity to the sequence set forth in SEQ ID NO:7, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO:7. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:4.Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants having various degrees of identity to the sequence set forth in SEQ ID NO:8, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO:8. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO: 8. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises the pyrin domain of E. coli FimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants having various degrees of identity to the sequence set forth in SEQ ID NO:24, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:24.Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to the sequence set forth in SEQ ID NO:26, such as at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to the sequence set forth in SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO:26.

[0100] In some embodiments, the composition comprises a polypeptide having at least n contiguous amino acids from any one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:26, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). Preferably, the fragment comprises an epitope from the sequence. In some embodiments, the composition comprises a polypeptide having at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of any one of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:26.

[0101] The location and length of the lectin domain of E. coli FimH or a homolog or variant thereof can be predicted based on a pairwise alignment of its sequence with any one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:26, for example, by aligning the amino acid sequence of FimH with SEQ ID NO:1 and identifying a sequence that aligns with residues 22 to 179 of SEQ ID NO:1.

[0102] D. Wild-type N-terminal signal sequence In some embodiments, the N-terminal wild-type signal sequence of full-length FimH is cleaved in the host cell to produce a mature FimH polypeptide. Thus, the FimH expressed by the host cell may not contain an N-terminal signal sequence. In a preferred embodiment, the polypeptide or fragment thereof derived from E. coli may be encoded by a nucleotide sequence that does not contain a coding sequence for the wild-type N-terminal signal sequence.

[0103] In some embodiments, the E. coli derived polypeptide or fragment thereof comprises a FimH-FimC complex-forming fragment of FimH, an N-terminal signal sequence (such as residues 1-21 of SEQ ID NO: 1), or a combination thereof. The complex-forming fragment of FimH can be any part or portion of the FimH protein that retains the ability to form a complex with FimC.

[0104] In some embodiments, the E. coli derived polypeptide or fragment thereof may lack between 1 and 21 amino acid residues at the N-terminus and / or C-terminus of the full-length FimH polypeptide, which may include the signal sequence, the lectin domain, and the pyrin domain (e.g., may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues, or 1-21 residues, 1-20 residues, 1-15 residues, 1-10 residues, 2-20 residues, 2-15 residues, 2-10 residues, 5-20 residues, 5-15 residues, or 5-10 residues).

[0105] II. Nucleic acids In one embodiment, a nucleic acid encoding a polypeptide or a fragment thereof derived from E. coli is disclosed. One or more nucleic acid constructs encoding a polypeptide or a fragment thereof derived from E. coli can be used for genomic integration and subsequent expression of the polypeptide or a fragment thereof derived from E. coli. For example, a single nucleic acid construct encoding a polypeptide or a fragment thereof derived from E. coli can be introduced into a host cell. Alternatively, the coding sequence for a polypeptide or a fragment thereof derived from E. coli can be carried by two or more nucleic acid constructs, which are then introduced into a host cell simultaneously or sequentially.

[0106] For example, in one exemplary embodiment, a single nucleic acid construct encodes the lectin domain and the pyrin domain of E. coli FimH. In another exemplary embodiment, one nucleic acid construct encodes the lectin domain of E. coli FimH and a second nucleic acid construct encodes the pyrin domain. In some embodiments, genomic integration is achieved.

[0107] The nucleic acid construct may comprise genomic DNA or cDNA containing one or more introns. Some genes are more efficiently expressed when introns are present. In some embodiments, the nucleic acid sequence is suitable for expressing an exogenous polypeptide in said mammalian cell.

[0108] In some embodiments, the nucleic acid encoding the polypeptide or fragment thereof is codon optimized to increase the level of expression in any particular cell.

[0109] In some embodiments, the nucleic acid construct comprises a signal sequence encoding a peptide that directs secretion of the E. coli derived polypeptide or fragment thereof. In some embodiments, the nucleic acid comprises the native signal sequence of the E. coli derived polypeptide FimH. In some embodiments, the E. coli derived polypeptide or fragment thereof comprises an endogenous signal sequence, the nucleic acid sequence encoding the signal sequence can be codon optimized to increase the level of expression of the protein in the host cell.

[0110] In some embodiments, the signal sequence is any one of the following lengths: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 amino acids in length. In some embodiments, the signal sequence is 20 amino acids in length. In some embodiments, the signal sequence is 21 amino acids in length.

[0111] In some embodiments, when a polypeptide or fragment thereof includes a signal sequence, the endogenous signal sequence naturally associated with the polypeptide can be replaced with a signal sequence not associated with the wild-type polypeptide in order to improve the level of expression of the polypeptide or fragment thereof in cultured cells. Thus, in some embodiments, the nucleic acid does not include the native signal sequence of the polypeptide or fragment thereof derived from E. coli. In some embodiments, the nucleic acid does not include the native signal sequence of the polypeptide derived from E. coli FimH. In some embodiments, the polypeptide or fragment thereof derived from E. coli can be expressed with a heterologous peptide, preferably a signal sequence, or other peptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide derived from E. coli or fragment thereof. For example, the polypeptide or fragment thereof derived from E. coli FimH can be expressed with a heterologous peptide, preferably a signal sequence (e.g., IgK signal sequence), or other peptide having a specific cleavage site at the N-terminus of the mature E. coli FimH protein. In a preferred embodiment, the specific cleavage at the N-terminus of the mature E. coli FimH protein occurs immediately before the first phenylalanine residue of the mature E. coli FimH protein. The heterologous sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.

[0112] In preferred embodiments, the signal sequence is an IgK signal sequence. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 22. In preferred embodiments, the signal sequence is a mouse IgK signal sequence.

[0113] Suitable mammalian expression vectors for producing polypeptides or fragments thereof from E. coli are known in the art and may be commercially available, such as the pSecTag2 expression vector from Invitrogen™. An ​​exemplary mouse Ig kappa signal peptide sequence comprises the sequence ETDTLLLWVLLLWVPGSTG (SEQ ID NO:54). In some embodiments, the vector comprises the pBudCE4.1 mammalian expression vector from Thermo Fisher. Additional exemplary suitable vectors include the pcDNA™ 3.1 mammalian expression vector (Thermo Fisher).

[0114] In some embodiments, the signal sequence does not include a hemagglutinin signal sequence.

[0115] In some embodiments, the nucleic acid comprises a native signal sequence of a polypeptide or fragment thereof derived from E. coli. In some embodiments, the signal sequence is not an IgK signal sequence. In some embodiments, the signal sequence comprises a hemagglutinin signal sequence.

[0116] In one aspect, a vector is disclosed herein that comprises a coding sequence of a polypeptide or a fragment thereof derived from E. coli. Exemplary vectors include plasmids that can replicate autonomously or in mammalian cells. Typical expression vectors include suitable promoters, enhancers, and terminators that are useful for regulating the expression of coding sequences in the expression construct. Vectors may also include selection markers that obtain phenotypic traits (such as resistance to antibiotics such as ampicillin or neomycin) for selecting transformed host cells.

[0117] Suitable promoters are known in the art. Exemplary promoters include, for example, CMV promoter, adenovirus, EF1a, GAPDH metallothionine promoter, SV40 early promoter, SV40 late promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, etc. The promoter may be a constitutive or inducible promoter. One or more vectors may be used (e.g., one vector encoding all subunits or domains or fragments thereof, or multiple vectors collectively encoding the subunits or domains or fragments thereof).

[0118] Internal ribosome entry sites (IRES) and 2A peptide sequences can also be used. IRES and 2A peptides provide an alternative approach for co-expression of multiple sequences. IRES is a nucleotide sequence that allows translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the larger process of protein synthesis. Normally, in eukaryotes, translation can only be initiated at the 5' end of an mRNA molecule. IRES elements allow the expression of multiple genes in one transcript. IRES-based polycistronic vectors that express multiple proteins from one transcript can reduce leakage of non-expressing clones from selection. 2A peptides allow multiple proteins in one open reading frame to be translated into a polyprotein that is subsequently cleaved into individual proteins via a ribosome-skipping mechanism. 2A peptides can provide a more balanced expression of multiple protein products. Exemplary IRES sequences include, for example, EV71 IRES, EMCV IRES, HCV IRES. With respect to genome integration, integration can be site-specific or random. Site-specific recombination can be achieved by introducing a homologous sequence into the nucleic acid construct described herein. Such a homologous sequence substantially matches an endogenous sequence at a specific target site in the host genome. Alternatively, random integration can be used. Sometimes, the expression level of a protein can vary depending on the integration site. Therefore, it may be desirable to select multiple clones according to the expression level of the recombinant protein to identify the clone that achieves the desired expression level.

[0119] Exemplary nucleic acid constructs are further described in the figures, such as any one of Figures 2A-2T.

[0120] In one aspect, the nucleic acid sequence encodes an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0121] III.Host cells In one aspect, the invention relates to a cell in which a sequence encoding a polypeptide or fragment thereof derived from E. coli is expressed in a mammalian host cell. In one embodiment, the polypeptide or fragment thereof derived from E. coli is transiently expressed in the host cell. In another embodiment, the polypeptide or fragment thereof derived from E. coli is stably integrated into the genome of the host cell, which expresses the polypeptide or fragment thereof derived from E. coli when cultured under suitable conditions. In a preferred embodiment, the polynucleotide sequence is expressed with high efficiency and genome stability.

[0122] Suitable mammalian host cells are known in the art. Preferably, the host cells are suitable for producing proteins on an industrial manufacturing scale. Exemplary mammalian host cells include any one of the following and their derivatives: Chinese hamster ovary (CHO) cells, COS cells (a cell line derived from monkey kidney (African green monkey), Vero cells, Hela cells, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, NSO cells (a mouse myeloma cell line), and C127 cells (a non-tumorigenic mouse cell line). Further exemplary mammalian host cells include mouse Sertoli (TM4), buffalo rat liver (BRL 3A), mouse mammary tumor (MMT), rat hepatoma (HTC), mouse myeloma (NSO), mouse hybridoma (Sp2 / 0), mouse thymoma (EL4), Chinese hamster ovary (CHO) and CHO cell derivatives, mouse embryonic (NIH / 3T3, 3T3 Li), rat cardiac muscle (H9c2), mouse myoblasts (C2C12), and mouse kidney (miMCD-3). Further examples of mammalian cell lines include NS0 / 1, Sp2 / 0, Hep G2, PER.C6, COS-7, TM4, CV1, VERO-76, MDCK, BRL 3A, W138, MMT 060562, TR1, MRC5, and FS4.

[0123] Any cell capable of cell culture can be used in accordance with the present invention, hi some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblastoma cells (PER.C6, Cru cells, Leiden, The Netherlands); monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the cells are CHO cells. In some preferred embodiments, the cells are GS cells.

[0124] In addition, any number of commercially available and non-commercially available hybridoma cell lines can be used according to the present invention. The term "hybridoma" as used herein refers to a cell or progeny cell resulting from the fusion of an immortalized cell and an antibody-producing cell. The hybridoma thus obtained is an immortalized cell that produces antibodies. The individual cells used to make hybridomas can be from any mammalian source, including, but not limited to, rat, pig, rabbit, sheep, pig, goat, and human. In some embodiments, hybridomas are trioma cell lines that arise when the progeny of a heterohybrid myeloma fusion, which is the product of the fusion between a human cell and a mouse myeloma cell line, is then fused with a plasma cell. In some embodiments, hybridomas are any immortalized hybrid cell line that produces antibodies, such as quadromas (see, for example, Milstein et al., Nature, 537:3053, 1983). One skilled in the art will know that hybridoma cell lines may have different nutritional requirements and / or require different culture conditions for optimal growth and will be able to modify conditions as necessary.

[0125] In some embodiments, the cell comprises a first gene of interest, wherein the first gene of interest is chromosomally integrated. In some embodiments, the first gene of interest comprises a reporter gene, a selection gene, a gene of interest (e.g., encoding a polypeptide or a fragment thereof derived from E. coli), a dependent gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a difficult-to-express (DtE) protein.

[0126] In some embodiments, the first gene of interest is located between two distinct recombination target sites (RTS) in a site-specific integration (SSI) mammalian cell, where the two RTSs are chromosomally integrated within the NL1 locus or the NL2 locus. For example, see US Patent Application No. 20200002727 for a description of the NL1 locus, NL2 locus, NL3 locus, NL4 locus, NL5 locus, and NL6 locus. In some embodiments, the first gene of interest is located within the NL1 locus. In some embodiments, the cell comprises a second gene of interest, where the second gene of interest is chromosomally integrated. In some embodiments, the second gene of interest comprises a reporter gene, a selection gene, a gene of therapeutic interest (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a DtE protein. In some embodiments, the second gene of interest is located between two RTSs. In some embodiments, the second gene of interest is located within the NL1 locus or the NL2 locus. In some embodiments, the first gene of interest is located within the NL1 locus and the second gene of interest is located within the NL2 locus. In some embodiments, the cell comprises a third gene of interest, wherein the third gene of interest is chromosomally integrated. In some embodiments, the third gene of interest comprises a reporter gene, a selection gene, a gene of therapeutic interest (such as a polypeptide or fragment thereof from E. coli), a dependent gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a DtE protein. In some embodiments, the third gene of interest is located between two RTSs. In some embodiments, the third gene of interest is located within the NL1 locus or the NL2 locus. In some embodiments, the third gene of interest is located within a locus separate from the NL1 locus and the NL2 locus. In some embodiments, the first gene of interest, the second gene of interest, and the third gene of interest are in three separate loci.In some embodiments, at least one of the first gene of interest, the second gene of interest, and the third gene of interest is in the NL1 locus, and at least one of the first gene of interest, the second gene of interest, and the third gene of interest is in the NL2 locus.In some embodiments, the cell comprises a site-specific recombinase gene.In some embodiments, the site-specific recombinase gene is chromosomally integrated.

[0127] In some embodiments, the disclosure provides a mammalian cell comprising at least four separate RTSs, wherein the cell comprises: (a) at least two separate RTSs chromosomally integrated within the NL1 locus or the NL2 locus; (b) a first gene of interest integrated between the at least two RTSs of (a), the first gene of interest comprising a reporter gene, a gene encoding a DtE protein, a dependent gene, or a combination thereof; and (c) a second gene of interest integrated within a second chromosomal locus separate from the locus of (a), the second gene of interest comprising a reporter gene, a gene encoding a DtE protein (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof. In some embodiments, the disclosure provides a mammalian cell comprising at least four distinct RTSs, wherein the cell comprises: (a) at least two distinct RTSs chromosomally integrated within the Fer1L4 locus; (b) at least two distinct RTSs chromosomally integrated within the NL1 locus or the NL2 locus; (c) a first gene of interest chromosomally integrated within the Fer1L4 locus, the first gene of interest comprising a reporter gene, a gene encoding a DtE protein, a dependent gene, or a combination thereof; and (d) a second gene of interest chromosomally integrated within the NL1 locus or the NL2 locus of (b), the second gene of interest comprising a reporter gene, a gene encoding a DtE protein (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof.

[0128] In some embodiments, the disclosure provides a mammalian cell comprising at least six distinct RTSs, wherein the cell comprises: (a) at least two distinct RTSs and a first gene of interest that is chromosomally integrated within the Fer1L4 locus; (b) at least two distinct RTSs and a second gene of interest that is chromosomally integrated within the NL1 locus; and (c) at least two distinct RTSs and a third gene of interest that is chromosomally integrated within the NL2 locus.

[0129] As referred to herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The terms also refer to the linking of amino acid sequences in such a manner that a functional protein is produced. In some embodiments, a gene of interest is operably linked to a promoter, where the gene of interest is chromosomally integrated into the host cell. In some embodiments, a gene of interest is operably linked to a heterologous promoter; where the gene of interest is chromosomally integrated into the host cell. In some embodiments, a dependent gene is operably linked to a promoter, where the dependent gene is chromosomally integrated into the host cell genome. In some embodiments, a dependent gene is operably linked to a heterologous promoter; where the dependent gene is chromosomally integrated into the host cell genome. In some embodiments, a gene encoding a DtE protein is operably linked to a promoter, where the gene encoding a DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the gene encoding the DtE protein is operably linked to a heterologous promoter, and the gene encoding the DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a promoter, and the recombinase gene is chromosomally integrated into the host cell. In some embodiments, the recombinase gene is operably linked to a promoter, and the recombinase gene is chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, and the recombinase gene is not chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, and the recombinase gene is not chromosomally integrated into the host cell genome.

[0130] As referred to herein, the term "chromosomally integrated" or "chromosomal integration" refers to the stable integration of a nucleic acid sequence into the chromosome of a host cell, e.g., a mammalian cell, i.e., the nucleic acid sequence is chromosomally integrated into the genomic DNA (gDNA) of a host cell, e.g., a mammalian cell. In some embodiments, the chromosomally integrated nucleic acid sequence is stable. In some embodiments, the chromosomally integrated nucleic acid sequence is not located on a plasmid or vector. In some embodiments, the chromosomally integrated nucleic acid sequence is not excised. In some embodiments, the chromosomal integration is mediated by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene editing system (CRISPR / CAS).

[0131] In some embodiments, the host cells are suitable for growth in suspension culture. Suspension-competent host cells are generally monodisperse or grow in loose aggregates without substantial aggregation. Suspension-competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., hematopoietic cells, lymphoid cells) and cells that have been made suspension-competent by modification or adaptation of anchorage-dependent cells (e.g., epithelial cells, fibroblasts).

[0132] In some embodiments, the expression level or activity of a polypeptide derived from E. coli or a fragment thereof is increased by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 90-fold, at least 100-fold compared to expression of a polypeptide derived from E. coli or a fragment thereof in a bacterial cell, such as, for example, an E. coli host cell.

[0133] The host cells described herein are suitable for large-scale cultivation. For example, the cell culture may be 10L, 30L, 50L, 100L, 150L, ​​200L, 300L, 500L, 1000L, 2000L, 3000L, 4000L, 5000L, 10,000L or more. In some embodiments, the cell culture size is 10L-5000L, 10L-10,000L, 10L-20,000L, 10L-50,000L, 40L-50,000L, 100L-50,000L, 500L-50,000L, 1000L-50,000L, 2000L-50,000L, 3000L-50,000L, 4000L-50,000L, 5 ... The volume of the medium may be in the range of 000L to 50,000L, 4500L to 50,000L, 1000L to 10,000L, 1000L to 20,000L, 1000L to 25,000L, 1000L to 30,000L, 15L to 2000L, 40L to 1000L, 100L to 500L, 200L to 400L, or any integer range therebetween. Media components for cell culture are known in the art and may include, for example, a buffer, an amino acid content, a vitamin content, a salt content, an inorganic salt content, a serum content, a carbon source content, a lipid content, a nucleic acid content, a hormone content, a trace element content, an ammonia content, a cofactor content, an indicator content, a small molecule content, a hydrolysate content, and an enzyme regulator content.

[0134] The terms "medium", "cell culture medium" and "culture medium" as used herein refer to a solution containing nutrients that support the growth of mammalian cells. Typically, such solutions provide the essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. Such solutions may contain supplemental components that enhance growth and / or survival beyond the minimal proportions, including, but not limited to, hormones and / or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds that are usually present at very low final concentrations), inorganic compounds (e.g., iron) that are present at high final concentrations, amino acids, lipids, and / or glucose or other energy sources. In some embodiments, the medium is advantageously formulated to an optimal pH and salt concentration for cell survival and growth. In some embodiments, the medium is a feed medium that is added after the initiation of cell culture.

[0135] In some embodiments, the cells can be grown in one of a variety of chemically defined media, where the components of the media are known and controlled. In some embodiments, the cells can be grown in complex media, where not all of the components of the media are known and / or controlled. Chemically defined growth media for culturing mammalian cells have been widely developed and published over the last several decades. All components of the defined media are well characterized, and such defined media do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to allow cell growth and survival with little or no concerns regarding protein production. More recently, media formulations have been developed with the express purpose of supporting the culture of highly proliferative recombinant protein producing cells. Such media are preferred for use in the methods of the invention. Such media generally contain large amounts of nutrients, particularly amino acids, that support the growth and / or maintenance of cells at high density. If necessary, these media can be modified by those skilled in the art for use in the methods of the invention. For example, one skilled in the art can reduce the amount of phenylalanine, tyrosine, tryptophan and / or methionine in these media for use as basal or feed media in the methods as disclosed herein.

[0136] Not all components of a complex medium are well characterized, and such a complex medium may contain additives such as simple and / or complex carbon sources, simple and / or complex nitrogen sources, and serum. In some embodiments, a complex medium suitable for the present invention contains additives such as hydrolysates in addition to other components of a defined medium as described herein. In some embodiments, a defined medium typically contains approximately 50 chemical entities at known concentrations in water. Many of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin, or BSA, while others do not require protein components and are therefore referred to as protein-free defined media. Typical chemical components of media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and a miscellaneous category that cannot be succinctly classified.

[0137] The cell culture medium may be optionally supplemented with supplementary components. The term "supplementary components" as used herein refers to components that enhance growth and / or survival beyond a minimal ratio, including, but not limited to, hormones and / or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In some embodiments, the supplementary components may be added to the initial cell culture. In some embodiments, the supplementary components may be added after the cell culture has been initiated. Typically, trace elements refer to various inorganic salts that are included at micromolar or lower levels. For example, commonly included trace elements are zinc, selenium, copper, etc. In some embodiments, iron (ferrous or ferric salts) may be included as a trace element in the initial cell culture medium at micromolar concentrations. Among the trace elements, manganese is also often added as a divalent cation (MnCl 2 or MnSO 4) in the nanomolar to micromolar range. Many less common trace elements are routinely added in nanomolar concentrations.

[0138] In some embodiments, the medium used in the methods of the present invention can be used to culture cells at a concentration of, for example, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5 x 10 8 It is a medium suitable for supporting high cell densities, such as 10 ...

[0139] In some embodiments, the cell culture medium comprises phenylalanine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises leucine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises serine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises threonine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises two of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and tyrosine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM.In some embodiments, the cell culture medium comprises phenylalanine and tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises three of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine and tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine and methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tryptophan and methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM.In some embodiments, the cell culture medium comprises tyrosine, tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises four of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises five of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises six of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM or between 0.5 and 1 mM.In some embodiments, the cell culture medium further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration of 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably more than 2mM. In some embodiments, the cell culture medium further comprises at least five of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration of 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably more than 2mM. In some embodiments, the cell culture medium further comprises glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration of 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably greater than 2mM. In some embodiments, the cell culture medium further comprises at least one, two, three, four, five, six, seven, eight or nine of valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration of 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably greater than 2mM. In some embodiments, the cell culture medium further comprises at least five of valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at concentrations of 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably more than 2mM. In some embodiments, the cell culture medium further comprises valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at concentrations of 2mM, 3mM, 4mM, 5mM, 10mM, 15mM, preferably more than 2mM. In some embodiments, the cell culture medium further comprises serine at concentrations of 3mM, 5mM, 7mM, 10mM, 15mM or 20mM, preferably more than 10mM.In some embodiments, the cell culture medium comprises valine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably more than 10 mM. In some embodiments, the cell culture medium comprises cysteine ​​at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably more than 10 mM. In some embodiments, the cell culture medium comprises isoleucine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably more than 10 mM. In some embodiments, the cell culture medium comprises leucine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably more than 10 mM. In some embodiments, the above cell culture medium is for use in the method as disclosed herein. In some embodiments, the above cell culture medium is used as a basal medium in the method as disclosed herein. In some embodiments, the above cell culture medium is used as a feed medium in the methods as disclosed herein.

[0140] IV. Production Method In one aspect, the invention includes a method for producing an E. coli derived polypeptide or fragment thereof. The method comprises culturing mammalian cells under suitable conditions, thereby expressing the E. coli derived polypeptide or fragment thereof. The method may further comprise collecting the E. coli derived polypeptide or fragment thereof from the culture. The process may further comprise purifying the E. coli derived polypeptide or fragment thereof.

[0141] In some embodiments, the method produces a yield of the polypeptide or fragment thereof of between 0.1 g / L and 0.5 g / L.

[0142] In some embodiments, the cells can be grown in batch or fed-batch culture, where the culture is stopped after sufficient expression of the polypeptide and the expressed polypeptide is then harvested and optionally purified. In some embodiments, the cells can be grown in perfusion culture, where the culture is not stopped, but rather fresh nutrients and other components are added to the culture periodically or continuously, while the expressed polypeptide is harvested periodically or continuously.

[0143] In some embodiments, the cells can be grown in small scale reaction vessels ranging in volume from a few milliliters to a few liters, hi some embodiments, the cells can be grown in large scale commercial bioreactors ranging in volume from approximately at least 1 liter to 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume therebetween.

[0144] The temperature of the cell culture will be selected primarily based on the range of temperatures at which the cell culture is still viable and high levels of polypeptides are produced, the temperature at which production or accumulation of metabolic waste products is minimized, and / or any combination of these or other factors deemed important by the practitioner. As one non-limiting example, CHO cells grow well and produce high levels of proteins or polypeptides at approximately 37°C. In general, most mammalian cells can grow well and / or produce high levels of proteins or polypeptides within the range of about 25°C to 42°C, but the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells can grow well and / or produce high levels of proteins or polypeptides within the range of about 35°C to 40°C. In certain embodiments, the cell culture is grown one or more times during the cell culture process at a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.

[0145] The terms "culture" and "cell culture" as used herein refer to a population of cells suspended in a medium under conditions suitable for the survival and / or growth of the population of cells. As will be apparent to one of ordinary skill in the art, in some embodiments, these terms as used herein refer to a combination comprising a population of cells and the medium in which the population is suspended. In some embodiments, the cells of the cell culture comprise mammalian cells.

[0146] The present invention can be used with any cell culture method that is applicable to the desired process (e.g., production of a recombinant protein (e.g., an antibody). As a non-limiting example, cells can be grown in batch or fed-batch culture, where the culture is stopped after sufficient expression of the recombinant protein (e.g., an antibody) and the expressed protein (e.g., an antibody) is then harvested. Alternatively, as another non-limiting example, cells can be grown in batch-refeed, where the culture is not stopped but rather new nutrients and other components are added to the culture periodically or continuously while the expressed recombinant protein (e.g., an antibody) is harvested periodically or continuously. Other suitable methods (e.g., spin tube culture) are known in the art and can be used to practice the present invention.

[0147] In some embodiments, the cell culture suitable for the present invention is a fed-batch culture. The term "fed-batch culture" as used herein refers to a method of culturing cells in which additional components are supplied one or more times after the start of the culture process. Such supplied components typically include nutritional components for cells that are depleted during the culture process. Fed-batch culture is typically stopped at multiple time points to collect cells and / or components in the medium and optionally purify them. In some embodiments, fed-batch culture includes a basal medium supplemented with a feeding medium.

[0148] The cells can be grown in any convenient volume selected by the practitioner. For example, the cells can be grown in small scale reaction vessels ranging in volume from a few milliliters to a few liters. Alternatively, the cells can be grown in large scale commercial bioreactors ranging in volume from approximately at least 1 liter to 10, 50, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000, 15000, 20000, or 25000 liters or more, or any volume therebetween.

[0149] The temperature of the cell culture will be selected primarily based on the range of temperatures at which the cell culture is still viable and at which high levels of the desired product (e.g., recombinant protein) are produced. In general, most mammalian cells can grow well and / or produce the desired product (e.g., recombinant protein) within a range of about 25°C to 42°C, although the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells can grow well and / or produce the desired product (e.g., recombinant protein or antibody) within a range of about 35°C to 40°C. In certain embodiments, the cell culture is grown one or more times during the cell culture process at a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. A person of ordinary skill in the art would be able to select a suitable temperature or temperatures to grow the cells at depending on the specific needs of the cells and the specific production requirements of the practitioner. The cells can be grown for any amount of time depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, the cells are grown at 37°C. In some embodiments, the cells are grown at 36.5°C.

[0150] In some embodiments, cells can be grown for a greater or lesser amount of time during the initial growth phase (or growth phase), depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, cells are grown for a sufficient period of time to achieve a predefined cell density. In some embodiments, cells are grown for a sufficient period of time to achieve a cell density that is a given percentage of the maximum cell density that the cells would eventually reach if grown undisturbed. For example, cells can be grown for a sufficient period of time to achieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent of the maximum cell density. In some embodiments, cells are grown until the cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% per day of culture. In some embodiments, the cells are grown until the cell density does not increase by more than 5% per day of culture.

[0151] In some embodiments, the cells are grown for a defined period of time. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells are grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more, preferably 4-10 days. In some cases, the cells can be grown for a month or more. The practitioner of the invention will be able to select the duration of the initial growth phase depending on the protein production requirements and the requirements of the cells themselves.

[0152] The cell culture can be agitated or shaken during the initial cultivation phase to increase oxygenation and distribution of nutrients to the cells. In accordance with the present invention, one skilled in the art will appreciate that it may be advantageous to control or regulate certain internal conditions of the bioreactor during the initial growth phase, including but not limited to pH, temperature, oxygenation, etc.

[0153] At the end of the initial growth phase, a second set of culture conditions can be applied to shift at least one of the culture conditions so that a metabolic shift occurs in the culture. The metabolic shift can be achieved, for example, by changing the temperature, pH, osmolality or chemical induction level of the cell culture. In one non-limiting embodiment, the culture conditions are shifted by shifting the temperature of the culture. However, as known in the art, temperature shift is not the only mechanism by which a suitable metabolic shift can be achieved. For example, such metabolic shift can also be achieved by shifting other culture conditions, including, but not limited to, pH, osmolality, and sodium butyrate level. The timing of the culture shift will be determined by the practitioner of the present invention based on the protein production requirements and the requirements of the cells themselves.

[0154] When the temperature of the culture is shifted, the temperature shift can be relatively gradual. For example, the temperature shift can take several hours or days to complete. Alternatively, the temperature shift can be relatively sudden. For example, the temperature shift can be completed in less than a few hours. Given suitable production and control equipment, such as is standard in commercial large-scale production of polypeptides or proteins, the temperature shift can be completed within less than an hour.

[0155] In some embodiments, once the cell culture conditions have been shifted as discussed above, the cell culture is maintained throughout the subsequent growth phase under a second set of culture conditions that are conducive to the survival and viability of the cell culture and suitable for expression of the desired polypeptide or protein at commercially relevant levels.

[0156] As discussed above, the culture can be shifted by shifting one or more of several culture conditions, including but not limited to temperature, pH, osmolality, and sodium butyrate level. In some embodiments, the temperature of the culture is shifted. In this embodiment, the culture is maintained at a lower temperature or temperature range during the subsequent growth phase than the temperature or temperature range of the initial growth phase. As discussed above, multiple separate temperature shifts can be used to increase cell density or viability, or to increase recombinant protein expression.

[0157] In some embodiments, the cells can be maintained in the subsequent production phase until a desired cell density or production titer is achieved. In another embodiment of the present invention, the cells are grown during the subsequent production phase for a predetermined period of time. For example, the cells can be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more, depending on the concentration of the cell culture at the start of the subsequent growth phase, the temperature at which the cells are grown, and the intrinsic growth rate of the cells. In some cases, the cells can be grown for a month or more. The practitioner of the present invention will be able to select the duration of the subsequent production phase depending on the polypeptide or protein production requirements and the needs of the cells themselves.

[0158] The cell culture can be agitated or shaken during the subsequent production phase to increase oxygenation and distribution of nutrients to the cells. In accordance with the present invention, one skilled in the art will appreciate that it may be advantageous to control or regulate certain internal conditions of the bioreactor during the subsequent growth phase, including but not limited to pH, temperature, oxygenation, etc.

[0159] In some embodiments, the cells express a recombinant protein and the cell culture methods of the invention comprise a growth phase and a production phase.

[0160] In some embodiments, step (ii) of any of the methods disclosed herein is applied during the entire cell culture method. In some embodiments, step (ii) of any of the methods disclosed herein is applied during a portion of the cell culture method. In some embodiments, step (ii) is applied until a predetermined viable cell density is obtained.

[0161] In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase, and step (ii) is applied during the growth phase. In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase, and step (ii) is applied during a portion of the growth phase. In some embodiments, the cell culture method of the invention comprises a growth phase and a production phase, and step (ii) is applied during the growth phase and the production phase.

[0162] In step (ii) of any of the methods disclosed herein, the term "maintaining" may refer to maintaining the concentration of amino acids or metabolites below C1 or C2 during the entire culture process (until harvest), or during a portion of the culture process, such as, for example, the growth phase, a portion of the growth phase, or until a predetermined cell density is obtained.

[0163] In some embodiments of any of the aforementioned methods, cell growth and / or productivity is increased compared to a control culture, where the control culture is identical except that it does not include step (ii).

[0164] In some embodiments of any of the methods described above, the method is a method for enhancing cell growth, hi some embodiments, the method is a method for enhancing cell growth at high cell densities in high density cell culture.

[0165] High cell density, as used herein, is greater than 1 x 10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5 x 10 8More than 1 x 10 cells / mL, preferably 7 More than 5 x 10 cells / mL, more preferably 7 Refers to cell density in excess of cells / mL.

[0166] In some embodiments, the method of the invention is a method for enhancing cell growth in cell culture, wherein the cell density is greater than or equal to 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5 x 10 8 In some embodiments, the method of the invention is a method for enhancing cell growth in cell culture, wherein the maximum cell density is greater than 1×10 cells / mL. 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5 x 10 8 More than cells / mL.

[0167] In some embodiments, cell proliferation is determined by viable cell density (VCD), maximum viable cell density, or integrated viable cell density (IVCC). In some embodiments, cell proliferation is determined by maximum viable cell density.

[0168] The term "viable cell density" as used herein refers to the number of cells present in a given volume of culture medium. Viable cell density can be measured by any method known to those skilled in the art. Preferably, viable cell density is measured using an automated cell counter such as Bioprofile Flex®. The term maximum cell density as used herein refers to the maximum cell density achieved during cell culture. The term "cell viability" as used herein refers to the likelihood that cells in culture will survive under a given set of culture conditions or experimental variations. Those of ordinary skill in the art will recognize that any of a number of methods for determining cell viability are encompassed by the present invention. For example, to determine cell viability, a dye (e.g., trypan blue) can be used that does not pass through the membrane of viable cells but can pass through the disrupted membrane of dead or dying cells.

[0169] The term "integrated viable cell count (IVCC)" as used herein refers to the area under the viable cell density (VCD) curve. IVCC is calculated according to the following formula: IVCC t+1 =IVCC t +(VCD t +VCD t+1 )×(Δt) / 2, where Δt is the time difference between time t and time t+1. t=0 can be assumed to be negligible. t and VCDs t+1 is the viable cell density at time points t and t+1.

[0170] The term "titer" as used herein refers to, for example, the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in grams of protein per liter of medium.

[0171] In some embodiments, cell proliferation is increased by at least 5%, 10%, 15%, 20% or 25% compared to a control culture. In some embodiments, cell proliferation is increased by at least 10% compared to a control culture. In some embodiments, cell proliferation is increased by at least 20% compared to a control culture.

[0172] In some embodiments, the production rate is determined by titer and / or volumetric production rate.

[0173] The term "titer" as used herein refers to, for example, the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in grams of protein per liter of medium.

[0174] In some embodiments, the productivity is determined by titer. In some embodiments, the productivity is increased by at least 5%, 10%, 15%, 20% or 25% compared to a control culture. In some embodiments, the productivity is increased by at least 10% compared to a control culture. In some embodiments, the productivity is increased by at least 20% compared to a control culture.

[0175] In some embodiments, the maximum cell density of the cell culture is 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL or 5 x 10 8 In some embodiments, the maximum cell density of the cell culture is greater than 5×10 6 In some embodiments, the maximum cell density of the cell culture is greater than 1×10 8 Higher than cells / mL.

[0176] V. Purification In some embodiments, the method for purifying an E. coli derived polypeptide or fragment thereof comprises isolating and / or purifying the E. coli derived polypeptide or fragment thereof. In some embodiments, the expressed E. coli derived polypeptide or fragment thereof is secreted into the medium so that cells and other solids can be removed by centrifugation and / or filtration.

[0177] The E. coli derived polypeptide or fragment thereof produced according to the methods described herein can be collected from the host cell and purified using any suitable method. Suitable methods for purifying the polypeptide or fragment thereof include precipitation and various types of chromatography such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion, all of which are known in the art. A suitable purification scheme may include two or more of these or other suitable methods. In some embodiments, one or more of the E. coli derived polypeptide or fragment thereof may include a "tag" that facilitates purification, such as an epitope tag or HIS tag, Strep tag, etc. Such tagged polypeptides can be conveniently purified, for example, from conditioned medium by chelation or affinity chromatography. Optionally, the tag sequence can be cleaved after purification.

[0178] In some embodiments, the polypeptide or fragment thereof derived from E. coli may comprise a tag for affinity purification. Affinity purification tags are known in the art. Examples include, for example, His tags (binds metal ions), antibodies, maltose binding protein (MBP) (binds amylose), glutathione-S-transferase (GST) (binds glutathione), FLAG tags, Strep tags (binds streptavidin or its derivatives).

[0179] In a preferred embodiment, the E. coli derived polypeptide or fragment thereof does not contain a purification tag.

[0180] In some embodiments, the yield of the polypeptide or fragment thereof derived from E. coli is at least about 1 mg / L, at least about 2 mg / L, at least about 3 mg / L, at least about 4 mg / L, at least about 5 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 11 mg / L, at least about 12 mg / L, at least about 13 mg / L, at least about 14 mg / L, at least about 15 mg / L, at least about 16 mg / L, at least about 17 mg / L , at least about 18 mg / L, at least about 19 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, or at least about 100 mg / L.

[0181] In some embodiments, the culture is at least about 10 liters in size, e.g., at least about 10 L, at least about 20 L, at least about 30 L, at least about 40 L, at least about 50 L, at least about 60 L, at least about 70 L, at least about 80 L, at least about 90 L, at least about 100 L, at least about 150 L, at least about 200 L, at least about 250 L, at least about 300 L, at least about 400 L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 800 L, at least about 900 L, at least about 1000 L, at least about 2000 L, at least about 3000 L, at least about 4000 L, At least about 5000 L, at least about 6000 L, at least about 10,000 L, at least about 15,000 L, at least about 20,000 L, at least about 25,000 L, at least about 30,000 L, at least about 35,000 L, at least about 40,000 L, at least about 45,000 L, at least about 50,000 L, at least about 55,000 L, at least about 60,000 L, at least about 65,000 L, at least about 70,000 L, at least about 75,000 L, at least about 80,000 L, at least about 85,000 L, at least about 90,000 L, at least about 95,000 L, at least about 100,000 L, etc.

[0182] VI. Compositions and Formulations In one aspect, the invention includes compositions comprising a polypeptide or fragment thereof derived from E. coli. In some embodiments, the compositions elicit an immune response including antibodies that can confer immunity against pathogenic species of E. coli.

[0183] In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli as the only antigen. In some embodiments, the composition does not comprise a conjugate.

[0184] In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an additional antigen. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an additional E. coli antigen. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and a glycoconjugate from E. coli.

[0185] In some embodiments, the polypeptide or fragment thereof is derived from E. coli FimH.

[0186] In some embodiments, the composition comprises a polypeptide or a fragment thereof derived from E. coli FimC.

[0187] In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli FimH; and a polypeptide or fragment thereof derived from E. coli FimC.

[0188] In one aspect, the invention includes a composition comprising a polypeptide or fragment thereof derived from E. coli FimH; and a saccharide, wherein the saccharide is selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180 / C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, Formula O18C, Formula O18D, Formula O18E, Formula O18F, Formula O18F, Formula O18G, Formula O18H, Formula O18I, Formula O18I, Formula O18J, Formula O18J, Formula O18J, Formula O18K ... , and formula O18B1), formula O19, formula O20, formula O21, formula O22, formula O23 (e.g., formula O23A), formula O24, formula O25 (e.g., formula O25a and formula O25b), formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O38, formula O39, formula O40, formula O41, formula O42, formula O43, formula O44, formula O45 (e.g., formula O45 and formula O45rel), formula O46, formula O48, formula O49, formula O50, formula O51, formula O52, formula O53, formula O54, formula O55, formula O56, formula O57, formula O58, formula O59, formula O60, formula O61, formula O62, formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73 (e.g., formula O73 (strain 73-1)), formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99 , formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O1 15, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O14 7, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O 163, formula O164, formula O165, formula O166, formula O167, formula O168, formula O169, formula O170, formula O171, formula O172, formula O173, formula O174, formula O175, formula O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, and formula O187 (wherein n is an integer of 1 to 100).

[0189] In some embodiments, the composition comprises one or more saccharides that are or are derived from one or more K. pneumoniae serotypes selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12. In some embodiments, the composition comprises saccharides from or derived from one or more of serotypes O1, O2, O3, and O5, or a combination thereof. In some embodiments, the composition comprises saccharides from or derived from each of K. pneumoniae serotypes O1, O2, O3, and O5.

[0190] In some embodiments, the composition further comprises at least one saccharide derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5. In some embodiments, the composition further comprises at least one saccharide derived from K. pneumoniae type O1. In some embodiments, the composition further comprises at least one saccharide derived from K. pneumoniae type O2. In some embodiments, the composition comprises a combination of saccharides derived from any one of K. pneumoniae types selected from the group consisting of O1, O2, O3, and O5. For example, in some embodiments, the composition comprises at least one saccharide derived from K. pneumoniae type O1 and at least one saccharide derived from K. pneumoniae type O2. In a preferred embodiment, a saccharide derived from Klebsiella pneumoniae is conjugated to a carrier protein; a saccharide derived from E. coli is conjugated to a carrier protein.

[0191] In some embodiments, the composition comprises any one of the sugars disclosed herein. In preferred embodiments, the composition comprises any one of the conjugates disclosed herein.

[0192] In some embodiments, the composition comprises at least one glycoconjugate from E. coli serotype O25, preferably serotype O25b. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O1, preferably serotype O1a. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O2. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O6.

[0193] In one embodiment, the composition comprises at least one glycoconjugate selected from any one of the following E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least two glycoconjugates selected from any one of the following E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least three glycoconjugates selected from any one of the following E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises a glycoconjugate from each of the following E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6.

[0194] In a preferred embodiment, the glycoconjugates of any of the above compositions individually comprise a CRM 197 is conjugated to

[0195] Thus, in some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from at least one E. coli serotype. In a preferred embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from more than one E. coli serotype. For example, the composition may comprise O antigens from 12 different serotypes (12v) from two different E. coli serotypes (or "v", valency). In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from three different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from four different E. coli serotypes. In one embodiment, the composition comprises O antigens from five different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from six different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from seven different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from eight different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from nine different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from ten different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen derived from eleven different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 12 different serotypes.In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 13 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 14 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 15 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 16 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 17 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 18 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from 19 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 20 different serotypes.

[0196] Preferably, the number of E. coli saccharides may range from 1 serotype (or "v", valency) to 26 different serotypes (26v). In one embodiment, 1 serotype is present. In one embodiment, 2 different serotypes are present. In one embodiment, 3 different serotypes are present. In one embodiment, 4 different serotypes are present. In one embodiment, 5 different serotypes are present. In one embodiment, 6 different serotypes are present. In one embodiment, 7 different serotypes are present. In one embodiment, 8 different serotypes are present. In one embodiment, 9 different serotypes are present. In one embodiment, 10 different serotypes are present. In one embodiment, 11 different serotypes are present. In one embodiment, 12 different serotypes are present. In one embodiment, 13 different serotypes are present. In one embodiment, 14 different serotypes are present. In one embodiment, 15 different serotypes are present. In one embodiment, 16 different serotypes are present. In one embodiment, 17 different serotypes are present. In one embodiment, 18 different serotypes are present. In one embodiment, 19 different serotypes are present. In one embodiment, there are 20 different serotypes. In one embodiment, there are 21 different serotypes. In one embodiment, there are 22 different serotypes. In one embodiment, there are 23 different serotypes. In one embodiment, there are 24 different serotypes. In one embodiment, there are 25 different serotypes. In one embodiment, there are 26 different serotypes. The saccharide is conjugated to the carrier protein to form a glycoconjugate as described herein.

[0197] In one aspect, the composition comprises a glycoconjugate comprising a polypeptide or fragment thereof derived from E. coli; and an O antigen from at least one E. coli serogroup, the O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from more than one E. coli serotype, each O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from two different E. coli serotypes, each O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and an O antigen from three different E. coli serotypes, each O antigen being conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from four different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from five different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from six different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from seven different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from eight different E. coli serotypes, each O antigen conjugated to a carrier protein.In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 9 different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 10 different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises an O antigen from a polypeptide or fragment thereof derived from E. coli; and 11 different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 12 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 13 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 14 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 15 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 16 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 17 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 18 different serotypes, each of which is conjugated to a carrier protein.In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 19 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and O antigens from 20 different serotypes, each O antigen conjugated to a carrier protein.

[0198] In another aspect, the composition comprises O polysaccharides from at least one E. coli serotype. In a preferred embodiment, the composition comprises O polysaccharides from more than one E. coli serotype. For example, the composition may comprise O polysaccharides from two different E. coli serotypes to 12 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from three different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from four different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from five different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from six different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from seven different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from eight different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 9 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 10 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 11 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes. In one embodiment, the composition comprises O polysaccharides from 13 different serotypes. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes.

[0199] In a preferred embodiment, the composition comprises O polysaccharides from at least one E. coli serotype, with each O polysaccharide conjugated to a carrier protein. In a preferred embodiment, the composition comprises O polysaccharides from more than one E. coli serotype, with each O polysaccharide conjugated to a carrier protein. For example, the composition may comprise O polysaccharides from two different E. coli serotypes to 12 different E. coli serotypes, with each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from three different E. coli serotypes, with each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from four different E. coli serotypes, with each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from five different E. coli serotypes, with each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from six different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from seven different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from eight different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from nine different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from ten different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from eleven different E. coli serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes, each O polysaccharide conjugated to a carrier protein.In one embodiment, the composition comprises O polysaccharides from 13 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes, each of which is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes, each of which is conjugated to a carrier protein.

[0200] In a most preferred embodiment, the composition comprises O polysaccharides from at least one E. coli serotype, where the O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In a preferred embodiment, the composition comprises O polysaccharides from more than one E. coli serotype, where each O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. For example, the composition may comprise O polysaccharides from 12 different E. coli serotypes from two different E. coli serotypes, where each O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from three different E. coli serotypes, where each O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from four different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from five different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from six different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from seven different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from eight different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from nine different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein, the O polysaccharide comprising an O antigen and a core saccharide.In one embodiment, the composition comprises O polysaccharides from 10 different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 11 different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 13 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes, each O polysaccharide being conjugated to a carrier protein, the O polysaccharide comprising an O antigen and a core saccharide. In a preferred embodiment, the carrier protein is a CRM. 197 It is.

[0201] In another preferred embodiment, the composition comprises a CRM comprising a polypeptide or fragment thereof derived from E. coli; and an O polysaccharide comprising the formula O25a, where n is at least 40, and a core saccharide. 197 In a preferred embodiment, the composition comprises an O polysaccharide conjugated to a CRM 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O25b, where n is at least 40, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O1a, where n is at least 40, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O2, where n is at least 40, and a core sugar. 197 and a core saccharide conjugated to the O polysaccharide, the O polysaccharide comprising formula O6, where n is at least 40.

[0202] In another embodiment, the composition comprises a CRM 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O17, where n is at least 40, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O15, where n is at least 40, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O18A, where n is at least 40, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O75, where n is at least 40, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O4, where n is at least 40, and a core sugar. 197In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O16, where n is at least 40, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM comprising the formula O13, where n is at least 40, and a core sugar. 197 and a core saccharide conjugated to the O polysaccharide, the O polysaccharide comprising formula O7, where n is at least 40.

[0203] In another embodiment, the composition comprises a CRM 197 and a core sugar. In another embodiment, the O polysaccharide comprises formula O8, where n is 1-20, preferably 2-5, more preferably 3. Formula O8 is shown, for example, in FIG. 10B. In another embodiment, the composition comprises a CRM 197 and a core saccharide. In another embodiment, the O polysaccharide comprises formula O9, where n is 1-20, preferably 4-8, more preferably 5. Formula O9 is shown, for example, in FIG. 10B. In another embodiment, the O polysaccharide comprises formula O9a, where n is 1-20, preferably 4-8, more preferably 5. Formula O9a is shown, for example, in FIG. 10B.

[0204] In some embodiments, the O polysaccharide is selected from any one of formulas O20ab, O20ac, O52, O97, and O101, where n is 1-20, preferably 4-8, more preferably 5. See, e.g., Figure 10B.

[0205] As mentioned above, the composition may comprise any combination of a polypeptide or fragment thereof derived from E. coli; and a conjugated O polysaccharide (antigen). In one exemplary embodiment, the composition comprises a polysaccharide comprising formula O25b, a polysaccharide comprising formula O1A, a polysaccharide comprising formula O2, and a polysaccharide comprising formula O6. More specifically, such a composition comprises: (i) a CRM 197 (ii) an O polysaccharide conjugated to CRM 197 (iii) an O polysaccharide conjugated to CRM 197 an O polysaccharide conjugated to a CRM comprising the formula O2, where n is at least 40, and a core sugar; and 197 wherein the O polysaccharide comprises a formula O6, where n is at least 40, and a core saccharide.

[0206] In one embodiment, a composition comprises a polypeptide or fragment thereof derived from E. coli; and at least one O polysaccharide derived from any E. coli serotype that is not O25a. For example, in one embodiment, the composition does not include a saccharide comprising the formula O25a. Such a composition may, for example, include an O polysaccharide comprising the formula O25b, an O polysaccharide comprising the formula O1A, an O polysaccharide comprising the formula O2, and an O polysaccharide comprising the formula O6.

[0207] In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli; and the respective O polysaccharide is a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from two different E. coli serotypes, the O polysaccharide comprising an O antigen and a core sugar, conjugated to a CRMP. In one embodiment, the composition comprises an O polysaccharide from an E. coli serotype, the O polysaccharide comprising an O antigen and a core sugar, the CRMP. 197In one embodiment, the composition comprises an O polysaccharide from three different E. coli serotypes, the O polysaccharide comprising an O antigen and a core sugar, the O polysaccharide being conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from four different E. coli serotypes, the O polysaccharide comprising an O antigen and a core sugar, the O polysaccharide being conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from five different E. coli serotypes, the O polysaccharide comprising an O antigen and a core sugar, the O polysaccharide being conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from six different E. coli serotypes, the O polysaccharide comprising an O antigen and a core sugar, conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from seven different E. coli serotypes, the O polysaccharide comprising an O antigen and a core saccharide conjugated to a CRMP. In one embodiment, the composition comprises an O polysaccharide from an E. coli serotype, the O polysaccharide comprising an O antigen and a core saccharide conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from eight different E. coli serotypes, the O polysaccharide comprising an O antigen and a core saccharide, the O polysaccharide being conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from nine different E. coli serotypes, the O polysaccharide comprising an O antigen and a core saccharide conjugated to a CRMP. In one embodiment, the composition comprises an O polysaccharide from an E. coli serotype, the O polysaccharide comprising an O antigen and a core saccharide conjugated to a CRMP. 197In one embodiment, the composition comprises an O polysaccharide from ten different E. coli serotypes, the O polysaccharide comprising an O antigen and a core saccharide conjugated to a CRMP. In one embodiment, the composition comprises an O polysaccharide from an E. coli serotype, the O polysaccharide comprising an O antigen and a core saccharide conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from eleven different E. coli serotypes, the O polysaccharide comprising an O antigen and a core sugar, conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from 12 different serotypes, the O polysaccharide comprising an O antigen and a core saccharide, the O polysaccharide being conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from 13 different serotypes, the O polysaccharide comprising an O antigen and a core saccharide, a polypeptide or fragment thereof from E. coli; 197 In one embodiment, the composition comprises an O polysaccharide from 14 different serotypes, the O polysaccharide comprising an O antigen and a core sugar, a polypeptide or fragment thereof from E. coli; 197 In one embodiment, the composition comprises an O polysaccharide from 15 different serotypes, the O polysaccharide comprising an O antigen and a core saccharide, a polypeptide or fragment thereof from E. coli; 197 In one embodiment, the composition comprises an O polysaccharide from 16 different serotypes, the O polysaccharide comprising an O antigen and a core sugar, a polypeptide or fragment thereof from E. coli; 197 In one embodiment, the composition comprises an O polysaccharide from 17 different serotypes, the O polysaccharide comprising an O antigen and a core sugar, a polypeptide or fragment thereof from E. coli; 197In one embodiment, the composition comprises an O polysaccharide from 18 different serotypes, the O polysaccharide comprising an O antigen and a core saccharide, a polypeptide or fragment thereof from E. coli; 197 In one embodiment, the composition comprises an O polysaccharide from 19 different serotypes, the O polysaccharide comprising an O antigen and a core saccharide, a polypeptide or fragment thereof from E. coli; 197 The O polysaccharides include O antigens and a core sugar conjugated to the O polysaccharide from 20 different serotypes.

[0208] In one aspect, the invention relates to a composition comprising a conjugate comprising a polypeptide or fragment thereof derived from E. coli; and a saccharide comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 15±2. In one aspect, the invention relates to a composition comprising a conjugate comprising a polypeptide or fragment thereof derived from E. coli; and a saccharide comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 17±2. In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a saccharide comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 55±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a saccharide comprising a saccharide covalently attached to a carrier protein comprising formula O25b, where n is 51±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli K12 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

[0209] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding to E. coli serotype O25B polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml or 0.5 pg / ml as determined by ELISA assay. Thus, a comparison of OPA activity of pre-immunization and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O25B to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coli serotype O25B as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coli serotype O25B as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention provide a method for the production of immunogenic antibodies that are able to inhibit or inhibit the proliferation of E. coli serotype O25B-responsive antibodies compared to a pre-immune population (i.e., in vitro In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O25B in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coli serotype O25B in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 against E. coli serotype O25B) compared to a pre-immune population ... In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O25B as compared to a pre-immune population.

[0210] In one aspect, the invention relates to a composition comprising a conjugate comprising a polypeptide or fragment thereof derived from E. coli; and a saccharide covalently attached to a carrier protein, the saccharide comprising formula O1a, where n is 39±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O1a, where n is 13±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

[0211] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding to E. coli serotype O1A polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml or 0.5 pg / ml as determined by ELISA assay. Thus, a comparison of OPA activity of pre-immunization and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O1A to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coli serotype O1A as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coli serotype O1A as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention provide a method for the production of immunogenic antibodies that are able to inhibit the proliferation and proliferation of E. coli serotype O1A-responsive individuals compared to a pre-immune population (i.e., in vitro In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O1A in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coli serotype O1A in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 against E. coli serotype O1A) compared to a pre-immune population ... In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O1A as compared to a pre-immune population.

[0212] In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 43±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 47±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 17±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O2, where n is 18±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli R4 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM197 In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

[0213] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding to E. coli serotype O2 polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml or 0.5 pg / ml as determined by ELISA assay. Thus, a comparison of OPA activity of pre-immunization and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O2 to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coli serotype O2 as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coli serotype O2 as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention provide a method for the production of immunogenic antibodies that inhibit the expression of E. coli serotype O2 in responders (i.e., in vitro In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O2 in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coli serotype O2 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 against E. coli serotype O2) compared to a pre-immune population ... In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O2 as compared to a pre-immune population.

[0214] In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O6, where n is 42±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli; and a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O6, where n is 50±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O6, where n is 17±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, the saccharide comprising formula O6, where n is 18±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent.

[0215] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of binding to E. coli serotype O6 polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml or 0.5 pg / ml as determined by ELISA assay. Thus, a comparison of OPA activity of pre-immunization and post-immunization sera with the immunogenic composition of the invention can be performed and compared for their response to serotype O6 to assess the possibility of increased responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, said antibodies being capable of killing E. coli serotype O6 as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, said antibodies being capable of killing E. coli serotype O6 as determined by in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention provide a method for the production of immunogenic antibodies that inhibit the expression of E. coli serotype O6 in responders (i.e., in vitro In one embodiment, the immunogenic composition elicits a titer of at least 1:8 against E. coli serotype O6 in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention elicits a titer of at least 1:8 against E. coli serotype O6 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 against E. coli serotype O6) compared to a pre-immune population ... In one embodiment, the immunogenic composition of the invention significantly increases the proportion of human subjects with sera having a titer of at least 1:8 as determined by OPA. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of human subjects against E. coli serotype O6 as compared to a pre-immune population.

[0216] In one aspect, a composition comprises a conjugate comprising a polypeptide or fragment thereof derived from E. coli; and a sugar covalently attached to a carrier protein, wherein the sugar is selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180 / C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O24, Formula O25, Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O31, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45, Formula O46, Formula O47, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O63, Formula O64, Formula O65, Formula O66, Formula O67, Formula O68, Formula O69, Formula O70, Formula O71, Formula O7 A1, formula O18B, and formula O18B1), formula O19, formula O20, formula O21, formula O22, formula O23 (e.g., formula O23A), formula O24, formula O25 (e.g., formula O25a and formula O25b), formula O26, formula O27, formula O28, formula O29, formula O30, formula O32, formula O33, formula O34, formula O35, formula O36, formula O37, formula O 38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D 1, formula O63, formula O64, formula O65, formula O66, formula O68, formula O69, formula O70, formula O71, formula O73 (e.g., formula O73 (strain 73-1)), formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99 , formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, formula O114, formula O1 15, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, formula O133, formula O134, formula O135, formula O136, formula O137, formula O138, formula O139, formula O140, formula O141, formula O142, formula O143, formula O144, formula O145, formula O146, formula O147 , formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159, formula O160, formula O161, formula O162, formula O1 63, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187, where n is an integer between 1 and 100. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli R2 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli R3 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli R4 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli K12 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197In one embodiment, the conjugate is prepared by single end-linked conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharma- ceutically acceptable diluent. In one embodiment, the composition further comprises at least 1, 2, 3, 4, 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, to at most 30, additional conjugates, each of which comprises a sugar covalently attached to a carrier protein, the sugar comprising a structure selected from any one of the formulas above.

[0217] A. Sugar In one embodiment, the sugar is produced by expression (not necessarily overexpression) of a different Wzz protein (eg, WzzB) to control the size of the sugar.

[0218] As used herein, the term "sugar" refers to a single sugar moiety or monosaccharide unit and combinations of two or more single sugar moieties or units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. Sugars may be linear or branched.

[0219] In one embodiment, the sugar is produced in a recombinant Gram-negative bacterium. In one embodiment, the sugar is produced in a recombinant E. coli cell. In one embodiment, the sugar is produced in a recombinant Salmonella cell. Exemplary bacteria include E. coli O25K5H1, E. coli BD559, E. coli GAR2831, E. coli GAR865, E. coli GAR868, E. coli GAR869, E. coli GAR872, E. coli GAR878, E. coli GAR896, E. coli GAR1902, E. coli O25a ETC NR-5, E. coli O157:H7:K-, Salmonella enterica Examples of suitable bacteria include E. coli GAR2401, Salmonella enterica serovar Typhimurium strain LT2, E. coli GAR2401, Salmonella enterica serovar Enteritidis CVD1943, Salmonella enterica serovar Typhimurium strain CVD1925, Salmonella enterica serovar Paratyphi A CVD1902, and Shigella flexneri CVD1208S. In one embodiment, the bacterium is not E. coli GAR2401. This genetic approach to sugar production allows for efficient production of O polysaccharide and O antigen molecules as vaccine components.

[0220] As used herein, the term "wzz protein" refers to, for example, wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepEAF011915 for C664-1992, AF011916 for C258-94, AF011917 for C722-89, and AF011919 for EDL933. The GenBank accession numbers for the G7 and Bi316-41 wzz gene sequences are U39305 and U39306, respectively. Additional GenBank accession numbers for exemplary wzz gene sequences are: NP_459581 for Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 FepE; AIG66859 for E. coli O157:H7 strain EDL933 FepE; NP_461024 for Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 WzzB; NP_416531 for E. coli K-12 substrain MG1655 WzzB; and NP_415119 for E. coli K-12 substrain MG1655 FepE. In a preferred embodiment, the wzz family proteins are selected from the group consisting of wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepE , wzz1 and wzz2, most preferably wzzB, and more preferably fepE.

[0221] Exemplary wzzB sequences include those set forth in SEQ ID NOs: 30 to 34. Exemplary FepE sequences include those set forth in SEQ ID NOs: 35 to 39.

[0222] In some embodiments, modified sugars (modified relative to the corresponding wild-type sugars) can be produced in Gram-negative bacteria by expressing (not necessarily overexpressing) a wzz family protein (e.g., fepE) from a Gram-negative bacterium and / or by switching off (i.e., suppressing, deleting, removing) a second wzz gene (e.g., wzzB) to generate high molecular weight sugars, such as lipopolysaccharides, containing intermediate or long O-antigen chains. For example, modified sugars can be produced by expressing (not necessarily overexpressing) wzz2 and switching off wzz1. Or, in another method, modified sugars can be produced by expressing (not necessarily overexpressing) wzzfepE and switching off wzzB. In another embodiment, modified sugars can be produced by expressing (not necessarily overexpressing) wzzB but switching off wzzfepE. In another embodiment, modified sugars can be produced by expressing fepE. Preferably, the wzz family protein is from a strain that is heterologous to the host cell.

[0223] In some embodiments, the sugar is produced by expressing a wzz family protein having an amino acid sequence having at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. In one embodiment, the wzz family protein comprises a sequence selected from any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. Preferably, the wzz family protein has at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. In some embodiments, the sugar is produced by expressing a protein having an amino acid sequence with at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the fepE protein.

[0224] In one aspect, the invention relates to saccharides produced by expressing a wzz family protein, preferably fepE, in a gram-negative bacterium to produce high molecular weight saccharides containing medium or long O antigen chains with at least 1, 2, 3, 4, or 5 increased repeat units compared to the corresponding wild-type O polysaccharide. In one aspect, the invention relates to saccharides produced by gram-negative bacteria expressing (but not necessarily overexpressing) a wzz family protein (e.g., wzzB) in culture to produce high molecular weight saccharides containing short, medium, or long O antigen chains with at least 1, 2, 3, 4, or 5 increased repeat units compared to the corresponding wild-type O antigen from the gram-negative bacterium. For further exemplary saccharides with an increased number of repeat units compared to the corresponding wild-type saccharide, see the description of O polysaccharides and O antigens below. The desired chain length is one that provides improved or maximal immunogenicity in the context of a given vaccine construct.

[0225] In another embodiment, the saccharide comprises any one of the formulas selected from Table 1, wherein the number of repeating units in the saccharide, n, is 1, 2, 3, 4, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 13, 14, , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and more than 100 repeating units. Preferably, the saccharide comprises an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 repeating units compared to the corresponding wild-type O polysaccharide. See, for example, Table 24. Methods for determining the length of a saccharide are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 13.

[0226] In a preferred embodiment, the invention relates to saccharides produced in a recombinant E. coli host cell in which the endogenous wzz O-antigen length regulator gene (e.g., wzzB) has been deleted and replaced with a (second) wzz gene from a Gram-negative bacterium heterologous to the recombinant E. coli host cell (e.g., Salmonella fepE), producing high molecular weight saccharides, such as lipopolysaccharides, containing intermediate or long O-antigen chains. In some embodiments, the recombinant E. coli host cell comprises a wzz gene from Salmonella, preferably Salmonella enterica. In other embodiments, the invention is applicable to all E. coli strains expressing an O-antigen regulated by wzzB. In one aspect, E. coli serotype O8 and O9 strains that produce homopolymeric mannan O antigens are not produced in this embodiment because they use different mechanisms for LPS chain length regulation and transport to the outer membrane (J Biol Chem 2009;284:30662-72; J Biol Chem 2012;287:35078-91; Proceedings of the National Academy of Sciences 2014;111:6407-12). In a further embodiment, homopolymeric galactan polysaccharides of Klebsiella serotypes O1 and O2 are produced by the methods described in this embodiment.

[0227] In one embodiment, the host cell comprises a heterologous gene for a wzz family protein as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for a wzz family protein as an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of a plasmid vector in an E. coli host cell and for integration of a heterologous gene into the chromosome of an E. coli host cell are known in the art. In one embodiment, the host cell comprises a heterologous gene for an O antigen as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for an O antigen as an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of a plasmid vector in E. coli and Salmonella host cells are known in the art. Methods for integration of a heterologous gene into the chromosome of an E. coli and Salmonella host cell are known in the art.

[0228] In one aspect, the recombinant host cell is cultured in a medium that includes a carbon source. Carbon sources for culturing E. coli are known in the art. Exemplary carbon sources include, but are not limited to, sugar alcohols, polyols, aldol sugars, or keto sugars, such as arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvate, succinate, and methylamine. In a preferred embodiment, the medium includes glucose. In some embodiments, the medium includes polyols or aldol sugars, such as mannitol, inositol, sorbose, glycerol, sorbitol, lactose, and arabinose, as carbon sources. All carbon sources may be added to the medium before starting the culture, or may be added in portions or continuously during the culture.

[0229] An exemplary culture medium for recombinant host cells is KH 2 PO4 , K 2 HPO 4 , (NH 4 ) 2 SO 4 , Sodium Citrate, Na 2 SO 4 , aspartic acid, glucose, MgSO 4 , FeSO 4 -7H 2 O, Na 2 MoO 4 -2H 2 O, H 3 BO 3 , CoCl 2 -6H 2 O, CuCl 2 -2H 2 O, MnCl 2 -4H 2 O, ZnCl 2 and CaCl 2 -2H 2 O. Preferably, the medium contains a component selected from KH 2 PO 4 , K 2 HPO 4 , (NH 4 ) 2 SO 4 , Sodium Citrate, Na 2 SO 4 , aspartic acid, glucose, MgSO 4 , FeSO 4 -7H 2 O, NaMoO 4 -2H 2 O, H 3 BO 3 , CoCl 2 -6H 2 O, CuCl 2 -2H 2 O, MnCl 2 -4H 2 O, ZnCl 2 and CaCl 2 -2H 2 Contains O.

[0230] As used herein, a medium may be solid or liquid, synthetic (i.e., artificial) or natural, and may contain sufficient nutrients for the cultivation of recombinant host cells. Preferably, the medium is a liquid medium.

[0231] In some embodiments, the medium may further comprise suitable inorganic salts. In some embodiments, the medium may further comprise micronutrients. In some embodiments, the medium may further comprise growth factors. In some embodiments, the medium may further comprise an additional carbon source. In some embodiments, the medium may further comprise suitable inorganic salts, micronutrients, growth factors, and additional carbon sources. Suitable inorganic salts, micronutrients, growth factors, and supplemental carbon sources for culturing E. coli are known in the art.

[0232] In some embodiments, the medium may optionally contain additional components such as peptone, NZ amine, soybean enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon source and various vitamins. In some embodiments, the medium does not optionally contain such additional components such as peptone, NZ amine, soybean enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon source and various vitamins.

[0233] Examples of suitable supplemental carbon sources include, but are not limited to, other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysates, cellulose hydrolysates, and molasses; organic acids such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols such as glycerol, inositol, mannitol, and sorbitol.

[0234] In some embodiments, the medium further comprises a nitrogen source. Suitable nitrogen sources for culturing E. coli are known in the art. Examples of suitable nitrogen sources include, but are not limited to, ammonia, including ammonia gas and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite, and other nitrogen-containing materials, including amino acids, meat extracts, peptones, fish meal, fish hydrolysates, corn infusions, casein hydrolysates, soybean meal hydrolysates, yeast extracts, dry yeast, ethanol-yeast distillates, soybean flour, cottonseed flour, and the like, in pure or unrefined preparations.

[0235] In some embodiments, the medium comprises inorganic salts. Examples of suitable inorganic salts include, but are not limited to, salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper, molybdenum, tungsten and other trace elements, and phosphate.

[0236] In some embodiments, the medium includes suitable growth factors. Examples of suitable micronutrients, growth factors, etc. include, but are not limited to, coenzyme A, pantothenic acid, pyridoxine-HCl, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, DL-6,8-thioctic acid, folic acid, vitamin B, either as pure or partially purified compounds or present in natural sources. 12 , other vitamins, amino acids such as cysteine ​​and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium thiosulfate, p- or r-aminobenzoic acid, niacinamide, nitriloacetate, etc. Those skilled in the art can empirically determine the amount according to methods and techniques known in the art.

[0237] In another embodiment, the modified sugars described herein (as compared to the corresponding wild-type sugars) are synthetically produced, e.g., in vitro. Synthetic production or synthesis of sugars can facilitate avoidance of costly and time-intensive production processes. In one embodiment, sugars are synthesized, e.g., by using a sequential glycosylation strategy or a combination of a sequential glycosylation strategy and a [3+2] block synthesis strategy from a suitably protected monosaccharide intermediate. For example, thiol glycosides and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, sugars synthesized in vitro have the same structure as sugars produced by recombinant means, such as the manipulation of wzz family proteins described above.

[0238] The sugars produced (by recombinant or synthetic means) can be, for example, from the following E. coli serotypes: O1 (e.g., O1A, O1B, and O1C), O2, O3, O4 (e.g., O4:K52 and O4:K6), O5 (e.g., O5ab and O5ac (180 / C3 strain)), O6 (e.g., O6:K2;K13;K15 and O6:K54), O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18 (e.g., O18A, O18ac, O18A1, O18B, O18C, O18D, O18E, O18F, O18G, O18H, O18I, O18I, O18J ... , and O18B1), O19, O20, O21, O22, O23 (e.g., O23A), O24, O25 (e.g., O25a and O25b), O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45 (e.g., O45 and O45rel), O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, 62D 1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g., O73 (73-1 strain)), O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, 0111, O112, O113, O114, O115, O116 , O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O132, O13 3, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O1 49, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, O163, O164, O and any one of O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

[0239] Individual polysaccharides are typically purified (enriched with respect to the amount of polysaccharide-protein conjugate) by methods known in the art, such as, for example, dialysis, concentration operations, diafiltration operations, tangential flow filtration, sedimentation, elution, centrifugation, sedimentation, ultrafiltration, depth filtration, and / or column chromatography (ion exchange chromatography, multimode ion exchange chromatography, DEAE, and hydrophobic interaction chromatography). Preferably, the polysaccharides are purified by methods that include tangential flow filtration.

[0240] The purified polysaccharides can be activated (e.g., chemically activated) to render them capable of reacting (e.g., directly to a carrier protein or via a linker such as an eTEC spacer) and then incorporated into a glycoconjugate of the invention, as further described herein.

[0241] In one preferred embodiment, the saccharide of the invention is derived from the E. coli serotype O25a. In another preferred embodiment, the serotype is O25b. In another preferred embodiment, the serotype is O1A. In another preferred embodiment, the serotype is O2. In another preferred embodiment, the serotype is O6. In another preferred embodiment, the serotype is O17. In another preferred embodiment, the serotype is O15. In another preferred embodiment, the serotype is O18A. In another preferred embodiment, the serotype is O75. In another preferred embodiment, the serotype is O4. In another preferred embodiment, the serotype is O16. In another preferred embodiment, the serotype is O13. In another preferred embodiment, the serotype is O7. In another preferred embodiment, the serotype is O8. In another preferred embodiment, the serotype is O9.

[0242] As used herein, reference to any of the above serotypes refers to a serotype that includes a repeating unit structure (O unit as described below) that is known in the art and unique to the corresponding serotype. For example, the term "O25a" serotype (also known in the art as serotype "O25") refers to a serotype that includes formula O25 as shown in Table 1. As another example, the term "O25b" serotype refers to a serotype that includes formula O25b as shown in Table 1.

[0243] As used herein, serotypes are generally referred to herein unless otherwise specified, such as, for example, the term "O18" is generally intended to include O18A, O18ac, O18A1, O18B, and O18B1.

[0244] As used herein, the term "O1" generally refers to encompassing species of formulae that include the generic designation "O1" in the formula name set forth in Table 1, such as any one of formulae O1A, O1A1, O1B, and O1C, respectively, as set forth in Table 1. Thus, "O1 serotype" generally refers to serotypes that include any one of formulae O1A, O1A1, O1B, and O1C.

[0245] As used herein, the term "O6" generally refers to species of formulae that include the generic name "O6" in the formula name set forth in Table 1, such as any one of the formulae O6:K2;K13;K15; and O6:K54, respectively, as set forth in Table 1. Thus, "O6 serotype" generally refers to serotypes that include any one of the formulae O6:K2;K13;K15; and O6:K54.

[0246] Other examples of terms generally referring to species of formulae that include a generic term in the formula name listed in Table 1 include "O4", "O5", "O18", and "O45".

[0247] As used herein, the term "O2" refers to formula O2 as shown in Table 1. The term "O2 O antigen" refers to a saccharide that encompasses formula O2 as shown in Table 1.

[0248] As used herein, references to O antigens from the above serotypes refer to the saccharide that includes the formula labeled with the corresponding serotype name. For example, the term "O25B O antigen" refers to the saccharide that includes the formula O25B as shown in Table 1.

[0249] As another example, the term "O1 O antigen" refers generally to saccharides that include formulas that include the term "O1," such as formula O1A, formula O1A1, formula O1B, and formula O1C, each of which is shown in Table 1.

[0250] As another example, the term "O6 O antigen" refers generally to saccharides that include formulas that include the term "O6," such as formula O6:K2; formula O6:K13; formula O6:K15 and formula O6:K54, each of which are shown in Table 1.

[0251] BO polysaccharide As used herein, the term "O polysaccharide" refers to any structure that contains an O antigen, provided that the structure does not contain whole cells or lipid A. For example, in one embodiment, the O polysaccharide comprises lipopolysaccharide that does not have lipid A attached. Steps for removing lipid A are known in the art and include, for example, heat treatment with the addition of acid. An exemplary process includes treatment with 1% acetic acid at 100°C for 90 minutes. This process is combined with a process for isolating the removed lipid A. An exemplary process for isolating lipid A includes ultracentrifugation.

[0252] In one embodiment, O polysaccharide refers to a structure consisting of O antigen, in which case O polysaccharide is synonymous with the term O antigen. In one preferred embodiment, O polysaccharide refers to a structure comprising repeating units of O antigen, without the core saccharide. Thus, in one embodiment, O polysaccharide does not comprise the E. coli R1 core portion. In another embodiment, O polysaccharide does not comprise the E. coli R2 core portion. In another embodiment, O polysaccharide does not comprise the E. coli R3 core portion. In another embodiment, O polysaccharide does not comprise the E. coli R4 core portion. In another embodiment, O polysaccharide does not comprise the E. coli K12 core portion. In another preferred embodiment, O polysaccharide refers to a structure comprising the O antigen and the core saccharide. In another embodiment, O polysaccharide refers to a structure comprising the O antigen, the core saccharide, and the KDO portion.

[0253] Methods for purifying O polysaccharide, including the core oligosaccharide, from LPS are known in the art. For example, after purification of LPS, the purified LPS can be hydrolyzed by heating in 1% (v / v) acetic acid at 100° C. for 90 minutes, followed by ultracentrifugation at 142,000×g for 5 hours at 4° C. The supernatant containing O polysaccharide is lyophilized and stored at 4° C. In certain embodiments, deletion of capsule synthesis genes to allow simple purification of O polysaccharide is described.

[0254] O polysaccharide can be isolated by methods including, but not limited to, mild acid hydrolysis to remove lipid A from LPS. Other embodiments may include the use of hydrazine as an agent for O polysaccharide preparation. Preparation of LPS can be accomplished by methods known in the art.

[0255] In certain embodiments, O polysaccharides purified from wild-type, modified, or attenuated Gram-negative bacterial strains expressing (but not necessarily overexpressing) a Wzz protein (e.g., wzzB) are provided for use in conjugate vaccines. In a preferred embodiment, O polysaccharide chains are purified from Gram-negative bacterial strains expressing (but not necessarily overexpressing) a wzz protein for use as a vaccine antigen as a conjugate or complexed vaccine.

[0256] In one embodiment, the O polysaccharide is about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, The O polysaccharide has a molecular weight that is increased by 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more times. In a preferred embodiment, the O polysaccharide has a molecular weight that is increased by at least 1 fold and at most 5 fold compared to the corresponding wild type O polysaccharide. In another embodiment, the O polysaccharide has a molecular weight that is increased by at least 2-fold and at most 4-fold compared to the corresponding wild-type O polysaccharide. The increase in molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increase in the number of O antigen repeating units. In one embodiment, the increase in molecular weight of the O polysaccharide is due to a wzz family protein.

[0257] In one embodiment, the O polysaccharide has about 1, 2, 3, 4, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 109, 109, 110 The O polysaccharide of the present invention has a molecular weight increased by 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 kDa or more. In one embodiment, the O polysaccharide of the present invention has a molecular weight increased by at least 1 kDa and at most 200 kDa compared to the corresponding wild-type O polysaccharide. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 21 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 22 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 100 kDa, In one embodiment, the molecular weight is increased by at least 15 kDa and at most 100 kDa.In one embodiment, the molecular weight is increased by at least 20 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 90 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 85 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 70 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 60 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 50 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 49 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 48 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 47 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 46 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 45 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 44 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 43 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 42 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 41 kDa.Such an increase in the molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increase in the number of O antigen repeating units. In one embodiment, the increase in the molecular weight of the O polysaccharide is due to a wzz family protein. See, e.g., Table 21.

[0258] In another embodiment, the O polysaccharide comprises any one of the formulas selected from Table 1, wherein the number of repeating units, n, in the O polysaccharide is 1, 2, 3, 4, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 110, 111, 1 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units. Preferably, the saccharide comprises an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 repeating units compared to the corresponding wild-type O polysaccharide. See, e.g., Table 21.

[0259] CO antigen O antigens are part of the lipopolysaccharide (LPS) in the outer membrane of gram-negative bacteria. O antigens are found on the cell surface and are variable cellular components. The variability of O antigens provides the basis for serotyping of gram-negative bacteria. The current Escherichia coli (E. coli) serotyping scheme includes O polysaccharides 1 to 181.

[0260] O antigens comprise oligosaccharide repeating units (O units), whose wild-type structures usually contain 2-8 residues from a wide range of sugars. Exemplary E. coli O antigen O units are shown in Table 1, see also Figures 9A-9C and 10A-10B.

[0261] In one embodiment, the saccharide of the invention may be one oligosaccharide unit. In one embodiment, the saccharide of the invention is one repeating oligosaccharide unit of the relevant serotype. In such an embodiment, the saccharide may comprise a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101.

[0262] In one embodiment, the saccharide of the present invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5-15 repeating units) and are typically derived synthetically or by hydrolysis of a polysaccharide. In such an embodiment, the saccharide may comprise a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101.

[0263] Preferably, all saccharides of the present invention and in the immunogenic compositions of the present invention are polysaccharides. High molecular weight polysaccharides can induce a certain antibody immune response due to the epitopes present on their antigenic surface. Isolation and purification of high molecular weight polysaccharides are preferably contemplated for use in the conjugates, compositions and methods of the present invention.

[0264] In some embodiments, the number of repeating O units in each individual O-antigen polymer (and thus the length and molecular weight of the polymer chain) depends on the wzz chain length regulator, an inner membrane protein. Different wzz proteins result in different ranges of mode lengths (from 4 to over 100 repeat units). The term "mode length" refers to the number of repeating O units. Gram-negative bacteria often have two different Wzz proteins that result in two different OAg mode chain lengths, one long and one short. Expression (not necessarily overexpression) of a wzz family protein (e.g., wzzB) in Gram-negative bacteria can allow for manipulation of O-antigen length to shift or bias bacterial production of a certain length range of O-antigens and enhance production of high yields of high molecular weight lipopolysaccharides. In one embodiment, a "short" mode length as used herein refers to a small number of repeating O units, e.g., 1 to 20. In one embodiment, a "long" mode length as used herein refers to a number of repeating O units greater than 20, up to 40. In one embodiment, a "very long" mode length as used herein refers to greater than 40 repeating O units.

[0265] In one embodiment, the saccharide produced has an increase of at least 10 repeat units, 15 repeat units, 20 repeat units, 25 repeat units, 30 repeat units, 35 repeat units, 40 repeat units, 45 repeat units, 50 repeat units, 55 repeat units, 60 repeat units, 65 repeat units, 70 repeat units, 75 repeat units, 80 repeat units, 85 repeat units, 90 repeat units, 95 repeat units, or 100 repeat units compared to the corresponding wild-type O polysaccharide.

[0266] In another embodiment, the saccharide of the invention has a saccharide sequence that is 1, 2, 3, 4, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 2 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more increase in repeat units. Preferably, the saccharide comprises an increase of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 repeating units compared to the corresponding wild-type O polysaccharide. See, for example, Table 21. Methods for determining the length of a saccharide are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 13.

[0267] Methods for determining the number of repeating units in a saccharide are also known in the art. For example, the number of repeating units (or "n" in the formula) can be calculated by dividing the molecular weight of the polysaccharide (not including the molecular weight of the core sugar or KDO residue) by the molecular weight of the repeating unit (i.e., the molecular weight of the structure in the corresponding formula shown in Table 1, which can theoretically be calculated as the sum of the molecular weights of each monosaccharide in the formula). The molecular weight of each monosaccharide in the formula is known in the art. For example, the molecular weight of the repeating unit of formula O25b is about 862 Da. For example, the molecular weight of the repeating unit of formula O1a is about 845 Da. For example, the molecular weight of the repeating unit of formula O2 is about 829 Da. For example, the molecular weight of the repeating unit of formula O6 is about 893 Da. When determining the number of repeating units in a conjugate, the molecular weight of the carrier protein and the protein:polysaccharide ratio are taken into account. As defined herein, "n" refers to the number of repeating units in a polysaccharide molecule (shown in parentheses in Table 1). As is known in the art, in biopolymers, the repeating structures may incorporate regions of imperfect repeats, e.g., missing branches. Furthermore, it is known in the art that polysaccharides isolated and purified from natural sources, such as bacteria, may be heterogeneous in size and branching. In such cases, n may represent the average or median value for n of the molecules in the population.

[0268] In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild type O polysaccharide. The repeating units of the O antigen are shown in Table 1. In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild type O polysaccharide. The repeating units of the O antigen are shown in Table 1. In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild type O polysaccharide. 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more total repeating units. Preferably, the saccharide has a total of at least 3 and at most 80 repeating units. In another embodiment, the O polysaccharide has a nucleotide sequence similar to that of the corresponding wild-type O polysaccharide, but is more similar to that of the corresponding wild-type O polysaccharide, and is 1, 2, 3, 4, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 109, 110, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more increase in repeat units.

[0269] In one embodiment, the saccharide is selected from the group consisting of O-antigen formulas (such as those shown in Table 1 (see also Figures 9A-9C and Figures 10A-10B)) in which n is at least 1, 2, 3, 4, 5, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and The O antigens include an integer of at most 200, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50. Any minimum value can be combined with any maximum value to define a range. Exemplary ranges include, for example, at least 1 to at most 1000; at least 10 to at most 500; and at least 20 to at most 80, preferably at most 90. In one preferred embodiment, n is at least 31 to at most 90. In a preferred embodiment, n is 40 to 90, more preferably 60 to 85.

[0270] In one embodiment the saccharide comprises an O antigen, wherein n in any one of the O antigen formulas is at least 1 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 5 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 10 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 25 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 50 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 75 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 100 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 125 and at most 200. In one embodiment n in any one of the O antigen formulas is at least 150 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 175 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 1 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 75 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 1 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 20 and at most 75.In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 40 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 90. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 85. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 70. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 60. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 50. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 49. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 48. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 47. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 46. In one embodiment, n in any one of the O antigen formulas is at least 36 and at most 45. In one embodiment, n in any one of the O antigen formulas is at least 37 and at most 44. In one embodiment, n in any one of the O antigen formulas is at least 38 and at most 43. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 42. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 41.

[0271] For example, in one embodiment, n in the sugar is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90, most preferably 40. In another embodiment, n is at least 35 and at most 60. For example, in one embodiment, n is any one of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, preferably 50. In another preferred embodiment, n is at least 55 and at most 75. For example, in one embodiment, n is 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, most preferably 60.

[0272] Sugar structures can be determined by methods and means known in the art, such as, for example, NMR, including 1D, 1H, and / or 13C, 2D TOCSY, DQF-COSY, NOESY, and / or HMQC.

[0273] In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight of between 5 kDa and 400 kDa. In other such embodiments, the saccharide has a molecular weight of between 10 kDa and 400 kDa; 5 kDa and 400 kDa; 5 kDa and 300 kDa; 5 kDa and 200 kDa; 5 kDa and 150 kDa; 10 kDa and 100 kDa; 10 kDa and 75 kDa; 10 kDa and 60 kDa; 10 kDa and 40 kDa; 10 kDa and 100 kDa; 10 kDa and 20 kDa. 00kDa; 15kDa to 150kDa; 12kDa to 120kDa; 12kDa to 75kDa; 12kDa to 50kDa; 12 to 60kDa; 35kDa to 75kDa; 40kDa to 60kDa; 35kDa to 60kDa; 20kDa to 60kDa; 12kDa to 20kDa; or 20kDa to 50kDa. In further embodiments, the polysaccharide has a molecular weight of 7 kDa to 15 kDa; 8 kDa to 16 kDa; 9 kDa to 25 kDa; 10 kDa to 100 kDa; 10 kDa to 60 kDa; 10 kDa to 70 kDa; 10 kDa to 160 kDa; 15 kDa to 600 kDa; 20 kDa to 1000 kDa; 20 kDa to 600 kDa; 20 kDa to 400 kDa; 30 kDa to 1,000 kDa; 30 kDa to 60 kDa; 30 kDa to 50 kDa or 5 kDa to 60 kDa. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0274] As used herein, the term "molecular weight" of a polysaccharide or a carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) in combination with a multi-angle laser light scattering detector (MALLS).

[0275] Polysaccharides may become slightly reduced in size during normal purification procedures. Additionally, polysaccharides may be subjected to sizing techniques prior to conjugation, as described herein. Mechanical or chemical sizing may be used. Chemical hydrolysis may be performed using acetic acid. Mechanical sizing may be performed using high pressure homogenization shear. The molecular weight ranges above refer to purified polysaccharides prior to conjugation (e.g., prior to activation).

[0276] [Table 1-1]

[0277] [Table 1-2]

[0278] [Table 1-3]

[0279] [Table 1-4]

[0280] [Table 1-5]

[0281] [Table 1-6]

[0282] [Table 1-7]

[0283] [Table 1-8]

[0284] [Table 1-9]

[0285] D. Core Oligosaccharides The core oligosaccharide is located between lipid A and the O-antigen outer region in wild-type E. coli LPS. More specifically, the core oligosaccharide is the portion of the polysaccharide that contains the bond between the O-antigen and lipid A in wild-type E. coli. This bond includes a ketosidic bond between the hemiketal function of the innermost 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue and the hydroxyl group of the GlcNAc residue of lipid A. The core oligosaccharide region shows a high degree of similarity among wild-type E. coli strains. It usually contains a limited number of sugars. The core oligosaccharide includes an inner core region and an outer core region.

[0286] More specifically, the inner core is composed primarily of L-glycero-D-manno-heptose (heptose) and KDO residues. The inner core is highly conserved. The KDO residue has the following formula KDO:

[0287] [ka] Includes.

[0288] The outer region of the core oligosaccharide shows greater variation than the inner core region, and differences in this region distinguish the five chemotypes in E. coli: R1, R2, R3, R4, and K-12. See Figure 24, which illustrates the general structure of the carbohydrate backbone of the outer core oligosaccharide of the five known chemotypes. HepII is the last residue of the inner core oligosaccharide. All outer core oligosaccharides contain (hexose) 3 They share a structural theme, with a carbohydrate backbone and two side chain residues, but the order of the hexoses in the backbone and the nature, position, and linkage of the side chain residues can all vary. The structures of the R1 and R4 outer core oligosaccharides are very similar, differing only in a single β-linked residue.

[0289] The core oligosaccharide of wild-type E. coli is classified in the art into five different chemotypes based on the structure of the distal oligosaccharide: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12.

[0290] In a preferred embodiment, the compositions described herein comprise a saccharide conjugate comprising a core oligosaccharide in which the O polysaccharide is bound to an O antigen. In one embodiment, the composition induces an immune response against at least one of the core E. coli chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12. In another embodiment, the composition induces an immune response against at least two core E. coli chemotypes. In another embodiment, the composition induces an immune response against at least three core E. coli chemotypes. In another embodiment, the composition induces an immune response against at least four core E. coli chemotypes. In another embodiment, the composition induces an immune response against all five core E. coli chemotypes.

[0291] In another preferred embodiment, the compositions described herein comprise saccharide conjugates in which the O polysaccharide does not comprise a core oligosaccharide bound to an O antigen. In one embodiment, such compositions induce an immune response against at least one of the core E. coli chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, even though the saccharide conjugate having an O polysaccharide does not comprise a core oligosaccharide.

[0292] E. coli serotypes can be characterized according to one of five chemotypes. Table 2 lists exemplary serotypes characterized according to chemotype. The bold serotypes represent the serotypes most commonly associated with the indicated core chemotype. Thus, in a preferred embodiment, the composition induces an immune response against at least any one of the core E. coli chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, including an immune response against any one of the respective corresponding E. coli serotypes.

[0293] [Table 2]

[0294] In some embodiments, a composition comprises a saccharide that comprises a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O8, formula O18, formula O9, formula O13, formula O20, formula O21, formula O91, and formula O163, where n is 1 to 100. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety, e.g., as shown in Figure 24.

[0295] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O18, formula O13, formula O20, formula O21, formula O91, and formula O163, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety in the saccharide.

[0296] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R2 chemotype, e.g., selected from saccharides having formula O21, formula O44, formula O11, formula O89, formula O162, and formula O9, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R2 core moiety, e.g., as shown in Figure 24.

[0297] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R3 chemotype, e.g., selected from saccharides having formula O25b, formula O15, formula O153, formula O21, formula O17, formula O11, formula O159, formula O22, formula O86, and formula O93, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R3 core moiety, e.g., as shown in Figure 24.

[0298] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R4 chemotype, e.g., selected from saccharides having formula O2, formula O1, formula O86, formula O7, formula O102, formula O160, and formula O166, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R4 core moiety, e.g., as shown in Figure 24.

[0299] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having a K-12 chemotype (e.g. selected from a saccharide having the formula O25b and a saccharide having the formula O16, where n is 1-100, preferably 31-100, more preferably 35-90, most preferably 35-65). In some embodiments, the saccharide in the composition further comprises an E. coli K-12 core moiety, e.g., as shown in Figure 24.

[0300] In some embodiments, the saccharide comprises a core saccharide. Thus, in one embodiment, the O polysaccharide further comprises an E. coli R1 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R2 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R3 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R4 core portion. In another embodiment, the O polysaccharide further comprises an E. coli K12 core portion.

[0301] In some embodiments, the saccharide does not include a core saccharide. Thus, in one embodiment, the O polysaccharide does not include an E. coli R1 core portion. In another embodiment, the O polysaccharide does not include an E. coli R2 core portion. In another embodiment, the O polysaccharide does not include an E. coli R3 core portion. In another embodiment, the O polysaccharide does not include an E. coli R4 core portion. In another embodiment, the O polysaccharide does not include an E. coli K12 core portion.

[0302] E. Conjugated O antigen Chemical conjugation of O antigens, or preferably O polysaccharides, to protein carriers can improve the immunogenicity of O antigens or O polysaccharides. However, variability in polymer size is a real challenge for production. In commercial use, the size of the sugar can affect compatibility with various conjugation synthesis strategies, uniformity of the product, and immunogenicity of the conjugate. Controlling the expression of Wzz family protein chain length regulators by engineering the O antigen synthesis pathway allows the production of O antigen chains of desired length in various Gram-negative bacterial strains, including E. coli.

[0303] In one embodiment, the purified sugar is chemically activated to produce an activated sugar that can react with a carrier protein. Once activated, each sugar is separately conjugated to a carrier protein to form a conjugate, i.e., a sugar conjugate. As used herein, the term "sugar conjugate" refers to a sugar covalently bound to a carrier protein. In one embodiment, the sugar is directly bound to the carrier protein. In another embodiment, the sugar is bound to the carrier protein via a spacer / linker.

[0304] Conjugates can be prepared by schemes in which a carrier is attached to the O antigen at one or more sites along the O antigen, or by schemes in which at least one residue of the core oligosaccharide is activated.

[0305] In one embodiment, each saccharide is conjugated to the same carrier protein.

[0306] Where the protein carrier is the same for two or more sugars in the composition, the sugars can be conjugated to the same molecule of carrier protein (e.g., a carrier molecule having two or more different sugars conjugated thereto).

[0307] In a preferred embodiment, the sugars are each individually conjugated to different molecules of the protein carrier (each molecule of the protein carrier has only one type of sugar conjugated to it), in which embodiment the sugars are said to be individually conjugated to the carrier protein.

[0308] Chemical activation of saccharides and subsequent conjugation to carrier proteins can be achieved by the activation and conjugation methods disclosed herein.After conjugation of polysaccharides to carrier proteins, the saccharide conjugates are purified (enriched in terms of the amount of polysaccharide-protein conjugates) by various techniques.These techniques include concentration / diafiltration operations, precipitation / elution, column chromatography, and depth filtration.After the individual saccharide conjugates are purified, they are mixed to formulate the immunogenic composition of the present invention.

[0309] Activation. The present invention further relates to an activated polysaccharide produced from any of the embodiments described herein, where the polysaccharide is activated with a chemical reagent that generates a reactive group for conjugation to a linker or carrier protein. In some embodiments, the saccharide of the present invention is activated prior to conjugation to a carrier protein. In some embodiments, the degree of activation does not substantially reduce the molecular weight of the polysaccharide. For example, in some embodiments, the degree of activation does not cleave the polysaccharide backbone. In some embodiments, the degree of activation is not a significant reduction in the molecular weight of the polysaccharide. 197 The degree of activation does not significantly affect the degree of conjugation as measured by the number of modified lysine residues in the carrier protein (determined by amino acid analysis), such as. For example, in some embodiments, the degree of activation does not significantly increase the number of modified lysine residues in the carrier protein (determined by amino acid analysis) by 3-fold compared to the number of modified lysine residues in the carrier protein of a conjugate with a reference polysaccharide at the same degree of activation. In some embodiments, the degree of activation does not increase the level of unconjugated free sugar. In some embodiments, the degree of activation does not reduce the optimal sugar / protein ratio.

[0310] In some embodiments, the activated saccharide has a percentage of activation in which the number of moles of thiol per saccharide repeat unit of the activated saccharide is 1-100%, such as 2-80%, 2-50%, 3-30%, and 4-25%. The degree of activation is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or about 100%. Preferably, the degree of activation is at most 50%, more preferably at most 25%. In one embodiment, the degree of activation is at most 20%. Any minimum value can be combined with any maximum value to define a range.

[0311] In one embodiment, the polysaccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then coupled to a carrier protein, preferably a CRMP. 197 or tetanus toxoid) directly or via a spacer (linker) group.

[0312] For example, the spacer may be cystamine or cysteamine to obtain a thiolated polysaccharide that can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy]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)). In one embodiment, a cyanate ester (which may be generated by CDAP chemistry) is coupled with hexanediamine or adipic dihydrazide (ADH) and the amino-derivatized sugar is coupled to a carrier protein (e.g., CRMP) using carbodiimide (e.g., EDAC or EDC) chemistry via a carboxyl group on the protein carrier. 197 ) to be conjugated.

[0313] Other suitable techniques for conjugation use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Conjugation may involve a carbonyl linker that can be formed by reaction of the free hydroxyl group of the sugar with CDI, followed by reaction with the protein to form a carbamate bond. This may involve reduction of the anomeric terminus to a 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 of the CDI carbamate intermediate with an amino group on the protein (CDI chemistry).

[0314] Molecular Weight. In some embodiments, the saccharide conjugate comprises a saccharide having a molecular weight between 10 kDa and 2,000 kDa. In other embodiments, the saccharide has a molecular weight between 50 kDa and 1,000 kDa. In other embodiments, the saccharide has a molecular weight between 70 kDa and 900 kDa. In other embodiments, the saccharide has a molecular weight between 100 kDa and 800 kDa. In other embodiments, the saccharide has a molecular weight between 200 kDa and 600 kDa. In a further embodiment, the sugars are from 100 kDa to 1000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 150 kDa to 8 00kDa;150kDa~700kDa;150kDa~600kDa;150kDa~500kDa;150kDa~400kDa;150kDa~300kDa;200kDa~ 1,000kDa;200kDa~900kDa;200kDa~800kDa;200kDa~700kDa;200kDa~600kDa;200kDa~500kDa;200k Da~400kDa;200kDa~300kDa;250kDa~1,000kDa;250kDa~900kDa;250kDa~800kDa;250kDa~700kDa;2 50kDa~600kDa;250kDa~500kDa;250kDa~400kDa;250kDa~350kDa;300kDa~1,000kDa;300kDa~900kDa a;300kDa-800kDa;300kDa-700kDa;300kDa-600kDa;300kDa-500kDa;300kDa-400kDa;400kDa-1,000kDa;400kDa-900kDa;400kDa-800kDa;400kDa-700kDa;400kDa-600kDa;500kDa-600kDa. In one embodiment, the sugar conjugates having such molecular weights are produced by single-end conjugation. In another embodiment, the sugar conjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in an aqueous buffer.Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0315] In some embodiments, the glycoconjugates of the present invention have a molecular weight of 400 kDa to 15,000 kDa; 500 kDa to 10,000 kDa; 2,000 kDa to 10,000 kDa; 3,000 kDa to 8,000 kDa; or 3,000 kDa to 5,000 kDa. In other embodiments, the glycoconjugates have a molecular weight of 500 kDa to 10,000 kDa. In other embodiments, the glycoconjugates have a molecular weight of 1,000 kDa to 8,000 kDa. In still other embodiments, the glycoconjugates have a molecular weight of 2,000 kDa to 8,000 kDa or 3,000 kDa to 7,000 kDa.In a further embodiment, the glycoconjugates of the invention are from 200 kDa to 20,000 kDa; from 200 kDa to 15,000 kDa; from 200 kDa to 10,000 kDa; from 200 kDa to 7,500 kDa; from 200 kDa to 5,000 kDa; from 200 kDa to 3,000 kDa; from 200 kDa to 1,000 kDa; from 500 kDa to 20,000 kDa; from 500 kDa to 15,000 kDa; from 500 kDa to 12,500 kDa; from 500 kDa to 10,000 kDa. Da;500kDa~7,500kDa;500kDa~6,000kDa;500kDa~5,000kDa;500kDa~4,000kDa;500kDa~3,000kDa;500kDa~2,000kDa;500 kDa~1,500kDa;500kDa~1,000kDa;750kDa~20,000kDa;750kDa~15,000kDa;750kDa~12,500kDa;750kDa~10,000kDa;750kDa ~7,500kDa;750kDa~6,000kDa;750kDa~5,000kDa;750kDa~4,000kDa;750kDa~3,000kDa;750kDa~2,000kDa;750kDa~1,500 kDa;1,000kDa~15,000kDa;1,000kDa~12,500kDa;1,000kDa~10,000kDa;1,000kDa~7,500kDa;1,000kDa~6,000kDa;1,000k Da to 5,000 kDa; 1,000 kDa to 4,000 kDa; 1,000 kDa to 2,500 kDa; 2,000 kDa to 15,000 kDa; 2,000 kDa to 12,500 kDa; 2,000 kDa to 10,000 kDa; 2,000 kDa to 7,500 kDa; 2,000 kDa to 6,000 kDa; 2,000 kDa to 5,000 kDa; 2,000 kDa to 4,000 kDa; or 2,000 kDa to 3,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein. In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC).In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in DMSO.

[0316] In further embodiments, the glycoconjugates of the invention have a molecular weight of from 1,000 kDa to 20,000 kDa; from 1,000 kDa to 15,000 kDa; from 2,000 kDa to 10,000 kDa; from 2000 kDa to 7,500 kDa; from 2,000 kDa to 5,000 kDa; from 3,000 kDa to 20,000 kDa; from 3,000 kDa to 15,000 kDa; from 3,000 kDa to 12,500 kDa; from 4,000 kDa to 10,000 kDa; from 4,000 kDa to 7,500 kDa; from 4,000 kDa to 6,000 kDa; or from 5,000 kDa to 7,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC). In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in DMSO. In another embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein.

[0317] In a further embodiment, the glycoconjugates of the invention have a molecular weight of from 5,000 kDa to 20,000 kDa; from 5,000 kDa to 15,000 kDa; from 5,000 kDa to 10,000 kDa; from 5,000 kDa to 7,500 kDa; from 6,000 kDa to 20,000 kDa; from 6,000 kDa to 15,000 kDa; from 6,000 kDa to 12,500 kDa; from 6,000 kDa to 10,000 kDa or from 6,000 kDa to 7,500 kDa.

[0318] The molecular weight of the glycoconjugate can be measured by SEC-MALLS. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure. The glycoconjugates of the present invention can also be characterized by the sugar to carrier protein ratio (w / w). In some embodiments, the polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is 0.5 to 3 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the sugar to carrier protein ratio (w / w) is 0.5-2.0, 0.5-1.5, 0.8-1.2, 0.5-1.0, 1.0-1.5, or 1.0-2.0. In further embodiments, the sugar to carrier protein ratio (w / w) is 0.8-1.2. In preferred embodiments, the polysaccharide to carrier protein ratio in the conjugate is 0.9-1.1. In some such embodiments, the carrier protein is a CRM. 197 It is.

[0319] The glycoconjugates were also analyzed to determine their molecular size distribution (K d ) can also be characterized. A size exclusion chromatography medium (CL-4B) can be used to determine the relative molecular size distribution of the conjugates. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugates. Large molecules that are excluded from small pores in the medium elute more quickly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically with a sugar assay. K d For the determination of 0 ), (K d =0), and the fraction showing maximum retention (V i ), (K d The column is calibrated to establish the fraction at which a particular sample characteristic is reached (V e ) into the formula Kd =(V e -V 0 ) / (V i -V 0 ) by K d Associate with.

[0320] Free saccharides. The saccharide conjugates and immunogenic compositions of the present invention may comprise free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the saccharide conjugate composition. The free saccharides may be non-covalently associated with the saccharide conjugate (i.e., non-covalently bound, adsorbed, or entrapped within or with it). In a preferred embodiment, the saccharide conjugate comprises at most 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises less than about 25% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises at most about 20% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises at most about 15% free polysaccharides relative to the total amount of polysaccharides. In another preferred embodiment, the saccharide conjugate comprises at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% free polysaccharides compared to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises less than about 8% free polysaccharides compared to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises at most about 6% free polysaccharides compared to the total amount of polysaccharides. In a preferred embodiment, the saccharide conjugate comprises at most about 5% free polysaccharides compared to the total amount of polysaccharides. See, for example, Table 19, Table 20, Table 21, Table 22, Table 23, Table 24, and Table 25.

[0321] Covalent bond. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide every 5-10 saccharide repeat units; every 2-7 saccharide repeat units; every 3-8 saccharide repeat units; every 4-9 saccharide repeat units; every 6-11 saccharide repeat units; every 7-12 saccharide repeat units; every 8-13 saccharide repeat units; every 9-14 saccharide repeat units; every 10-15 saccharide repeat units; every 2-6 saccharide repeat units; every 3-7 saccharide repeat units; every 4-8 saccharide repeat units; every 6-10 saccharide repeat units; every 7-11 saccharide repeat units; every 8-12 saccharide repeat units; every 9-13 saccharide repeat units; every 10-14 saccharide repeat units; every 10-20 saccharide repeat units; every 4-25 saccharide repeat units, or every 2-25 saccharide repeat units. In a frequent embodiment, the carrier protein is a CRM 197 In another embodiment, at least one bond between the carrier protein and the saccharide is present every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units of the polysaccharide. 197 Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0322] Another method of characterizing the glycoconjugates of the present invention is to characterize the extent of conjugated lysine (degree of conjugation) by the amount of carrier protein (e.g., CRMP) that becomes conjugated to the saccharide. 197) depending on the number of lysine residues in the polysaccharide. Evidence for lysine modification of the carrier protein due to covalent attachment to the polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugate material. In preferred embodiments, the conjugation degree of the glycoconjugates of the invention is 2-15, 2-13, 2-10, 2-8, 2-6, 2-5, 2-4, 3-15, 3-13, 3-10, 3-8, 3-6, 3-5, 3-4, 5-15, 5-10, 8-15, 8-12, 10-15 or 10-12. In one embodiment, the conjugation degree of the glycoconjugates of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 or about 15. In preferred embodiments, the degree of conjugation of the glycoconjugates of the invention is between 4 and 7. In some such embodiments, the carrier protein is a CRM 197 It is.

[0323] The frequency of attachment of glycans to lysines on the carrier protein is another parameter for characterizing the glycoconjugates of the invention. For example, in some embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs for every 4 saccharide repeat units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 10 saccharide repeat units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 15 saccharide repeat units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 25 saccharide repeat units of the polysaccharide. O-acetylation. In some embodiments, the saccharides of the invention are O-acetylated. In some embodiments, the glycoconjugate comprises a sugar having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90% or 80-90%. In other embodiments, the degree of O-acetylation is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or about 100%. Percent O-acetylation refers to the percentage of a given sugar relative to 100% (each repeat unit is fully acetylated relative to its acetylated structure).

[0324] In some embodiments, the saccharide conjugate is prepared by reductive amination. In some embodiments, the saccharide conjugate is a single-end-linked conjugated saccharide in which the saccharide is directly covalently attached to the carrier protein. In some embodiments, the saccharide conjugate is covalently attached to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

[0325] Reductive amination. In one embodiment, the sugar is conjugated to the carrier protein by reductive amination (such as described in U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 0231340, 2007 / 0184071 and 2007 / 0184072, WO2006 / 110381, WO2008 / 079653, and WO2008 / 143709).

[0326] Reductive amination involves (1) oxidation of the sugar, and (2) reduction of the activated sugar and the carrier protein to form a conjugate. Prior to oxidation, the sugar may be hydrolyzed. Mechanical or chemical hydrolysis may be used. Chemical hydrolysis may be carried out using acetic acid.

[0327] The oxidation step may include reaction with periodate. As used herein, the term "periodate" refers to both periodate and periodic acid. The term also refers to metaperiodate (IO 4 - ) and orthoperiodate (IO 6 5- ), and various salts of periodic acid (e.g., sodium periodate and potassium periodate). In one embodiment, the polysaccharide is reacted with the polysaccharide in the presence of metaperiodate, preferably sodium periodate (NaIO 4 In another embodiment, the polysaccharide is oxidized in the presence of an orthoperiodate, preferably in the presence of periodic acid.

[0328] In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxide radical compound, such as a piperidine-N-oxy or pyrrolidine-N-oxy compound, in the presence of an oxidizing agent to selectively oxidize the primary hydroxyl. In the reaction, the actual oxidizing agent is an N-oxoammonium salt in the catalytic cycle. In one aspect, the stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one aspect, the stable nitroxyl or nitroxide radical compound bears a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, the stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, the oxidizing agent is a molecule bearing an N-halo moiety. In one embodiment, the oxidizing agent is selected from any one 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 oxidizing agent is N-chlorosuccinimide.

[0329] After the sugar oxidation step, the sugar is said to be activated and is referred to hereinafter as "activated". The activated sugar and the carrier protein can be lyophilized (freeze-dried) independently (individual lyophilization) or together (co-lyophilization). In one embodiment, the activated sugar and the carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and the carrier protein are lyophilized independently.

[0330] In one embodiment, lyophilization is carried out in the presence of a non-reducing sugar, possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol and palatinit.

[0331] The next step of the conjugation process is the reduction of the activated sugar and carrier protein with a reducing agent (so-called reductive amination) to form a conjugate.Suitable reducing agents include cyanoborohydrides such as sodium cyanoborohydride, sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of Bronsted or Lewis acid, amine boranes such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB), borane-pyridine, or borohydride exchange resin.In one embodiment, the reducing agent is sodium cyanoborohydride.

[0332] In certain embodiments, 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 at pH 6.0-8.5, 7.0-8.0 or 7.0-7.5), and in other embodiments, the reaction is carried out in an aprotic solvent. In certain embodiments, the reduction reaction is carried out in DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. DMSO or DMF solvent can be used to reconstitute the lyophilized activated sugar and carrier protein.

[0333] 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 ). After conjugation (reduction reaction and, if necessary, capping), the glycoconjugate can be purified (enriched with respect to the amount of polysaccharide-protein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration sedimentation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. The glycoconjugate can be purified by diafiltration and / or ion exchange chromatography and / or size exclusion chromatography. In an embodiment, the glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography. In one embodiment, the glycoconjugate is sterile filtered.

[0334] In a preferred embodiment, glycoconjugates derived from E. coli serotypes selected from any one of O25B, O1, O2, and O6 are prepared by reductive amination. In a preferred embodiment, glycoconjugates derived from E. coli serotypes O25B, O1, O2, and O6 are prepared by reductive amination.

[0335] In one aspect, the present invention provides a carrier protein, e.g.

[0336] [ka] [In the formula, n is an integer equal to or greater than 1] A CRM linked to a sugar of formula O25B, represented by 197In a preferred embodiment, n is an integer of at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and at most 200, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50. Any minimum value can be combined with any maximum value to define a range. Exemplary ranges include, for example, at least 1 and at most 1000; at least 10 and at most 500; and at least 20 and at most 80. In one preferred embodiment, n is at least 31 and at most 90, more preferably 40-90, and most preferably 60-85.

[0337] In another aspect, the present invention provides a carrier protein, e.g., CRMP, conjugated to a saccharide having any one of the following structures shown in Table 1 (see also Figures 9A-9C and Figures 10A-10B), where n is an integer greater than or equal to 1: 197 The present invention relates to a conjugate comprising:

[0338] Without being bound by theory or mechanism, it is believed that in some embodiments, a stable conjugate requires a level of antigen modification that balances preservation of the structural integrity of the antigen's important immunogenic epitopes.

[0339] Activation and formation of aldehydes. In some embodiments, the sugars of the present invention are activated, resulting in the formation of aldehydes. In such embodiments in which the sugars are activated, the percentage (%) of activation (or degree of oxidation (DO)) (see, e.g., Example 31) refers to the moles of sugar repeating units per mole of aldehyde of the activated polysaccharide. For example, in some embodiments, the sugars are activated by periodate oxidation of vicinal diols on the repeating units of the polysaccharide, resulting in the formation of aldehydes. Varying the molar equivalent (meq) of sodium periodate relative to the sugar repeating units and the temperature during oxidation results in varying levels of degree of oxidation (DO).

[0340] Sugar and aldehyde concentrations are typically determined by colorimetric assays. An alternative reagent is the TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl radical)-N-chlorosuccinimide (NCS) mixture, which leads to the formation of aldehydes from primary alcohol groups.

[0341] In some embodiments, the activated saccharide has a degree of oxidation between 1 and 100, such as between 2 and 80, between 2 and 50, between 3 and 30, and between 4 and 25 moles of saccharide repeating units per mole of aldehyde of the activated saccharide. The degree of activation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more, or about 100. Preferably, the degree of oxidation (DO) is at least 5 and at most 50, more preferably at least 10 and at most 25. In one embodiment, the degree of activation is at least 10 and at most 25. Any minimum value and any maximum value can be combined to define a range. The value of the degree of oxidation can be expressed as a percentage (%) of activation. For example, in one embodiment, a DO value of 10 refers to 1 activated saccharide repeat unit out of a total of 10 saccharide repeat units in the activated saccharide, in which case a DO value of 10 can be expressed as 10% activation.

[0342] In some embodiments, the conjugates prepared by reductive amination chemistry include a carrier protein and a sugar, the sugar being selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180 / C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18C), Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O3 9, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O62D 1, formula O63, formula O64, formula O65, for...

Claims

Claim 1: A composition comprising a saccharide derived from E. coli serotype O9a conjugated to CRM 197, wherein the saccharide is represented by formula I Formula I 20. A composition for eliciting an immune response against Klebsiella pneumoniae serotype O3b, comprising a repeating unit structure of:

2. 2. The composition of claim 1, wherein the sugar further comprises a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) moiety.

3. 3. The composition of claim 1 or 2, wherein the immune response comprises opsonophagocytic antibodies against Klebsiella pneumoniae.

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