Escherichia coli compositions and methods thereof

Abstract

In one aspect, the present application relates to immunogenic compositions comprising modified O-polysaccharide molecules derived from E. coli lipopolysaccharide and conjugates thereof. A multivalent vaccine can be prepared by combining two or more monovalent immunogenic compositions for different E. coli serotypes. In one embodiment, the modified O-polysaccharide molecules are produced by recombinant bacteria comprising a wzz gene.

Description

[0001] Cross-Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 722,370, filed August 24, 2018, U.S. Provisional Application Serial No. 62 / 784,940, filed December 26, 2018, and U.S. Provisional Application Serial No. 62 / 881,361, filed July 31, 2019, each of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present invention relates to compositions of Escherichia coli (E. coli) and methods thereof. Escherichia coli SUMMARY BACKGROUND

[0004] The cell wall of Gram-negative bacteria includes an outer membrane and a peptidoglycan layer located inside the outer membrane. The outer membrane includes phospholipids, lipopolysaccharides (LPS), lipoproteins, and membrane proteins. Lipopolysaccharides are present on the outer layer of the membrane, and phospholipids are present on the inner layer thereof.

[0005] LPS includes a lipid A membrane anchor point that links a core oligosaccharide to an O- polysaccharide polymer containing repeating sugar monomer units that form short, long, or very long O-chains. While the core oligosaccharide is mostly conserved across bacterial species, the O- polysaccharide can vary between serotypes.

[0006] Escherichia coli (E. coli) is a Gram-negative bacterium known to cause life-threatening bacterial sepsis. Both the capsule (K) and the lipopolysaccharide (LPS) O-antigen are important virulence factors. E. coli SUMMARY

[0007] There is significant interest in using O-polysaccharides as the basis for a vaccine against E. coli. However, previous attempts to develop an E. coli complex saccharide vaccine using conventional chemical conjugation or bioconjugation methods have failed to generate enhanced functional immune responses for all serotypes. Thus, there is an unmet need for immunogenic compositions against E. coli that generate enhanced functional immune responses. SUMMARY

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

[0009] In one embodiment, the present application relates to sugars, conjugates, and compositions comprising the same produced by E. coli strains that have been artificially engineered to express longer O-antigen lipopolysaccharide (LPS) with improved properties for bioprocessing development and higher immunogenic epitope density. For example, in one embodiment, an E. coli strain is artificially engineered to express S. enterica O-antigen from a high copy plasmid Salmonella enterica ) fepE gene, which results in the production of long chain LPS in E. coli strains of different O-antigen serotypes.

[0010] The initial strain development focused on the O-antigen of serotype O25b, which is associated with emerging multi-antibiotic resistant isolates that are difficult to treat. The rfaA gene was deleted from a clinical E. coli O25b strain. wzzB After transformation with Salmonella sp. Salmonella ) fepE The artificially engineered E. coli O25b strain now produces only long chain LPS. After chemical extraction with acetic acid to release the O-antigen from the bacterial surface, the resulting long chain O25b polysaccharide is amenable to routine purification and conjugation techniques. Unlike O-antigens produced by bioconjugation, these longer chain O-antigens retain both the inner and outer core oligosaccharides, which independently have the potential to elicit functional antibodies that are both bacteriocidal and capable of blocking sepsis. Surprisingly, the exemplary O25b complex sugar is significantly more immunogenic in mammals than the unconjugated polysaccharide.

[0011] Thus, in one aspect, the present application relates to a sugar comprising an increase of at least 5 repeat units compared to the corresponding wild-type O-polysaccharide of E. coli.

[0012] In one embodiment, the composition comprises a compound selected from Formula O1, Formula O2, Formula O3, Formula O4, Formula O5, Formula O6, 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 O19, Formula O20, Formula O21, Formula O22, Formula O23, Formula O24, Formula O25, 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, 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 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, 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 0111, 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 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 O163, 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,structures of Formula 0186 and Formula 0187, wherein n is an integer from 1 to 1000. See Table 1 and Figure 9A -C and Figure 10A -B.

[0013] As used herein, unless expressly stated otherwise, the term "wherein n is" means "one or more of the formulae selected from the group of n". In one embodiment, the composition comprises a compound selected from 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 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 62D1, 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 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 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 O163, 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 from 1 to 1000. See Table 1 and Figure 9A -C and Figure 10A -B.

[0014] In one embodiment, the composition comprises a structure selected from 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 O10, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O21, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O28, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O55, Formula O56, Formula O58, Formula O64, Formula O69, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O75, Formula O77, Formula O78, Formula O86, Formula O88, Formula O90, Formula O98, Formula O104, Formula 0111, Formula O113, Formula O114, Formula O119, Formula O121, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O136, Formula O138, Formula O141, Formula O142, Formula O143, Formula O147, Formula O149, Formula O152, Formula O157, Formula O158, Formula O159, Formula O164, Formula O173, Formula 62D1, Formula O22, Formula O35, Formula O65, Formula O66, Formula O83, Formula O91, Formula O105, Formula O116, Formula O117, Formula O139, Formula O153, Formula O167, and Formula O172, wherein n is an integer from 1 to 1000. See Table 1 and Figure 9A -C and Figure 10A .

[0015] In one embodiment, the composition comprises a structure selected from the group consisting of 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 O10, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O21, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O28, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O55, Formula O56, Formula O58, Formula O64, Formula O69, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O75, Formula O77, Formula O78, Formula O86, Formula O88, Formula O90, Formula O98, Formula O104, Formula 0111, Formula O113, Formula O114, Formula O119, Formula O121, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O136, Formula O138, Formula O141, Formula O142, Formula O143, Formula O147, Formula O149, Formula O152, Formula O157, Formula O158, Formula O159, Formula O164, Formula O173, and Formula 62D1, wherein n is an integer from 1 to 1000. See Table 1 and Figure 9A -C.

[0016] In one embodiment, the composition does not comprise a structure selected from the group consisting of Formula O8, Formula O9a, Formula O9, Formula O20ab, Formula O20ac, Formula O52, Formula O97, and Formula O101. In another embodiment, the composition comprises a structure selected from the group consisting of Formula O8, Formula O9a, Formula O9, Formula O20ab, Formula O20ac, Formula O52, Formula O97, and Formula O101, wherein n is an integer from 1 to 10. See Table 1 and Figure 10B .

[0017] In one embodiment, the E. coli is of any one of E. coli serotypes selected from the group consisting of O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O19, O20, O21, O22, O23, O24, O25, O25b, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45, O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, 62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73, 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, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, 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.

[0018] In one embodiment, the E. coli is an E. coli serotype selected from the group consisting of O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18, O19, O20, O21, O22, O23, O24, O25, O25b, O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45, O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, 62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73, 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, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, 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.

[0019] In one embodiment, the sugar is produced by expressing a wzz family protein in a gram-negative bacterium to produce the sugar. In one embodiment, the sugar is produced by increasing the number of repeating units of O-polysaccharide produced by a gram-negative bacterium in culture, including expressing (not necessarily overexpressing) a wzz family protein in a gram-negative bacterium to produce the sugar. In preferred embodiments, the wzz family protein is selected from any one of wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepE , wzzi, and wzz2. In preferred embodiments, the expressed (not necessarily overexpressed) wzz family protein is selected from wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepE , wzzi, and wzz2. In alternative embodiments, the sugar is synthetically synthesized. In one embodiment, the sugar is covalently bound (conjugated) to a carrier protein. Preferably, the carrier protein is CRM 197 .

[0020] In one aspect, the present application relates to a composition comprising a sugar and / or conjugate thereof and a pharmaceutically acceptable diluent.

[0021] In another aspect, the present application relates to a method of inducing an immune response in a subject, comprising administering to the subject an effective amount of a composition. In one embodiment, the immune response comprises the induction of anti-E. coli O-specific polysaccharide antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1A -B- Figure 1A Plasmid depicting pUC replicon, 500-700x copies per cell, chain length regulator (clpX) and P15a replicon - 10-12x copies per cell, O-antigen operon (wecA) plasmid. Figure 1A Figure 1B

[0023] Figure 2 -WzzB amino acid sequence alignment showing K12\W3110\WzzB (SEQ ID NO: 23); O25a\ETEC\ATCC\WzzB (SEQ ID NO: 22); O25a:K5:H1\WzzB (SEQ ID NO: 21); O25b\2401\WzzB (SEQ ID NO: 20); and O25b\2401\WzzB (SEQ ID NO: 20).

[0024] Figure 3A -B: by plasmid-based heterologous wzzB and​​fepE Expression of chain length regulators to modulate O-antigen chain length in serotypes O25a and O25b strains. Genetic complementation of LPS expression in plasmid transformants of wzzB knockout strains O25K5H1 (O25a) and GAR2401 (O25b) is shown. LPS profiles of plasmid transformants of O25a O25K5HΔwzzB are shown on the left; and similar profiles of O25b GAR2401ΔwzzB transformants are shown on the right. Immunoblots of replicate gels probed with O25-specific serum (Statens Serum Institut) are shown in Figure 3A Figure 3B wxxB The (knockout) background is associated with lanes 1-7; O25b 2401 Δ wzzB The (knockout) background is associated with lanes 8-15.

[0025] Figure 4 - E. coli and Salmonella fepE plasmids confer long chain O-antigen expression in host O25K5H1ΔwzzB.

[0026] Figure 5 - FepE amino acid sequence alignment. O157 FepE amino acid sequence is SEQ ID NO: 18; O25a ETECATCC FepE amino acid sequence is SEQ ID NO: 17; O25a:K5:H1 FepE amino acid sequence is SEQ ID NO: 16; O25b 2401 FepE amino acid sequence is SEQ ID NO: 15; Salmonella LT2 FepE amino acid sequence is SEQ ID NO: 19.

[0027] Figure 6 - Salmonella fepE expression produces long O-antigen LPS in multiple clinical isolates.

[0028] Figure 7A - Plasmid-mediated arabinose-inducible expression of O25b long O-antigen LPS in B- O25b O-antigen knockout host strain. Results from SPS PAGE are shown in Figure 7A and results from O25 immunoblot are shown in Figure 7B wherein in both Figure 7A and Figure 7B 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. ​​

[0029] Figure 8 - Plasmid-mediated arabinose-inducible expression of long O-antigen LPS in a common host strain.

[0030] Figure 9A - Structure of O-antigen synthesized by a polymerase-dependent pathway with four or fewer residues in the backbone.

[0031] Figure 10A - B- Structure of O-antigen synthesized by a polymerase-dependent pathway with five or six residues in the backbone. Figure 10A - Structure of O-antigen believed to be synthesized by an ABC-transporter dependent pathway. Figure 10B

[0032] - Expression of O25 O-antigen LPS in Exploratory Bioprocess strains. Figure 11

[0033] - SEC profiles and properties of short (Strain 1 O25b wt2831) and long O25b O-antigen (Strain 2 O25b 2401A Figure 12A / LT2 FepE) purified from strains GAR2831 and ‘2401A wzzB / fepE. Figure 12A Figure 12B wzzB

[0034] Figure 13A - B- (a) Information on the vaccination schedule for Rabbit Study 1 VAC-2017-PRL-EC-0723; (b) Vaccination schedule for Rabbit Study 2 VAC-2018-PRL-EC-077. Figure 13A Figure 13B - C- O25b complex saccharide IgG responses, where - · - represents results from Prebleed; -■- Bleed 1 (6 weeks); -▲- Bleed 2 (8 weeks); -♦- Bleed 3 (12 weeks).

[0035] - Depicts results from Rabbits 1-3 (Moderate activation); Figure 14A - Depicts results from Rabbits 2-3 (Low activation); Figure 14A - Depicts results from Rabbit 3-1 (High activation). Figure 14B Figure 14C

[0036] Figure 15A ​​​​​- F - and unconjugated polysaccharide, i.e., free O25b polysaccharide (Fig. A-C, where -•- represents pre-bleed results from rabbit A-1, -■- week 6 antisera from rabbit A-1, -A- week 8 antisera from rabbit A-1) compared to O25b long O-antigen complex saccharide, i.e., hypo-activated O25b-CRM 197 conjugate (F) Figure 15D - F, where -•- represents pre-bleed results from rabbit 2-1, -■- week 12 antisera from rabbit 2-1). Note that MFIs are plotted on a log scale to highlight the difference between pre-immune antibodies and immune antibodies in the <1000 MFI range. Figure 15A Results from rabbit A-1 (unconjugated polysaccharide (Poly)) are depicted; Figure 15B Results from rabbit A-3 (unconjugated polysaccharide) are depicted; Figure 15C Results from rabbit A-4 (unconjugated polysaccharide) are depicted; Figure 15D Results from rabbit 2-1 (hypo-activated) are depicted; Figure 15E Results from rabbit 2-2 (hypo-activated) are depicted; and Figure 15F Results from rabbit 2-3 (hypo-activated) are depicted.

[0037] Figure 16A - C - Native surface expression of long O25b O-antigen with O25b antisera. Figure 16A Results are depicted, where -•- represents results from O25b 2831 against PD3 antisera; -■- O25b 2831 wt against pre-bleed; -A- O25b 2831 / fepE against PD3 antisera; -▼- O25b 2831 / fepE against pre-bleed. Figure 16B Results are depicted, where -•- represents results from O25b 2401 against PD3 antisera; -■- O25b 2401 against pre-bleed; -A- O25b 2401 / fepE against PD3 antisera; -▼- O25b 2401 / fepE against pre-bleed. Figure 16C Results are depicted, where -•- represents results from E. coli K12 against PD3 antisera; -■- E. coli K12 against pre-bleed.

[0038] Figure 17 - Five General structure of the carbohydrate backbone of an outer core oligosaccharide of a known serotype. Unless otherwise noted, all glycoses are in the a- anomeric configuration. Dashed arrows indicate the genes that catalyze the formation of each bond of its product. Asterisks indicate the residue of the core oligosaccharide to which O-antigen is attached.

[0039] Figure 18- unconjugated free O25b polysaccharide is not immunogenic (dLIA), where - · - represents results from week 18 (1 wk = PD4) antisera from 4-1 ; -■- week 18 (1 wk = PD4) antisera from 4-2; -▲- week 18 (1 wk = PD4) antisera from 5-1 ; -▼- week 18 (1 wk = PD4) antisera from 5-2; week 18 (1 wk = PD4) antisera from 6-1 ; week 18 (1 wk = PD4) antisera from 6-2.

[0040] Figure 19A - C - illustrates a graph of specificity of BRC rabbit O25b RAC conjugate immune serum OPA titers. Figure 19A Rabbit 2-3 preimmune serum - · - and post-immune serum wk 13 -■- OPA titers are shown. Figure 19B Rabbit 1-2 preimmune serum - · - and post-immune serum wk 19 -■- OPA titers are shown. Figure 19C Rabbit 1-2 wk 19 OPA titer specificity is shown, where OPA activity of rabbit 1-2 immune serum was blocked by pre-incubation with 100 µg / mL purified unconjugated O25b long O-antigen polysaccharide, where -■- represents results from rabbit 1-2 immune serum wk 19; and -▼- represents results from rabbit 1-2 wk 19 w / R1 long-OAg.

[0041] Figure 20A - C - Figure 20A Graphical illustration of an exemplary administration schedule. Figure 20B and Figure 20C A graph showing O-antigen O25b IgG levels elicited by unconjugated O25b long O-antigen polysaccharide ( Figure 20B , O25b free polysaccharide (2 µg)) and derivatized O25b RAC / DMSO long O-antigen complex saccharide ( Figure 20C , O25b-CRM 197 RAC long (2 µg)) is shown, where -... - (dashed line) represents naive CD1 O25b IgG levels.

[0042] Figure 21A - B - depicts 2 ( Figure 21A ) and 3 ( Figure 21B ) post-dose.Figure depicting OPA immunogenicity of RAC, eTEC O25b long complex saccharide and monovalent complex saccharide, where -o- represents results from monovalent short 2 ug; -•- monovalent long 2 ug; -A- RAC / DMSO long 2 ug; -V- eTEC long 2 ug; * background control (n = 20).†Responder rate is % of mice with > 2x unvaccinated baseline titer.

[0043] Figure 22 Figure depicting OPA immunogenicity of eTEC chemistry and modified polysaccharide activation levels.†Responder rate is % of mice with > 2x unvaccinated baseline titer.

[0044] Figure 23A -B- Figure 23A Graphical illustration of an exemplary administration schedule, and Figure 23B Figure depicting protection of mice immunized with E. coli eTEC conjugate doses from lethal challenge with O25b isolates, where -◇- represents eTEC long chain 17% activation; -A- eTEC long chain 10% activation; -V- eTEC long chain 4% activation; -□- O25b polysaccharide; -o- unvaccinated control.

[0045] Figure 24 Schematic diagram illustrating exemplary preparation of monovalent conjugates, where the conjugation process includes selective activation of 2-keto-3-deoxyoctonic acid (KDO) with a disulfide amine linker upon unmasking of the thiol functionality. KDO is then conjugated to bromo-activated CRM 197 protein, such as Figure 24 as depicted in

[0046] Figure 25A -B shows an exemplary process flow diagram for the activation ( 197 ) and conjugation ( Figure 25A ) processes for preparing E. coli complex saccharides with CRM Figure 25B .

[0047] Sequence Identifier

[0048] SEQ ID NO: 1 shows the primer sequence for LT2 wzzB_S described in Table 4.

[0049] SEQ ID NO: 2 shows the primer sequence for LT2 wzzB_AS described in Table 4.

[0050] SEQ ID NO: 3 shows the primer sequence for O25b FepE_S described in Table 4.

[0051] SEQ ID NO: 4 shows the primer sequence for O25b FepE_A described in Table 4.

[0052] SEQ ID NO: 5 shows the primer sequence of wzzB P1_S described in Table 4.

[0053] SEQ ID NO: 6 shows the primer sequence of wzzB P2_AS described in Table 4.

[0054] SEQ ID NO: 7 shows the primer sequence of wzzB P3_S described in Table 4.

[0055] SEQ ID NO: 8 shows the primer sequence of wzzB P4_AS described in Table 4.

[0056] SEQ ID NO: 9 shows the primer sequence for O157 FepE_S described in Table 4.

[0057] SEQ ID NO: 10 shows the primer sequence for O157 FepE_AS described in Table 4.

[0058] SEQ ID NO: 11 shows the primer sequence of pBAD33_connector_S described in Table 4.

[0059] SEQ ID NO: 12 shows the primer sequence of pBAD33_interchangeor_AS described in Table 4.

[0060] SEQ ID NO: 13 shows the primer sequence for JUMPSTART_r described in Table 4.

[0061] SEQ ID NO: 14 shows the primer sequence for gnd_f described in Table 4.

[0062] SEQ ID NO: 15 shows Figure 5 The O25b 2401 FepE amino acid sequence is shown.

[0063] SEQ ID NO: 16 shows Figure 5 The O25a:K5:H1 FepE amino acid sequence is shown.

[0064] SEQ ID NO: 17 shows Figure 5 The amino acid sequence of O25a ETEC ATCC FepE is shown.

[0065] SEQ ID NO: 18 shows Figure 5 The O157 FepE amino acid sequence is shown.

[0066] SEQ ID NO: 19 shows Figure 5Salmonella LT2 FepE amino acid sequence shown in Table 1.

[0067] SEQ ID NO: 20 sets forth the O25b 2401 WzzB amino acid sequence shown in Table 1. Figure 2

[0068] SEQ ID NO: 21 sets forth the O25a:K5:H1 WzzB amino acid sequence shown in Table 1. Figure 2

[0069] SEQ ID NO: 22 sets forth the O25a ETEC ATCC WzzB amino acid sequence shown in Table 1. Figure 2

[0070] SEQ ID NO: 23 sets forth the K12 W3110 WzzB amino acid sequence shown in Table 1. Figure 2

[0071] SEQ ID NO: 24 sets forth the Salmonella LT2 WzzB amino acid sequence shown in Table 1. Figure 2 DETAILED DESCRIPTION

[0072] The inventors have surprisingly discovered E. coli antigens produced from the artificial engineering of different Wzz proteins (e.g., WzzB), including immunogenic conjugates thereof and methods of use. The inventors have further surprisingly discovered complex sugars containing such sugars covalently conjugated to carrier proteins, such as, for example, via a bivalent, heterobifunctional linker of (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. The inventors have also surprisingly discovered complex sugars containing sugars conjugated to carrier proteins, such as, for example, via reductive amination (RAC). In particular, RAC in an aprotic solvent, preferably DMSO (RAC / DMSO) or aqueous solution. The present invention further relates to immunogenic compositions comprising such complex sugars, as well as methods for using such complex sugars and immunogenic compositions. Additionally, the inventors have surprisingly discovered that, in some embodiments, complex sugars containing high molecular weight sugars with intermediate or long O-antigen chains can be more immunogenic than complex sugars with relatively short O-antigen chains. Moreover, the inventors have discovered that, in some embodiments, complex sugars produced by multi-armed activation of sugars prior to conjugation to carrier proteins, such as, for example, via methods including RAC or mono-armed conjugation or eTEC, can be more immunogenic than complex sugars produced by single-armed activation of sugars prior to conjugation to carrier proteins.

[0073] In one embodiment, sugars are produced by expressing (not necessarily overexpressing) different Wzz proteins (e.g., WzzB) to control the size of the sugars.​​​​​

[0074] 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 monosaccharide units covalently linked to form a disaccharide, oligosaccharide, and polysaccharide. Sugars can be linear or branched.

[0075] In one embodiment, the sugar is produced in a recombinant Gram-negative bacterium. In one embodiment, the sugar is produced in a recombinant Escherichia coli cell. In one embodiment, the sugar is produced in a recombinant Salmonella cell. Exemplary bacteria include Escherichia coli O25K5H1 、 Escherichia coli BD559, Escherichia coli GAR2831, Escherichia coli GAR865, Escherichia coli GAR868, Escherichia coli GAR869, Escherichia coli GAR872, Escherichia coli GAR878, Escherichia coli GAR896, Escherichia coli GAR1902, Escherichia coli O25a ETC NR-5, Escherichia coli O157:H7:K-, Salmonella enterica serovar Typhimurium strain LT2, Escherichia coli GAR2401, Salmonella enterica serovar Enteritidis (S. Salmonella enterica erovar Typhimurium) CVD 1925, Salmonella enterica serovar Paratyphi A (S. Salmonella enterica erovar Typhimurium) CVD 1925, Salmonella enterica serovar Paratyphi A (S. Salmonella enterica erovar Typhimurium) CVD 1925, Salmonella enterica serovar Paratyphi A (S. Salmonella enterica erovar Typhimurium) CVD 1925, Salmonella enterica serovar Paratyphi A (S. Shigella flexneri ) CVD 1208S. In one embodiment, the bacterium is not Escherichia coli GAR2401. The genetic approach to sugar production allows for efficient production of O- polysaccharide and O-antigen molecules as vaccine components.

[0076] As used herein, the term "wzz protein" refers to a chain length determinant polypeptide such as, for example, wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepEThe GenBank search numbers for exemplary wzz gene sequences are AF011910 for E4991 / 76, AF011911 for F186, AF011912 for M70 / 1-1, AF011913 for 79 / 311, AF011914 for Bi7509-41, AF011915 for C664-1992, AF011916 for C258-94, AF011917 for C722-89, and AF011919 for EDL933. The GenBank search numbers for G7 and Bi316-41 wzz gene sequences are U39305 and U39306, respectively. Further GenBank search numbers for exemplary wzz gene sequences are for Salmonella enterica subsp. enterica typhoid serological variants (Salmonella enterica). Salmonella enterica NP_459581 for subsp. enterica serovar Typhimurium str. LT2 FepE; AIG66859 for Escherichia coli O157:H7 strain EDL933 FepE; NP_461024 for Salmonella enterica subsp. typhoid serological variant LT2 WzzB; NP_416531 for Escherichia coli K-12 substrain MG1655 WzzB; and NP_415119 for Escherichia coli K-12 substrain MG1655 FepE. In a preferred embodiment, the wzz family proteins are wzzB, wzz, and wzz. SF wzz ST ,fepE,wzz fepE Any one of wzz1 and wzz2, with wzzB being the most preferred and fepE being the most preferred.

[0077] Exemplary wzzB sequences include:

[0078]

[0079]

[0080] Salmonella spp. LT2 WzzB

[0081]

[0082] Exemplary FepE sequences include:

[0083]

[0084]

[0085] Salmonella spp. LT2 FepE

[0086]

[0087] In some embodiments, the modified sugar (modified as compared to the corresponding wild-type sugar) can be produced by expressing (not necessarily overexpressing) a wzz family protein from a Gram-negative bacterium (e.g., fepE) in the Gram-negative bacterium and / or turning off (i.e., repressing, deleting, removing) a second wzz gene (e.g., wzzB) to produce high molecular weight sugars, such as lipopolysaccharides, containing intermediate or long O-antigen chains. For example, the modified sugar can be produced by expressing (not necessarily overexpressing) wzz2 and turning off wzzl. Alternatively, the modified sugar can be produced by expressing (not necessarily overexpressing) wzzfepE and turning off wzzB. In another embodiment, the modified sugar can be produced by expressing (not necessarily overexpressing) wzzB and turning off wzzfepE. In another embodiment, the modified sugar can be produced by expressing fepE. Preferably, the wzz family protein is derived from a strain that is heterologous to the host cell.

[0088] 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: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In one embodiment, the wzz family protein comprises a sequence selected from any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. 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: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In some embodiments, the sugar is produced by expressing a protein having an amino acid sequence having at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to a fepE protein.

[0089] In one aspect, the application relates to a sugar produced by expressing a wzz family protein, preferably fepE, in a gram-negative bacterium to produce a high molecular weight sugar containing intermediate or long O-antigen chains having at least a 1, 2, 3, 4, or 5 repeat unit increase compared to the corresponding wild-type O-polysaccharide. In one aspect, the application relates to a sugar produced by a gram-negative bacterium in culture expressing (not necessarily overexpressing) a wzz family protein from a gram-negative bacterium (e.g., wzzB) to produce a high molecular weight sugar containing intermediate or long O-antigen chains having at least a 1, 2, 3, 4, or 5 repeat unit increase compared to the corresponding wild-type O-antigen. For additional exemplary sugars having an increased number of repeat units compared to the corresponding wild-type sugar, see the description of O-polysaccharides and O-antigens below. The desired chain length is the chain length that produces improved or maximal immunogenicity in the context of a given vaccine construct.

[0090] In another embodiment, the saccharide comprises any one of the formulae selected from Table 1, wherein the number of repeating units in the saccharide is n one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty-nine, sixty, sixty-one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixty-seven, sixty-eight, sixty-nine, seventy, seventy-one, seventy-two, seventy-three, seventy-four, seventy-five, seventy-six, seventy-seven, seventy-eight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty-seven, eighty-eight, eighty-nine, ninety, ninety-one, ninety-two, ninety-three, ninety-four, ninety-five, ninety-six, ninety-seven, ninety-eight, ninety-nine, one hundred or more repeating units than in the corresponding wild-type O-polysaccharide. Preferably, the saccharide comprises an increase of at least twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, or fifty repeating units compared to the corresponding wild-type O-polysaccharide. See, e.g., Table 21. Methods of determining saccharide length are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 5.

[0091] In preferred embodiments, the application relates to saccharides produced in recombinant E. coli host cells, wherein the gene for an endogenous wzz O-antigen length regulator (e.g., wzzB) is deleted and replaced by a (second) wzz gene from a Gram-negative bacterium that is heterologous to the recombinant E. coli host cell (e.g., Salmonella fepE) to produce 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 from Salmonella enterica.

[0092] 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 a gene integrated in the chromosomal DNA of the host cell. Methods of stably expressing plasmid vectors in E. coli host cells and methods of integrating heterologous genes into the chromosome of E. coli host cells 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 a gene integrated in the chromosomal DNA of the host cell. Methods of stably expressing plasmid vectors in E. coli host cells and Salmonella host cells are known in the art. Methods of integrating heterologous genes into the chromosome of E. coli host cells and Salmonella host cells are known in the art.

[0093] In one aspect, the recombinant host cell is cultured in a culture medium comprising a carbon source. Carbon sources for culturing E. coli are known in the art. Exemplary carbon sources include sugar alcohols, polyols, aldohexose sugars, or ketose sugars, including but not limited to arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbose, sucrose, trehalose, pyruvic acid, succinic acid, and methylamine. In preferred embodiments, the culture medium comprises glucose. In some embodiments, the culture medium comprises a polyol or aldohexose sugar, such as mannitol, inositol, sorbose, glycerol, sorbitol, lactose, and arabinose as a carbon source. All of the carbon source can be added to the culture medium prior to the start of the culture, or all of the carbon source can be added stepwise or continuously during the culture.

[0094] Exemplary culture media for the recombinant host cell comprise an element selected from any one of KH2PO4, K2HPO4, (NH4)2SO4, sodium citrate, Na2SO4, aspartic acid, glucose, MgSO4, FeSO4-7H2O, Na2MoO4-2H2O, H3BO3, CoCl2-6H2O, CuCl2-2H2O, MnCl2-4H2O, ZnCl2, and CaCl2-2H2O. Preferably, the culture medium comprises KH2PO4, K2HPO4, (NH4)2SO4, sodium citrate, Na2SO4, aspartic acid, glucose, MgSO4, FeSO4-7H2O, Na2MoO4-2H2O, H3BO3, CoCl2-6H2O, CuCl2-2H2O, MnCl2-4H2O, ZnCl2, and CaCl2-2H2O.

[0095] The culture medium used herein can be solid or liquid, synthetic (i.e., artificial) or natural, and can contain sufficient nutrients to support the growth of the recombinant host cell. Preferably, the culture medium is a liquid medium.

[0096] In some embodiments, the culture medium can further comprise suitable inorganic salts. In some embodiments, the culture medium can further comprise trace nutrients. In some embodiments, the culture medium can further comprise growth factors. In some embodiments, the culture medium can further comprise additional carbon sources. In some embodiments, the culture medium can further comprise suitable inorganic salts, trace nutrients, growth factors, and supplemental carbon sources. Suitable inorganic salts, trace nutrients, growth factors, and supplemental carbon sources for culturing E. coli are known in the art.

[0097] In some embodiments, the culture medium can comprise additional components, such as peptone, N-Z amine, enzymatic soy hydrolysate, additional yeast extract, malt extract, supplemental carbon sources, and various vitamins, as appropriate. In some embodiments, the culture medium does not comprise such additional components, such as peptone, N-Z amine, enzymatic soy hydrolysate, additional yeast extract, malt extract, supplemental carbon sources, and various vitamins.

[0098] Illustrative examples of suitable supplemental carbon sources include, but are not limited to, other carbohydrates, such as glucose, fructose, mannitol, starch or starch hydrolysate, cellulose hydrolysate, 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.

[0099] In some embodiments, the culture medium further comprises a nitrogen source. Suitable nitrogen sources for culturing E. coli are known in the art. Illustrative 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; nitrates or nitrites, and other nitrogen-containing materials, including amino acids as pure or crude preparations, meat extract, peptone, fish meal, fish hydrolysate, corn steep liquor, casein hydrolysate, soybean meal hydrolysate, yeast extract, dried yeast, ethanol-yeast distillate, soybean flour, cottonseed meal, and the like.

[0100] In some embodiments, the culture medium comprises inorganic salts. Illustrative 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 phosphoric acid.

[0101] In some embodiments, the culture medium comprises appropriate growth factors. Illustrative examples of appropriate trace nutrients, growth factors, and the like include, but are not limited to, coenzyme A, pantothenic acid, pyridoxal hydrochloride, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, DL-6,8- thioctic acid, folic acid, vitamin B 12 , other vitamins, amino acids (e.g., cysteine and hydroxyproline), bases (e.g., adenine, uracil, guanine, thymine, and cytosine), sodium thiothiocyanate, p- or r-aminobenzoic acid, nicotinamide, nitrosoguanidine, and the like, as pure or partially purified compounds, or present in natural materials. Amounts can be determined empirically by one of skill in the art according to methods and techniques known in the art.

[0102] In another embodiment, the modified saccharide described herein (as compared to the corresponding wild-type saccharide) is synthetically produced, e.g., in vitro. Synthetic production or synthesis of saccharides can facilitate avoidance of cost and time intensive production processes. In one embodiment, the saccharide is synthetically synthesized from an appropriately protected monosaccharide intermediate, such as, for example, by using a sequential glycosylation strategy or a combination of sequential glycosylation and [3+2] block synthesis strategies. For example, thioglycosides and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, the saccharide synthetically synthesized in vitro has the same structure as a saccharide produced by a recombinant method, e.g., by manipulation of a wzz family protein described above.

[0103] The produced saccharide (by recombinant or synthetic methods) comprises a structure derived from any E. coli serotype, including, for example, any of 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 (strain 180 / C3)), 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, 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, 62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g., O73 (strain 73-1)), 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, O111, O112, O113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, 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.

[0104] Each polysaccharide (enriched for the amount of polysaccharide-protein conjugate) is purified, generally by methods known in the art, such as, for example, dialysis, concentration operations, diafiltration operations, tangential flow filtration, precipitation, elution, centrifugation, sedimentation, ultrafiltration, depth filtration, and / or column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE, and hydrophobic interaction chromatography). Preferably, the polysaccharides are purified by methods including tangential flow filtration.

[0105] The purified polysaccharides can be activated (e.g., chemically activated) to enable it to react (e.g., directly with a carrier protein or through a linker, such as an eTEC spacer), and then incorporated into the glycoconjugate of the application, as further described herein.

[0106] In a preferred embodiment, the polysaccharide of the application is derived from an E. coli serotype, wherein the serotype is 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.

[0107] As used herein, reference to any of the serotypes listed above refers to a serotype comprising the repeat unit structure (O-unit, as described below) known in the art and specific to the respective serotype. For example, the term "O25a" serotype (also referred to in the art as serotype "O25") refers to a serotype comprising formula O25 as shown in Table 1. As another example, the term "O25b" serotype refers to a serotype comprising formula O25b as shown in Table 1.

[0108] As used herein, reference to a serotype is generally made herein, unless otherwise specified, such that, for example, the term formula "O18" generally refers to includes formula O18A, formula O18ac, formula 18A1, formula O18B, and formula O18B1.

[0109] As used herein, the term "01" generally refers to a class of formulas included in the formula names according to Table 1 that include the general term "01", such as any one of Formula 01A, Formula 01A1, Formula 01B, and Formula 01C, each of which is shown in Table 1. Thus, an "01 serotype" generally refers to a serotype that includes any one of Formula 01A, Formula 01A1, Formula 01B, and Formula 01C.

[0110] As used herein, the term "06" generally refers to a class of formulas included in the formula names according to Table 1 that include the general term "06", such as any one of Formula 06:K2; K13; K15; and 06:K54, each of which is shown in Table 1. Thus, an "06 serotype" generally refers to a serotype that includes any one of Formula 06:K2; K13; K15; and 06:K54.

[0111] Other examples of terms that generally refer to a class of formulas included in the formula names according to Table 1 that include the general term include: "04", "05", "018", and "045".

[0112] As used herein, the term "02" refers to Formula 02 shown in Table 1. The term "02 0-antigen" refers to a saccharide that includes Formula 02 shown in Table 1.

[0113] As used herein, reference to an 0-antigen from a serotype listed above refers to a saccharide that includes the formula labeled with the corresponding serotype name. For example, the term "025B 0-antigen" refers to a saccharide that includes Formula 025B shown in Table 1.

[0114] As another example, the term "01 0-antigen" generally refers to a saccharide that includes a formula that includes the term "01", such as Formula 01A, Formula 01A1, Formula 01B, and Formula 01C, each of which is shown in Table 1.

[0115] As another example, the term "06 0-antigen" generally refers to a saccharide that includes a formula that includes the term "06", such as Formula 06:K2; Formula 06:K13; Formula 06:K15; and Formula 06:K54, each of which is shown in Table 1.

[0116] O-polysaccharide

[0117] As used herein, the term "O-polysaccharide" refers to any structure that includes an 0-antigen, provided that the structure does not include whole cells or Lipid A. For example, in one embodiment, an O-polysaccharide includes a lipopolysaccharide in which the Lipid A is not bound. Steps for removing Lipid A are known in the art, and include, by way of example, heat treatment in the presence of added acid. An exemplary method includes treatment with 1% acetic acid at 100°C for 90 minutes. The method is combined with a method for isolating the removed Lipid A. An exemplary method for isolating Lipid A includes ultracentrifugation.

[0118] In one embodiment, O-polysaccharide refers to a structure consisting of O-antigen, in which case the O-polysaccharide is synonymous with the term O-antigen. In a preferred embodiment, O-polysaccharide refers to a structure comprising repeating units of O-antigen without the core saccharide. Thus, in one embodiment, the O-polysaccharide does not comprise the E. coli Rl core portion. In another embodiment, the O-polysaccharide does not comprise the E. coli R2 core portion. In another embodiment, the O-polysaccharide does not comprise the E. coli R3 core portion. In another embodiment, the O-polysaccharide does not comprise the E. coli R4 core portion. In another embodiment, the O-polysaccharide does not comprise the E. coli K12 core portion. In another preferred embodiment, O-polysaccharide refers to a structure comprising O-antigen and core saccharide. In another embodiment, O-polysaccharide refers to a structure comprising O-antigen, core saccharide, and KDO portion.

[0119] Methods for purifying O-polysaccharide comprising core oligosaccharide from LPS are known in the art. For example, after purification of LPS, the purified LPS can be hydrolyzed by heating at 100 degrees Celsius in 1% (v / v) acetic acid for 90 minutes followed by ultracentrifugation at 142,000 x g at 4 degrees Celsius for 5 hours. The supernatant containing O-polysaccharide is freeze-dried and stored at 4 degrees Celsius. In certain embodiments, deletion of the capsule synthesis genes is described to enable simple purification of O-polysaccharide.

[0120] O-polysaccharide can be isolated by methods including, but not limited to, mild acid hydrolysis to remove lipid A from LPS. Other embodiments can include the use of hydrazine as a reagent for O-polysaccharide preparation. Preparation of LPS can be accomplished by methods known in the art.

[0121] In certain embodiments, O-polysaccharide purified from a wild-type, modified, or attenuated Gram-negative bacterial strain expressing (not necessarily overexpressing) a Wzz protein (e.g., wzzB) is provided for use in conjugate vaccines. In preferred embodiments, O-polysaccharide chains are purified from a Gram-negative bacterial strain expressing (not necessarily overexpressing) a wzz protein for use as a vaccine antigen as a conjugate or complex vaccine.

[0122] In one embodiment, the O-poly saccharide has a molecular weight that is increased by 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, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64-fold, 65-fold, 66-fold, 67-fold, 68-fold, 69-fold, 70-fold, 71-fold, 72-fold, 73-fold, 74-fold, 75-fold, 76-fold, 77-fold, 78-fold, 79-fold, 80-fold, 81-fold, 82-fold, 83-fold, 84-fold, 85-fold, 86-fold, 87-fold, 88-fold, 89-fold, 90-fold, 91-fold, 92-fold, 93-fold, 94-fold, 95-fold, 96-fold, 97-fold, 98-fold, 99-fold, 100-fold or more compared to the corresponding wild-type O-poly saccharide. In a preferred embodiment, the O-poly saccharide has a molecular weight that is increased by at least 1-fold and at most 5-fold compared to the corresponding wild-type O-poly saccharide. In another embodiment, the O-poly saccharide has a molecular weight that is increased by at least 2-fold and at most 4-fold compared to the corresponding wild-type O-poly saccharide. The increase in the molecular weight of the O-poly saccharide compared to the corresponding wild-type O-poly saccharide is preferably associated with an increase in the number of O-antigen repeat units. In one embodiment, the increase in the molecular weight of the O-poly saccharide is due to a wzz family protein.

[0123] In one embodiment, the O-polysaccharide has a molecular weight that is increased by 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 kDa or more compared to the corresponding wild-type O-polysaccharide. In one embodiment, the O-polysaccharide of the application has a molecular weight that is increased by at least 1 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 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 15 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 18 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 21 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 22 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 30 and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 1 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 5 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 15 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 20 and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 1 and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 5 and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 12 and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 15 and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 18 and at most 75 kDa.In one embodiment, the molecular weight is increased by at least 20 and up to 75 kDa. In one embodiment, the molecular weight is increased by at least 30 and up to 75 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 90 kDa. In one embodiment, the molecular weight is increased by at least 12 and up to 85 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 75 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 70 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 60 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 50 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 49 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 48 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 47 kDa. In one embodiment, the molecular weight is increased by at least 10 and up to 46 kDa. In one embodiment, the molecular weight is increased by at least 20 and up to 45 kDa. In one embodiment, the molecular weight is increased by at least 20 and up to 44 kDa. In one embodiment, the molecular weight is increased by at least 20 and up to 43 kDa. In one embodiment, the molecular weight is increased by at least 20 and up to 42 kDa. In one embodiment, the molecular weight is increased by at least 20 and up to 41 kDa. This 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 repeat 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.

[0124] In another embodiment, the O- polysaccharide comprises any one of the formulae selected from Table 1, wherein the number of repeat units in the O- polysaccharide is none, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, fifty-four, fifty-five, fifty-six, fifty-seven, fifty-eight, fifty-nine, sixty, sixty-one, sixty-two, sixty-three, sixty-four, sixty-five, sixty-six, sixty-seven, sixty-eight, sixty-nine, seventy, seventy-one, seventy-two, seventy-three, seventy-four, seventy-five, seventy-six, seventy-seven, seventy-eight, seventy-nine, eighty, eighty-one, eighty-two, eighty-three, eighty-four, eighty-five, eighty-six, eighty-seven, eighty-eight, eighty-nine, ninety, ninety-one, ninety-two, ninety-three, ninety-four, ninety-five, ninety-six, ninety-seven, ninety-eight, ninety-nine, one hundred or more repeat units than the number of repeat units in the corresponding wild-type O-polysaccharide. 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 repeat units compared to the corresponding wild-type O-polysaccharide. See, e.g., Table 21.

[0125] O-antigen

[0126] O-antigen is part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. O-antigen is on the cell surface and is a variable cell component. The variability of O-antigen provides the basis for serotyping of Gram-negative bacteria. The current E. coli serotyping scheme includes O-polysaccharides 1-181.

[0127] O-antigen comprises oligosaccharide repeat units (O-units) whose wild-type structure typically comprises 2 to 8 residues from a variety of sugars. The O-units of exemplary E. coli O-antigens are shown in Table 1, also see Figure 9A -C and Figure 10A -B.

[0128] In one embodiment, the saccharide of the application can be one oligosaccharide unit. In one embodiment, the saccharide of the application is one repeat oligosaccharide unit of the relevant serotype. In such an embodiment, the saccharide can 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.

[0129] In one embodiment, the saccharide of the application can be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5-15 repeating units) and are generally obtained synthetically or by hydrolysis of polysaccharides. In this embodiment, the saccharide can 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.

[0130] Preferably, all saccharides of the application and all saccharides in immunogenic compositions of the application are polysaccharides. High molecular weight polysaccharides can induce certain antibody immune responses due to epitopes present on the antigenic surface. Isolation and purification of high molecular weight polysaccharides are preferably contemplated for use in conjugates, compositions, and methods of the application.

[0131] 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) is dependent on the wzz chain length regulator, an inner membrane protein. Different wzz proteins confer different ranges of modal lengths (4 to > 100 repeating units). The term "modal length" refers to the number of repeating O-units. Gram-negative bacteria often have two different Wzz proteins, which confer two different OAg modal chain lengths, one longer and one shorter. Expression (not necessarily overexpression) of a wzz family protein (e.g., wzzB) in a Gram-negative bacterium can allow manipulation of the length of O-antigen to alter or bias bacterial production of O-antigens of certain length ranges, as well as to increase production of high-yield, high molecular weight lipopolysaccharides. In one embodiment, "short" modal length as used herein refers to a small number of repeating O-units, e.g., 1-20. In one embodiment, "long" modal length as used herein refers to a number of repeating O-units greater than 20 and up to a maximum of 40. In one embodiment, "very long" modal length as used herein refers to greater than 40 repeating O-units.

[0132] In one embodiment, the saccharide produced has an increase of at least 10 repeating units, 15 repeating units, 20 repeating units, 25 repeating units, 30 repeating units, 35 repeating units, 40 repeating units, 45 repeating units, 50 repeating units, 55 repeating units, 60 repeating units, 65 repeating units, 70 repeating units, 75 repeating units, 80 repeating units, 85 repeating units, 90 repeating units, 95 repeating units, or 100 repeating units compared to the corresponding wild-type O-polysaccharide.

[0133] In another embodiment, the sugars of the present invention have the following ratios compared to the corresponding wild-type O-polysaccharides: 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. An increase of 100 or more repeating units. Preferably, the sugar 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 sugar length are known in the art. As described in Example 5, such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography.

[0134] Methods for determining the number of repeating units in a sugar are also known in the art. For example, the number of repeating units (or the number of repeating units in the formula ""). n The number of repeating units in a conjugate can be calculated by dividing the molecular weight of the polysaccharide (excluding the core sugar or KDO residues) 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 the conjugate, the molecular weight of the carrier protein and the protein:polysaccharide ratio are taken into account in the calculation. As defined herein, " n "" refers to the number of repeating units (indicated in parentheses in Table 1) in the polysaccharide molecule. As is known in the art, in biomacromolecules, repeating structures can be interspersed with regions of imperfect repetition, such as, for example, missing branches. Furthermore, it is known in the art that polysaccharides isolated and purified from natural sources, such as bacteria, can be non-uniform in size and branching. In such cases, nmay represent the average or median of the molecules in the population. n

[0135] In one embodiment, the O-polysaccharide has an increase of at least one repeat unit of O-antigen compared to the corresponding wild-type O-polysaccharide. Table 1 shows the repeat units of O-antigen. In one embodiment, the O-polysaccharide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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 or more total repeat units. Preferably, the sugar has a total of at least 3 to at most 80 repeat units. In another embodiment, the O-polysaccharide has an increase of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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 or more repeat units compared to the corresponding wild-type O-polysaccharide.

[0136] In one embodiment, the sugar comprises an O-antigen, wherein the O-antigen is in any of the O-antigen formulae (as for example, the formulae shown in Table 1 (see also Figure 9A -C and Figure 10A -B). n ​is an integer of 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 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 and any maximum value can be combined 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 a 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.

[0137] In one embodiment, the saccharide comprises an O-antigen, wherein the n is at least 1 and at most 200. In one embodiment, the n is at least 5 and at most 200. In one embodiment, the n is at least 10 and at most 200. In one embodiment, the n is at least 25 and at most 200. In one embodiment, the n is at least 50 and at most 200. In one embodiment, the n is at least 75 and at most 200. In one embodiment, the n is at least 100 and at most 200. In one embodiment, the n is at least 125 and at most 200. In one embodiment, the n is at least 150 and at most 200. In one embodiment, the n is at least 175 and at most 200. In one embodiment, the n is at least 1 and at most 100. In one embodiment, the n is at least 5 and at most 100. In one embodiment, the n is at least 10 and at most 100. In one embodiment, the nis at least 25 and at most 100. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 25 and at most 100. n is at least 50 and at most 100. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 50 and at most 100. n is at least 75 and at most 100. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 75 and at most 100. n is at least 1 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 1 and at most 75. n is at least 5 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 5 and at most 75. n is at least 10 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 10 and at most 75. n is at least 20 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 20 and at most 75. n is at least 25 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 25 and at most 75. n is at least 30 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 30 and at most 75. n is at least 40 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 40 and at most 75. n is at least 50 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 50 and at most 75. n is at least 30 and at most 90. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 30 and at most 90. n is at least 35 and at most 85. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 85. n is at least 35 and at most 75. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 75. n is at least 35 and at most 70. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 70. n is at least 35 and at most 60. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 60. n is at least 35 and at most 50. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 50. n is at least 35 and at most 49. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 49. n is at least 35 and at most 48. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 48. n is at least 35 and at most 47. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 47. n is at least 35 and at most 46. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 35 and at most 46. n is at least 36 and at most 45. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 36 and at most 45. n is at least 37 and at most 44. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 37 and at most 44. n is at least 38 and at most 43. In one embodiment, the number of O-antigen formulas in any of the above embodiments is at least 38 and at most 43.n is at least 39 and at most 42. In one embodiment, the O-antigen formula in any of n is at least 39 and at most 41.

[0138] For example, in one embodiment, the sugar in the O-antigen formula n 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 to at most 60. For example, in one embodiment, n is 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 to 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.

[0139] The sugar structure can be determined by methods and tools known in the art, such as, for example, NMR, including 1D, 1H and / or 13C, 2D TOCSY, DQF-COSY, NOESY and / or HMQC.

[0140] In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight of 5 kDa to 400 kDa. In other such embodiments, the saccharide has a molecular weight of 10 kDa to 400 kDa; 5 kDa to 400 kDa; 5 kDa to 300 kDa; 5 kDa to 200 kDa; 5 kDa to 150 kDa; 10 kDa to 100 kDa; 10 kDa to 75 kDa; 10 kDa to 60 kDa; 10 kDa to 40 kDa; 10 kDa to 100 kDa; 10 kDa to 200 kDa; 15 kDa to 150 kDa; 12 kDa to 120 kDa; 12 kDa to 75 kDa; 12 kDa to 50 kDa; 12 to 60 kDa; 35 kDa to 75 kDa; 40 kDa to 60 kDa; 35 kDa to 60 kDa; 20 kDa to 60 kDa; 12 kDa to 20 kDa; or 20 kDa to 50 kDa. 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; 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 integer within any of the above ranges is contemplated as an embodiment of the disclosure.

[0141] As used herein, the term "molecular weight" of a polysaccharide or 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).

[0142] During normal purification procedures, the size of the polysaccharide can become slightly reduced. Additionally, the polysaccharide can be subjected to sizing techniques prior to conjugation as described herein. Mechanical or chemical sizing can be employed. Chemical hydrolysis can be performed using acetic acid. Mechanical sizing can be performed using high pressure homogenization shearing. The above molecular weight ranges refer to the purified polysaccharide prior to conjugation (e.g., prior to activation).

[0143] Table 1: E. coli serogroups / serotypes and O-unit moieties

[0144] Table 1: E. coli serogroups / serotypes and O-unit moieties

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] β-D-6d man Hep2Ac is 2- O - acetyl-6-deoxy-β-D- mannose - pyranohexosyl.

[0154] β-D-Xul f is β-D- threose - furanopentosyl.

[0155] Core oligosaccharide

[0156] The core oligosaccharide is located between the lipid A and the outer region of the O-antigen in wild-type E. coli LPS. More specifically, the core oligosaccharide is part of the polysaccharide that includes the linkage between the O-antigen and the lipid A in wild-type E. coli. The linkage includes a ketoside bond between the hemiketal function of the innermost 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue and the hydroxyl group of a GlcNAc-residue of the lipid A. The core oligosaccharide region shows a high degree of similarity between wild-type E. coli strains. It usually contains a limited number of sugars. The core oligosaccharide comprises an inner core region and an outer core region.

[0157] More specifically, the inner core is mainly composed of L-glycero-D-manno-heptose (heptose) and KDO residues. The inner core is highly conserved. The KDO residues include KDO of the following formula:

[0158]

[0159] The outer region of the core oligosaccharide displays more variation than the inner core region, and the differences in this region distinguish five chemotypes in E. coli: Rl, R2, R3, R4, and K-12. See Figure 17which illustrates the general structure of the carbohydrate backbone of the five known chemical types of outer core oligosaccharides. Hep II is the last residue of the inner core oligosaccharide. Although all outer core oligosaccharides share the structural theme of having a (hexose)3 carbohydrate backbone and two side chain residues, the order of the hexoses in the backbone and the identity, location, and linkage of the side chain residues can vary. The structures of the Rl and R4 outer core oligosaccharides are highly similar, differing only in a single β-linked residue.

[0160] Based on the structure of the distal oligosaccharide, the core oligosaccharides of wild-type E. coli are classified in the art into five different chemical types: E. coli Rl 、 E. coli R2 、 E. coli R3 、 E. coli R4 and E. coli K12.

[0161] In preferred embodiments, the compositions described herein comprise a complex saccharide, wherein the O-polysaccharide comprises a core oligosaccharide bound to an O-antigen. In one embodiment, the composition induces an immune response against at least any one of the core E. coli chemical types E. coli Rl 、 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 of the core E. coli chemical types. In another embodiment, the composition induces an immune response against at least three of the core E. coli chemical types. In another embodiment, the composition induces an immune response against at least four of the core E. coli chemical types. In another embodiment, the composition induces an immune response against all five of the core E. coli chemical types.

[0162] In another preferred embodiment, the compositions described herein comprise a complex saccharide, wherein the O-polysaccharide does not comprise a core oligosaccharide bound to an O-antigen. In one embodiment, such a composition induces an immune response against at least any one of the core E. coli chemical types E. coli Rl 、 E. coli R2 、 E. coli R3 、 E. coli R4 and E. coli K12, despite the complex saccharide having an O-polysaccharide that does not comprise a core oligosaccharide.

[0163] E. coli serotypes can be characterized according to one of the five chemical types. Table 2 lists exemplary serotypes characterized according to chemical type. The serotypes indicated in bold represent the serotypes most commonly associated with the indicated core chemical type. Thus, in preferred embodiments, the composition induces an immune response against at least any one of the core E. coli chemical types E. coli Rl 、 E. coli R2 、 E. coli R3、 immune response against each respective E. coli serotype of any one of the group.

[0164] Table 2: Core chemotypes and related E. coli serotypes

[0165]

[0166] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, for example, a saccharide selected from the group of 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, wherein n is 1 to 100. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety, for example, as shown in Figure 17

[0167] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, for example, a saccharide selected from the group of 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, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety in the saccharide.

[0168] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R2 chemotype, for example, a saccharide selected from the group of saccharides having formula O21, formula O44, formula O11, formula O89, formula O162, and formula O9, wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli R2 core moiety, for example, as shown in Figure 17

[0169] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R3 chemotype, for example, a saccharide selected from the group of saccharides having formula O25b, formula O15, formula O153, formula O21, formula O17, formula O11, formula O159, formula O22, formula O86, and formula O93, wherein n ​​from 1 to 100, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the sugars in the composition further comprise an E. coli R3 core portion, e.g., as shown in Figure 17

[0170] In some embodiments, the composition comprises sugars comprising structures derived from serotypes having the R4 chemotype, e.g., sugars selected from the group consisting of sugars having formula O2, formula Oi, formula O86, formula O7, formula O102, formula O160, and formula O166, wherein n from 1 to 100, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the sugars in the composition further comprise an E. coli R3 core portion, e.g., as shown in Figure 17

[0171] In some embodiments, the composition comprises sugars comprising structures derived from serotypes having the K-12 chemotype, e.g., sugars selected from the group consisting of sugars having formula O25b and sugars having formula O16, wherein n from 1 to 1000, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65. In some embodiments, the sugars in the composition further comprise an E. coli K-12 core portion, e.g., as shown in Figure 17

[0172] In some embodiments, the sugars comprise core sugars. Thus, in one embodiment, the O- polysaccharide further comprises an E. coli Rl 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.

[0173] In some embodiments, the sugars do not comprise core sugars. Thus, in one embodiment, the O- polysaccharide does not comprise an E. coli Rl core portion. In another embodiment, the O- polysaccharide does not comprise an E. coli R2 core portion. In another embodiment, the O- polysaccharide does not comprise an E. coli R3 core portion. In another embodiment, the O- polysaccharide does not comprise an E. coli R4 core portion. In another embodiment, the O- polysaccharide does not comprise an E. coli K12 core portion.

[0174] Conjugated O-antigens

[0175] ​​​Chemical linkage of O-antigen or preferably O-polysaccharide to a protein carrier can improve the immunogenicity of the O-antigen or O-polysaccharide. However, variability in polymer size represents a practical challenge for production. In commercial use, the size of the saccharide can affect compatibility with different conjugation synthesis strategies, product uniformity, and conjugate immunogenicity. Manipulation of the expression of Wzz family protein chain length regulators by manipulating the O-antigen synthesis pathway creates conditions for production of O-antigen chains of desired length in a variety of Gram-negative bacterial strains, including E. coli.

[0176] In one embodiment, the purified saccharides are chemically activated to produce activated saccharides capable of reacting with carrier proteins. Once activated, each saccharide is separately conjugated to a carrier protein to form a conjugate, i.e., a glycoconjugate. As used herein, the term "glycoconjugate" refers to a saccharide covalently linked to a carrier protein. In one embodiment, the saccharide is directly linked to the carrier protein. In another embodiment, the saccharide is linked to the protein through a spacer / linker.

[0177] The conjugates can be prepared by a protocol that binds the carrier to the O-antigen at one or more sites along the O-antigen or by a protocol that activates at least one residue of the core oligosaccharide.

[0178] In one embodiment, each saccharide is conjugated to the same carrier protein.

[0179] If the protein carrier is the same for 2 or more saccharides in the composition, the saccharides can be conjugated to the same carrier protein molecule (e.g., a carrier molecule with 2 or more different saccharides conjugated thereto).

[0180] In a preferred embodiment, the saccharides are each individually conjugated to a different protein carrier molecule (each protein carrier molecule has only one type of saccharide conjugated thereto). In the described embodiment, the saccharides are considered to be individually conjugated to carrier proteins.

[0181] Chemical activation of the saccharides and subsequent conjugation to carrier proteins can be achieved by the activation and conjugation methods disclosed herein. Following conjugation of the polysaccharides to carrier proteins, the glycoconjugates (enriched relative to the amount of polysaccharide-protein conjugate) are purified by a variety of techniques. These techniques include concentration / diafiltration operations, precipitation / elution, column chromatography, and depth filtration. Following purification of the individual glycoconjugates, they are combined to formulate the immunogenic compositions of the present application.

[0182] Activation. This invention further relates to activated polysaccharides produced by any of the embodiments described herein, wherein the polysaccharides are activated with chemical reagents to generate reactive groups for conjugation to linker or carrier proteins. In some embodiments, the sugars of this invention are activated prior to conjugation to a carrier protein. In some embodiments, the degree of activation does not significantly 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 does not significantly affect the degree of conjugation, such as through conjugation to a carrier protein such as a CRM. 197 The number of modified lysine residues is measured (as determined by amino acid analysis). For example, in some embodiments, the activation level does not significantly increase the number of modified lysine residues in the carrier protein (as determined by amino acid analysis) by 3-fold compared to the number of modified lysine residues in a carrier protein having a reference polysaccharide conjugate at the same activation level. In some embodiments, the activation level does not increase the level of unconjugated free sugars. In some embodiments, the activation level does not decrease the optimal sugar / protein ratio.

[0183] In some embodiments, the activated sugar has an activation percentage, wherein the molar number of thiols / sugar repeating unit of the activated sugar is 1-100%, such as, for example, 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%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, or ≥90%, 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 and any maximum value can be combined to define a range.

[0184] In one embodiment, the polysaccharide is treated with pyridine 1-cyano-4-dimethylaminotetrafluoroborate. (CDAP) activation to form cyanate ester. The activated polysaccharide is then coupled directly or via a spacer (linker) group to a carrier protein (preferably CRM). 197 The amino group on the tetanus toxoid (or tetanus toxoid).

[0185] For example, the spacer can be cystamine or cysteamine to produce a thiolated polysaccharide which can be coupled to a carrier via a thioether linkage after reaction with a maleimide-activated carrier protein (e.g. using N-[Y-maleimidobutyryloxy] succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g. using iodoacetimide, bromoacetic acid N-succinimidyl ester (SBA; SIB), (4-iodoacetyl)aminobenzoic acid N-succinimidyl ester (SIAB), (4-iodoacetyl)aminobenzoic acid sulfo-succinimidyl ester (sulfo-SIAB), iodoacetic acid N-succinimidyl ester (SIA) or 3-[bromoacetylamino]propionic acid succinimidyl ester (SBAP)). In one embodiment, a cyanate ester (optionally prepared by CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatized sugar is conjugated to the carrier protein (e.g. CRM 197 ).

[0186] Other suitable conjugation techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Conjugation can involve a carbonyl linker which can be formed by reaction of a free hydroxyl group of the sugar with CDI followed by reaction with a protein to form a carbamate linkage. This can include reduction of an anomeric end group 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 to an amino group on the protein (CDI chemistry).

[0187] Molecular weight. In some embodiments, the complex saccharide comprises a saccharide having a molecular weight of 10 kDa - 2,000 kDa. In other embodiments, the saccharide has a molecular weight of 50 kDa - 1,000 kDa. In other embodiments, the saccharide has a molecular weight of 70 kDa - 900 kDa. In other embodiments, the saccharide has a molecular weight of 100 kDa - 800 kDa. In other embodiments, the saccharide has a molecular weight of 200 kDa - 600 kDa. In further embodiments, the saccharide has a molecular weight of 100 kDa - 1000 kDa; 100 kDa - 900 kDa; 100 kDa - 800 kDa; 100 kDa - 700 kDa; 100 kDa - 600 kDa; 100 kDa - 500 kDa; 100 kDa - 400 kDa; 100 kDa - 300 kDa; 150 kDa - 1,000 kDa; 150 kDa - 900 kDa; 150 kDa - 800 kDa; 150 kDa - 700 kDa; 150 kDa - 600 kDa; 150 kDa - 500 kDa; 150 kDa - 400 kDa; 150 kDa - 300 kDa; 200 kDa - 1,000 kDa; 200 kDa - 900 kDa; 200 kDa - 800 kDa; 200 kDa - 700 kDa; 200 kDa - 600 kDa; 200 kDa - 500 kDa; 200 kDa - 400 kDa; 200 kDa - 300; 250 kDa - 1,000 kDa; 250 kDa - 900 kDa; 250 kDa - 800 kDa; 250 kDa - 700 kDa; 250 kDa - 600 kDa; 250 kDa - 500 kDa; 250 kDa - 400 kDa; 250 kDa - 350 kDa; 300 kDa - 1,000 kDa; 300 kDa - 900 kDa; 300 kDa - 800 kDa; 300 kDa - 700 kDa; 300 kDa - 600 kDa; 300 kDa - 500 kDa; 300 kDa - 400 kDa; 400 kDa - 1,000 kDa; 400 kDa - 900 kDa; 400 kDa - 800 kDa; 400 kDa - 700 kDa; 400 kDa - 600 kDa; 500 kDa - 600 kDa. In one embodiment, the complex saccharide having such a molecular weight is produced by mono- conjugation. In another embodiment, the complex saccharide having such a molecular weight is produced by reductive amination chemistry (RAC) prepared in aqueous buffer.Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0188] In some embodiments, the complex carbohydrates of the present application have a molecular weight of 400 kDa - 15,000 kDa; 500 kDa - 10,000 kDa; 2,000 kDa - 10,000 kDa; 3,000 kDa - 8,000 kDa; or 3,000 kDa - 5,000 kDa. In other embodiments, the complex carbohydrates have a molecular weight of 500 kDa - 10,000 kDa. In other embodiments, the complex carbohydrates have a molecular weight of 1,000 kDa - 8,000 kDa. In yet other embodiments, the complex carbohydrates have a molecular weight of 2,000 kDa - 8,000 kDa or 3,000 kDa - 7,000 kDa.In further embodiments, the complex carbohydrates of the application have a molecular weight of 200 kDa - 20,000 kDa; 200 kDa - 15,000 kDa; 200 kDa - 10,000 kDa; 200 kDa - 7,500 kDa; 200 kDa - 5,000 kDa; 200 kDa - 3,000 kDa; 200 kDa - 1,000 kDa; 500 kDa - 20,000 kDa; 500 kDa - 15,000 kDa; 500 kDa - 12,500 kDa; 500 kDa - 10,000 kDa; 500 kDa - 7,500 kDa; 500 kDa - 6,000 kDa; 500 kDa - 5,000 kDa; 500 kDa - 4,000 kDa; 500 kDa - 3,000 kDa; 500 kDa - 2,000 kDa; 500 kDa - 1,500 kDa; 500 kDa - 1,000 kDa; 750 kDa - 20,000 kDa; 750 kDa - 15,000 kDa; 750 kDa - 12,500 kDa; 750 kDa - 10,000 kDa; 750 kDa - 7,500 kDa; 750 kDa - 6,000 kDa; 750 kDa - 5,000 kDa; 750 kDa - 4,000 kDa; 750 kDa - 3,000 kDa; 750 kDa - 2,000 kDa; 750 kDa - 1,500 kDa; 1,000 kDa - 15,000 kDa; 1,000 kDa - 12,500 kDa; 1,000 kDa - 10,000 kDa; 1,000 kDa - 7,500 kDa; 1,000 kDa - 6,000 kDa; 1,000 kDa - 5,000 kDa; 1,000 kDa - 4,000 kDa; 1,000 kDa - 2,500 kDa; 2,000 kDa - 15,000 kDa; 2,000 kDa - 12,500 kDa; 2,000 kDa - 10,000 kDa; 2,000 kDa - 7,500 kDa; 2,000 kDa - 6,000 kDa; 2,000 kDa - 5,000 kDa; 2,000 kDa - 4,000 kDa; or 2,000 kDa - 3,000 kDa. In one embodiment, the complex carbohydrates having such molecular weights are produced by eTEC conjugation as described herein. In another embodiment, the complex carbohydrates having such molecular weights are produced by reductive amination chemistry (RAC).In another embodiment, the complex carbohydrate having this molecular weight is produced by reductive amination chemistry (RAC) prepared in DMSO.

[0189] In a further embodiment, the complex carbohydrate of the present application has a molecular weight of 1,000 kDa-20,000 kDa; 1,000 kDa-15,000 kDa; 2,000 kDa-10,000 kDa; 2000 kDa-7,500 kDa; 2,000 kDa-5,000 kDa; 3,000 kDa-20,000 kDa; 3,000 kDa-15,000 kDa; 3,000 kDa-12,500 kDa; 4,000 kDa-10,000 kDa; 4,000 kDa-7,500 kDa; 4,000 kDa-6,000 kDa; or 5,000 kDa-7,000 kDa. In one embodiment, the complex carbohydrate having this molecular weight is produced by reductive amination chemistry (RAC). In another embodiment, the complex carbohydrate having this molecular weight is produced by reductive amination chemistry (RAC) prepared in DMSO. In another embodiment, the complex carbohydrate having this molecular weight is produced by eTEC conjugation as described herein.

[0190] In a further embodiment, the complex carbohydrate of the present application has a molecular weight of 5,000 kDa-20,000 kDa; 5,000 kDa-15,000 kDa; 5,000 kDa-10,000 kDa; 5,000 kDa-7,500 kDa; 6,000 kDa-20,000 kDa; 6,000 kDa-15,000 kDa; 6,000 kDa-12,500 kDa; 6,000 kDa-10,000 kDa or 6,000 kDa-7,500 kDa.

[0191] The molecular weight of the glycoconjugate can be measured by SEC-MALLS. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure. The glycoconjugate of the present invention can also be characterized by the ratio of saccharide to carrier protein (weight / weight). In some embodiments, the ratio of polysaccharide to carrier protein (w / w) in the glycoconjugate is 0.5-3 (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 ratio of saccharide to carrier protein (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 ratio of saccharide to carrier protein (w / w) is 0.8-1.2. In preferred embodiments, the ratio of polysaccharide to carrier protein in the conjugate is 0.9-1.1. In some such embodiments, the carrier protein is CRM 197 .

[0192] The glycoconjugate can also be characterized by its molecular size distribution (K d ). Size exclusion chromatography matrix (CL-4B) can be used to determine the relative molecular size distribution of the conjugate. Size exclusion chromatography (SEC) is used in a gravity flow feed column to graphically represent the molecular size distribution of the conjugate. Larger molecules are eluted faster than smaller molecules from the pores of the media. A fraction collector is used to collect the column eluate. Fractions are tested by saccharide assay colorimetry. For K d determination, the column is calibrated to establish the fraction where molecules are completely excluded (V0), (K d = 0), and the fraction representing maximum retention (V i ), (K d = 1). The fraction (V e ) that achieves the specified sample attribute is related to Kd by the expression K d = (V e - V o ) / (V i - V0).

[0193] Free sugar. The complex saccharide and immunogenic compositions of the present application can comprise free saccharide that is not covalently conjugated to a carrier protein but is still present in the complex saccharide composition. The free saccharide can be non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in) the complex saccharide. In preferred embodiments, the complex saccharide comprises at most 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide compared to the total amount of polysaccharide. In preferred embodiments, the complex saccharide comprises less than about 25% free polysaccharide compared to the total amount of polysaccharide. In preferred embodiments, the complex saccharide comprises at most about 20% free polysaccharide compared to the total amount of polysaccharide. In preferred embodiments, the complex saccharide comprises at most about 15% free polysaccharide compared to the total amount of polysaccharide. In another preferred embodiment, the complex saccharide 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 polysaccharide compared to the total amount of polysaccharide. In preferred embodiments, the complex saccharide comprises less than about 8% free polysaccharide compared to the total amount of polysaccharide. In preferred embodiments, the complex saccharide comprises at most about 6% free polysaccharide compared to the total amount of polysaccharide. In preferred embodiments, the complex saccharide comprises at most about 5% free polysaccharide compared to the total amount of polysaccharide. See, e.g., Tables 12, 13, 14, 15, 16, 17, and 18.

[0194] Covalent bond. In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for 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 6-1 1 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 common embodiments, the carrier protein is CRM 197 . In another embodiment, there is at least one bond between the carrier protein and the saccharide for 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. In one embodiment, the carrier protein is CRM 197 . Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0195] Lysine residues. Another way of characterizing the complex saccharides of the present invention is by the number of lysine residues in the carrier protein (e.g., CRM 197 ) that become conjugated to the saccharide, which can be characterized as a range of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent bonding to the polysaccharide can be obtained by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a decrease in the number of lysine residues recovered compared to the starting material of the carrier protein used to produce the conjugate material. In preferred embodiments, the degree of conjugation of the complex saccharides of the present 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 degree of conjugation of the complex saccharides of the present 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 complex saccharides of the present invention is 4 to 7. In some such embodiments, the carrier protein is CRM 197 .

[0196] The frequency of attachment of the saccharide chain to lysines on the carrier protein is another parameter that characterizes the complex saccharides of the present invention. For example, in some embodiments, there is at least one covalent linkage between the carrier protein and the polysaccharide for every 4 saccharide repeat units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 10 saccharide repeat units of the polysaccharide. In another embodiment, the covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 15 saccharide repeat units of the polysaccharide. In a further embodiment, the covalent linkage 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 present invention are O-acetylated. In some embodiments, the complex saccharides comprise saccharides 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%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, or >90% or about 100%. By % O-acetylated is meant the percentage of a given saccharide relative to 100% where each repeat unit relative to its acetylated structure is fully acetylated.

[0197] In some embodiments, the complex carbohydrate is prepared by reductive amination. In some embodiments, the complex carbohydrate is a mono-end linked conjugated sugar in which the sugar is directly covalently bound to the carrier protein. In some embodiments, the complex carbohydrate is covalently bound to the carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

[0198] Reductive amination. In one embodiment, the sugar is conjugated to the carrier protein by reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 0231340, 2007 / 0184071, and 2007 / 0184072, WO 2006 / 110381, WO 2008 / 079653, and WO 2008 / 143709).

[0199] Reductive amination includes (1) oxidation of the sugar, (2) reduction of the activated sugar and carrier protein to form the conjugate. Prior to oxidation, the sugar is optionally hydrolyzed. Mechanical or chemical hydrolysis can be employed. Chemical hydrolysis can be performed using acetic acid.

[0200] The oxidation step can include reaction with periodic acid. The term "periodic acid" as used herein refers to both periodic acid and metaperiodic acid. The term also includes metaperiodate (IO4 − ) and orthoperiodate (IO6 5− ) as well as various salts of periodic acid (e.g., sodium periodate and potassium periodate). In one embodiment, the polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate (NalO4). In another embodiment, the polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid.

[0201] In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxyl radical compound that selectively oxidizes primary hydroxyl groups in the presence of the oxidizing agent, such as a piperidine-N-oxyl or pyrrolidine-N-oxyl compound. In the reaction, the actual oxidizing agent is an oxoammonium salt in a catalytic cycle. In one aspect, the stable nitroxyl or nitroxyl radical compound is a piperidine-N-oxyl or pyrrolidine-N-oxyl compound. In one aspect, the stable nitroxyl or nitroxyl radical compound bears a TEMPO (2,2,6,6-tetramethyl-l-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-l-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 aspect, the oxidizing agent is selected from any one of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-l,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-l,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-l,3,5-triazinane-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

[0202] Following the oxidation step of the sugar, the sugar is considered activated and is referred to hereinafter as "activated". The activated sugar and the carrier protein can be lyophilized (freeze-dried) independently (separate lyophilization) or together (co-lyophilized). In one embodiment, the activated sugar and the carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and the carrier protein are independently lyophilized.

[0203] In one embodiment, the lyophilization occurs in the presence of a non-reducing sugar, possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit.

[0204] The next step in the conjugation process is the reduction of the activated sugar and the carrier protein using a reducing agent to form the conjugate (so-called reductive amination). Suitable reducing agents include cyanoborohydride, such as sodium cyanoborohydride, sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of a Brønsted or Lewis acid, aminoborane, such as pyridine borane, 2-methylpyridine borane, 2,6-dimethylpyridine-borane, dimethylamine-borane, t-BuMe'PrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), borane-pyridine, or a borohydride exchange resin. In one embodiment, the reducing agent is sodium cyanoborohydride.

[0205] In the implementation scheme, the reduction reaction is carried out in an aqueous solvent (e.g., PBS, MES, HEPES, 2-[bis(2-hydroxyethyl)amino]-2-hydroxymethyl-propane-1,3-diol (Bistris), ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, etc.) at pH 6.0-8.5, 7.0-8.0, or 7.0-7.5. N The reaction is carried out in a solvent containing 2-[hydroxyethyl]glycine or HEPB, or in another embodiment, in an aprotic solvent. In another embodiment, the reduction reaction is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. DMSO or DMF solvents can be used to reconstruct lyophilized activated polysaccharides and carrier proteins.

[0206] At the end of the reduction reaction, unreacted aldehyde groups may remain 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 optional capping), the complex sugar (enriched relative to the amount of the polysaccharide-protein conjugate) can be purified using a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / percolation operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. The complex sugar can be purified by percolation and / or ion exchange chromatography and / or size exclusion chromatography. In an embodiment, the complex sugar is purified by percolation or ion exchange chromatography or size exclusion chromatography. In one embodiment, the complex sugar is sterilely filtered.

[0207] In a preferred embodiment, the complex sugar from any one of the Escherichia coli serotypes selected from O25B, O1, O2, and O6 is prepared by reductive amination.

[0208] In one aspect, the present invention relates to conjugates comprising, and composed of, by means of,

[0209]

[0210] The glycocarrier protein represented by formula O25B, such as CRM 197 ,in n It is any integer greater than or equal to 1. In the preferred embodiment, nis an integer greater than or equal to 1. n is at least 31 to at most 90, more preferably 40 to 90, most preferably 60 to 85.

[0211] In another aspect, the present application relates to conjugates comprising a carrier protein, e.g., CRM197, linked to a saccharide having any one of the following structures shown in Table 1 (see also Figure 9A -C and Figure 10A -B) is a carrier protein, e.g., CRM197. 197 wherein n is an integer greater than or equal to 1.

[0212] Without being bound by theory or mechanism, in some embodiments, it is believed that stable conjugates require a certain level of saccharide antigen modification that is balanced with the structural integrity of key immunogenic epitopes of the antigen.

[0213] Activation and formation of aldehydes. In some embodiments, the saccharides of the present application are activated and result in the formation of aldehydes. In such embodiments where the saccharides are activated, the percentage (%) of activation (or degree of oxidation (DO)) refers to the moles of saccharide repeating units / moles of aldehyde of the activated polysaccharide. For example, in some embodiments, the saccharides are activated by periodate oxidation of vicinal diols on the repeating units of the polysaccharide, resulting in the formation of aldehydes. Different levels of degree of oxidation (DO) result from varying the molar equivalents (meq) of sodium periodate to saccharide repeating units and temperature during oxidation.

[0214] The concentration of saccharides and aldehydes is generally determined by colorimetric assays. An alternative reagent is the TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl radical)-N-chlorosuccinimide (NCS) combination, which results in the formation of aldehydes from primary alcohol groups.

[0215] In some embodiments, the activated sugar has a degree of oxidation such that the number of moles of sugar repeat units per number of moles of aldehyde of the activated sugar is 1-100, such as, for example, 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, >30, >40, >50, >60, >70, >80, or >90, 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 degree of oxidation value can be expressed as a percentage (%) of activation. For example, in one embodiment, a DO value of 10 means that one activated sugar repeat unit out of a total of 10 sugar repeat units in the activated sugar, in which case the DO value of 10 can be expressed as 10% activation.

[0216] In some embodiments, conjugates prepared by reductive amination chemistry comprise a carrier protein and a saccharide, wherein the saccharide comprises a saccharide selected from 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 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 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g.,the structure of any one of 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 0111, 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 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 O163, 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. In some embodiments, the sugar in the conjugate comprises the structure of any one of 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 0111, 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 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 O163, 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 from 1 to 1000, 5 to 1000, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65.

[0217] Single-end linked conjugates. In some embodiments, the conjugate is a single-end linked conjugated saccharide, wherein the saccharide is covalently bound to the carrier protein at one end of the saccharide. In some embodiments, the single-end linked conjugated polysaccharide has a terminal saccharide. For example, if one of the termini of the polysaccharide (a terminal saccharide residue) is covalently bound to the carrier protein, then the conjugate is single-end linked. In some embodiments, the conjugate is single-end linked if the terminal saccharide residue of the polysaccharide is covalently bound to the carrier protein via a linker. Such linkers can include, for example, a cystamine linker (Al), a 3,3'-dithio bis(propanoic dihydrazide) linker (A4), and a 2,2'-dithio-N,N'-bis(ethane-2, 1 -diyl)bis(2- (aminooxy)acetamide) linker (A6).

[0218] In some embodiments, the saccharide is conjugated to the carrier protein via a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue to form a single-end linked conjugate. See, for example, Example 18, Example 19, Example 20, and Figure 24 .

[0219] In some embodiments, the conjugate is preferably not a bioconjugate. The term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an antigen, such as an O antigen (e.g., O25B), prepared in a host cell context, wherein the host cell means links (e.g., N-links) the antigen to the protein. Complex saccharides include bioconjugates, as well as saccharide antigen (e.g., oligosaccharides and polysaccharides)-protein conjugates prepared by means other than requiring preparation of the conjugate in a host cell, such as, for example, conjugation by chemical bonding of the protein and saccharide.

[0220] Thiol-activated saccharides. In some embodiments, the saccharides of the present application are thiol-activated. In such embodiments where the saccharide is thiol-activated, the percentage (%) of activation refers to the moles of thiol per saccharide repeating unit of the activated polysaccharide. Saccharide and thiol concentrations are generally determined by the Ellman's assay for thiol quantitation. For example, in some embodiments, the saccharide includes 2-keto-3-deoxyoctonic acid (KDO) activated with a disulfide amine linker. See, for example, Example 18 and Figure 24 In some embodiments, the saccharide is covalently bound to the carrier protein via a bivalent, heterobifunctional linker (also referred to herein as a "spacer"). The linker preferably provides a thioether bond between the saccharide and the carrier protein, resulting in a complex saccharide referred to herein as a "thioether complex saccharide." In some embodiments, the linker further provides a carbamate and amide bond, such as, for example, (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC). See, for example, Example 13.

[0221] In some embodiments, the single-end linked conjugate comprises a carrier protein and a saccharide, wherein the saccharide comprises a saccharide selected from 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 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 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g.,the structure of any one of 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 0111, 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 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 O163, 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. In some embodiments, the sugar in the conjugate comprises the structure of any one of 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 0111, 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 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 O163, 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 from 1 to 1000, 5 to 1000, preferably 31 to 100, more preferably 35 to 90, most preferably 35 to 65.

[0222] For example, in one embodiment, the single-end linked conjugate comprises a carrier protein and a sugar having a structure selected from the group consisting of Formula O8, Formula O9a, Formula O9, Formula O20ab, Formula O20ac, Formula O52, Formula O97, and Formula O101, wherein n is an integer from 1 to 10

[0223] eTEC conjugate

[0224] In one aspect, the present invention relates generally to complex saccharides comprising a saccharide derived from E. coli as described above (as described, for example, in U.S. Patent 9517274 and International Patent Application Publication WO2014027302, which are incorporated herein by reference in their entireties) covalently conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl) carbamate (eTEC) spacer, including immunogenic compositions comprising such complex saccharides, and methods for making and using such complex saccharides and immunogenic compositions. The complex saccharide comprises a saccharide covalently conjugated to a carrier protein via one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer via a carbamate linkage, and wherein the carrier protein is covalently conjugated to the eTEC spacer via an amide linkage. The eTEC spacer comprises seven linear atoms (i.e., -C(0)NH(CH2)2SCH2C(0)-) and provides stable thioether and amide linkages between the saccharide and the carrier protein.

[0225] The eTEC-linked complex saccharides of the present invention can be represented by the general formula (I):

[0226]

[0227] wherein the atoms comprising the eTEC spacer are contained in the central box.

[0228] In the complex saccharides of the present invention, the saccharide can be a polysaccharide or an oligosaccharide.

[0229] The carrier protein incorporated into the complex saccharides of the present invention is selected from carrier proteins generally suitable for such purposes, as further described herein or known to those skilled in the art. In particular embodiments, the carrier protein is CRM197 197 .

[0230] In another aspect, the present invention provides a method of making a complex saccharide comprising a saccharide described herein conjugated to a carrier protein via an eTEC spacer, comprising the steps of: a) reacting the saccharide with a carbonic acid derivative in an organic solvent to produce an activated saccharide; b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free thiol residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more a-haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated a-haloacetamide groups of the activated carrier protein; and / or (ii) a second capping reagent capable of capping unconjugated free thiol residues of the activated thiolated saccharide; thereby producing an eTEC-linked complex saccharide.

[0231] In common embodiments, the carbonic acid derivative is l,l'-carbonyl-di-(l,2,4- triazole) (CDT) or l,l'-carbonyldiimidazole (CDI). Preferably, the carbonic acid derivative is CDT, and the organic solvent is a polar aprotic solvent, such as dimethyl sulfoxide (DMSO). In preferred embodiments, the thiolated sugar is produced by reaction of the activated sugar with a bifunctional symmetrical thioalkyl amine reagent, cystamine or a salt thereof. In another aspect, the thiolated sugar can be formed by reaction of the activated sugar with cysteamine or a salt thereof. The eTEC-linked complex sugars produced by the methods of the present application can be represented by general formula (I).

[0232] In common embodiments, the first capping reagent is N-acetyl-L-cysteine, which reacts with unconjugated a-haloacetamide groups on lysine residues of the carrier protein to form S-carboxymethylcysteine (CMC) residues covalently linked to the activated lysine residues via a thioether linkage.

[0233] In other embodiments, the second capping reagent is iodoacetamide (IAA), which reacts with unconjugated free thiol groups of the activated thiolated sugar to provide a capped thioacetamide. Step e) often includes capping with both the first capping reagent and the second capping reagent. In certain embodiments, step e) includes capping with N-acetyl-L-cysteine as the first capping reagent and IAA as the second capping reagent.

[0234] In some embodiments, the capping step e) further includes reaction with a reducing agent, such as DTT, TCEP, or mercaptoethanol, after reaction with the first and / or second capping reagent.

[0235] The eTEC-linked complex sugars and immunogenic compositions of the present application can comprise free thiol residues. In some cases, the activated thiolated sugar formed by the methods provided herein will comprise a plurality of free thiol residues, some of which can not undergo covalent conjugation to the carrier protein during the conjugation step. Such remaining free thiol residues are capped by reaction with a thiol-reactive capping reagent (e.g., iodoacetamide (IAA)) to cap the potential reactivity functionality. Other thiol-reactive capping reagents are also contemplated, such as maleimide-containing reagents, and the like.

[0236] Additionally, the eTEC-linked complex sugars and immunogenic compositions of the present application can comprise remaining unconjugated carrier protein, which can include activated carrier protein that has undergone modification during the capping process step.

[0237] In some embodiments, step d) further comprises providing an activated carrier protein comprising one or more a-haloacetamide groups prior to reacting the activated thiolated saccharide with the activated carrier protein. In a common embodiment, the activated carrier protein comprises one or more a-bromoacetamide groups.

[0238] In another aspect, the present application provides an eTEC-linked complex saccharide produced according to any one of the methods disclosed herein, comprising a saccharide described herein conjugated to a carrier protein via an eTEC spacer.

[0239] In some embodiments, the carrier protein is CRM 197 and occurs at least once in every 4, 10, 15, or 25 saccharide repeat units of the polysaccharide, covalent bonding between the CRM 197 and the polysaccharide via an eTEC spacer.

[0240] For each aspect of the present application, in particular embodiments of the methods and compositions described herein, the eTEC-linked complex saccharide comprises a saccharide described herein, e.g., derived from E. coli.

[0241] In another aspect, the present application provides a method of preventing, treating, or ameliorating a bacterial infection, disease, or condition in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present application, wherein the immunogenic composition comprises an eTEC-linked complex saccharide comprising a saccharide described herein. In some embodiments, the saccharide is derived from E. coli.

[0242] In some embodiments, the eTEC-linked complex saccharide comprises a carrier protein and a saccharide, wherein the saccharide comprises a saccharide selected from 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 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 62D1, 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 O10 4. Equations O105, O106, O107, O108, O109, O110, O111, O112, O113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O132, O13 3. Equations O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O1 61. The structure of any one of formulas O162, 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. In some embodiments, the sugar in the conjugate comprises the formula, wherein, n It is an integer from 1 to 1000, 5 to 1000, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65.

[0243] The number of lysine residues in a carrier protein that becomes sugar-conjugated can be characterized as the range of conjugated lysine residues. For example, in some embodiments of the immunogenic composition, CRM 197 It may contain 4 to 16 lysine residues out of 39 covalently linked to the sugar. Another way to represent these parameters is as approximately 10% to approximately 41% CRM. 197 Lysine is covalently linked to the sugar. In other implementations, CRM 197 It may contain 2 to 20 lysine residues from 39 covalently linked to the sugar. Another way to represent these parameters is as approximately 5% to approximately 50% CRM.197 Lysine is covalently linked to sugar.

[0244] In a common implementation, the carrier protein is CRM. 197 And at least once in every 4, 10, 15 or 25 sugar repeating units of the polysaccharide in CRM 197 Covalent bonding between polysaccharides and eTEC spacers.

[0245] In other embodiments, for every 5-10 sugar repeat units; every 2-7 sugar repeat units; every 3-8 sugar repeat units; every 4-9 sugar repeat units; every 6-11 sugar repeat units; every 7-12 sugar repeat units; every 8-13 sugar repeat units; every 9-14 sugar repeat units; every 10-15 sugar repeat units; every 2-6 sugar repeat units; every 3-7 sugar repeat units; every 4-8 sugar repeat units; every 6-10 sugar repeat units; every 7-11 sugar repeat units; every 8-12 sugar repeat units; every 9-13 sugar repeat units; every 10-14 sugar repeat units; every 10-20 sugar repeat units; or every 4-25 sugar repeat units, the conjugate contains at least one covalent bond between the carrier protein and the sugar.

[0246] In another embodiment, for 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 sugar repeating units of the polysaccharide, there is at least one bond between the carrier protein and the sugar.

[0247] carrier proteins

[0248] The component of the complex sugar of this invention is a carrier protein conjugated with the sugar. The terms "protein carrier," "carrier protein," or "carrier" are used interchangeably herein. The carrier protein should be modifiable for standard conjugation procedures.

[0249] One component of the conjugate is a carrier protein conjugated to the O-polysaccharide. In one embodiment, the conjugate comprises a carrier protein conjugated to the core oligosaccharide of the O-polysaccharide (see [link to previous embodiment]). Figure 17 In one embodiment, the conjugate comprises a carrier protein conjugated to the O-antigen of the O-polysaccharide.

[0250] The terms “protein carrier”, “carrier protein”, or “carrier” are used interchangeably in this document. The carrier protein should be modifiable for standard conjugation procedures.

[0251] In a preferred embodiment, the carrier protein of the conjugate is independently selected from TT, DT, DT mutants (e.g., CRM). 197 Haemophilus influenzae ( ) H. influenzaePhtD protein, PhtX, PhtD, PhtDE fusions (especially those described in WO 01 / 98334 and WO 03 / 54007), detoxifying pneumococcal hemolysin, PorB, N19 protein, PspA, OMPC, Clostridium difficile ( C. Difficile The conjugate contains either toxin A or B and PsaA. In one embodiment, the carrier protein of the conjugate is DT (diphtheria toxoid). In another embodiment, the carrier protein of the conjugate is TT (tetanus toxoid). In yet another embodiment, the carrier protein of the conjugate is PD (Haemophilus influenzae D protein – see, for example, EP 0 594 610 B).

[0252] In a preferred embodiment, sugar is combined with CRM. 197 Protein conjugation. CRM 197 The protein is a non-toxic form of diphtheria toxin, but it is immunologically indistinguishable from diphtheria toxin. (CRM) 197 It is a non-toxigenic phage β197tox produced by mutagenesis of toxigenic Corynebacterium phage β via nitrosoguanidine. - Corynebacterium diphtheriae infection ( C. diphtheriae This is generated by CRM. 197 The protein has the same molecular weight as diphtheria toxin, but differs from it in its structural genes by a single base change (guanine to adenine). This single base change results in the substitution of glycine for glutamic acid in the mature protein, eliminating the toxic properties of diphtheria toxin. 197 Proteins are safe and effective T-cell-dependent carriers for glucose.

[0253] Therefore, in some embodiments, the conjugate of the present invention comprises CRM as a carrier protein. 197 Sugar and CRM 197 Covalent connection.

[0254] In preferred embodiments, the carrier protein of the complex sugar is selected from DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM197 (a non-toxic but antigenically identical variant of diphtheria toxin), other DT mutants (e.g. CRM176, CRM228, CRM 45 (Uchida et al., J. Biol. Chem. 218; 3838-3844, 1973), CRM9, CRM45, CRM102, CRM103 or CRM107; and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gin or Ser and / or Ala 158 to Gly and other mutations disclosed in US 4709017 or US 4950740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations disclosed in US 5917017 or US 6455673; or fragments disclosed in US 5843711), pneumococcal pneumolysin (Kuo et al. (1995) Infect lmmun 63; 2706-13), including ply detoxed in some way, e.g. dPLY-GMBS (WO 04081515, PCT / EP2005 / 010258) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00 / 37105 or WO 00 / 39299) and fusions of Pht proteins, e.g. PhtDE fusions, PhtBE fusions, Pht A-E (WO 01 / 98334, WO 03 / 54007, WO 2009 / 000826), OMPC (meningococcal outer membrane protein - typically from Neisseria meningitidis (N.meningitidis) serogroup B extract - EP 0 372 501), PorB (from Neisseria meningitidis), PD (influenzae D protein - see, e.g., EP 0 594 610 B) or immunologically functional equivalents thereof, synthetic peptides (EP 0 378 881, EP 0 427 347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP 0 471 177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), artificial proteins comprising various human CD4+ T cell epitopes derived from antigens from various pathogens (Falugi et al. (2001) Eur J Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al. (2004) Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO 02 / 091998), iron uptake proteins (WO 01 / 72337), toxin A or B of C. difficile (WO 00 / 61761), transferrin binding proteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (in particular a non-toxic mutant thereof (e.g. exotoxin A with a substitution at glutamic acid 553 (Uchida Cameron DM, RJ Collier. 1987. J. Bacteriol. 169:4967-4971)). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxin (e.g. as described in International Patent Application No. WO 2004 / 083251), E. coli LT, E. coli ST and exotoxin A from Pseudomonas aeruginosa.

[0255] In some embodiments, the carrier protein is selected from, for example, CRM 197 197, 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) of Pseudomonas aeruginosa, maltose binding protein (MBP), flagellin, detoxified hemolysin A of Staphylococcus aureus (S. aureus), clumping factor A, clumping factor B, cholera toxin B subunit (CTB), pneumolysin of Streptococcus pneumoniae (S. pneumoniae) and detoxified variants thereof, heat shock proteins of Campylobacter jejuni (C. jejuni), and others. S. aureus Streptococcus pneumoniae ​​C. jejuni ) AcrA and any of the natural glycoproteins of C. jejuni. In one embodiment, the carrier protein is detoxified Pseudomonas exotoxin (EPA). In another embodiment, the carrier protein is not detoxified Pseudomonas exotoxin (EPA). In one embodiment, the carrier protein is flagellin. In another embodiment, the carrier protein is not flagellin.

[0256] In preferred embodiments, the carrier protein of the complex saccharide is independently selected from TT, DT, a DT mutant (e.g. CRM 197 197), influenza haemophilus D protein, PhtX, PhtD, PhtDE fusion (in particular those described in WO 01 / 98334 and WO 03 / 54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B of C. difficile and PsaA. In embodiments, the carrier protein of the complex saccharide of the application is DT (diphtheria toxoid). In another embodiment, the carrier protein of the complex saccharide of the application is TT (tetanus toxoid). In another embodiment, the carrier protein of the complex saccharide of the application is PD (Haemophilus influenzae D protein - see, for example, EP 0 594 610 B).

[0257] In preferred embodiments, the capsular saccharide of the application is conjugated to CRM 197 197 protein. CRM 197 197 protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. CRM 197 197 was produced by Corynebacterium diphtheriae infected with the non-toxic phage beta 197 tox- produced by nitrosoguanidine mutagenesis of the producer phage beta (Uchida, T. et al., 1971, Nature New Biology 233:8-11). CRM 197 197 protein has the same molecular weight as diphtheria toxin, but differs from it by a single base change (guanine to adenine) in the structural gene. This single base change results in an amino acid substitution of glutamic acid for glycine in the mature protein and abolishes the toxic properties of diphtheria toxin. CRM 197 197 protein is a safe and effective T-cell dependent carrier for saccharides. Further details regarding CRM 197 197 and its production can be found, for example, in US 5,614,382.

[0258] Thus, in common embodiments, the complex saccharide of the application comprises CRM 197 197 as the carrier protein, wherein the capsular polysaccharide is covalently linked to CRM 197 197.

[0259] Compositions and vaccines

[0260] The inventors have further discovered compositions comprising at least one of the above sugars and compositions comprising at least one of the above conjugates. In a preferred embodiment, the composition is an immunogenic composition. In another embodiment, the composition is a vaccine.

[0261] In one aspect, the immunogenic composition comprises any one of the sugars disclosed herein. In a preferred aspect, the immunogenic composition comprises any one of the conjugates disclosed herein.

[0262] In one embodiment, the immunogenic composition comprises at least one complex sugar from E. coli serogroup O25, preferably serogroup O25b. In one embodiment, the immunogenic composition comprises at least one complex sugar from E. coli serogroup O1, preferably serogroup O1a. In one embodiment, the immunogenic composition comprises at least one complex sugar from E. coli serogroup O2. In one embodiment, the immunogenic composition comprises at least one complex sugar from E. coli serogroup O6.

[0263] In one embodiment, the immunogenic composition comprises at least one complex sugar from any one of the following E. coli serogroups O25, O1, O2 and O6, preferably O25b, O1a, O2 and O6. In one embodiment, the immunogenic composition comprises at least two complex sugars from any one of the following E. coli serogroups O25, O1, O2 and O6, preferably O25b, O1a, O2 and O6. In one embodiment, the immunogenic composition comprises at least three complex sugars from any one of the following E. coli serogroups O25, O1, O2 and O6, preferably O25b, O1a, O2 and O6. In one embodiment, the immunogenic composition comprises a complex sugar from each of the following E. coli serogroups O25, O1, O2 and O6, preferably O25b, O1a, O2 and O6.

[0264] In a preferred embodiment, the complex sugars of any one of the above immunogenic compositions are each conjugated to CRM 197

[0265] ​Accordingly, the composition comprises O-antigen from at least one E. coli serotype. In preferred embodiments, the composition comprises O-antigen from more than one E. coli serotype. For example, the composition can comprise O-antigen from two different E. coli serotypes (or "v", valency) up to 12 different serotypes (12v). In one embodiment, the composition comprises O-antigen from 3 different serotypes. In one embodiment, the composition comprises O-antigen from 4 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 5 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 6 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 7 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 8 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 9 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 10 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 11 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 12 different serotypes. In one embodiment, the composition comprises O-antigen from 13 different serotypes. In one embodiment, the composition comprises O-antigen from 14 different serotypes. In one embodiment, the composition comprises O-antigen from 15 different serotypes. In one embodiment, the composition comprises O-antigen from 16 different serotypes. In one embodiment, the composition comprises O-antigen from 17 different serotypes. In one embodiment, the composition comprises O-antigen from 18 different serotypes. In one embodiment, the composition comprises O-antigen from 19 different serotypes. In one embodiment, the composition comprises O-antigen from 20 different serotypes.

[0266] Preferably, the number of E. coli saccharides can range from 1 serotype (or "v", valency) up to 26 different serotypes (26v). In one embodiment, there is one serotype. In one embodiment, there are 2 different serotypes. In one embodiment, there are 3 different serotypes. In one embodiment, there are 4 different serotypes. In one embodiment, there are 5 different serotypes. In one embodiment, there are 6 different serotypes. In one embodiment, there are 7 different serotypes. In one embodiment, there are 8 different serotypes. In one embodiment, there are 9 different serotypes. In one embodiment, there are 10 different serotypes. In one embodiment, there are 11 different serotypes. In one embodiment, there are 12 different serotypes. In one embodiment, there are 13 different serotypes. In one embodiment, there are 14 different serotypes. In one embodiment, there are 15 different serotypes. In one embodiment, there are 16 different serotypes. In one embodiment, there are 17 different serotypes. In one embodiment, there are 18 different serotypes. In one embodiment, there are 19 different serotypes. 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 saccharides are conjugated to a carrier protein to form a conjugate saccharide as described herein.

[0267] In one aspect, the composition comprises a complex saccharide comprising an O-antigen from at least one E. coli serogroup, wherein the O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from more than one E. coli serotype, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 3 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 4 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 5 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 6 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 7 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 8 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 9 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 10 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 11 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 12 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 13 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 14 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 15 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 16 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 17 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 18 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 19 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises O-antigen from 20 different serotypes, wherein each O-antigen is conjugated to a carrier protein.

[0268] In another aspect, the composition comprises O-polysaccharides from at least one E. coli serotype. In preferred embodiments, the composition comprises O-polysaccharides from more than one E. coli serotype. For example, the composition can comprise O-polysaccharides from two different E. coli serotypes to 12 different E. coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 3 different E. coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 4 different E. coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 5 different E. coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 6 different E. coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 7 different E. coli serotypes. In one embodiment, the composition comprises O-polysaccharides from 8 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.

[0269] In preferred embodiments, the composition comprises an O-polysaccharide from at least one E. coli serotype, wherein the O-polysaccharide is conjugated to a carrier protein. In preferred embodiments, the composition comprises O-polysaccharides from more than one E. coli serotype, wherein each O-polysaccharide is conjugated to a carrier protein. For example, the composition can comprise O-polysaccharides from two different E. coli serotypes to 12 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 3 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 4 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 5 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 6 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 7 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 8 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 9 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 10 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 11 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 12 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 13 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 14 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 15 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 16 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 17 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 18 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharides from 19 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein.In one embodiment, the composition comprises O-polysaccharides from 20 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein.

[0270] In the most preferred embodiment, the composition comprises O-polysaccharides from at least one E. coli serotype, wherein the O-polysaccharides are conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In a preferred embodiment, the composition comprises O-polysaccharides from more than one E. coli serotype, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. For example, the composition can comprise O-polysaccharides from two different E. coli serotypes to 12 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 3 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 4 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 5 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 6 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 7 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 8 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 9 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 10 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 11 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 12 different E. coli serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharides comprise an O-antigen and a core saccharide.In one embodiment, the composition comprises O-polysaccharides from 13 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O- polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 14 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 15 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 16 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 17 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 18 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 19 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 20 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In a preferred embodiment, the carrier protein is CRM. 197 .

[0271] In another preferred embodiment, the composition comprises O-polysaccharides conjugated to CRM 197 , wherein the O-polysaccharide comprises the formula O25a, wherein n is at least 40, and a core saccharide. In a preferred embodiment, the composition further comprises O-polysaccharides conjugated to CRM 197 , wherein the O-polysaccharide comprises the formula O25b, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises O-polysaccharides conjugated to CRM 197 , wherein the O-polysaccharide comprises the formula Ola, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises O-polysaccharides conjugated to CRM 197 , wherein the O-polysaccharide comprises the formula O2, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises O-polysaccharides conjugated to CRM 197conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O6, wherein n is at least 40, and a core saccharide.

[0272] In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O17, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O15, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O18A, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O75, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O4, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O16, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O13, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O7, wherein n is at least 40, and a core saccharide.

[0273] In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O8, wherein n is at least 40, and a core saccharide. In another embodiment, the O-polysaccharide comprises Formula O8, wherein n is 1-20, preferably 2-5, more preferably 3. Formula O8 is shown, for example, in Figure 10B In another embodiment, the composition further comprises a CRM 197 conjugated O-polysaccharide, wherein the O-polysaccharide comprises Formula O9, whereinn is at least 40, and a core saccharide. In another embodiment, the O- polysaccharide comprises Formula O9, wherein n is 1-20, preferably 4-8, more preferably 5. Formula O9 is shown, for example, in Figure 10B . In another embodiment, the O-poly saccharide comprises Formula O9a, wherein n is 1-20, preferably 4-8, more preferably 5. Formula O9a is shown, for example, in Figure 10B .

[0274] In some embodiments, the O-poly saccharide comprises any one selected from Formula O20ab, Formula O20ac, Formula O52, Formula O97, and Formula O101, wherein n is 1-20, preferably 4-8, more preferably 5. See, for example, Figure 10B .

[0275] As described above, the composition can comprise any combination of conjugated O- polysaccharides (antigens). 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, for example, a composition comprising: (i) an O-poly saccharide conjugated to CRM 197 , wherein the O-poly saccharide comprises Formula O25b, wherein n is at least 40, and a core saccharide; (ii) an O-poly saccharide conjugated to CRM 197 , wherein the O-poly saccharide comprises Formula O1a, wherein n is at least 40, and a core saccharide; (iii) an O-poly saccharide conjugated to CRM 197 , wherein the O-poly saccharide comprises Formula O2, wherein n is at least 40, and a core saccharide; and (iv) an O-poly saccharide conjugated to CRM 197 , wherein the O-poly saccharide comprises Formula O6, wherein n is at least 40, and a core saccharide.

[0276] In one embodiment, the composition comprises at least one O-poly saccharide derived from any E. coli serotype, wherein the serotype is not O25a. For example, in one embodiment, the composition does not comprise a saccharide comprising Formula O25a. Such a composition can comprise, for example, an O-poly saccharide comprising Formula O25b, an O-poly saccharide comprising Formula O1A, an O-poly saccharide comprising Formula O2, and an O-poly saccharide comprising Formula O6.

[0277] In one embodiment, the composition comprises O-poly saccharides from 2 different E. coli serotypes, wherein each O-poly saccharide is conjugated to CRM 197conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 3 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 4 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 5 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 6 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 7 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 8 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 9 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 10 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 11 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 12 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 13 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197 conjugated, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-polysaccharides from 14 different E. coli serotypes, wherein each O-polysaccharide is conjugated to CRM 197conjugated, and wherein the O-poly saccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-poly saccharides from 15 different serotypes, wherein each O-poly saccharide is conjugated to CRM 197 conjugated, and wherein the O-poly saccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-poly saccharides from 15 different serotypes, wherein each O-poly saccharide is conjugated to CRM 197 conjugated, and wherein the O-poly saccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-poly saccharides from 15 different serotypes, wherein each O-poly saccharide is conjugated to CRM 197 conjugated, and wherein the O-poly saccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-poly saccharides from 15 different serotypes, wherein each O-poly saccharide is conjugated to CRM 197 conjugated, and wherein the O-poly saccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-poly saccharides from 15 different serotypes, wherein each O-poly saccharide is conjugated to CRM 197 conjugated, and wherein the O-poly saccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises O-poly saccharides from 15 different serotypes, wherein each O-poly saccharide is conjugated to CRM 197 conjugated, and wherein the O-poly saccharide comprises an O-antigen and a core saccharide.

[0278] In one aspect, the present application relates to a composition comprising a conjugate, said conjugate comprising a saccharide covalently bound to a carrier protein, wherein said saccharide comprises the formula O25b, wherein n is 15 ± 2. In one aspect, the present application relates to a composition comprising a conjugate, said conjugate comprising a saccharide covalently bound to a carrier protein, wherein said saccharide comprises the formula O25b, wherein n is 17 ± 2. In one aspect, the present application relates to a composition comprising a conjugate, said conjugate comprising a saccharide covalently bound to a carrier protein, wherein said saccharide comprises the formula O25b, wherein n is 55 ± 2. In another aspect, the present application relates to a composition comprising a conjugate, said conjugate comprising a saccharide covalently bound to a carrier protein, wherein said saccharide comprises the formula O25b, wherein n is 51 ± 2. In one embodiment, the saccharide further comprises an E. coli Rl 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 CRM 197In one embodiment, the conjugate is prepared by conjugation of a single end. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0279] In one embodiment, the immunogenic composition elicits in humans IgG antibodies capable of binding 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- and post-immunization sera with the immunogenic composition of the application can be performed and their response to serotype O25B compared to assess potential increase in responders. In one embodiment, the immunogenic composition elicits in humans IgG antibodies capable of killing E. coli serotype O25B as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits in humans functional antibodies capable of killing E. coli serotype O25B as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition of the application increases the proportion of responders (i.e. individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to E. coli serotype O25B compared to the pre-immunization population. In one embodiment, the immunogenic composition elicits a titer of at least 1 :8 to E. coli serotype O25B in at least 50% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the application elicits a titer of at least 1 :8 to E. coli serotype O25B in at least 60%, 70%, 80% or at least 90% of the subjects as determined by in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the application significantly increases the proportion of responders (i.e. individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to E. coli serotype O25B compared to the pre-immunization population. In one embodiment, the immunogenic composition of the application significantly increases the OPA titer to E. coli serotype O25B in human subjects compared to the pre-immunization population.

[0280] In one aspect, the application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises the formula Ola, wherein nis 39 ± 2. In another aspect, the present application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula Ol a, wherein n is 13 ± 2. In one embodiment, the saccharide further comprises an E. coli Rl core saccharide moiety. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM 197 . In one embodiment, the conjugate is prepared by single-end ligation conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein by a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0281] In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of binding 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- and post-immunization sera with the immunogenic compositions of the application can be made and their responses to serotype O1A compared to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing E. coli serotype O1A as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing E. coli serotype O1A as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic compositions of the application increase the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to E. coli serotype O1A compared to the pre-immunization population. In one embodiment, the immunogenic composition elicits a titer of at least 1 :8 to E. coli serotype O1A in at least 50% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the application elicit a titer of at least 1 :8 to E. coli serotype O1A in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the application significantly increase the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to E. coli serotype O1A compared to the pre-immunization population. In one embodiment, the immunogenic compositions of the application significantly increase the OPA titer of human subjects to E. coli serotype O1A compared to the pre-immunization population.

[0282] In one aspect, the application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O2, wherein n is 43 ± 2. In another aspect, the application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O2, wherein n is 47 ± 2. In another aspect, the application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O2, wherein n is 17 ± 2. In another aspect, the application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O2, wherein nis 18 ± 2. In one embodiment, the saccharide further comprises an E. coli Rl 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 CRM 197 In one embodiment, the conjugate is prepared by single end conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0283] In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are 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. Thus, a comparison of OPA activity of pre- and post-immunization sera with the immunogenic compositions of the application can be made, and their responses to serotype O2 compared, to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing E. coli serotype O2, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing E. coli serotype O2, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic compositions of the application increase the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8, as determined by in vitro OPA) to E. coli serotype O2 compared to the pre-immunization population. In one embodiment, the immunogenic composition elicits a titer of at least 1 :8 to E. coli serotype O2 in at least 50% of the subjects, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the application elicit a titer of at least 1 :8 to E. coli serotype O2 in at least 60%, 70%, 80%, or at least 90% of the subjects, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the application significantly increase the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8, as determined by in vitro OPA) to E. coli serotype O2 compared to the pre-immunization population. In one embodiment, the immunogenic compositions of the application significantly increase the OPA titer of human subjects to E. coli serotype O2 compared to the pre-immunization population.

[0284] In one aspect, the present application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O6, wherein n is 42 ± 2. In another aspect, the present application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O6, wherein n is 50 ± 2. In another aspect, the present application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O6, wherein n is 17 ± 2. In another aspect, the present application relates to a composition comprising a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises Formula O6, wherein 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 CRM 197 . In one embodiment, the conjugate is prepared by single-end ligation conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in a DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0285] In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of binding 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- and post-immunization sera with the immunogenic composition of the application can be performed and their responses to serotype O6 compared to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing E. coli serotype O6 as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing E. coli serotype O6 as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition of the application increases the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to E. coli serotype O6 compared to the pre-immunization population. In one embodiment, the immunogenic composition elicits a titer of at least 1 :8 to E. coli serotype O6 in at least 50% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the application elicits a titer of at least 1 :8 to E. coli serotype O6 in at least 60%, 70%, 80%, or at least 90% of the subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the application significantly increases the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) to E. coli serotype O6 compared to the pre-immunization population. In one embodiment, the immunogenic composition of the application significantly increases OPA titers to E. coli serotype O6 in human subjects compared to the pre-immunization population.

[0286] In one aspect, the composition comprises a conjugate comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises a structure selected from 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 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 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g.,any one of 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 0111, 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 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 O163, 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 from 1 to 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 CRM 197In one embodiment, the conjugate is prepared by single-end ligation conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically 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 additional conjugates up to 30 additional conjugates, each comprising a saccharide covalently bound to a carrier protein, wherein the saccharide comprises a structure selected from any one of the described formulae.

[0287] Dosage of the composition

[0288] The amount of each complex saccharide in a dose is selected to be an amount that induces an immunoprotective response without significant adverse side effects in a typical vaccine. The amount will vary depending on which specific immunogen is employed and how it is presented.

[0289] The amount of a particular complex saccharide in an immunogenic composition can be calculated based on the total polysaccharide (conjugated and unconjugated) of the conjugate. For example, a complex saccharide with 20% free polysaccharide would have about 80 g of conjugated polysaccharide and about 20 g of unconjugated polysaccharide in a 100 g polysaccharide dose. The amount of complex saccharide can vary depending on the E. coli serotype. Saccharide concentration can be determined by uronic acid assay.

[0290] The "immunogenic amount" of the different polysaccharide components in an immunogenic composition can vary and each can comprise about 1.0 g, about 2.0 g, about 3.0 g, about 4.0 g, about 5.0 g, about 6.0 g, about 7.0 g, about 8.0 g, about 9.0 g, about 10.0 g, about 15.0 g, about 20.0 g, about 30.0 g, about 40.0 pg, about 50.0 pg, about 60.0 pg, about 70.0 pg, about 80.0 pg, about 90.0 pg, or about 100.0 g of any particular polysaccharide antigen. Typically, for a given serotype, each dose will comprise 0.1 g to 100 g of polysaccharide, particularly 0.5 g to 20 g, more particularly 1 g to 10 g, and even more particularly 2 g to 5 g. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure. In one embodiment, for a given serotype, each dose will comprise 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, or 20 g of polysaccharide.

[0291] Carrier protein amount. Typically, each dose will comprise 5 g to 150 g of carrier protein, in particular 10 g to 100 g of carrier protein, more particularly 15 g to 100 g of carrier protein, more particularly 25 to 75 g of carrier protein, more particularly 30 g to 70 g of carrier protein, more particularly 30 to 60 g of carrier protein, more particularly 30 g to 50 g of carrier protein, and even more particularly 40 to 60 g of carrier protein. In one embodiment, the carrier protein is CRM 197 . In one embodiment, each dose will comprise about 25 g, about 26 g, about 27 g, about 28 g, about 29 g, about 30 g, about 31 g, about 32 g, about 33 g, about 34 g, about 35 g, about 36 g, about 37 g, about 38 g, about 39 g, about 40 g, about 41 g, about 42 g, about 43 g, about 44 g, about 45 g, about 46 g, about 47 g, about 48 g, about 49 g, about 50 g, about 51 g, about 52 g, about 53 g, about 54 g, about 55 g, about 56 g, about 57 g, about 58 g, about 59 g, about 60 g, about 61 g, about 62 g, about 63 g, about 64 g, about 65 g, about 66 g, about 67 g, 68 g, about 69 g, about 70 g, about 71 g, about 72 g, about 73 g, about 74 g, or about 75 g of carrier protein. In one embodiment, the carrier protein is CRM 197 .

[0292] Adjuvant

[0293] In some embodiments, the immunogenic compositions disclosed herein can further comprise at least one, two, or three adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. Antigens can act primarily as a delivery system, primarily as an immunomodulator, or have strong features of both. Suitable adjuvants include those suitable for use in mammals, including humans.

[0294] Examples of known suitable delivery system type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions, such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)), water-in-oil emulsions, such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0295] In embodiments, the immunogenic compositions disclosed herein comprise an aluminum salt (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide). In preferred embodiments, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as an adjuvant. In embodiments, the immunogenic compositions disclosed herein comprise 0.1 mg / mL to 1 mg / mL or 0.2 mg / mL to 0.3 mg / mL of elemental aluminum in the form of aluminum phosphate. In embodiments, the immunogenic compositions disclosed herein comprise about 0.25 mg / mL of elemental aluminum in the form of aluminum phosphate. Examples of known suitable immunomodulatory type adjuvants that can be used in humans include, but are not limited to, saponin extract from the bark of the Aquilla tree (QS21, Quil A), TLR4 agonists such as MPL (monophosphoryl lipid A), 3DMPL (3-O-deacylated MPL) or GLA-AQ, LT / CT mutants, cytokines such as various interleukins (e.g., IL-2, IL-12) or GM-CSF, AS01, and the like.

[0296] Examples of known suitable immunomodulatory type adjuvants that can be used in humans that have both delivery and immunomodulatory features include, but are not limited to, ISCOMS (see, e.g., Sjölander et al. (1998) J. Leukocyte Biol. 64:713; WO 90 / 03184, WO 96 / 11711, WO 00 / 48630, WO 98 / 36772, WO 00 / 41720, WO 2006 / 134423, and WO 2007 / 026190) or GLA-EM, which is a combination of a TLR4 agonist and an oil-in-water emulsion.

[0297] For veterinary applications including, but not limited to, animal experiments, one can use Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), Emulsigen, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween 80 emulsion.

[0298] Further exemplary adjuvants that enhance the effectiveness of the immunogenic compositions disclosed herein include, but are not limited to, (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents, such as muramyl peptide (see below) or bacterial cell wall components), such as, for example, (a) SAF, which contains 10% squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, which is either microfluidized into a submicron emulsion or vortexed to create a larger particle size emulsion, and (b) RIBI™ adjuvant system (RAS) (Ribi Immunochem, Hamilton, Mont.), which contains 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components, such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (2) saponin adjuvants, such as QS21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.) or ISCOMATRIX® (Commonwealth Serum Laboratories, Australia), or particles produced therefrom, such as ISCOMs (immunostimulating complexes), which can be free of additional detergents (e.g., WO 00 / 07621); (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO 99 / 44636)), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), and the like; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB 2220211, EP 0689454) (see, e.g., WO 00 / 56358); (6) 3dMPL in combination with, e.g., QS21 and / or oil-in-water emulsion (see, e.g., EP 0835318, EP 0735898, EP 0761231); (7) polyoxyethylene ethers or esters (see, e.g., WO 99 / 52549); (8) polyoxyethylene sorbitan ester surfactants in combination with octoxynol (e.g., WO 01 / 21207), or polyoxyethylene alkyl ether or ester surfactants in combination with at least one additional non-ionic surfactant such as octoxynol (e.g., WO 01 / 21152); (9) saponins and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (e.g., WO 00 / 62800); (10) immunostimulants and metal salt particles (see, e.g., WO 00 / 23105); (11) saponins and oil-in-water emulsions (e.g., WO 99 / 11241); (12) saponin (e.g., QS21) + 3dMPL + IM2 (optionally + cholesterol) (e.g., WO 98 / 57659); (13) other substances that act as immunostimulants to enhance the efficacy of the compositions. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-muramyl-L-alanyl-D-isoglutamine (murf-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl- sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), and the like.

[0299] In embodiments of the application, the immunogenic compositions as disclosed herein comprise a CpG oligonucleotide as an adjuvant. CpG oligonucleotides as used herein refer to immunostimulatory CpG oligodeoxynucleotides (CpG ODNs) and thus the terms are used interchangeably unless otherwise indicated. Immunostimulatory CpG oligodeoxynucleotides contain one or more immunostimulatory CpG motifs, which are unmethylated cytosine-guanine dinucleotides, optionally within a certain preferred base context. The methylation status of a CpG immunostimulatory motif generally refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide containing a 5' unmethylated cytosine linked by a phosphate bond to a 3' guanine and which activates the immune system through binding to Toll-like Receptor 9 (TLR-9). In another embodiment, an immunostimulatory oligonucleotide can contain one or more methylated CpG dinucleotides which will activate the immune system through TLR9, but not as strongly as if the CpG motif(s) were unmethylated. CpG immunostimulatory oligonucleotides can contain one or more palindromic sequences, which in turn can comprise CpG dinucleotides. CpG oligonucleotides have been described in a number of issued patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068.

[0300] In embodiments of the application, the immunogenic compositions as disclosed herein comprise any one of the CpG oligonucleotides described in WO 2010 / 125480 on page 3 line 22 to page 12 line 36.

[0301] Different classes of CpG immunostimulatory oligonucleotides have been identified. These are referred to as classes A, B, C, and P, and are described in more detail in WO 2010 / 125480 on page 3 line 22 to page 12 line 36. The methods of the application include the use of these different classes of CpG immunostimulatory oligonucleotides.

[0302] Formulations

[0303] The immunogenic compositions of the application can be formulated in liquid form (i.e., a solution or suspension) or lyophilized form. Liquid formulations can be advantageously administered directly from their packaging form without the need for reconstitution in an aqueous medium as otherwise required for lyophilized compositions of the application, and thus are ideal for injection.

[0304] Formulation of the immunogenic compositions of the present application can be accomplished using art-recognized methods. For example, various conjugates can be formulated with physiologically acceptable vehicles to prepare compositions. Examples of such vehicles include, but are not limited to, water, buffered saline, polyhydric alcohol (e.g., glycerol, propylene glycol, liquid polyetheylene glycol), and dextrose solutions. The present disclosure provides immunogenic compositions comprising any one of the combinations of complex carbohydrates disclosed herein and pharmaceutically acceptable excipients, carriers, or diluents.

[0305] In certain embodiments, the immunogenic composition formulation comprises a pharmaceutically acceptable diluent, excipient, or pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable diluent includes sterile water, water for injection, sterile isotonic saline, or a biological buffer. The polysaccharide-protein conjugate and / or protein immunogen is mixed with such diluents or carriers in the conventional manner. As used herein, the expression pharmaceutically acceptable "carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to a human or other vertebrate host. Suitable carriers will be apparent to those skilled in the art, and will depend in large part on the route of administration.

[0306] For example, excipients that can be present in the immunogenic composition formulation include preservatives, chemical stabilizers, and suspending or dispersing agents. Generally, stabilizers, preservatives, and the like are optimized to determine the optimal formulation for efficacy in the targeted recipient (e.g., a human subject). Examples of preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and p-chloro-phenol. Examples of stabilizing ingredients include casein amino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium phosphate, lactose, whey protein hydrolysate, and milk powder.

[0307] In embodiments, the immunogenic compositions of the present application are in liquid form, preferably in aqueous liquid form.

[0308] The immunogenic compositions of the present disclosure can comprise one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an antioxidant such as a free radical scavenger or a chelator, or any combination of multiple thereof.

[0309] In embodiments, the immunogenic compositions of the present application comprise a buffer. In embodiments, the buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is a phosphate, succinate, histidine, or citrate. In certain embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM. In a particular embodiment, the succinate buffer is at a final concentration of about 5 mM.

[0310] In embodiments, the immunogenic compositions of the application comprise a salt. In some embodiments, the salt is selected from any one of magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In some embodiments, the salt is selected from magnesium chloride, potassium chloride, sodium chloride, and combinations thereof. In a particular embodiment, the salt is sodium chloride. In a particular embodiment, the immunogenic compositions of the application comprise 150 mM sodium chloride.

[0311] In embodiments, the immunogenic compositions of the application comprise a surfactant. In some embodiments, the compositions comprise a non-ionic surfactant, including but not limited to polyoxyethylene sorbitan fatty acid esters. In embodiments, the surfactant is selected from any one of polysorbate 20 (TWEEN™ 20), polysorbate 40 (TWEEN™ 40), polysorbate 60 (TWEEN™ 60), polysorbate 65 (TWEEN™ 65), polysorbate 80 (TWEEN™ 80), polysorbate 85 (TWEEN™ 85), TRITON™ N-101, TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine oleate polypeptide, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soybean lecithin, and poloxamer. In embodiments, the surfactant is selected from polysorbate 20 (TWEEN™ 20), polysorbate 40 (TWEEN™ 40), polysorbate 60 (TWEEN™ 60), polysorbate 65 (TWEEN™ 65), polysorbate 80 (TWEEN™ 80), polysorbate 85 (TWEEN™ 85), TRITON™ N-101, TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine oleate polypeptide, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soybean lecithin, and poloxamer. In some embodiments, the compositions further comprise any one of the polyoxyethylene alkyl ethers, including but not limited to BRIJ 58, BRIJ 35, TRITON X-100, TRITON X-114, NP40, SPAN 85, and the PLURONIC series of non-ionic surfactants, e.g., PLURONIC 121. In a particular embodiment, the surfactant is polysorbate 80. In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.0001% to 10% polysorbate 80 by weight by weight (w / w). In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001% to 1% polysorbate 80 by weight by weight (w / w). In some such embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01% to 1% polysorbate 80 by weight by weight (w / w).In other embodiments, the final concentration of polysorbate 80 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1% polysorbate 80 (w / w). In another embodiment, the final concentration of polysorbate 80 in the formulation is 1% polysorbate 80 (w / w).

[0312] In certain embodiments, the immunogenic composition of the application has a pH of 5.5 to 7.5, more preferably a pH of 5.6 to 7.0, even more preferably a pH of 5.8 to 6.0.

[0313] In one embodiment, the application provides a container filled with any one of the immunogenic compositions disclosed herein. In one embodiment, the container is selected from any one of a vial, a syringe, a bottle, a fermenter, a bioreactor, a bag, a flask, an ampoule, a cartridge, and a disposable pen. In one embodiment, the container is selected from a vial, a syringe, a bottle, a fermenter, a bioreactor, a bag, a flask, an ampoule, a cartridge, and a disposable pen. In certain embodiments, the container is siliconized. In embodiments, the container of the application is made of glass, metal (e.g., steel, stainless steel, aluminum, etc.), and / or polymer (e.g., thermoplastic, elastomer, thermoplastic-elastomer). In embodiments, the container of the application is made of glass.

[0314] In one embodiment, the application provides a syringe filled with any one of the immunogenic compositions disclosed herein. In certain embodiments, the syringe is siliconized and / or made of glass.

[0315] A typical dose of the immunogenic composition of the application for injection has a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, even more preferably a volume of about 0.5 mL.

[0316] Thus, the container or syringe as defined hereinabove is filled with any one of the immunogenic compositions as defined herein in a volume of 0.1 mL to 2 mL, more preferably 0.2 mL to 1 mL, even more preferably a volume of about 0.5 mL.

[0317] The compositions of the application can be administered to a subject by one or more known methods, for example parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally, and formulated accordingly.

[0318] In one embodiment, the composition of the application is administered by epicutaneous injection of a liquid formulation, intramuscular injection, intravenous, intra-arterial, subcutaneous injection, or respiratory mucosal injection. The composition of the application can be formulated as a single-dose vial, a multi-dose vial, or a pre-filled syringe.

[0319] In another embodiment, the composition of the application is administered orally and is thus formulated in a form suitable for oral administration, i.e. as a solid or liquid formulation. Solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic esters such as ethyl oleate.

[0320] Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of animal, vegetable or synthetic origin such as, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil or lipids from milk or eggs.

[0321] The pharmaceutical composition can be isotonic, hypotonic or hypertonic. The pharmaceutical composition for infusion or injection is preferably substantially isotonic when it is administered. For storage, the pharmaceutical composition can preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it can be diluted to become an isotonic solution before administration. The isotonic agent can be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate. Examples of ionic isotonic agents include, but are not limited to, NaCl, CaCl3, KC1 and MgCl2. Examples of non-ionic isotonic agents include, but are not limited to, sucrose, trehalose, mannitol, sorbitol and glycerol.

[0322] It is also preferred that the at least one pharmaceutically acceptable additive is a buffer. For some purposes, e.g. when the pharmaceutical composition is intended for infusion or injection, it is often desirable that the composition comprises a buffer which is able to buffer the solution to a pH in the range of 4 to 10, e.g. 5 to 9, e.g. 6 to 8. The buffer can e.g. be selected from any one of the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, aminoacetate, L-histidine, glycine, succinate and triethanolamine buffers. The buffer can in addition e.g. be selected from USP compatible buffers for parenteral use, in particular when the pharmaceutical preparation is for parenteral use. For example, the buffer can be selected from any one of the group consisting of monobasic acids, e.g. acetic acid, benzoic acid, gluconic acid, glyceric acid and lactic acid; dibasic acids, e.g. aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid, polybasic acids, e.g. citric acid and phosphoric acid; and bases, e.g. ammonia, diethanolamine, glycine, triethanolamine and tris-hydroxymethyl aminomethane. Parenteral vehicles (for subcutaneous, intravenous, intra-arterial or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluids and nutrient supplements, electrolyte supplements, e.g. those based on Ringer's dextrose, and the like. Examples are sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Generally, water, saline, aqueous dextrose and related sugar solutions, such as glycols or polyethylene glycols, polysorbate 80 (PS-80), polysorbate 20 (PS-20) and poloxamer 188 (PI 88) are preferred liquid carriers, in particular for injectable solutions. Examples of oils are those of animal, vegetable or synthetic origin, e.g. peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil or lipids from milk or eggs.

[0323] The formulation can further comprise a surfactant. Preferred surfactants include, but are not limited to, polyoxyethylene sorbitan ester surfactants (commonly known as TWEENs), especially PS-20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the DOWFAX™ trade name, such as linear EO / PO block copolymers; octoxynols, the number of repeating ethoxy (oxy-1,2-ethanediyl) groups of which can vary, with octoxynol-9 (TRITON X-100 or t-octylphenoxy polyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates such as the TERGITOL™ NP series; polyoxyethylene fatty ether derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (known as BRIJ surfactants), such as triethylene glycol monolauryl ether (BRIJ 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (SPAN 85) and sorbitan monolaurate. A preferred surfactant for inclusion in the emulsion is PS-20 or PS-80. Mixtures of surfactants can be used, such as a PS-80 / SPAN 85 mixture. Combinations of polyoxyethylene sorbitan esters such as polyoxyethylene sorbitan monooleate (PS-80) and octoxynols such as t-octylphenoxy polyethoxyethanol (TRITON X-100) are also suitable. Another useful combination includes laureth 9 plus polyoxyethylene sorbitan ester and / or octoxynol.

[0324] The formulation can also include a pH-buffered saline solution. The buffer can be, for example, selected from any of the following: Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, aminoacetate, L-histidine, glycine, succinate, HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), and triethanolamine buffers. The buffer can buffer the solution to a pH in the range of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspects, the buffer is selected from any of the following: phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate, or citrate. The buffer can additionally be, for example, selected from USP compatible buffers for parenteral use, particularly when the pharmaceutical formulation is for parenteral use. The concentration of the buffer ranges from 1 mM up to 50 mM or from 5 mM up to 50 mM. In certain aspects, the buffer is L-histidine at a final concentration of 5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM. In certain aspects, the L-histidine is at a final concentration of 20 mM ± 2 mM.

[0325] Although a saline solution (i.e., a solution comprising NaCl) is preferred, other salts suitable for the formulation include, but are not limited to, CaCl3, KCl, and MgCl2, and combinations thereof. Non-ionic isotonic agents can be used in place of salts, including, but not limited to, sucrose, trehalose, mannitol, sorbitol, and glycerol. Suitable salt ranges include, but are not limited to, 25 mM to 500 mM or 40 mM to 170 mM. In one aspect, the saline is NaCl, optionally present at a concentration of 20 mM to 170 mM. In a preferred embodiment, the formulation comprises an L-histidine buffer with sodium chloride.

[0326] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the agent can be administered using intravenous infusion, transdermal patch, liposome, or other modes of administration. In another embodiment, a polymeric material is used; for example, in a spherulite or an implant.

[0327] Activity

[0328] In one embodiment, the saccharide described herein is capable of inducing opsonic activity. In another embodiment, the saccharide described herein is capable of inducing opsonic and phagocytic activity (e.g., opsonophagocytic activity).

[0329] The present inventors have surprisingly found that sugars having increased repeat units compared to the corresponding wild-type sugar induce an increase in the immune response in a mammal compared to wild-type sugars. The present inventors have also surprisingly found that a composition comprising a conjugate comprising a carrier protein and a sugar having increased repeat units compared to the corresponding wild-type sugar induces an increase in the immune response in a mammal compared to a mammal administered a composition comprising a conjugate comprising a carrier protein and the corresponding wild-type sugar. Without being bound by mechanism or theory, sugars having increased repeat units can have an increase in the retained epitopes, which can result in an increase in the immune response.

[0330] Opsonic activity or opsonization refers to the process by which opsonins (e.g., antibodies or complement factors) bind to antigens (e.g., sugars or conjugates thereof described herein) that facilitate the attachment of the antigens to phagocytic or phagocytic cells (e.g., macrophages, dendritic cells, and polymorphonuclear leukocytes (PMNL). Some bacteria, such as encapsulated bacteria that are generally not phagocytosed due to the presence of a capsule, become more likely to be recognized by phagocytic cells when coated with opsonic antibodies. In one embodiment, the sugar induces an immune response, such as, for example, an antibody, that is opsonic. In one embodiment, the opsonic activity is directed against Gram-negative bacteria, preferably against Escherichia species, more preferably against at least one E. coli strain. Echerichia ) species, more preferably against at least one E. coli strain.

[0331] Phagocytic activity or phagocytosis refers to the process by which phagocytic cells engulf material and enclose the material in their cytoplasm. In one embodiment, the sugar induces an immune response, such as, for example, an antibody, that facilitates phagocytosis. In one embodiment, the phagocytic activity is directed against Gram-negative bacteria, preferably against Escherichia species, more preferably against at least one E. coli strain. For example, rabbit antibodies raised against an isolated sugar described herein can be able to mediate specific opsonophagocytosis of strains expressing the sugar in the presence of complement, as indicated by, for example, in vitro phagocytosis assays.

[0332] In still another embodiment, the sugars described herein are capable of inducing a bactericidal immune response. In one embodiment, the bactericidal activity is directed against Gram-negative bacteria, preferably against Escherichia species, more preferably against at least one E. coli strain.

[0333] Methods for measuring opsonization, phagocytosis, and / or bactericidal activity are known in the art, such as, for example, by measuring reduction in bacterial load in vivo (e.g., by measuring bacteremia levels in mammals challenged with E. coli) and / or by measuring bacterial cell killing in vitro (e.g., in vitro opsonophagocytic assays). In one embodiment, the saccharide is capable of inducing opsonization, phagocytosis, and / or bactericidal activity as compared to a suitable control, such as, for example, antiserum raised against heat-killed Gram-negative bacteria.

[0334] An opsonophagocytic assay that measures killing of E. coli cells by phagocytic effector cells in the presence of functional antibody and complement can be a surrogate for evaluating the effectiveness of an E. coli vaccine against a particular E. coli serotype (such as, for example, E. coli serotype O25B). An in vitro opsonophagocytic assay can be performed by incubating a mixture of E. coli cells, heat-inactivated human serum to be tested, differentiated HL-60 cells (phagocytes), and an exogenous source of complement (e.g., rabbit complement). Opsonophagocytosis proceeds during incubation and kills antibody- and complement-coated bacterial cells as they are opsonophagocytosed. Colony forming units (cfu) of surviving bacteria that escape opsonophagocytosis are determined by plating the assay mixture. The OPA titer is defined as the reciprocal dilution that results in a 50% reduction in bacterial count compared to control wells without test serum. The OPA titer is interpolated from the two dilutions that include the 50% killing cutoff.

[0335] In these types of killing OPA, an endpoint titer of 1 :8 or greater is considered a positive result.

[0336] In some embodiments, a subject can have a serotype-specific OPA titer prior to vaccination due to, for example, natural exposure to E. coli (e.g., in the case of adult subjects).

[0337] Accordingly, a comparison of OPA activity of pre- and post-immune sera with the immunogenic composition of the application can be performed and compared for their response to a particular E. coli serotype, such as, for example, E. coli serotype O25B, to assess potential increases in responders.

[0338] In one embodiment, the immunogenic composition of the application significantly increases the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8 as determined by in vitro OPA) as compared to the pre-immune population.

[0339] A comparison of OPA activity of pre- and post-immune sera with the immunogenic composition of the application can also be performed by comparing the potential increase in OPA titer.

[0340] Thus, a comparison of the OPA activity of pre- and post-immune sera with the immunogenic composition of the application can be performed and their response to a specific E. coli serotype, such as for example E. coli serotype O25B, compared to assess the potential to increase the OPA titre. In one embodiment, the immunogenic composition of the application is capable of significantly increasing the OPA titre of a human subject compared to the pre-immune population.

[0341] In one aspect, the composition induces an immune response against at least one E. coli serotype. The E. coli serotype can be any serotype, including, for example, any of 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 (strain 180 / C3)), 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, 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, 62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g.,O73 (strain 73-1), 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, O111, O112, O113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, 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. In one embodiment, the serotype is O25a. In another embodiment, the serotype is O25b. In another embodiment, the serotype is O1A. In another embodiment, the serotype is O2. In another embodiment, the serotype is O6. In another embodiment, the serotype is O6. In another embodiment, the serotype is O17. In another embodiment, the serotype is O15. In another embodiment, the serotype is O18A. In another embodiment, the serotype is O75. In another embodiment, the serotype is O4. In another embodiment, the serotype is O16. In another embodiment, the serotype is O13. In another embodiment, the serotype is O7. In another embodiment, the serotype is O8.

[0342] In preferred embodiments, the composition induces an immune response against at least two E. coli serotypes. In one embodiment, the composition induces an immune response against at least 3 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 4 different E. coli serotypes. In one embodiment, the composition comprises O-antigen from 5 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 6 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 7 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 8 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 9 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 10 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 11 different E. coli serotypes. In one embodiment, the composition induces an immune response against at least 12 different serotypes. In one embodiment, the composition induces an immune response against at least 13 different serotypes. In one embodiment, the composition induces an immune response against at least 14 different serotypes. In one embodiment, the composition induces an immune response against at least 15 different serotypes. In one embodiment, the composition induces an immune response against at least 16 different serotypes. In one embodiment, the composition induces an immune response against at least 17 different serotypes. In one embodiment, the composition induces an immune response against at least 18 different serotypes. In one embodiment, the composition induces an immune response against at least 19 different serotypes. In one embodiment, the composition induces an immune response against at least 20 different serotypes.

[0343] In one embodiment, the immunogenic composition of the application elicits IgG antibodies in humans that are capable of binding to a specific E. coli seroglycan, such as, for example, the E. coli serogroup O25B glycan, as determined by an ELISA assay. Preferably, the enhanced immune response can include an increase in IgGl and / or IgG2a and / or IgM production.

[0344] In the ELISA (enzyme-linked immunosorbent assay) method, antibodies from the serum of inoculated subjects are incubated with polysaccharides that have been adsorbed onto a solid support. Bound antibodies are detected using an enzyme-conjugated secondary detection antibody. The ELISA measures type-specific IgG anti-E. coli polysaccharide antibodies present in human serum. When human serum dilutions are added to type-specific polysaccharide-coated microtiter plates, antibodies specific for the polysaccharide bind to the microtiter plate. Antibodies bound to the plate are detected using a goat anti-human IgG alkaline phosphatase-labeled antibody followed by a p-nitrophenyl phosphate substrate. The optical density of the colored end product is directly proportional to the amount of anti-0-antigen polysaccharide antibodies present in the serum.

[0345] In one embodiment, the immunogenic composition of the application significantly increases OPA titers in human subjects against E. coli serotypes O25B, O1, O2, and O6 compared to the pre-immune population.

[0346] In some embodiments, the compositions of the application are effective to increase the production of antibodies in a mammal against E. coli to a geometric mean titer (GMT) level of at least 1,000, preferably at least 5,000 and up to 200,000, for an initial administration, such as, for example, about 30 days after the initial administration, preferably about 60 days after the initial administration. In preferred embodiments, the compositions are effective to increase the production of antibodies in a mammal against E. coli serotype O25B to a geometric mean titer (GMT) level of at least 1,000, preferably at least 5,000 and up to 200,000, for an initial administration.

[0347] In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of binding to E. coli serotypes corresponding to the repeating units of the saccharide in the composition. For example, the composition can elicit IgG antibodies 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- and post-immunization sera with the immunogenic compositions of the application can be made, and their responses to each serotype compared, to assess potential increases in responders. In one embodiment, the immunogenic composition elicits IgG antibodies in humans that are capable of killing E. coli serotypes corresponding to the repeating units of the saccharide in the composition, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans that are capable of killing E. coli serotypes corresponding to the repeating units of the saccharide in the composition, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic compositions of the application increase the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8, as determined by in vitro OPA) to E. coli serotypes corresponding to the repeating units of the saccharide in the composition, as compared to a pre-immunization population. In one embodiment, the immunogenic composition elicits a titer of at least 1 :8 in at least 50% of subjects to E. coli serotypes corresponding to the repeating units of the saccharide in the composition, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the application elicit a titer of at least 1 :8 in at least 60%, 70%, 80%, or at least 90% of subjects to E. coli serotypes corresponding to the repeating units of the saccharide in the composition, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the application significantly increase the proportion of responders (i.e., individuals with sera having a titer of at least 1 :8, as determined by in vitro OPA) to E. coli serotypes corresponding to the repeating units of the saccharide in the composition, as compared to a pre-immunization population. In one embodiment, the immunogenic compositions of the application significantly increase OPA titers in human subjects to E. coli serotypes corresponding to the repeating units of the saccharide in the composition, as compared to a pre-immunization population.

[0348] Methods

[0349] In another aspect, the application relates to a method of preventing infection in a subject, e.g., a human subject, comprising administering to the subject an effective amount of a composition described herein (e.g., an immunogenic composition).

[0350] In another aspect, the present application relates to a method of treating an infection in a subject, e.g., a human subject, comprising administering to the subject an effective amount of a composition described herein (e.g., an immunogenic composition).

[0351] In another aspect, the present application relates to a method of inducing an immune response in a subject, e.g., a human subject, comprising administering to the subject an effective amount of a composition described herein (e.g., an immunogenic composition).

[0352] In another aspect, the present application relates to a method of inducing the production of opsonophagocytic antibodies in a subject, e.g., a human subject, comprising administering to the subject an effective amount of a composition described herein (e.g., an immunogenic composition).

[0353] As used herein, the term "effective amount" in the context of administering a composition described herein to a subject means an amount of the composition that has one or more prophylactic and / or therapeutic effects. In certain embodiments, an "effective amount" means an amount of the composition sufficient to achieve at least one, two, three, four or more of the following effects: (i) reduce or ameliorate the severity of an E. coli infection or a symptom associated therewith; (ii) reduce the duration of an E. coli infection or a symptom associated therewith; (iii) prevent the progression of an E. coli infection or a symptom associated therewith; (iv) cause regression of an E. coli infection or a symptom associated therewith; (v) prevent the development or onset of an E. coli infection or a symptom associated therewith; (vi) prevent the recurrence of an E. coli infection or a symptom associated therewith; (vii) reduce organ failure associated with an E. coli infection; (viii) reduce hospitalization of a subject having an E. coli infection; (ix) reduce the duration of hospitalization of a subject having an E. coli infection; (x) increase survival of a subject having an E. coli infection; (xi) eliminate an E. coli infection in a subject; (xii) inhibit or reduce E. coli replication in a subject; and / or (xiii) enhance or improve one or more prophylactic or therapeutic effects of another therapy.

[0354] In embodiments, the immunogenic compositions disclosed herein are used as a medicament. The immunogenic compositions described herein can be used in various therapeutic or prophylactic methods for preventing, treating, or ameliorating a bacterial infection, disease, or condition in a subject. In particular, the immunogenic compositions described herein can be used for preventing, treating, or ameliorating an E. coli infection, disease, or condition in a subject.

[0355] Thus, in one aspect, the present application provides a method of preventing, treating, or ameliorating an infection, disease, or condition associated with E. coli in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present application.

[0356] In one aspect, the present application provides a method of preventing, treating or ameliorating an infection, disease or condition associated with E. coli in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present application.

[0357] In one aspect, the present application provides a method of inducing an immune response to E. coli in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present application.

[0358] In one aspect, the immunogenic composition of the present application is for use in a method of preventing, treating or ameliorating an infection, disease or condition caused by E. coli in a subject. In another aspect, the composition of the present application is for use in a method of protecting a mammal from an infection, disease or condition caused by E. coli.

[0359] In one embodiment, any one of the immunogenic compositions disclosed herein is for use in a method of immunizing a subject against an E. coli infection.

[0360] In one aspect, the present application relates to the use of an immunogenic composition disclosed herein in the manufacture of a medicament for preventing, treating or ameliorating an infection, disease or condition caused by E. coli in a subject.

[0361] In embodiments, the present application relates to the use of an immunogenic composition disclosed herein in the manufacture of a medicament for immunizing a subject against an E. coli infection.

[0362] In embodiments, the immunogenic composition disclosed herein is used as a vaccine. More particularly, the immunogenic composition described herein can be used to prevent an E. coli infection in a subject. Thus, in one aspect, the present application provides a method of preventing an E. coli infection in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present application.

[0363] In some such embodiments, the infection is selected from any one of urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intra-abdominal infection, meningitis, complicated pneumonia, wound infection, infection associated with prostate biopsies, neonatal / infant sepsis, neutropenic fever, pneumonia, bacteremia and sepsis, and other bloodstream infections. For example, E. coli serotypes associated with neonatal meningitis include serotypes O1, O6, O7, O16, O18, and O83. Thus, in one embodiment, the composition comprises conjugates comprising saccharides having the structure of any one selected from the group consisting of Formula O1, Formula O6, Formula O7, Formula O16, Formula O18, Formula O83, and any combination of conjugates thereof. As another example, E. coli serotypes associated with bacteremia include serotypes O1, O2, O6, O15, and O75. Thus, in another embodiment, the composition comprises at least one conjugate comprising saccharides having the structure of any one selected from the group consisting of Formula O1, O2, O6, O15, and O75, and any combination of conjugates thereof. As a further example, E. coli O1 and O2 strains, such as O1:K1:H7 or O2:K1:H4, O2:K1:H5, O2:K1:H6, and O2:K1:H7, are identified as pathogens of urinary tract infections, septicemia, and neonatal meningitis in humans and animals. Both O1 and O2 serogroups make up the majority of strains that cause bacteremia and sepsis in human patients. Thus, in one embodiment, the composition comprises a conjugate comprising saccharides of Formula O1 and a conjugate comprising saccharides of Formula O2.

[0364] Thus, by generating an immune response in a mammal with these uses and methods, the mammal can be protected from E. coli infection, including ExPEC and non-ExPEC strains. The present application is particularly useful for providing broad protection against pathogenic E. coli, including enteropathogenic types such as EPEC, EAEC, EIEC, ETEC, and DAEC pathotypes. Thus, the mammal can be protected from disease, including, but not limited to, peritonitis, pyelonephritis, cystitis, endocarditis, prostatitis, urinary tract infections (UTIs), meningitis (particularly neonatal meningitis), sepsis (or SIRS), dehydration, pneumonia, diarrhea (infantile diarrhea, traveler's diarrhea, acute diarrhea, persistent diarrhea, etc.), bacillary dysentery, hemolytic uremic syndrome (HUS), pericarditis, bacteriuria, and the like.

[0365] In one aspect, the subject to be vaccinated is a mammal, such as a human, cat, sheep, pig, horse, cow, or dog. Preferably, the subject to be vaccinated is a human. Where the vaccine is for prophylactic use, the human is preferably a child (e.g., a toddler or infant) or an adolescent or adult; where the vaccine is for therapeutic use, the human is preferably an adolescent or adult. Vaccines intended for children can also be administered to adults, e.g., to assess safety, dose, immunogenicity, etc.

[0366] The vaccines of the application can be used to treat both children and adults. Thus, the human patient can be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients to receive the vaccines are the elderly (e.g., > 50 years old, > 60 years old, and preferably > 65 years old), the young (e.g., < 5 years old), hospitalized patients, health care workers, armed forces and military personnel, pregnant women, patients with chronic diseases or immunodeficiency. However, the vaccines are not only suitable for these groups, and can be used more widely in the population.

[0367] The vaccines of the application are particularly useful for patients who are scheduled for surgery or other hospitalization. They are also useful in patients who are to undergo catheterization. They can also be used in adolescent females (e.g., 11-18 years old) and patients with chronic urinary tract infections.

[0368] In embodiments, the immunogenic compositions disclosed herein are administered by intramuscular, intraperitoneal, intradermal, or subcutaneous routes. In embodiments, the immunogenic compositions disclosed herein are administered by intramuscular, intraperitoneal, intradermal, or subcutaneous injection. In embodiments, the immunogenic compositions disclosed herein are administered by intramuscular or subcutaneous injection.

[0369] In embodiments, the application relates to a method comprising the steps of: (i) injecting an immunologically effective amount of any one of the immunogenic compositions defined herein into a subject; (ii) collecting a serum sample from the subject; (iii) testing the serum sample for opsonophagocytic killing activity against E. coli serotype O25B by an in vitro opsonophagocytic killing assay (OPA).

[0370] Subject

[0371] As disclosed herein, the immunogenic compositions described herein can be used in various therapeutic or prophylactic methods to prevent, treat, or ameliorate a bacterial infection, disease, or condition in a subject.

[0372] In preferred embodiments, the subject is a human. In most preferred embodiments, the subject is a neonate (i.e., less than three months old), an infant (i.e., from three months to one year old), or a toddler (i.e., from one to four years old).

[0373] In embodiments, the immunogenic compositions disclosed herein are used as vaccines.

[0374] In embodiments, the subject to be vaccinated can be less than 1 year of age. For example, the subject to be vaccinated can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 months of age. In embodiments, the subject to be vaccinated is about 2, 4, or 6 months of age. In another embodiment, the subject to be vaccinated is less than 2 years of age. For example, the subject to be vaccinated can be about 12 to about 15 months of age. In some cases, as few as one dose of the immunogenic composition according to the application is required, but in some cases, a second, third, or fourth dose can be administered.

[0375] In embodiments, the subject to be vaccinated can be less than 18 years of age. For example, the subject to be vaccinated can be about 1, 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, about 15, about 16, or about 17 years of age. For example, the subject to be vaccinated can be about 10 to about 18 years of age. In some cases, as few as one dose of the immunogenic composition according to the application is required, but in some cases, a second, third, or fourth dose can be administered.

[0376] In another embodiment, the subject to be vaccinated can be a human 18 years of age or older. For example, the subject to be vaccinated can be about 18 to about 50 years of age. In some cases, as few as one dose of the immunogenic composition according to the application is required, but in some cases, a second, third, or fourth dose can be administered.

[0377] In embodiments of the application, the subject to be vaccinated is a human 50 years of age or older, more preferably a human 55 years of age or older. In embodiments, the subject to be vaccinated is a human 65 years of age or older, 70 years of age or older, 75 years of age or older, or 80 years of age or older. In embodiments, the subject to be vaccinated is an immunocompromised individual, human. An immunocompromised individual is generally defined as a human who exhibits a diminished or decreased ability to mount a normal humoral or cellular defense against challenge by an infectious agent.

[0378] In embodiments of the application, the immunocompromised subject to be vaccinated has a disease or condition that impairs the immune system and results in an antibody response that is insufficient to protect against or treat urinary tract infections.

[0379] In an embodiment, the disease is a primary immunodeficiency disease. Preferably, the primary immunodeficiency disease is selected from any one of combined T- and B-cell immunodeficiencies, antibody deficiencies, well-defined syndromes, immunological disorders, phagocyte disorders, congenital immunodeficiencies, autoinflammatory disorders and complement deficiencies.

[0380] In a particular embodiment of the application, the immunocompromised subject to be vaccinated suffers from a disease selected from any one of HIV-infection, acquired immune deficiency syndrome (AIDS), cancer, chronic heart or lung conditions, congestive heart failure, diabetes, chronic liver disease, alcoholism, cirrhosis, spinal fluid leaks, cardiomyopathy, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), splenic dysfunction (e.g. sickle cell disease), asplenia, hematological malignancies, leukemia, multiple myeloma, Hodgkin's disease, lymphoma, renal failure, nephrotic syndrome and asthma.

[0381] In an embodiment of the application, the subject to be vaccinated suffers from malnutrition.

[0382] In an embodiment of the application, the subject to be vaccinated is receiving a drug or treatment that reduces the body's resistance to infection.

[0383] In an embodiment of the application, the subject to be vaccinated is a smoker.

[0384] In an embodiment of the application, the subject to be vaccinated has a white blood cell count (leukocyte count) of less than 5 x 10 9 cells per liter, or less than 4 x 10 9 cells per liter, or less than 3 x 10 9 cells per liter, or less than 2 x 10 9 cells per liter, or less than 1 x 10 9 cells per liter, or less than 0.5 x 10 9 cells per liter, or less than 0.3 x 10 9 cells per liter, or less than 0.1 x 10 9 cells per liter.

[0385] White blood cell count (WBC): The number of white blood cells (WBCs) in the blood. WBC is often measured as part of the CBC (complete blood cell count). White blood cells are infection-fighting cells in the blood and are different from red (oxygen-carrying) cells called red blood cells. There are different types of white blood cells, including neutrophils (polymorphonuclear leukocytes; PMNs), band neutrophils (slightly immature neutrophils), T-cells, B-cells, monocytes, eosinophils, and basophils. All types of white blood cells are reflected in the white blood cell count. The normal range for a white blood cell count is typically 4,300 to 10,800 cells per cubic millimeter of blood. This is also known as the white blood cell count and can be expressed in international units as 4.3 - 10.8 x 10⁻⁶. 9 Cells / liter.

[0386] In the implementation plan, the subject to be vaccinated suffers from neutropenia. For example, the subject to be vaccinated may have a neutropenia level below 2 x 10⁻⁶. 9 cells / liter, or less than 1 x 10 9 1 cell / L, or less than 0.5 x 10⁻⁶ 9 1 cell / L, or less than 0.1 x 10 9 1 cell / L, or less than 0.05 x 10⁻⁶ 9 Neutrophil count per cell / liter.

[0387] A low white blood cell count, or "neutropenia," is a condition characterized by an abnormally low level of neutrophils in the circulating blood. Neutrophils are a special type of white blood cell that helps prevent and fight infections. The most common reason cancer patients experience neutropenia is as a side effect of chemotherapy. Chemotherapy-induced neutropenia increases the risk of infection and can disrupt cancer treatment.

[0388] In the implementation plan, the subject to be inoculated has a density of less than 500 / mm. 3 CD4+ cell count, or less than 300 / mm 3 CD4+ cell count, or less than 200 / mm 3 CD4+ cell count, below 100 / mm 3 CD4+ cell count, below 75 / mm 3 CD4+ cell count, or less than 50 / mm 3 CD4+ cell count.

[0389] CD4 cell test results are usually reported in mm. 3Normal CD4 counts are 500-1600, and CD8 counts are 375-1100. CD4 counts in people with HIV drop dramatically.

[0390] In embodiments of the application, any immunocompromised subject disclosed herein is a human male or a human female.

[0391] Regimen

[0392] In some cases, as few as one dose of the immunogenic composition according to the application is effective, in other cases, for example, conditions of greater immunodeficiency, a second, third or fourth dose can be administered. Following the initial inoculation, the subject can receive one or several booster immunizations sufficiently spaced apart from each other.

[0393] In embodiments, the inoculation schedule of the immunogenic composition according to the application is a single dose. In another embodiment, the single dose schedule is for healthy humans at least 2 years of age.

[0394] In embodiments, the inoculation schedule of the immunogenic composition according to the application is a multiple dose schedule. In another embodiment, the multiple dose schedule comprises a series of 2 doses with a time interval of about 1 month to about 2 months apart. In one embodiment, the multiple dose schedule comprises a series of 2 doses with a time interval of about 1 month apart, or a series of 2 doses with a time interval of about 2 months apart.

[0395] In another embodiment, the multiple dose schedule comprises a series of 3 doses with a time interval of about 1 month to about 2 months apart. In another embodiment, the multiple dose schedule comprises a series of 3 doses with a time interval of about 1 month apart, or a series of 3 doses with a time interval of about 2 months apart.

[0396] In another embodiment, the multiple dose schedule comprises a series of 3 doses with a time interval of about 1 month to about 2 months apart, plus a fourth dose about 10 months to about 13 months after the first dose. In another embodiment, the multiple dose schedule comprises a series of 3 doses with a time interval of about 1 month apart, plus a fourth dose about 10 months to about 13 months after the first dose, or a series of 3 doses with a time interval of about 2 months apart, plus a fourth dose about 10 months to about 13 months after the first dose.

[0397] In embodiments, the multiple dose schedule comprises at least one dose (e.g., 1, 2 or 3 doses) at the first year of life, plus at least one toddler dose.

[0398] In embodiments, the multiple dose schedule comprises a series of 2 or 3 doses (e.g., 28-56 days between doses) at intervals of about 1 month to about 2 months starting at 2 months of age, plus a toddler dose at 12 to 18 months of age. In embodiments, the multiple dose schedule comprises a series of 3 doses (e.g., 28-56 days between doses) at intervals of about 1 to 2 months starting at 2 months of age, plus a toddler dose at 12 to 15 months of age. In another embodiment, the multiple dose schedule comprises a series of 2 doses at intervals of about 2 months starting at 2 months of age, plus a toddler dose at 12 to 18 months of age.

[0399] In embodiments, the multiple dose schedule comprises a 4-dose vaccine series at 2, 4, 6, and 12-15 months of age.

[0400] In embodiments, a prime dose is given at day 0, and one or more boosts are given at intervals ranging from about 2 weeks up to about 24 weeks, preferably with an administration interval of 4-8 weeks.

[0401] In embodiments, a prime dose is given at day 0, and a boost is given about 3 months later.

[0402] Kits and methods

[0403] In embodiments, the present invention relates to a kit comprising an immunogenic composition and an information leaflet disclosed herein.

[0404] In embodiments, the information leaflet comprises the ability of the composition to elicit functional antibodies against E. coli.

[0405] In embodiments, the information leaflet comprises the ability of the composition to elicit OPA titers against E. coli serotype O25B in a human population. In embodiments, the information leaflet comprises the ability of the composition to elicit OPA titers against E. coli serotype O1 in a human population. In embodiments, the information leaflet comprises the ability of the composition to elicit OPA titers against E. coli serotype O2 in a human population. In embodiments, the information leaflet comprises the ability of the composition to elicit OPA titers against E. coli serotype O6 in a human population.

[0406] In embodiments, the present invention relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of: (i) producing an immunogenic composition of the present disclosure, and (ii) combining the immunogenic composition and information leaflet in the same kit, wherein the information leaflet documents the ability of the composition to elicit functional antibodies against E. coli.

[0407] In embodiments, the present application relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of: (i) producing an immunogenic composition of the present disclosure, and (ii) combining the immunogenic composition and an information leaflet in the same kit, wherein the information leaflet documents the ability of the composition to elicit anti-0-antigen antibodies against E. coli in a human population.

[0408] In embodiments, the present application relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of: (i) producing an immunogenic composition of the present disclosure, and (ii) combining the immunogenic composition and an information leaflet in the same kit, wherein the information leaflet documents the ability of the composition to elicit OPA titers against E. coli in a human population.

[0409] In embodiments, the present application relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of: (i) producing an immunogenic composition of the present disclosure; (ii) printing an information leaflet, wherein the information leaflet documents the ability of the composition to elicit functional antibodies against E. coli; (iii) combining the immunogenic composition and the information leaflet in the same kit.

[0410] In embodiments, the present application relates to a method for producing a kit comprising an immunogenic composition and an information leaflet, the method comprising the steps of: (i) producing an immunogenic composition of the present disclosure; (ii) printing an information leaflet, wherein the information leaflet documents the ability of the composition to elicit OPA titers against E. coli in a human population; (iii) combining the immunogenic composition and the information leaflet in the same kit.

[0411] Example

[0412] In order that the application can be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the application in any manner. The following examples illustrate some embodiments of the application.

[0413] Example 1: E. coli and Salmonella enterica strains

[0414] Clinical strains and derivatives are listed in Table 3. Additional reference strains include: O25K5H1, a clinical O25a serotype strain; and Salmonella enterica serovar typhimurium strain LT2.

[0415] Gene knockouts in E. coli strains were constructed that removed the targeted open reading frame but left a short scar sequence.

[0416] For simplicity, the hydrolyzed O-antigen chains and core saccharides are subsequently denoted as O-polysaccharides (OPS).

[0417]

[0418] Example 2: O-antigen gene cluster cloning wzzB , fepE Oligonucleotide primers used for O-antigen gene cluster cloning

[0419] Table 4 Oligonucleotide primers

[0420]

[0421] Example 3: Plasmids

[0422] The plasmid vectors and subclones are listed in Table 5. PCR fragments harboring various E. coli and Salmonella wzzB and fepE genes were amplified from purified genomic DNA and subcloned into high copy number plasmids provided in the Invitrogen PCR® Blunt cloning kit (Figure 1). The plasmids are based on the pUC replicon. Primers P3 and P4 were used to amplify the E. coli wzzB genes with their native promoters and were designed to bind to regions in the proximal and distal genes encoding UDP-glucose-6-dehydrogenase and phosphoribosyl-adenine nucleotide hydrolase, respectively (annotated in Genbank MG1655 NC_000913.3). PCR fragments containing the Salmonella fepE genes and promoters were amplified using primers described previously. Similar E. coli fepE primers were designed based on available Genbank genomic sequences or internally generated whole genome data (in the case of GAR2401 and O25K5H1). The low copy number plasmid pBAD33 was used to express the O-antigen biosynthesis genes under the control of the arabinose promoter. The plasmid was first modified to facilitate cloning (by Gibson method) of long PCR fragments amplified using universal primers homologous to the 5' promoter and 3' 6-phosphogluconate dehydrogenase gnd genes. The pBAD33 subclone containing the Q25b biosynthesis operon is specified in Figure 1.

[0423] Table 5

[0424] Plasmids

[0425]

[0426] Example 4: O-antigen purification

[0427] The fermentation broth was treated with acetic acid to a final concentration of 1-2% (final pH of 4.1). Extraction and delipidation of OAg was achieved by heating the acid treated broth to 100°C for 2 hours. At the end of the acid hydrolysis, the batch was cooled to ambient temperature and 14% NH4OH was added to a final pH of 6.1. The neutralized broth was centrifuged and the centrate was collected. CaCl2in sodium phosphate was added to the centrate and the resulting slurry was incubated at room temperature for 30 minutes. The solids were removed by centrifugation and the centrate was concentrated 12-fold using a 10 kDa membrane followed by two diafiltrations against water. The retentate containing OAg was then purified using a carbon filter. The carbon filtrate was diluted 1:1 (v / v) with 4.0 M ammonium sulfate. The final ammonium sulfate concentration was 2 M. The ammonium sulfate treated carbon filtrate was further purified using a membrane using 2 M ammonium sulfate as the running buffer. OAg was collected in the flow through. For long OAg, the HIC filtrate was concentrated and then buffer exchanged (20 diavolumes) against water using a 5 kDa membrane. For short (native) OAg polysaccharide, the MWCO was further reduced to improve yield.

[0428] Example 5: O25b long O-antigen conjugation to CRM197 197 Example 5: O25b long O-antigen conjugation to CRM197

[0429] First set of long chain O25b polysaccharide-CRM conjugates 197 Conjugates were produced using periodate oxidation followed by conjugation using reductive amination chemistry (RAC) (Table 7). By varying the level of oxidation, conjugation variants had three levels of activation (low, medium, and high). Lyophilized activated polysaccharide reconstituted in DMSO media was reacted with lyophilized CRM197 197 to produce conjugates. The conjugation reaction was carried out at 23°C for 24 hours followed by capping with sodium borohydride for 3 hours. After the conjugation quench step, the conjugates were purified by ultrafiltration / diafiltration using 5 mM succinate / 0.9% NaCl, pH 6.0, with 100K MWCO regenerated cellulose membranes. Final filtration of the conjugates was performed using a 0.22 µm membrane.

[0430] Unless otherwise explicitly stated, the conjugates disclosed throughout the following examples comprise a core saccharide moiety.

[0431] 1.1. Long O-antigen expression conferred by heterologous polymerase chain length regulators

[0432] Initial E. coli strain construction focused on the O25 serogroup. The goal was to overexpress heterologous wzzB or fepE genes to see if they conferred O25 wzzBLonger chain length in knockout strains. First, blood isolates were screened by PCR to identify O25a and O25b subtypes of strains. Next, strains were screened for sensitivity to ampicillin. A single ampicillin-sensitive O25b isolate, GAR2401, was identified in which wzzB deletions were introduced wzzB deletions were introduced. For genetic complementation of these mutations, the wzzB genes from GAR 2401 and O25K5H1 were subcloned into a high copy PCR-Blunt II cloning vector and introduced into both strains by electroporation. Additional wzzB genes from E. coli K-12 and Salmonella enterica serovar Typhi LT2 were cloned and transferred similarly; likewise, the fepE genes from E. coli O25K5H1, GAR 2401, O25a ETEC NR-5, O157:H7:K-, and Salmonella enterica serovar Typhi LT2.

[0433] wzzB Genetic complementation of LPS expression in plasmid transformants of knockout strains O25K5H1 (O25a) and GAR2401 (O25b) is shown in Figure 2 B. Bacteria were grown overnight in LB media and LPS was extracted with phenol, separated by SDS PAGE (4-12% acrylamide) and stained. Each well of the gel was loaded with LPS extracted from the same number of bacterial cells (about 2 OD 600 units). The size of the LPS was estimated according to internal native E. coli LPS standards and by counting the discernible ladders from a subpopulation of the sample showing a broad chain length distribution (differing by one repeat unit). On the left side of panel A of Figure 3, the LPS profile of the plasmid transformant of O25a O25K5H1 is shown; and on the right, the similar profile of the O25b GAR 2401 transformant. Immunoblots of duplicate gels probed with O25-specific serum are shown in panel B of Figure 3. wzzB wzzB

[0434] The results from the experiments indicate that introduction of homologous wzzB genes into E. coli O25a wzzB hosts restores expression of short O25 LPS (10-20x), and Salmonella LT2 wzzB as well. Introduction of O25b wzzB ​​Genes, not, suggesting that the WzzB enzyme from the strain is defective. Comparison of the E. coli WzzB amino acid sequence suggests that it is likely the A210E and P253S substitutions that are responsible. Importantly, Salmonella LT2 fepE and E. coli from O25a O25K5H1 fepE confer the ability to express very long (VL) OAg LPS, while Salmonella LT2 fepE results in OAg sizes that exceed those conferred by E. coli fepE O25a.

[0435] Similar expression patterns were observed with GAR2401 Δ wzzB transformants: E. coli O25a or K12 strains wzzB restored the ability to produce short LPS. Salmonella LT2 fepE produced the longest LPS, E. coli fepE produced slightly shorter LPS, while Salmonella LT2 wzzB produced intermediate size long LPS (L). The ability of other E. coli wzzB genes to produce very long LPS was assessed in independent experiments with GAR2401 Δ fepE transformants. The genes from GAR2401, the O25a ETEC strain, and the strain that produces the O157 Shigella Shigella ) toxin also conferred the ability to produce very long LPS, but not as long as that produced by Salmonella fepE ( ). An alignment of the FepE amino acid sequences is shown in LT2 fepE Figure 4 Figure 5

[0436] Having established that Salmonella LT2 fepE produces the longest LPS of the evaluated polymerase regulators, we next sought to determine if it would produce very long LPS in other E. coli serotypes. Wild-type bacteremic isolates of O1, O2, O6, O15, and O75 serotypes were transformed with the Salmonella fepE plasmid and LPS was extracted. The results shown in Figure 6 demonstrate that Salmonella fepE can confer the ability to produce very long LPS in other prevalent serotypes associated with blood infections. The results also suggest that plasmid-based expression of Salmonella fepE appears to exceed the chain length control normally imposed by the endogenous wzzB in these strains.

[0437] 1.2. Plasmid-based expression of O-antigen in a common E. coli host strain. ​​​

[0438] From a bioprocessing development perspective, the ability to generate O-antigens from different serotypes within a common *E. coli* host, rather than from multiple strains, would greatly simplify the production of individual antigens. To this end, O-antigen gene clusters from different serotypes were amplified by PCR and cloned into a low-copy-number plasmid (pBAD33) controlled by an arabinose-regulated promoter. This plasmid is compatible with *Salmonella* serotypes in *E. coli*. LT2 fepE The plasmids are compatible (can coexist) because they carry different (p15a) replicons and different selection markers (chloramphenicol versus kanamycin). In the first experiment, the pBAD33O25b operon plasmid subclones were used with Salmonella spp. LT2 fepE The plasmid was co-transfected into GAR2401ΔwzzB, and the transformants grew with or without 0.2% arabinose. The results shown in Figure 7 demonstrate the production of very long O-antigen LPS in an arabinose-dependent manner.

[0439] Similarly, O-antigen gene clusters from other serotype clones were evaluated, and the results are shown in... Figure 8 Salmonella spp. LT2 fepE Co-expression of the pBAD33-OAg plasmid resulted in detectable long LPS corresponding to serotypes O1, O2 (for two of the four clones), O16, O21, and O75. For unknown reasons, the pBAD33-O6 plasmid failed to produce detectable LPS in all four isolates tested. Although expression levels are variable, the results indicate that expressing long O-antigens in a common host is feasible. However, in some cases, further optimization may be required to improve expression, for example, by modifying the plasmid promoter sequence.

[0440] From those with or without Salmonella LT2 fepE LPS profiles of different serotypes of Escherichia coli strains with plasmids were shown in Figure 11 The fermentation, extraction, and purification of O-antigen from two strains were studied: GAR2831, for the production of natural short O25b OAg; and GAR2401Δ. wzzB / fepE Used in the production of long O25b OAg. (Shown...) Figure 11 The corresponding short and long forms of LPSs in the SDS-PAGE gel are highlighted in red. Polysaccharides were extracted directly from the fermenting bacteria using acetic acid and purified. Size exclusion chromatography spectra of the purified short and long, or very long, O25b polysaccharides are shown in Figure 12. The properties of two batches of short polysaccharides (from GAR2831) are compared with those of a single very long polysaccharide formulation (from strain GAR2401Δ). wzzB / fepEwere compared. The long O-antigen has a molecular weight that is 3.3-fold greater than the short O-antigen, and the number of repeat units is estimated to be ~65 (very long) versus ~20. See Table 6.

[0441] Table 6

[0442]

[0443] The very long O25b O-antigen polysaccharide was conjugated to diphtheria toxoid CRM 197 using a conventional reductive amination method. Three different batches of conjugate sugar were prepared with different degrees of periodate activation: medium (5.5%), low (4.4%), and high (8.3%). The resulting preparations and unconjugated polysaccharide were shown to be free of endotoxin contamination (Table 7).

[0444] According to the schedule shown in Figure 13A , groups of four rabbits (New Zealand White females) were each inoculated with 10 meg of conjugate sugar and 20 meg of QS21 adjuvant, and serum samples were taken (VAC-2017-PRL-EC-0723). It is noted that the 10 meg dose is at the lower end of the range typically given to rabbits (20-50 meg is more typical) in the evaluation of bacterial conjugate sugars. In a separate study (VAC-2017-PRL-GB-0698), a group of rabbits was also inoculated with unconjugated polysaccharide using the same dose (10 meg polysaccharide + 20 meg QS21 adjuvant) and the same schedule of administration.

[0445] Rabbit antibody responses to the three O25b conjugate sugar preparations were evaluated in a LUMINEX assay in which the carboxyl beads were coated with methylated human serum albumin pre-bound with unconjugated O25b long polysaccharide. The presence of O25b-specific IgG antibodies in the serum samples was detected with a phycoerythrin (PE)-labeled anti-IgG secondary antibody. The observed immune response profile in the sera sampled at week 0 (pre-immune), week 6 (post-dose 2, PD2), week 8 (post-dose 3, PD3), and week 12 (post-dose 4, PD4) in the best responder rabbit (one from each of the four groups) is shown in Figure 14. No significant pre-immune serum IgG titers were detected in any of the 12 rabbits. In contrast, O25b antigen-specific antibody responses were detected in the post-inoculation sera from all three groups of rabbits, with the low-activation conjugate sugar group tending to respond slightly higher than the medium or high-activation conjugate sugar groups. The maximum response was observed at the 3 post-dose time point. One rabbit in the low-activation group and one rabbit from the high-activation group failed to respond to the inoculation (non-responders).

[0446] To assess the CRM 197The effect of carrier protein conjugation on the immunogenicity of long O25b OAg polysaccharide was compared in sera from rabbits immunized with unconjugated polysaccharide to sera from rabbits immunized with low activation CRM 197 Complex carbohydrate immunized rabbit sera were compared in Figure 15. Strikingly, free polysaccharide was not immunogenic, eliciting virtually no IgG response in the immunization compared to pre-immune sera (panel A). In contrast, O25b OAg-CRM 197 O25b OAg-specific IgG mean fluorescence intensity values (MFIs) approximately ten-fold higher than pre-immune serum levels were observed in PD4 sera from three of the four rabbits immunized. These results demonstrate the necessity of carrier protein conjugation for the generation of IgG antibodies against O25b OAg polysaccharide at the 10 mcg dose level.

[0447] Bacteria grown on TSA plates were suspended in PBS, adjusted to an OD 600 of 2.0 and fixed in 4% paraformaldehyde in PBS. After 1 hour blocking in 4% BSA / PBS, bacteria were incubated with serial dilutions of pre-immune and PD3 immune sera in 2% BSA / PBS and bound IgG was detected with PE-labeled secondary F(ab) antibody.

[0448] The specificity of O25b antibodies elicited by O25b OAg-CRM 197 was demonstrated in flow cytometry experiments with whole bacteria. Binding of IgG to whole cells was detected with PE-conjugated F(ab')2 fragment goat anti-rabbit IgG in an Accuri flow cytometer.

[0449] As shown in Figure 16, pre-immune rabbit antibodies failed to bind wild-type serotype O25b isolates GAR2831 and GAR2401 or the K-12 E. coli strain, while the matching PD3 antibodies stained the O25b bacteria in a concentration-dependent manner. The negative control K-12 strain, which lacks the ability to express OAg, showed only very weak binding of PD3 antibodies, most likely due to the presence of exposed internal core oligosaccharide epitopes on its surface. Introduction of the Salmonella fepE plasmid into the wild-type O25b isolates resulted in significantly enhanced staining, consistent with the higher immunogenic epitope density provided by the longer OAg polysaccharide.

[0450] Conclusion: The results described demonstrate that Salmonella fepENot only are the determinants of very long O-antigen polysaccharides in Salmonella species, but they can also confer the ability of E. coli strains of different O-antigen serotypes to produce very long OAg. This property can be exploited to produce O-antigen vaccine polysaccharides with improved properties for bioprocess development by facilitating purification and chemical conjugation to suitable carrier proteins, as well as potentially enhancing immunogenicity by forming higher molecular weight complexes.

[0451] Example 6: Initial rabbit studies produced first polyclonal antibody reagent and IgG responses to RAC O25b OAg-CRM 197

[0452] Long chain O25b polysaccharide-CRM 197 Conjugates were produced using periodate oxidation followed by conjugation using reductive amination chemistry (RAC) (Table 7). See also Table 17.

[0453] Table 7

[0454]

[0455] In rabbit study 1 (VAC-2017-PRL-EC-0723) (also described in Example 5 above) - five (5) rabbits / group received compositions according to the schedule shown in Figure 13A Example 5 above, at 10 ug of L-, M- or H-activated RAC (+ QS21). In a subsequent rabbit study (VAC-2017-PRL-GB-0698), it was observed that unconjugated free O25b polysaccharide was not immunogenic (see Example 6 below). Figure 18

[0456] In rabbit study 2 (VAC-2018-PRL-EC-077) - two (2) rabbits / group received compositions according to the schedule shown in Example 5 above, at L-RAC (AIOH3, QS21 or no adjuvant). Figure 13B

[0457] Rabbits 4-1, 4-2, 5-1, 5-2, 6-1 and 6-2 received the very long unconjugated O25b polysaccharide described in Example 5, and test week 18 sera.

[0458] ​​​More specifically, a composition comprising 50 ug unconjugated O25b, 100 ug AlOH3 adjuvant was administered to rabbit 4-1. A composition comprising 50 ug unconjugated O25b, 100 ug AlOH3 adjuvant was administered to rabbit 4-2. A composition comprising 50 ug unconjugated O25b, 50 ug QS-21 adjuvant was administered to rabbit 5-1. A composition comprising 50 ug unconjugated O25b, 50 ug QS-21 adjuvant was administered to rabbit 5-2. A composition comprising 50 ug unconjugated O25b, no adjuvant was administered to rabbit 6-1. A composition comprising 50 ug unconjugated O25b, no adjuvant was administered to rabbit 6-2.

[0459] Example 7: Rabbit study with O25b RAC conjugate: dLIA serum dilution titers

[0460] Rabbit study 2 (VAC-2018-PRL-EC-077) O25b dLIA serum dilution titers The best responding rabbits from study 1 (VAC-2017-PRL-EC-0723) were used for these experiments. For these experiments, a modified direct binding Luminex assay was performed in which a polylysine conjugate of the long O-antigen of O25b was passively adsorbed onto Luminex carboxyl beads instead of the previously described methylated serum albumin long O-antigen mix. The use of the polylysine-O25b conjugate improved the sensitivity of the assay and the quality of the IgG concentration dependent response, allowing the determination of serum dilution titers by using curve fitting (four parameter non-linear equation). The O25b IgG titers in the sera of the highest titer rabbits from the first study were compared to the sera from the second study rabbits in Table 8.

[0461] Table 8

[0462]

[0463] Higher doses (50 / 20 ug vs 10 ug) did not improve IgG titers in the second rabbit study.

[0464] A two month rest enhanced the IgG response (not observed with shorter intervals).

[0465] Alum appears to enhance the IgG response in rabbits compared to QS21 or no adjuvant.

[0466] A neutrophil opsonophagocytosis assay (OPA) was established using baby rabbit complement (BRC) and HL60 cells as a source of neutrophils to measure the functional immunogenicity of O-antigen complex carbohydrates. Pre-frozen stocks of E. coli GAR2831 were grown in LB media at 37°C. Cells were pelleted and suspended to 1 OD 600 unit concentration in PBS supplemented with 20% glycerol and frozen. Pre-titered thawed bacteria were diluted to 0.5 X 10 5 CFU / ml in 1% gelatin in HBSS (Hank's Balanced Salt Solution) and 10 μΐ (10 3 CFU) were combined with 20 μΐ of serially diluted serum in U-bottom tissue culture microplates and the mixture was shaken at 700 rpm BELLCO shaker) for 30 minutes at 37°C in a 5% CO2 incubator. 10 μΐ of 2.5% complement (Baby Rabbit Serum, PEL-FREEZ 31061-3, pre-diluted in HBG) and 20 μΐ of HL-60 cells (0.75 X 10 7 / ml) and 40 μΐ of HBG were added to the U-bottom tissue culture microplates and the mixture was shaken at 700 rpm BELLCO shaker) for 45 minutes at 37°C in a 5% CO2 incubator. Next, 10 μΐ from each of the 100 μΐ reactions was transferred to the corresponding well of a pre-wetted MILLIPORE MULTISCREEN HTS HV filter plate prepared by applying 100 μΐ of water, vacuum filtered and applying 150 μΐ of 50% LB. The filter plate was vacuum filtered and incubated overnight at 37°C in a 5% CO2 incubator. On the following day, colonies were counted after fixation, staining and destaining with COOMASSIE stain and destain solution using an IMMUNOSPOT® analyzer and IMMUNOCAPTURE software. To establish the specificity of OPA activity, immune sera were pre-incubated with 100 μg / mL of purified long O25b O-antigen prior to combining with the other assay components in the OPA reaction. The OPA assay included control reactions without HL60 cells or complement to demonstrate the dependence of any observed killing on these components.

[0467] Matched pre-immune and post-vaccination serum samples from representative rabbits from two rabbit studies were evaluated in the assay and serum dilution titers were determined (Table 9, Figure 19A -B). Pre-incubation with unconjugated O25b long O-antigen polysaccharide blocked the bactericidal activity, demonstrating the specificity of the OPA.Figure 19C Table 9 OPA titers

[0468] Rabbits 2-3 were dosed as follows: 10 / 10 / 10 / 10 ug RAC conjugate + QS21, with a bleed at post-dose (PD) 4. Rabbits 1-2 were dosed as follows: 50 / 20 / 20 / 20 ug RAC conjugate + Al(OH)3, with a bleed at PD 4.

[0469]

[0470] Example 8: O-antigen O25b IgG levels elicited by unconjugated O25b long O-antigen polysaccharide and derivatized O25b RAC / DMSO long O-antigen complex saccharide.

[0471] Groups of ten CD-1 mice were dosed at weeks 0, 5, and 13 with 0.2 or 2.0 ug / animal of O25b RAC / DMSO long O-antigen complex saccharide by subcutaneous injection, with bleeds for immunogenicity testing at week 3 (post-dose 1, PD1), week 6 (post-dose 2, PD2), and week 13 (post-dose 3, PD3) time points. Levels of antigen-specific IgG were determined by quantitative Luminex assay with O25b-specific mouse mAb as an internal standard (see details in Example 7). Baseline IgG levels were determined in pooled sera from 20x randomly selected unvaccinated mice (dashed line). Free unconjugated O25b long O-antigen polysaccharide immunogen did not induce IgG above baseline levels at any time point. In contrast, an IgG response was observed after two doses of O25b-CRM197 RAC long conjugate complex saccharide: an enhanced uniform IgG response was observed through PD3, while intermediate and more variable IgG levels were seen at PD2. GMT IgG values (ng / ml) are indicated with 95% CI error bars. See Figure 20.

[0472] Example 9: Specificity of O25b rabbit complement (BRC) OPA. A-B) Post-immune sera from rabbits 2-3 and 1-2 (but not the mismatched pre-immune control sera) from O25b RAC / DMSO long O-antigen immunization showed bactericidal OPA activity. C) OPA activity of immune sera from rabbit 1-2 was blocked by pre-incubation with 100 ug / mL long O-antigen O25b polysaccharide. Strain GAR2831 bacteria were incubated with HL60s, 2.5% BRC, and serial dilutions of sera at 37°C for 1 hour, and surviving bacteria were counted by plating on filter plates for small colony counts (CFUs). See Figure 19.

[0473] Example 10: RAC and eTEC O25b long conjugate is more immunogenic than single ended conjugate. BRC OPA assay with carbapenem resistant fluoroquinolone resistant MDR strain Atlas 187913. Groups of 20 CD-1 mice were vaccinated with 2 μg of conjugate according to the same schedule as shown in Figure 21 and OPA responses were measured at post-dose 2 (PD2) (panel A) and post-dose 3 (PD3) (panel B) time points. Bars represent GMTs with 95% CI. The proportion of responders above the unvaccinated baseline is shown. Log-transformed data from different groups were evaluated for statistical significance using unpaired t-test with Welch's correction (Graphpad Prism) to assess if the difference was statistically significant. Results are summarized in Table 10. See Figure 21. In mice vaccinated with 2 μg of eTEC Ola long conjugate, OPA titers against Ola, PD2 and PD3 were observed to be greater than O25b, PD2 and PD3, respectively (data not shown) as shown in Table 10.

[0474] Table 10

[0475]

[0476] Example 11: OPA immunogenicity of eTEC chemistry can be improved by varying the level of polysaccharide activation. BRC OPA assay with carbapenem resistant fluoroquinolone resistant MDR strain Atlas 187913. Groups of 20 CD-1 mice were vaccinated with 0.2 μg or 2 μg of the indicated long O25b eTEC conjugate and OPA responses were measured at the PD2 time point. Log-transformed data from the combined 4% activation vs 17% activation groups were evaluated for statistical significance using unpaired t-test with Welch's correction (Graphpad Prism) to assess if the difference in OPA response was statistically significant. GMTs and the proportion of responders for each group are summarized in Table 11. See Figure 22 .

[0477] Table 11

[0478]

[0479] Example 12: Challenge studies indicate that long E. coli O25b eTEC conjugates elicit protection after three doses. Groups of 20x CD-1 mice immunized with 2 μg doses according to the indicated schedule were challenged with 1 x 10 9Bacterial strains GAR2831 of E. coli were subjected to IP challenge. Survival was monitored for six days. Groups of mice inoculated with eTEC complex saccharide activated at 4%, 10%, or 17% levels were protected from lethal infection, while control mice not inoculated or mice inoculated with 2 μg of unconjugated O25b long polysaccharide were not. See Figure 23.

[0480] Example 13: Methods for preparing eTEC-linked complex saccharides

[0481] Activation of saccharide and thiolation with cystamine dihydrochloride. Saccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The water content of the solution was determined by Karl Fischer (KF) analysis and adjusted to achieve a moisture content of 0.1 and 1.0%, typically 0.5%.

[0482] To initiate activation, a solution of 1,1'-carbonyl-di-1,2,4-triazole (CDT) or 1,1'- carbonyldiimidazole (CDI) was freshly prepared at a concentration of 100 mg / mL in DMSO. The saccharide was activated with various amounts of CDT / CDI (1-10 molar equivalents) and the reaction was allowed to proceed at room temperature (rt) or 35°C for 1-5 hours. Water was added to quench any residual CDT / CDI in the activated reaction solution. Calculations were performed to determine the amount of water to add and to achieve a final moisture content of 2-3% total aqueous. The reaction was allowed to proceed at room temperature for 0.5 hours. Cystamine dihydrochloride was freshly prepared at a concentration of 50 mg / mL in anhydrous DMSO. The activated saccharide was reacted with 1-2 molar equivalents (mol. eq.) of cystamine dihydrochloride. Alternatively, the activated saccharide was reacted with 1-2 molar equivalents of cysteamine hydrochloride. The thiolation reaction was allowed to proceed at room temperature for 5-20 hours to produce the thiolated saccharide. The level of thiolation was determined by the amount of CDT / CDI added.

[0483] Reduction and purification of activated thiolated saccharide. To the thiolated saccharide reaction mixture was added 3-6 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) solution and allowed to proceed at room temperature for 3-5 hours. The reaction mixture was then diluted 5-10-fold by addition to pre-chilled 10 mM sodium phosphate monobasic and filtered through a 5 μm filter. The thiolated saccharide was diafiltered against a pre-chilled 10 mM sodium phosphate monobasic for a 30-40-fold volume of dialysis. An aliquot of the activated thiolated saccharide retentate was obtained to determine saccharide concentration and thiol content (Ellman) assay.

[0484] Activation and purification of bromoacetylated carrier protein. Free amino groups of the carrier protein are bromoacetylated by reaction with a bromoacetylation agent such as bromoacetic acid N-hydroxysuccinimidyl ester (BAANS), bromoacetyl bromide, or another suitable reagent.

[0485] Prior to activation, the carrier protein (in 0.1 M sodium phosphate, pH 8.0 ± 0.2) is first held at 8 ± 3 °C, approximately pH 7. To the protein solution, N-hydroxysuccinimidyl ester of bromoacetate (BAANS) is added as a stock solution in dimethyl sulfoxide (DMSO) at a ratio of 0.25-0.5 BAANS: protein (w / w). The reaction is gently mixed at 5 ± 3 °C for 30-60 minutes. The resulting bromoacetylated (activated) protein is purified by ultrafiltration / diafiltration using a 10 kDa MWCO membrane, for example, using 10 mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein is estimated by the Lowry protein assay.

[0486] The extent of activation is determined by total bromide assay coupled to ion exchange liquid chromatography with conductivity detection (ion chromatography). The bound bromide on the activated bromoacetylated protein is cleaved from the protein in the assay sample preparation and quantified along with any free bromide that can be present. Any remaining covalently bound bromine on the protein is liberated by heating the sample in alkaline 2-mercaptoethanol by conversion to ionic bromide.

[0487] Activation and purification of bromoacetylated CRM 197 197 Diluted to 5 mg / mL with 10 mM phosphate buffered 0.9% NaCl pH 7 (PBS) and in addition 0.1 M NaHCO3 pH 7.0 made from a 1 M stock solution. BAANS was added using a 20 mg / mL DMSO stock of BAANS at a CRM 197 : BAANS ratio of 1 :0.35 (w:w). The reaction mixture was incubated at 3 °C to 11 °C for 30 minutes to 1 hour and then purified by ultrafiltration / diafiltration using a 10K MWCO membrane and 10 mM sodium phosphate / 0.9% NaCl, pH 7.0. The purified activated CRM 197 was determined by the Lowry assay to determine protein concentration and then diluted to 5 mg / mL with PBS. Sucrose was added to 5% wt / vol as a cryoprotectant and the activated protein was frozen and stored at -25 °C until conjugation was required.

[0488] Activation of bromoacetylated CRM 197 The bromoacetylation of lysine residues of CRM

[0489] ​Conjugation of activated thiolated saccharide to bromoacetylated carrier protein. Bromoacetylated carrier protein and activated thiolated saccharide were then added. The saccharide / protein input ratio was 0.8 ± 0.2. The reaction pH was adjusted to 9.0 ± 0.1 with 1 M NaOH solution. The conjugation reaction was allowed to proceed for 20 ± 4 hours at 5 °C.

[0490] Quenching of residual reactive functional groups. Unreacted bromoacetylated residues on the carrier protein were quenched by reacting with 2 molar equivalents of N-acetyl-L-cysteine as a quenching reagent for 3-5 hours at 5 °C. Residual free thiol groups were capped with 4 molar equivalents of iodoacetamide (IAA) for 20-24 hours at 5 °C.

[0491] Purification of eTEC-linked conjugated saccharide. The conjugation reaction (post-IAA capping) mixture was filtered through a 0.45 pm filter. Ultrafiltration / diafiltration of the conjugated saccharide was performed against 5 mM succinate-0.9% saline, pH 6.0. The conjugated saccharide retentate was then filtered through a 0.2 pm filter. An aliquot of the conjugated saccharide was obtained for assay. The remaining conjugated saccharide was stored at 5 °C. See Tables 14, 15, 16, 17, and 18.

[0492] Example 14: Preparation of E. coli O25B ETEC conjugate

[0493] Activation process - activation of E. coli - O25b lipopolysaccharide. Lyophilized E. coli - O25b polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the lyophilized O25b / DMSO solution was determined by Karl Fischer (KF) analysis. The moisture content was adjusted by adding WFI to the O25b / DMSO solution to achieve a moisture content of 0.5%.

[0494] To initiate the activation, 1,1'-carbonyldiimidazole (CDI) was freshly prepared in DMSO solution at 100 mg / mL. Prior to the thiolation step, E. coli - O25b polysaccharide was activated with various amounts of CDI. The CDI reaction was carried out at room temperature or 35 °C for 1-3 hours. Water was added to quench any residual CDI in the activation reaction solution. Calculations were performed to determine the amount of water to add and to achieve a final moisture content of 2-3% total aqueous. The reaction was allowed to proceed at room temperature for 0.5 hours.

[0495] Thiolation of activated E. coli - O25b polysaccharide. Cystamine dihydrochloride was freshly prepared in anhydrous DMSO and 1-2 molar equivalents of cystamine dihydrochloride were added to the activated polysaccharide reaction solution. The reaction was allowed to proceed at room temperature for 20 ± 4 hours.

[0496] Reduction and purification of activated thiolized *E. coli*-O25b polysaccharide. 3-6 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) solution were added to the thiolized sugar reaction mixture, and the mixture was allowed to stand at room temperature for 3-5 hours. The reaction mixture was then diluted 5-10 times by adding to pre-cooled 10 mM sodium dihydrogen phosphate and filtered through a 5 µm filter. The thiolized sugar was percolated using a 5 kW WCO ultrafiltration membrane cartridge with 40-fold dialysis volume of pre-cooled 10 mM sodium dihydrogen phosphate. The thiolized O25b polysaccharide residue was obtained for both sugar concentration and thiol (Ellman) determination. Figure 25A The activation process flowchart is provided in the document.

[0497] Conjugation process - Thiolized E. coli O25b polysaccharide and bromoacetylated CRM 197 The combination. CRM 197 The carrier protein was independently activated via bromoacetylation, as described in Example 13, and then reacted with activated *E. coli*-O25b polysaccharide for a conjugation reaction. The bromoacetylated CRM was placed in a reaction vessel... 197 Mix with thiolized O25b polysaccharide. The sugar / protein input ratio is 0.8 ± 0.2. Adjust the reaction pH to 8.0–10.0. Initiate the conjugation reaction at 5°C for 20 ± 4 hours.

[0498] Bromoacetylated CRM 197 End-capping of reactive groups on thiolated E. coli-O25b polysaccharides. CRM 197 Unreacted bromoacetylated residues on the protein are capped by reacting with 2 molar equivalents of N-acetyl-L-cysteine ​​at 5°C for 3-5 hours, followed by capping any remaining free thiol groups of the thiolated O25b-polysaccharide with 4 molar equivalents of iodoacetamide (IAA) at 5°C for 20-24 hours.

[0499] Purification of eTEC-linked *E. coli*-O25b complex sugars. The conjugated solution was filtered through 0.45 µm or 5 µm filters. Perfiltration of the O25b complex sugars was performed using a 100 K MWCO ultrafiltration membrane cartridge. Perfiltration was performed against 5 mM succinate-0.9% saline at pH 6.0. The 100 K retention of the *E. coli*-O25b complex sugars was then filtered through a 0.22 µm filter and stored at 5 °C.

[0500] Figure 25B The document provides a flowchart of the joining process.

[0501] result

[0502] Reaction parameters and characterization data for several batches of E. coli-025b eTEC complexed saccharides are shown in Table 12. CDI activation-thiolation with cystamine dihydrochloride produced complexed saccharides with 41 to 92% saccharide yield and <5 to 14% free saccharide. See also Tables 14, 15, 16, 17, and 18.

[0503] Table 12 Experimental parameters and characterization data for E. coli-025b eTEC conjugates

[0504]

[0505] Example 15: Procedure for preparing E. coli O-antigen polysaccharide-CRM197 eTEC conjugates (applicable to O-antigens from E. coli serotypes 025b, Ola, O2, and O6

[0506] Activation of the polysaccharide.

[0507] The E. coli O-antigen polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). To initiate activation, various amounts of 1,1'- carbonyldiimidazole (CDI) (1-10 molar equivalents) were added to the polysaccharide solution and the reaction was allowed to proceed at room temperature or 35°C for 1-5 hours. Then, water (2-3%, v / v) was added to quench any residual CDI in the activated reaction solution. After allowing the reaction to proceed at room temperature for 0.5 hours, 1-2 molar equivalents of cystamine dihydrochloride were added. The reaction was allowed to proceed at room temperature for 5-20 hours and then treated with 3-6 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) to produce the thiolated saccharide. The level of thiolation was determined by the amount of CDI added.

[0508] The reaction mixture was then diluted 5-10-fold by addition to pre-chilled 10 mM sodium phosphate monobasic and filtered through a 5 pm filter. The thiolated saccharide was diafiltered against a pre-chilled 10 mM sodium phosphate monobasic for a 30-40-fold volume of the dialysis volume. An aliquot of the activated thiolated saccharide retentate was taken to determine the saccharide concentration and thiol content (Ellman) assay.

[0509] Activation of the carrier protein (CRM 197

[0510] Prior to activation, the CRM 197 ​Maintained at 8 ± 3 °C, about pH 8 in 0.1 M sodium phosphate, pH 8.0 ± 0.2. To the protein solution, N-hydroxysuccinimidyl ester of bromoacetate (BAANS) (20 mg / mL) as a stock solution in dimethyl sulfoxide (DMSO) was added at a ratio of 0.25-0.5 BAANS: protein (w / w). The reaction was gently mixed at 5 ± 3 °C for 30-60 minutes. The resulting bromoacetylated (activated) protein was purified, for example, by ultrafiltration / diafiltration using a 10 kDa MWCO membrane using 10 mM phosphate (pH 7.0) buffer. After purification, the protein concentration of the bromoacetylated carrier protein was estimated by the Lowry protein assay.

[0511] Conjugation

[0512] The activated CRM 197 and activated E. coli O-antigen polysaccharide were added to the reactor and mixed. The sugar / protein input ratio was 1 ± 0.2. The reaction pH was adjusted to 9.0 ± 0.1 with 1 M NaOH solution. The conjugation reactio...

Claims

1. E. coli sugars containing any of the following structures: Contains [→3)-α-L-Rha-(1→3)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-GlcNAc-(1→ |β-D-ManNAc-(1→2) ] n The structure is O1A; Contains [→3)-α-L-Rha-(1→2)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-GlcNAc-(1→ |α-D-Fuc3NAc-(1→2) ] n The structure of O2; Otherwise [→4)-α-D-GalNAc-(1→3)-β-D-Man-(1→4)-β-D-Man-(1→3)-α-D-GlcNAc-(1→|β-D-Glc-(1→2) n [→4)-α-D-GalNAc-(1→3)-β-D-Man-(1→4)-β-D-Man-(1→3)-α-D-GlcNAc-(1→|β-D-GlcNAc-(1→2) n Effective antioxidant O6: Include[ ] n The structural formula is O25b. For each sugar molecule, in its formula... n It is an integer consisting of 31 to 100.

2. The sugar of claim 1, wherein the sugar is produced in a recombinant host cell expressing a wzz family protein, the wzz family protein comprising any one of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:

19.

3. The sugar of claim 2, wherein the protein comprises any one of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:

19.

4. The sugar according to claim 1, wherein the sugar is synthetically synthesized.

5. A conjugate comprising a carrier protein covalently bound to an E. coli sugar, wherein the sugar comprises a structure selected from any of the following: Contains [→3)-α-L-Rha-(1→3)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-GlcNAc-(1→ |β-D-ManNAc-(1→2) ] n The structure is O1A; Contains [→3)-α-L-Rha-(1→2)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-GlcNAc-(1→ |α-D-Fuc3NAc-(1→2) ] n The structure of O2; Otherwise [→4)-α-D-GalNAc-(1→3)-β-D-Man-(1→4)-β-D-Man-(1→3)-α-D-GlcNAc-(1→|β-D-Glc-(1→2) n [→4)-α-D-GalNAc-(1→3)-β-D-Man-(1→4)-β-D-Man-(1→3)-α-D-GlcNAc-(1→|β-D-GlcNAc-(1→2) n Effective antioxidant O6: Include[ ] n The structural formula is O25b. For each sugar molecule, in its formula... n It is an integer consisting of 31 to 100.

6. The conjugate of claim 5, wherein the sugar further comprises any one of the Escherichia coli R1 moiety, Escherichia coli R2 moiety, Escherichia coli R3 moiety, Escherichia coli R4 moiety, and Escherichia coli K-12 moiety.

7. The conjugate of claim 5, wherein the sugar does not further comprise any one of the Escherichia coli R1 portion, Escherichia coli R2 portion, Escherichia coli R3 portion, Escherichia coli R4 portion, and Escherichia coli K-12 portion.

8. The conjugate of claim 5, wherein the sugar does not further comprise the Escherichia coli R2 portion.

9. The conjugate of claim 5, wherein the sugar further comprises a 3-deoxy-d-manno-oct-2-ketoglycolic acid (KDO) moiety.

10. The conjugate of claim 5, wherein the carrier protein is selected from CRM. 197 Diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, exotoxin A from Pseudomonas aeruginosa, exotoxin A (EPA) from Pseudomonas aeruginosa, maltose-binding protein (MBP), Staphylococcus aureus ( S. aureus The detoxifying agents include hemolysin A, aggregation factor A, aggregation factor B, cholera toxin B subunit (CTB), and Streptococcus pneumoniae. Streptococcus pneumoniae ) pneumococcal hemolysin or its antidote variant, Campylobacter jejuni ( C. jejuni (AcrA and any of the natural glycoproteins of Campylobacter jejuni) 11. The conjugate of claim 5, wherein the carrier protein is CRM. 197 .

12. The conjugate of claim 5, wherein the carrier protein is tetanus toxoid.

13. The conjugate of claim 5, wherein the ratio of the sugar to the carrier protein is at least 0.5 to at most 2.

14. The conjugate according to claim 5, wherein the conjugate is prepared by reductive amination.

15. The conjugate of claim 5, wherein the sugar is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer group.

16. The conjugate of claim 5, wherein the sugar is a single-end linked conjugate sugar.

17. The conjugate of claim 9, wherein the sugar is conjugated to the carrier protein via a 3-deoxy-d-manno-oct-2-ketogluconic acid (KDO) residue.

18. The conjugate of claim 5, wherein the conjugate is prepared by CDAP chemistry.

19. A composition comprising (a) A conjugate comprising a carrier protein covalently bound to an E. coli sugar comprising the formula O25b, wherein the formula O25b comprises the following structure: [ ] n For each sugar molecule, in its formula... n It is an integer consisting of 31 to 90. (b) A conjugate comprising a carrier protein covalently bound to an E. coli sugar comprising the formula O1A, wherein the formula O1A comprises the following structure: [→3)-α-L-Rha-(1→3)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-GlcNAc-(1→ | β-D-ManNAc-(1→2) ] n For each sugar molecule, in its formula... n It is an integer consisting of 31 to 90. (c) A conjugate comprising a carrier protein covalently bound to an E. coli sugar comprising the formula O2, wherein the formula O2 comprises the following structure: [→3)-α-L-Rha-(1→2)-α-L-Rha-(1→3)-β-L-Rha-(1→4)-β-D-GlcNAc-(1→ | α-D-Fuc3NAc-(1→2) ] n For each sugar molecule, in its formula... n It consists of integers from 31 to 90, and (d) A conjugate comprising a carrier protein covalently bound to an E. coli sugar comprising formula O6, wherein formula O6 comprises the following structure: [→4)-α-D-GalNAc-(1→3)-β-D-Man-(1→4)-β-D-Man-(1→3)-α-D-GlcNAc-(1→ |β-D-Glc-(1→2) n [→4)-α-D-GalNAc-(1→3)-β-D-Man-(1→4)-β-D-Man-(1→3)-α-D-GlcNAc-(1→|β-D-GlcNAc-(1→2) n For each sugar molecule, in its formula... n It is an integer consisting of 31 to 90.

20. The composition of claim 19, comprising up to 25% free sugars compared to the total amount of sugars in the composition.

21. Use of the composition according to any one of claims 19 to 20 in the preparation of a medicament for inducing an immune response against Escherichia coli in mammals.

22. The use according to claim 21, wherein the immune response comprises opsonization antibodies against Escherichia coli.

23. The use according to claim 21, wherein the immune response protects the mammal from Escherichia coli infection.

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