Methods for making saccharide-protein glycoconjugates

Abstract

The invention provides a process for the reductive amination of a carbonyl group at the reducing terminus of a polysaccharide, wherein the reductive amination is carried out at a pH between 4 and 5. The invention also provides a process for preparing a conjugate of a polysaccharide and a carrier molecule, comprising the steps of: (a) coupling the polysaccharide to a linker, to form a polysaccharide-linker compound in which the free terminus of the linker is an ester group; and (b) reacting the ester group with a primary amine group in the carrier molecule, to form a polysaccharide-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage. The invention also provides a process for reducing contamination of a polysaccharide-linker compound with unreacted linker, comprising a step of precipitating unreacted linker under aqueous conditions at a pH of less than 5. The invention also provides polysaccharide-linker-carrier molecule conjugates and intermediate compounds obtained or obtainable by these processes.

Claims

1. A polysaccharide-linker intermediate of the formula: ##STR00002## wherein OAg is an O-antigen-core from a lipopolysaccharide which has been coupled using a carbonyl group at the reducing terminus.

2. The polysaccharide-linker intermediate of claim 1, wherein the lipopolysaccharide is from a Salmonella bacterium.

3. The polysaccharide-linker intermediate of claim 2, wherein the lipopolysaccharide is from Salmonella serogroups A, B or D.

4. A polysaccharide antigen conjugate prepared by a process comprising the steps of: (a1) reacting a carbonyl group at a reducing terminus of a polysaccharide with Y.sub.1 of an additional linker of Formula 1, wherein Formula 1 is Y.sub.1-L-Y.sub.2, wherein L is a linking moiety, Y.sub.1 and Y.sub.2 are each NHNH.sub.2 groups; (a2) reacting Y.sub.2 of the additional linker with a linker, to form a polysaccharide-additional linker-linker compound in which the free terminus of the linker is an ester group; and (b) reacting the ester group of the linker with a primary amine group in a carrier molecule, to form a polysaccharide-additional linker-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage.

5. The polysaccharide antigen conjugate of claim 4, wherein the polysaccharide comprises an O-antigen from a lipopolysaccharide.

6. The polysaccharide antigen conjugate of claim 5, wherein the polysaccharide also comprises a core domain.

7. The polysaccharide antigen conjugate of claim 5, wherein the lipopolysaccharide is from a Gram negative bacterium.

8. The polysaccharide antigen conjugate of claim 6, wherein the lipopolysaccharide is from a Gram negative bacterium.

9. The polysaccharide antigen conjugate of claim 7, wherein the lipopolysaccharide is from a Salmonella bacterium.

10. The polysaccharide antigen conjugate of claim 8, wherein the lipopolysaccharide is from a Salmonella bacterium.

11. The polysaccharide antigen conjugate of claim 4, wherein the carrier molecule is CRM.sub.197.

12. The polysaccharide antigen conjugate of claim 4, wherein the additional linker is adipic acid dihydrazide (ADH).

13. The polysaccharide antigen conjugate of claim 4, wherein the polysaccharide comprises an O-antigen and a core from a lipopolysaccharide from a Salmonella bacterium, wherein the carrier molecule is CRM.sub.197, wherein the additional linker is adipic acid dihydrazide (ADH), wherein the linker is adipic acid N-hydroxysuccinimide diester (SIDEA) and wherein the additional linker is coupled using a carbonyl group at the reducing terminus of the polysaccharide.

14. A process for preparing a conjugate of a polysaccharide and a carrier molecule comprising the steps of: (a1) reacting a carbonyl group at a reducing terminus of the polysaccharide with Y.sub.1 of an additional linker of Formula 1, wherein Formula 1 is Y.sub.1-L-Y.sub.2, wherein L is a linking moeity, Y.sub.1 and Y.sub.2 are each NHNH.sub.2 groups; (a2) reacting Y.sub.2 with the linker SIDEA, to form a polysaccharide-additional linker-linker compound in which the free terminus of the linker is an ester group; and (b) reacting the ester group of the linker with a primary amine group in a carrier molecule, to form a polysaccharide-additional linker-linker-carrier molecule conjugate in which the linker is coupled to the carrier molecule via an amide linkage.

15. The process of claim 14, wherein the polysaccharide comprises an O-antigen from a lipopolysaccharide.

16. The process of claim 15, wherein the polysaccharide also comprises a core domain.

17. The process of claim 15, wherein the lipopolysaccharide is from a Gram negative bacterium.

18. The process of claim 16, wherein the lipopolysaccharide is from a Gram negative bacterium.

19. The process of claim 17, wherein the lipopolysaccharide is from a Salmonella bacterium.

20. The process of claim 18, wherein the lipopolysaccharide is from a Salmonella bacterium.

21. The process of claim 14, wherein the carrier molecule is CRM.sub.197.

22. The process of claim 14, wherein the additional linker is adipic acid dihydrazide (ADH).

23. The process of claim 14, wherein the polysaccharide comprises an O-antigen and a core from a lipopolysaccharide from a Salmonella bacterium, wherein the carrier molecule is CRM.sub.197, wherein the additional linker is adipic acid dihydrazide (ADH), wherein the linker is adipic acid N-hydroxysuccinimide diester (SIDEA) and wherein the additional linker is coupled using a carbonyl group at the reducing terminus of the polysaccharide.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates published structures of the repeating units of the O-antigens from a) Salmonella Paratyphi A; b) Salmonella Typhimurium; and c) Salmonella Enteritidis.

(2) FIG. 2 illustrates the structure of the Salmonella Paratyphi A O-antigen repeating unit and core domain.

(3) FIG. 3 shows the separation of O:2-CRM.sub.197 from unconjugated components by Sephacryl S300HR.

(4) FIG. 4 shows the immunogenicity of different O:2-CRM.sub.197 conjugates, as measured by an ELISA immunoassay for anti-O:2 antibodies.

(5) FIG. 5 is a schematic of a conjugation method of the invention applied to O-antigen-core.

(6) FIG. 6 illustrates a large scale method for the conjugation of O-antigen from Salmonella Paratyphi A to CRM.sub.197.

(7) FIG. 7 is a schematic of a conjugation method of the invention applied to the capsular polysaccharide from N. meningitidis serogroup X.

(8) FIG. 8 shows an SDS-PAGE analysis of conjugates of capsular polysaccharide from N. meningitidis serogroup X and CRM.sub.197.

(9) FIG. 9 shows the result of Serum Bactericidal Activity (SBA) assays performed using day 42 pooled sera from mice immunized with 8 ug of conjugated or unconjugated O:2 and S. Paratyphi A strain. Data are presented as percentage of CFU recovered in test sera with active BRC (and in control serum with inactive BRC) compared with CFU present in negative control, per anti-O:2 ELISA antibody unit of each serum pool.

MODES FOR CARRYING OUT THE INVENTION

(10) Summary of Conjugate Production

(11) Different methods were compared for conjugating the O-antigen-core from S. Paratyphi to CRM.sub.197. In particular, conjugates obtained by activating the O:2 chain randomly, or specifically through the terminal KDO subunit, were compared. Theoretically, activation of only the KDO would not modify the structure of the repeating saccharides and would therefore result in a better defined and easier to characterize conjugate.

(12) Two methods were based on prior art methods. In the first of these methods, the O-antigen was randomly activated with ADH using CDAP and then conjugated to CRM.sub.197 using EDAC. In the second method, the core region of the O-antigen was activated with ADH (using the COOH group of KDO by EDAC) and then conjugated to CRM.sub.197 using EDAC.

(13) Alternative methods were also tested. A first method involved KDO activation with ADH through the ketone group by reductive amination, followed by activation with SIDEA, and then conjugation with CRM.sub.197. A second method substituted the ADH with CDH. A third method involved activation of O-antigen with SIDEA (without activation of KDO with ADH) using the pyrophosphoethanolamine group on the core region.

(14) Conjugation of O-Antigen-Core to CRM.sub.197

(15) (Comparative) Method A: Random Activation of the O-Antigen-Core Chain with ADH by CDAP and Conjugation with CRM.sub.197 by EDAC

(16) This conjugate was synthesized according to ref. 2, as described in detail below.

(17) O-Antigen-Core Derivatisation with ADH Through CDAP.

(18) 30 Cpl of CDAP (100 mg/mL in acetonitrile) was added per ml of 10 mg/ml O-antigen-core in 150 mM NaCl at room temperature. The pH was maintained at 5.8 to 6.0 for 30 seconds, then 0.2 M TEA was added to increase the pH to 7.0 and the solution mixed at room temperature for 2 minutes. 1 ml of 0.8 M ADH in 0.5 M NaHCO.sub.3 was then added per 10 mg of O-antigen-core. The reaction was carried out for 2 hours at room temperature, and pH maintained at 8.0 to 8.5 with 0.1 N NaOH. The reaction mixture was then desalted using a G-25 column against water and the product, designated as OAg-(CDAP)ADH, characterized.

(19) Conjugation of OAg-(CDAP)ADH with CRM.sub.197.

(20) The OAg-(CDAP)ADH was dissolved in 100 mM MES at pH 5.8. An equal weight of protein was added to give an O-antigen-core:CRM.sub.197 ratio of 1:1 by weight, with an O-antigen-core concentration of 5 mg/ml. The reaction mixture was placed on ice and EDAC added to a final concentration of 50 mM, The reaction was mixed on ice for a further 4 h. The resulting conjugate was designated OAg-(CDAP)ADH-CRM.sub.197.

(21) (Comparative) Method B: Activation of Terminal KDO with ADH by EDAC and Conjugation with CRM.sub.9 by EDAC

(22) This conjugate was synthesized according to ref. 9.

(23) O-Antigen-Core_Derivatization with ADH at KDO Through EDAC.

(24) The O-antigen-core was solubilized at 3 mg/ml in 100 mM MES at pH 5.8. ADH was then added at a w/w ratio ADH:O-antigen-core of 1.36, followed by EDAC to a final concentration of 3.7 mM. The reaction was mixed at room temperature for 4 hours. The reaction mixture was then desalted using a G-25 column against water and the product, designated as OAg-(EDAC)ADH, characterized.

(25) Conjugation of OAg-(EDAC)ADH with CRM.sub.197.

(26) The conjugate was prepared according to the method described in Method A above for OAg-(CDAP)ADH. The conjugate was designated as OAg-(EDAC)ADH-CRM.sub.197.

(27) Methods C and D: Activation of the Terminal KDO with ADH (Method C) or CDH (Method D) by Reductive Amination and Conjugation with CRM.sub.197 Via SIDEA Linker

(28) O-Antigen-Core Derivatization with ADH or CDH at KDO by Reductive Amination.

(29) After testing different conditions, an optimized protocol for the O-antigen-core derivatization was identified. 0-antigen-core was solubilized at 40 mg/ml in 100 mM AcONa at pH 4.5. Either ADH or CDH was added at a w/w ratio of 1:2 with respect to the O-antigen-core. NaBH.sub.3CN was then added at a w/w ratio of 1:2 with respect to the O-antigen-core. The solution was mixed at 30 C. for 1 hour. The reaction mixture was then desalted using a G-25 column against water and the product, designated as OAg-ADH or OAg-CDH characterized.

(30) OAg-ADH and OAg-CDH Derivatization with SIDEA.

(31) Either OAg-ADH or OAg-CDH was dissolved in 1:9 (vol/vol) water/DMSO to a final O-antigen-core concentration of 50 mg/ml. Once the polysaccharide was in solution, TEA was added to give a molar ratio of TEA/total NH.sub.2 groups of 5 and then SIDEA to give a molar ratio of SIDEA/total NH.sub.2 groups of 12. The solution was mixed at room temperature for 3 hours. In preliminary attempts to purify the SIDEA-derivatised O-antigen-core, the O-antigen-core was precipitated by addition of AcOEt or dioxane (90% volume in the resulting solution) and washing the pellet times with the same organic solvent (ten times using of the volume added for the precipitation) in order to remove unreacted SIDEA. This process was then adapted to avoid the use of toxic AcOEt and dioxane reagents. A volume of 100 mM sodium citrate at pH 3 equal to two times the volume of the SIDEA-derivatised O-antigen-core reaction mixture was added and mixed at 4 C. for 30 min. Unreacted SIDEA was precipitated by the low pH and separated by centrifugation. The SIDEA-derivatised O-antigen-core was then recovered from the supernatant by precipitation with absolute ethanol (80% volume in the resulting solution). The pellet was washed with ethanol twice (using of the volume added for the precipitation) and dried. The product, designated as OAg-ADH-SIDEA or OAg-CDH-SIDEA was characterized.

(32) Conjugation of OAg-ADH-SIDEA and OAg-CDH-SIDEA with CRM.sub.197.

(33) The OAg-ADH-SIDEA or OAg-CDH-SIDEA was solubilized in NaH.sub.2PO.sub.4 buffer at pH 7.2 and CRM.sub.197 added to a final protein concentration of 20 mg/ml, final buffer capacity of 100 mM and molar ratio of active ester groups to CRM.sub.197 of 30 to 1. The reaction was mixed at room temperature for 2 hours.

(34) Method E: Direct Conjugation with CRM.sub.197 Via SIDEA Linker

(35) The reaction conditions used in the above OAg-ADH and OAg-CDH derivatization with SIDEA was also applied to native O-antigen-core (i.e. O-antigen-core that had not previously been derivatized with ADH or CDH). The resulting product was designated as as OAg-SIDEA. The OAg-SIDEA was then conjugated to CRM.sub.197 by the reaction conditions used in the above Conjugation of OAg-ADH-SIDEA and OAg-CDH-SIDEA with CRM.sub.197.

(36) Purification of the O-Antigen-Core Conjugates

(37) Conjugates made according to methods A-E above were purified by size exclusion chromatography on a 1.6 cm90 cm S-300 HR column eluted at 0.5 ml/min in 50 mM NaH.sub.2PO.sub.4, 0.15 M NaCl at pH 7.2. Different pools were collected according to free O-antigen-core and free CRM.sub.197 profiles on the same column in the same eluting conditions (FIG. 3). The first pool at high molecular weight (corresponding to the purified conjugate) did not contain free saccharide or free protein.

(38) Analysis of Conjugates

(39) The conjugates were analysed by SDS-PAGE and showed an expected high molecular weight population smear compared to free CRM.sub.197. The conjugates were separated from free O:2 and CRM.sub.197 by Sephacryl S300HR size exclusion (1.690 cm column; 50 mM NaH.sub.2PO.sub.4, 150 mM NaCl pH 7.2; 0.5 mL/min flow). Results are shown in FIG. 3. Purified conjugates were then characterized by the phenol sulfuric assay of ref. 209 (total sugar), microBCA (total protein), HPAEC-PAD (sugar composition) and HPLC-SEC (size determination, Kd). Results are shown in Table 1 below.

(40) TABLE-US-00001 TABLE 1 Wt/wt Total Presence ratio Kd sugar, of free Protein O:2/ (HPLC- Conjugate g/mL O:2 g/mL CRM.sub.197 SEC) O:2 yes 0.549 CRM.sub.197 0.690 O:2-(CDAP)ADH- 32.88 no 62.98 0.52 0.439 CRM.sub.197 pool 1 O:2-(CDAP)ADH- 101.09 yes 60.44 1.67 0.534 CRM.sub.197 pool 2 O:2-CDH-SIDEA- 82.82 no 36.67 2.26 0.403 CRM.sub.197 Lot A O:2-CDH-SIDEA- 167.61 no 38.19 4.39 2 peaks CRM.sub.197 Lot B 0.128 0.413 O:2-ADH-SIDEA- 51.54 no 29.56 1.74 2 peaks CRM.sub.197 Lot A 0.115 0.388 O:2-ADH-SIDEA- 215.13 no 50.89 4.23 2 peaks CRM.sub.197 Lot B 0.118 0.421 O:2-(EDAC)ADH- 50.19 yes 31.31 1.60 0.52 CRM.sub.197 O:2-SIDEA- 108.57 no 52.79 2.06 0.376 CRM.sub.197, pool 1 O:2-SIDEA- 185.31 yes 68.18 2.72 0.457 CRM.sub.197, pool 2

(41) Imunogenicity Studies

(42) An ELISA immunoassay was used to detect anti-O:2 antibodies elicited by O:2-CRM.sub.197 immunized mice. For the assay, MaxiSorp microtiter plates were coated with 15 g/mL O:2 in a carbonate coating buffer (pH 9.6) overnight at 4 C.

(43) The O:2-CRM.sub.197 conjugates were compared to unconjugated O:2 antigen. Briefly, groups of female CD1 mice (8 per group at 5 weeks of age) were injected subcutaneously with 200 L of conjugates as set out in Table 2. Mice received immunizations on days 0, 14 and 28. Sera were collected from the mice during the course of the study and tested by the ELISA assay.

(44) TABLE-US-00002 TABLE 2 Group Vaccine O:2 antigen dose, g 1 O:2-(CDAP)ADH-CRM.sub.197, pool 1 1 2 8 3 O:2-(CDAP)ADH-CRM.sub.197, pool 2 1 4 8 5 O:2-CDH-SIDEA-CRM.sub.197 1 6 8 7 O:2-ADH-SIDEA-CRM.sub.197 1 8 8 9 O:2-(EDAC)ADH-CRM.sub.197 1 10 8 11 Unconjugated O:2 antigen 8

(45) ELISA results through day 42 of the study showed that all the conjugates, except O:2-(EDAC)ADH-CRM.sub.197 were able to elicit high serum levels of anti-O:2 IgG antibodies in mice when delivered at the 1 g dose (FIG. 4). Increases in antibody were observed following the second vaccination with conjugate, whereas repeated immunization with 8 g of unconjugated O:2-antigen did not result in specific IgG antibodies. Delivery of 8 g doses was no better than 1 g doses at generating a humoral immune response in mice (FIG. 4).

(46) Serum Bactericidal Activity

(47) Serum Bactericidal Activity (SBA) assays were performed using day 42 pooled sera from mice immunized with 8 ug of conjugated or unconjugated O:2 and S. Paratyphi A (see Table 2). The result is shown in FIG. 9. Inhibition of S. Paratyphi A growth in vitro correlated with increasing anti-O:2 ELISA units present in the sera pools. The strongest growth inhibition was observed with those conjugates produced using a selective chemistry; both SIDEA conjugates and O:2-(EDAC)ADH-CRM197 resulted in increased inhibition compared to O:2-(CDAP)ADH-CRM197, whose O:2 was randomly modified prior to conjugation. Even unconjugated O:2, although far from reaching high levels of bacterial growth inhibition due to the lower amount of antibody present in the sera, presented an inhibition profile similar to the conjugates prepared with unmodified O:2 chains. No bacterial growth inhibition was detected using control serum in the presence of iBRC (the same sera in the presence of active BRC is shown as control), indicating a role for complement mediated killing.

(48) Larger-Scale Processing

(49) Based on the immunogenicity study and ease of conjugate characterization, the conjugation method based on O:2-activation with ADH and then SIDEA and reaction with CRM.sub.197 (method C, FIG. 5) was selected for further development. Conjugates made by this method were more immunogenic than conjugates made by the method B, for example. Although conjugates made by method A gave comparable ELISA titers (FIG. 4), they resulted in considerably lower Serum Bactericidal Activity (see Example and FIG. 9). Furthermore these conjugates had a cross-linked structure, with multiple possible points of linkage on the polysaccharide chain to one or more protein molecules, a with disadvantages in terms of reproducibility and characterization of the product. In contrast, the conjugates of method C contain more polysaccharide chains per protein molecule, with only one point of linkage on the polysaccharide chain. The remainder of the polysaccharide chain is left unchanged. Derivatization of the O-antigen-core through CDAP (method A) can result in crosslinking of the sugar chains and the overall conjugation method was more complicated. The use of EDAC in methods A and B can also result in cross-linking of the protein.

(50) A scaled-up process was developed for method C for the production of greater amounts of conjugate (FIG. 6). In particular, the step of O:2 reductive amination with ADH was optimized to reduce the reaction time to 1 hour (instead of 7-14 days as previous reported for this kind of reaction [13]) with good % of O:2 activation (>65%). This step was scaled to 300 mg of O:2 and repeated several times with reproducible results in terms of yield and derivatisation degree. Recovery was >75% after tangential flow filtration, with >70% of O-antigen-core activation (calculated as molar ratio of linked ADH groups per GlcNAc groups on the O-antigen-core) and good purity (ratio of free ADH/linked ADH <1%). The stability of the O:2-ADH intermediate in aqueous solution at 4 C. was verified to be >1 month. The inventors envisage replacing the drying step by O:2-ADH precipitation in 85% ethanol.

(51) The reaction of O:2-ADH with SIDEA was optimized to avoid the use of AcOEt during O:2-ADH-SIDEA purification. Removal of free SIDEA by precipitation at pH 4-5 and then precipitation of O:2-ADH-SIDEA in 85% EtOH showed better precipitate formation that was easier to wash compared with AcOEt. Recoveries >85% were obtained working on a 100 mg scale with activated NH.sub.2 groups >80%.

(52) The inventors have found that in addition to O:2 from S. Paratyphi A the conjugation method based on O-antigen-core activation with ADH and then SIDEA and reaction with CRM.sub.197 (method C, FIG. 5), works equally well for O-antigen-core from S. Typhimurium and O-antigen-core from S. Enteritidis.

(53) Conjugation of MenX Capsular Polysaccharide to CRM.sub.197

(54) Method C was also applied to the conjugation of capsular polysaccharide from N. meningitidis serogroup X (FIG. 7), resulting in conjugate formation with no free protein in the reaction mixture (FIG. 8).

(55) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

(56) Effect of pH and Temperature on the Reductive Amination of S. Paratyphi a O-Antigen-Core with ADH as Linker

(57) Experiments summarized in Table 3 were performed working with S. Paratyphi A O-antigen-core. The effect of different pH and different temperatures was evaluated. The best activation was obtained at lower pH and was temperature independent. Reactions were performed in 100 mM buffer, with O-antigen-core concentration of 20 mg/mL and a ratio of ADH to O-antigen-core and NaBH.sub.3CN to O-antigen-core both of 1.2 to 1 (w/w). ADH and NaBH.sub.3CN were added at the same time and solutions were mixed for 1 hour.

(58) TABLE-US-00003 TABLE 3 Reductive animation of O:2-KDO with ADH is pH dependent and temperature independent. Buffer Temperature % activated O:2 Sodium acetate pH 4.5 30 C. 65.8 Sodium acetate pH 4.5 50 C. 65.4 Sodium acetate pH 4.5 60 C. 68.3 MES pH 6.0 30 C. 43.9 Phosphate pH 8.0 30 C. 26.2

(59) Derivatization of S. Typhimurium O-Antigen-Core with ADH by Reductive Amination Comparing Different Reaction Conditions (Table 4).

(60) Reaction conditions described in this application (Table 4, method 1) for performing the reductive amination with ADH were compared with the traditional method reported in literature [13] (Table 4, method 2), working with S. Typhimurium O-antigen-core (strain D23580). Results are summarized in Table 5, showing that the process at lower pH is faster and also more efficient.

(61) TABLE-US-00004 TABLE 4 Reaction conditions used for performing reductive animation with ADH comparing NVGH procedure with the classical procedure reported in literature. Temper- OAg ADH NaBH.sub.3CN ature Method (mg/mL) (mg/mL) (mg/mL) Buffer ( C.) 1 40 48 48 AcONa 30 100 mM pH 4.5 2 20 100 100 NaHCO.sub.3 37 100 mM pH 8.3

(62) TABLE-US-00005 TABLE 5 Reductive amination of O:4,5-KDO with ADH using method 1 is more efficient and faster than using the classical method reported in literature (method 2). Reaction % activated Method time O:4,5 1 3 h 88 2 3 h 31.6 2 24 h 28.5 2 5 d 28.5

REFERENCES

(63) [1] Watson et al. (1992) Infect Immun. 60(11):4679-86 [2] Konadu et al. (1996) Infect Immun. (7):2709-15. [3] Petri et al. (2008) J Clin Invest. 118(4):1277-90. [4] Ashkenazi et al. (1999) J Infect Dis. 179(6):1565-8 [5] Cohen et al. (1997) Lancet 349(9046):155-9 [6] Passwell et al. (2003) Pediatr Infect Dis J. 22(8):701-6 [7] Pozsgay et al. (2007) Proc NatlAcadSci USA. 104(36):14478-82 [8] Robbins et al. (2009) Proc Natl Acad Sci USA. 106(19):7974-8 [9] Taylor et al. (1993) Infect Immun. 61(9):3678-87 [10] Konadu et al. (1994) Infect Immun. 62(11):5048-54. [11] Ahmed et al. (2006) J Infect Dis. 193(4):515-21 [12] Cox et al. (2011) Glycoconj J 28:165-182 [13] Chu et al. (1991) Infect Immun. 59(12):4450-58. [14] WO96/40242. [15] Lei et al. (2000) Dev Biol (Basel) 103:259-264. [16] WO00/38711; U.S. Pat. No. 6,146,902. [17] Westphal and Jann (1965) Methods Carbohydr. Chem. 5:83-91. [18] Micoli et al., 2012 PlosOne, in press. [19] Knirel et al. (2011) Glycobiology. (10):1362-72, [20] Chhibber et al. (2005) Indian J Exp Biol. 43(1):40-5 [21] Gupta et al. (1992) Infect Immun. 60(8):3201-8 [22] Cox et al. (2005) Vaccine. 23(43):5045-54 [23] Lemercinier and Jones (1996) Carbohydrate Res. 296:83-96. [24] Hestrin (1994) J Biol Chem 180(1):249-61. [25] Kao and Tasi (2004) Vaccine. 22(3-4):335-44 [26] Ramsay et al. (2001) Lancet 357(9251):195-196. [27] Lindberg (1999) Vaccine 17 Suppl 2:S28-36. [28] Buttery & Moxon (2000) J R Coil Physicians Lond 34:163-168. [29] Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-33, vii. [30] Goldblatt (1998) J. Med. Microbiol. 47:563-567. [31] European patent 0477508. [32] U.S. Pat. No. 5,306,492. [33] WO98/42721. [34] Dick et al. in Conjugate Vaccines (eds. Cruse et al.) Karger, Basel, 1989, 10:48-114. [35] Hermanson Bioconjugate Techniques, Academic Press, San Diego (1996) ISBN: 0123423368. [36] Research Disclosure, 453077 (January 2002) [37] EP-A-0372501. [38] EP-A-0378881. [39] EP-A-0427347. [40] WO93/17712 [41] WO94/03208. [42] WO98/58668. [43] EP-A-0471177. [44] WO91/01146 [45] Falugi et al. (2001) Eur J Immunol 31:3816-3824. [46] Baraldo et al. (2004) Infect Immun 72(8):4884-7. [47] EP-A-0594610. [48] Ruan et al. (1990) J Immunol 145:3379-3384. [49] WO00/56360. [50] Kuo et al. (1995) Infect Immun 63:2706-13. [51] Michon et al. (1998) Vaccine. 16:1732-41. [52] WO02/091998. [53] WO01/72337 [54] WO00/61761. [55] WO00/33882 [56] WO99/42130 [57] Canh et al. (2004) Infect Immun. 72(11):6586-8 [58] Rondini et al. (2011) Clin Vaccine Immunol. 18(3):460-8 [59] Micoli et al. (2011) Vaccine. 29(4):712-20 [60] WO 2009/150543 [61] Watson (2000) Pediatr Infect Dis J 19:331-332. [62] Rubin (2000) Pediatr Clin North Am 47:269-285, v. [63] Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207. [64] Vaccines (2004) eds. Plotkin & Orenstein. ISBN 0-7216-9688-0. [65] Bell (2000) Pediatr Infect Dis J 19:1187-1188. [66] Iwarson (1995) APMIS 103:321-326. [67] Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80. [68] Hsu et al. (1999) Clin Liver Dis 3:901-915. [69] Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355. [70] Rappuoli et al. (1991) TIBTECH 9:232-238. [71] WO02/02606. [72] Kalman et al. (1999) Nature Genetics 21:385-389. [73] Read et al. (2000) Nucleic Acids Res 28:1397-406. [74] Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527. [75] WO99/27105. [76] WO00/27994. [77] WO00/37494. [78] WO99/28475. [79] Ross et al. (2001) Vaccine 19:4135-4142. [80] Sutter et al. (2000) Pediatr Clin North Am 47:287-308. [81] Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126. [82] Dreesen (1997) Vaccine 15 Suppl:S2-6. [83] MMWR Morb Mortal Wkly Rep 1998 Jan. 16; 47(1):12, 19. [84] McMichael (2000) Vaccine 19 Suppl 1:S101-107. [85] WO02/34771. [86] Dale (1999) Infect Dis Clin North Am 13:227-43, viii. [87] Ferretti et al. (2001) PNAS USA 98: 4658-4663. [88] WO03/093306. [89] WO2004/018646. [90] WO2004/041157. [91] Ichiman and Yoshida (1981) J. Appl. Bacteriol. 51:229. [92] U.S. Pat. No. 4,197,290 [93] Ichiman et al. (1991) J. Appl. Bacteriol. 71:176. [94] Robinson & Torres (1997) Seminars in Immunology 9:271-283. [95] Donnelly et al. (1997) Annu Rev Immunol 15:617-648. [96] Scott-Taylor & Dalgleish (2000) Expert Opin Investig Drugs 9:471-480. [97] Apostolopoulos & Plebanski (2000) Curr Opin Mol Ther 2:441-447. [98] Ilan (1999) Curr Opin Mol Ther 1:116-120. [99] Dubensky et al. (2000) Mol Med 6:723-732. [100] Robinson & Pertmer (2000) Adv Virus Res 55:1-74. [101] Donnelly et al. (2000) Am J Respir Crit Care Med 162(4 Pt 2):S190-193. [102] Davis (1999) Mt. SinaiJ. Med. 66:84-90. [103] Paoletti et al. (2001) Vaccine 19:2118-2126. [104] WO00/56365. [105] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [106] WO03/009869. [107] Almeida & Alpar (1996) J. Drug Targeting 3:455-467. [108] Agarwal & Mishra (1999) Indian J Exp Biol 37:6-16. [109] WO00/53221. [110] Jakobsen et al. (2002) Infect Immun 70:1443-1452. [111] Bergquist et al. (1998) APMIS 106:800-806. [112] Baudner et al. (2002) Infect Immun 70:4785-4790. [113] Ugozzoli et al. (2002) Jlnfect Dis 186:1358-1361. [114] WO90/14837. [115] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203. [116] Podda (2001) Vaccine 19: 2673-2680. [117] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X). [118] Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan. [119] Allison & Byars (1992) Res Immunol 143:519-25. [120] Hariharan et al. (1995) Cancer Res 55:3486-9. [121] US-2007/014805. [122] WO95/11700. [123] U.S. Pat. No. 6,080,725. [124] WO2006/113373. [125] WO2005/097181. [126] U.S. Pat. No. 5,057,540. [127] WO96/33739. [128] EP-A-0109942. [129] WO96/11711. [130] WO00/07621. [131] Barr et al. (1998) Advanced Drug Delivery Reviews 32:247-271. [132] Sjolanderet et al. (1998) Advanced Drug Delivery Reviews 32:321-338. [133] EP-A-0689454. [134] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278. [135] Evans et al. (2003) Expert Rev Vaccines 2:219-229. [136] Meraldi et al. (2003) Vaccine 21:2485-2491. [137] Pajak et al. (2003) Vaccine 21:836-842. [138] Kandimalla et al. (2003) Nucleic Acids Research 31:2393-2400. [139] WO02/26757. [140] WO99/62923. [141] Krieg (2003) Nature Medicine 9:831-835. [142] McCluskie et al. (2002) FEMS Immunology and Medical Microbiology 32:179-185. [143] WO98/40100. [144] U.S. Pat. No. 6,207,646. [145] U.S. Pat. No. 6,239,116. [146] U.S. Pat. No. 6,429,199. [147] Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-658. [148] Blackwell et al. (2003) J Immunol 170:4061-4068. [149] Krieg (2002) Trends Immunol 23:64-65. [150] WO01/95935. [151] Kandimalla et al. (2003) BBRC 306:948-953. [152] Bhagat et al. (2003) BBRC 300:853-861. [153] WO03/035836. [154] Schellack et al. (2006) Vaccine 24:5461-72. [155] Lingnau et al. (2007) Expert Rev Vaccines 6:741-6. [156] WO2004/084938. [157] WO95/17211. [158] WO98/42375. [159] Beignon et al. (2002) Infect Immun 70:3012-3019. [160] Pizza et al. (2001) Vaccine 19:2534-2541. [161] Pizza et al. (2000) Int J Med Microbiol 290:455-461. [162] Scharton-Kersten et al. (2000) Infect Immun 68:5306-5313. [163] Ryan et al. (1999) Infect Immun 67:6270-6280. [164] Partidos et al. (1999) Immunol Lett 67:209-216. [165] Peppoloni et al. (2003) Expert Rev Vaccines 2:285-293. [166] Pine et al. (2002) J Control Release 85:263-270. [167] Tebbey et al. (2000) Vaccine 18:2723-34. [168] Domenighini et al. (1995) Mol Microbiol 15:1165-1167. [169] WO99/40936. [170] WO99/44636. [171] Singh et al] (2001) J Cont Release 70:267-276. [172] WO99/27960. [173] U.S. Pat. No. 6,090,406. [174] U.S. Pat. No. 5,916,588. [175] EP-A-0626169. [176] Stanley (2002) Clin Exp Dermatol 27:571-577. [177] Jones (2003) Curr Opin Investig Drugs 4:214-218. [178] WO99/11241. [179] WO94/00153. [180] WO98/57659. [181] European patent applications 0835318, 0735898 and 0761231. [182] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472. [183] Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.) [184] Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds, 1986, Blackwell Scientific Publications) [185] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition (Cold Spring Harbor Laboratory Press). [186] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed., CRC Press, 1997) [187] Ausubel et al. (eds) (2002) Short protocols in molecular biology, 5th edition (Current Protocols). [188] Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press) [189] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag) [190] Geysen et al. (1984) PNAS USA 81:3998-4002. [191] Carter (1994) Methods Mol Biol 36:207-23. [192] Jameson, B A et al. 1988, CABIOS 4(1):181-186. [193] Raddrizzani & Hammer (2000) Brief Bioinform 1(2):179-89. [194] Bublil et al. (2007) Proteins 68(1):294-304. [195] De Lalla et al. (1999) J. Immunol. 163:1725-29. [196] Kwok et al. (2001) Trends Immunol 22:583-88. [197] Brusic et al. (1998) Bioinformatics 14(2):121-30 [198] Meister et al. (1995) Vaccine 13(6):581-91. [199] Roberts et al. (1996) AIDS Res Hum Retroviruses 12(7):593-610. [200] Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-7. [201] Feller & de la Cruz (1991) Nature 349(6311):720-1. [202] Hopp (1993) Peptide Research 6:183-190. [203] Welling et al. (1985) FEBS Lett. 188:215-218. [204] Davenport et al. (1995) Immunogenetics 42:392-297. [205] Tsurui & Takahashi (2007) J Pharmacol Sci. 105(4):299-316. [206] Tong et al. (2007) Brief Bioinform. 8(2):96-108. [207] Schirle et al. (2001) J Immunol Methods. 257(1-2):1-16. [208] Chen et al. (2007) Amino Acids 33(3):423-8. [209] Dubois et al. (1956) Analytical Chemistry 28:350.