Modified saccharides

Abstract

Modified capsular saccharides comprising a blocking group at a hydroxyl group position on at least one of the monosaccharide units of the corresponding native capsular saccharide, wherein the blocking group is of the formula (Ia) or (Ib): —OX—Y (Ia) or —O—R.sup.1 (Ib) wherein X is C(O), S(O) or SO.sub.2; Y is NR.sup.1R.sup.2 or R.sup.3; R.sup.1 is C.sub.1-6 alkyl substituted with 1, 2 or 3 groups independently selected from hydroxyl, sulphydryl and amine; R.sup.2 is H or C.sub.1-6 alkyl; and R.sup.3 is C.sub.1-6 alkyl; processes for modifying a capsular saccharide with the blocking groups; saccharide-protein conjugates comprising the modified capsular saccharide; processes for making the saccharide-protein conjugates, pharmaceutical compositions comprising the modified capsular saccharides and/or saccharide-protein conjugates; and methods and uses of the same.

Claims

1. A saccharide-protein conjugate, wherein the saccharide is a modified Neisseria meningitidis serogroup A capsular saccharide wherein at least 90% of the hydroxyl groups at position 3 and at least 90% of the hydroxyl groups at position 4 of the monosaccharide units of the saccharide comprise a blocking group of the formula (Ia):
—O—X—Y  (Ia) wherein X is C(O); Y is R.sup.3; and R.sup.3 is CH.sub.3, wherein the capsular saccharide comprises four or more monosaccharide units, and an effective amount of the modified capsular saccharide is able to induce a protective immune response in mammals.

2. The conjugate according to claim 1, wherein all the monosaccharide units of the saccharide have blocking groups, at both the position 3 hydroxyl groups and the position 4 hydroxyl groups.

3. The conjugate according to claim 1, wherein the modified capsular saccharide is an oligosaccharide.

4. The conjugate according to claim 1, wherein there is at least one monosaccharide unit of the modified capsular saccharide where two vicinal hydroxyl groups of the saccharide do not comprise blocking groups.

5. The conjugate of claim 1, wherein the protein is a bacterial toxin or toxoid.

6. The conjugate of claim 5, wherein the bacterial toxin or toxoid is diphtheria toxin or toxoid.

7. The conjugate of claim 5, wherein the bacterial toxin or toxoid is CRM197.

8. A pharmaceutical composition comprising (a) the conjugate according to claim 1, and (b) a pharmaceutically acceptable carrier.

9. The composition according to claim 8, further comprising a saccharide antigen from one or more of semigroups C, W135 and Y of N. meningitidis.

10. The composition according to claim 8 or claim 9, further comprising a vaccine adjuvant.

11. The composition according to claim 10, wherein the adjuvant is an aluminium phosphate.

12. The composition according to claim 8, which is a vaccine against a disease caused by N. meningitidis.

13. A method for raising an antibody response in a mammal, comprising administering the pharmaceutical composition according to claim 8 to the mammal.

14. The composition according to claim 9, wherein the saccharide, in the conjugate, is an oligosaccharide.

15. A molecule comprising a saccharide moiety of formula: ##STR00019## wherein T is of the formula (A) or (B): ##STR00020## n is an integer from 2 to 100; each Z group is independently selected from OH or a blocking group of the formula (1a):
—O—X—Y  (Ia); each Q group is independently selected from OH or a blocking group of the formula (1a):
—O—X—Y  (Ia); W is selected from OH or a blocking group of the formula (Ia):
—O—X—Y  (Ia): L is O, NH, NE, S or Se, wherein the free covalent bond of L is joined to a protein carrier; and wherein the protein carrier is a bacterial toxin or toxoid; wherein at least 90% of the Z groups and at least 90% of the Q groups comprise a blocking group of formula (1a); wherein in formula (Ia); X is C(O); Y is R.sup.3; and R.sup.3 is CH.sub.3.

16. A pharmaceutical composition comprising (a) a molecule according to claim 15, and (b) a pharmaceutically acceptable carrier.

17. The composition according to claim 16, further comprising a saccharide antigen from one or more of semigroups C, W135 and Y of N. meningitidis.

18. The composition according to claim 17, wherein the saccharide, in the molecule, is an oligosaccharide.

19. The composition according to claim 16 or claim 17, further comprising a vaccine adjuvant.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 provides a scheme for the chemical synthesis of CRM.sub.197-MenA conjugates. The prevalent structures of “MenA10/90” (MenA oligosaccharide comprising a 4,5-dihydroxypentylcarbamate blocking group on approximately 10% of the monosaccharide units and a 2-hydroxyethylcarbamate blocking group on approximately 90% of the monosaccharide units) and “MenA10/0” (MenA oligosaccharide comprising a 4,5-dihydroxypentylcarbamate blocking group on approximately 10% of the monosaccharide units) are represented.

(2) FIG. 2 provides the anion exchange analytical profile at 214 nm of MenA oligosaccharides before (panel A) and after (panel B) sizing.

(3) FIG. 3 provides the 600 MHz .sup.1H NMR spectrum at 25° C. of MenA oligosaccharide without chemical modification (panel A), MenA10/90 oligosaccharide (panel B) and MenA10/0 oligosaccharide (panel C).

(4) FIG. 4 compares the percentage of phosphomonoester developed during storage at 37° C. by MenA oligosaccharide without chemical modification, MenA10/90 oligosaccharide and MenA10/0 oligosaccharide.

(5) FIG. 5 provides the .sup.31P NMR spectrum of MenA oligosaccharide.

(6) FIG. 6 compares the degree of polymerisation (DP), measured by .sup.31P NMR, during storage at 37° C. of MenA oligosaccharide without chemical modification, MenA10/90 oligosaccharide and MenA10/0 oligosaccharide.

(7) FIG. 7 provides the SDS-Page profile of CRM-MenA oligosaccharide conjugates: Lane M—Mw Markers; Lane 1—CRM; Lane 2—CRM-MenA10/90; and Lane 3—CRM-MenA10/0.

(8) FIG. 8 compares the free saccharide (FS) released during storage at 37° C. from CRM-MenA10/90 oligosaccharide conjugates and CRM-MenA10/0 oligosaccharide conjugates.

(9) FIG. 9 compares the percentage of phosphomonoester developed during storage at 37° C. by CRM-MenA10/90 oligosaccharide conjugates and CRM-MenA10/0 oligosaccharide conjugates.

MODES FOR CARRYING OUT THE INVENTION

Example 1

Modification of Men A Oligosaccharide

(10) Controlled Hydrolysis of MenA Polysaccharide

(11) MenA oligosaccharides were generated by chemical hydrolysis of a MenA polysaccharide solution. Briefly, MenA polysaccharide was solubilised at a final concentration of 10 mg/ml in 50 mM acetate buffer, pH 4.75. The solution was heated at 73° C. until a degree of polymerization (DP) of approximately 10 was reached. The hydrolysis was controlled by monitoring the variation of the optical activity of the solution (α Hg 365 nm) over time in accordance with the following equation: DP=1/{0.5817[1−(α.sub.t/α.sub.m)]}, where α.sub.m is the average value of the optical rotatory power of 6 samples when the temperature solution is 50° C., and α.sub.t is the optical rotatory power at time t. The hydrolysis was stopped when the α value corresponding to a DP of 10 was reached. At the end of the hydrolysis reaction the solution was cooled at room temperature and the pH corrected to about 6.5.

(12) Size Fractionation of MenA Oligosaccharide

(13) Controlled acidic hydrolysis of MenA polysaccharide generates a polydispersion with the target average DP. For conjugate preparation, the oligosaccharide polydispersion may be further restricted using a two-step size fractionation. These sizing steps typically change the DP of the MenA oligosaccharides from a value of about 10 to a value between 15 and 20, as measured by the molar ratio between total phosphorus (Pt) and terminal monoester phosphate (Pm) values. Pt concentration was determined according to the method described in reference 259 and Pm was determined by measuring the inorganic phosphate released by enzymatic reaction with potato acid phosphatase [260].

(14) Briefly, the MenA hydrolysate was first ultrafiltered through a 30 KDa tangential flow membrane to remove high molecular weight species. During this procedure the product was concentrated about 10-fold and then diafiltered against 13 volumes of 5 mM acetate buffer, pH 6.5. The permeate, containing the desired oligosaccharides, was collected while the retentate was discarded.

(15) In the second step, the permeate was fractionated by anionic exchange column chromatography. This step is designed to remove low Mw species characterized by a DP of less than 6, which may be poorly immunogenic [261]. The oligosaccharide mixture obtained from the 30 KDa ultrafiltration was loaded onto a column packed with Q-Sepharose Fast Flow previously equilibrated with 5 mM sodium acetate, pH 6.5. The ratio oligosaccharide/packed volume was 17 mg/ml packed resin. The column was then washed with 5 column volumes (cv) of the equilibration buffer. A wash of 10 cv of 5 mM sodium acetate buffer/125 mM NaCl, pH 6.5 was then applied to the column to elute oligosaccharides of DP≦6. The desired oligosaccharide fraction was then recovered by elution with 5 mM sodium acetate buffer/500 mM NaCl, pH 6.5. Stripping with 5 cv of 2M NaCl and sanitization with 1M NaOH completed the procedure.

(16) Analytical anion exchange chromatography was used to measure the oligosaccharide polydispersion before and after the fractionation. Briefly, the polydispersions of MenA oligosaccharide were analyzed by HPLC using a Mono-Q HR 5/5 column. After equilibration with water, 1 ml of sample containing about 1 mg of saccharide was loaded onto the column, which was then developed with a linear gradient form 0 to 60% of NaCl 1 M at the flow rate of 0.5 ml. The chromatogram was monitored at 214 nm. A standard preparation of a monodispersed MenA oligosaccharide having a defined DP of 5 and 6 respectively as evidenced by Mass Spectrometry and .sup.1H NMR, was used to identify the presence or removal of oligosaccharides having a DP lower than 6 in the tested polydispersion samples. FIG. 2 shows the analytical profiles of the hydrolysate (panel A) as compared to the sized MenA oligosaccharide (panel B).

(17) Counter Ion Exchange

(18) The Q-Sepharose eluate from the two-step size fractionation procedure was ultrafiltered on a 3 KDa membrane in order to exchange the sodium counter ion with tetrabutylammonium, which confers solubility to the oligosaccharide in non-aqueous solvents. Briefly, the MenA oligosaccharide solution was diafiltered against 4 volumes of 10 mM tetrabutylammoniumbromide followed by 10 volumes of water. The retentate, containing the desired product, was collected and the permeate discarded. Water was removed from the retentate by rotary evaporation.

(19) Chemical Modification of MenA Oligosaccharide

(20) The MenA oligosaccharide was modified using 1,1′-carbonyldiimidazole (CDI) activation followed by reaction with either 1-amino-4,5-pentandiol (APD) alone or APD and 2-aminoethanol (ETA), in order to obtain two different target structures (FIG. 1): i) MenA oligosaccharide comprising a 4,5-dihydroxypentylcarbamate blocking group on approximately 10% of the monosaccharide units and a 2-hydroxyethylcarbamate blocking group on approximately 90% of the monosaccharide units (MenA10/90); and ii) MenA oligosaccharide comprising a 4,5-dihydroxypentylcarbamate blocking group on approximately 10% of the monosaccharide units (MenA10/0).

(21) Briefly, the MenA oligosaccharide derived from the 3 KDa membrane ultrafiltration described above was solubilised in DMSO to a final concentration of about 10 mg/ml. To this solution a 20-fold molar excess of CDI (relative to the number of moles of MenA monosaccharide units) was added and the solution stirred at room temperature for 2 hrs. The activated oligosaccharide solution was then added to 9 volumes of cold (−20° C.) ethyl acetate followed by a 2 M solution of CaCl.sub.2 to a final concentration equimolar with the MenA monosaccharide units. The mixture was stirred for 30 minutes and, after sedimentation of the oligosaccharide, the majority of the supernatant was removed by suction and the pellet recovered by centrifugation, washed 3 times with ethyl acetate and dried under vacuum.

(22) For addition of blocking groups, the activated oligosaccharide was solubilised in DMSO to a final concentration of 10 mg/ml. To obtain the “MenA10/0” oligosaccharide, a 0.1-fold molar excess (relative to the number of moles of MenA monosaccharide units) of APD was added, and the reaction was stirred for 2 hrs at room temperature. After this time, nineteen volumes of 0.25 M sodium phosphate buffer, pH 6.5 were added under stirring. Any opalescence formed during this operation was removed by filtration through a 0.2 μm membrane. To obtain the “MenA10/90” oligosaccharide, a 0.6-fold molar excess of triethylamine and a 0.1-fold molar excess of APD were added and the reaction was stirred for 2 hrs at room temperature. Subsequently, a 50-fold molar excess (relative to the number of moles of MenA monosaccharide units) of ETA was added and the reaction continued under stirring for a further 2 hrs. Once again, after this time nineteen volumes of 0.25 M sodium phosphate buffer, pH 6.5 were added under stirring and any opalescence removed by filtration through a 0.2 μm membrane.

(23) The crude solutions of derivatised oligosaccharide were purified from the excess of low molecular weight reagents by ultrafiltration on a 3 KDa membrane. The solutions were first concentrated about 20-fold and then diafiltered against 10 volumes of 0.1 M sodium phosphate buffer, pH 7.2, followed by 10 volumes of distilled water. The purified products were recovered from the retentates, with the permeates being discarded.

(24) Confirmation of Chemical Modifications by .sup.1H NMR

(25) The chemically-modified MenA oligosaccharides were characterized by NMR to confirm that the desired chemical modifications had taken place.

(26) The .sup.1H NMR spectrum of the native MenA oligosaccharide is shown in FIG. 3, panel A. The spectrum is in agreement with the published literature [262, 263]. .sup.1H NMR conducted on pure APD and ETA gave the following signals: APD signals: HOCH.sub.2.sup.ACH.sup.B(OH)CH.sub.2.sup.CCH.sub.2.sup.DCH.sub.2.sup.ENH.sub.2 (H.sup.A at 3.6 ppm, H.sup.B at 3.7 ppm, H.sup.C at 1.5 ppm, H.sup.D at 1.6 ppm, H.sup.E at 2.7 ppm); ETA signals: HOCH.sub.2.sup.FCH.sub.2.sup.GNH.sub.2 (H.sup.F at 4.4 ppm, H.sup.G at 3.6 ppm). These assignments were used as a guide to identify the APD and ETA signals in the spectra of the derivatised oligosaccharides. The .sup.1H NMR spectra of the MenA10/90 oligosaccharide is reported in FIG. 3, panel B. The .sup.1H NMR spectrum of the MenA10/0 oligosaccharide is reported in FIG. 3, panel C. The covalent linkage between the ETA or the APD groups and the carbonyl groups introduced in position 4 and/or 3 of N-acetyl-mannosamine was confirmed by the (.sup.1H, .sup.13C) heteronuclear correlation detected in the HSQC spectra. Long-range correlation peaks between the carbonyl groups and the H.sup.G of ETA or H.sup.E of APD were detected. Similarly, the carbonyl groups gave long range correlation with the geminal protons in position 3/4 of N-acetyl-mannosamine. The percentages of APD groups introduced by the chemical treatment were estimated by integration of selected signals coming from APD and MenA. H.sup.D+H.sup.C overlapped signals at 1.5 ppm (APD groups) were integrated versus the H.sub.2 peak at 4.6 ppm (MenA oligosaccharide). In different experiments from 6% to 14% of MenA monosaccharide units were substituted with APD groups. Following the same approach, ETA groups were estimated by the ratio with H.sup.F overlapped signals at 3.6 ppm (ETA groups) against the H.sub.2 peak at 4.6 ppm (MenA oligosaccharide). Due the partial overlapping with the APD signals (H.sup.A at 3.6 ppm and H.sup.B at 3.7 ppm) the integral of H.sup.F was subtracted by the ¾ of H.sup.D+H.sup.C value. In different experiments from 66% to 85% of MenA monosaccharide units were substituted with ETA groups. As expected, in FIG. 3, panel C signals related to ETA groups are not present, which confirms the proposed structure and the suitability of NMR as a tool for structure elucidation and identity assessment. FIG. 3, panel A indicates that O-acetylation is preserved after acidic hydrolysis of serogroup A meningococcal polysaccharide and, although the carbamate groups change the local magnetic field and make the assignment more complicated, the O-acetylation status appears to be maintained after chemical modification (FIG. 3, panels B and C).

(27) Stability of MenA Oligosaccharides

(28) Degradation of MenA oligosaccharide, a consequence of hydrolysis at phosphodiester bonds, results in newly formed phosphomonoester groups. The stability of MenA10/90 and MenA10/0 oligosaccharides was compared with the stability of a native oligosaccharide.

(29) Briefly, solutions of the MenA oligosaccharides, in a concentration range from 1.4 to 3 mg/ml, were incubated at 37° C. in 10 mM histidine buffer, pH 7.2. At different time points over a period of 42 days, the oligosaccharides were analysed for the amount of phosphomonoester generated during storage.

(30) FIG. 4 shows the increment of phosphomonoester groups during storage at 37° C. for the three oligosaccharides mentioned above. The percentage of phosphomonoester was calculated as [Pm(t)−Pm(0)]×100/[(Pt(0)−Pm(0)], where Pm(t) and Pt(t) are the concentrations of phosphomonoester groups and total phosphorus at time t; and Pm(0) and Pt(0) are the concentrations of phosphomonoester groups and total phosphorus at time 0. Total phosphorus (Pt) concentration was determined according to the method described in reference 259 and terminal monoester phosphate (Pm) was determined by measuring the inorganic phosphate released by enzymatic reaction with potato acid phosphatase [260].

(31) The MenA10/90 and MenA10/0 oligosaccharides showed improved stability compared to the native oligosaccharide, as evidenced by the reduced trend to release phosphomonoester groups over the time. These results show that the stability of the MenA oligosaccharide can be enhanced by blocking the hydroxyl groups in position 4 and 3 of N-acetylmannosamine with a blocking group according to the present invention.

(32) Similarly, .sup.31P NMR analysis [264] was used to evaluate the stability of the modified MenA oligosaccharides in comparison to the native oligosaccharide at 37° C. for 42 days in 10 mM histidine buffer pH 7.2. Briefly, the average degree of depolymerisation (avDP) was determined by the molar ratio between the phosphodiester in chain groups (P.sub.in chain) and the phosphomonoester non-reducing end groups (P.sub.non-red end) (FIG. 5).
avDP=[P.sub.in chain+1]/P.sub.non-red end

(33) Once again, the MenA10/90 and MenA10/0 oligosaccharides showed improved stability compared to the native oligosaccharide, as evidenced by the greater degree of polymerisation at all time points (FIG. 6).

(34) TABLE-US-00001 TABLE I avDP Sample 0 d 7 d 14 d 21 d 28 d 35 d 42 d Oligo MenA Native 19.2 17.5 17.1 14.7 13.8 12.8 11.5 Oligo MenA10/0 24.9 23.8 22.6 20.5 19.1 18.2 16.8 Oligo MenA10/90 24.3 24.3 24.1 23.5 23.5 23.6 23.1
CRM.sub.197-MenA Conjugates
Generation of Reactive Aldehydic Groups by Controlled Periodate Oxidation

(35) The vicinal hydroxyl groups of the 4,5-dihydroxypentylcarbamate blocking groups derived from APD in the MenA10/90 and MenA10/0 oligosaccharides were oxidized by limited sodium periodate treatment to generate reactive aldehydic groups. Briefly, solutions of MenA10/90 and MenA10/0 oligosaccharides in 0.1 M sodium phosphate buffer, pH 7.2, were reacted with 0.1 moles of NaIO.sub.4 per mole of MenA monosaccharide units. The reactions was carried out in the dark with stirring, and monitored spectrophotometrically at 225 nm. After about 2 hrs the 225 nm absorbance reached a plateau. The amount of aldehydic groups generated by the reaction was determined by analyzing the equimolar amount of formaldehyde released during oxidation [265]. The reactions were stopped by addition of ethylene glycol to a final concentration equimolar with the NaIO.sub.4.

(36) The generation of aldehydic groups was almost quantitative as compared to the initial number of 4,5-dihydroxypentylcarbamate blocking groups.

(37) Purification of Oxidized Oligosaccharides

(38) The oxidized oligosaccharides were purified by ultrafiltration on a 3 KDa membrane. The solutions were concentrated 2-fold and then diafiltered against 10 volumes of 0.5 M NaCl followed by 10 volumes of distilled water. The retentate, containing the desired product, was collected and the permeate discarded. Water was removed from the retentate by rotary evaporation.

(39) Conjugation to CRM.sub.197

(40) The oxidized MenA oligosaccharides were conjugated to CRM.sub.197, a non-toxic mutant of the diphtheria toxin [266], via reductive amination to obtain CRM-MenA10/90 and CRM-MenA10/0 respectively (FIG. 1).

(41) Briefly, the oxidized MenA oligosaccharides were solubilised in a 50 mg/ml solution of CRM.sub.197 at a ratio of 13 moles of aldehydic groups per mole of protein. 100 mM sodium phosphate buffer, pH 7.2, was added to obtain a final protein concentration of 30 mg/ml. A 2M solution of NaBH.sub.3CN in 10 mM sodium phosphate buffer, pH 7.2, was then added to obtain a 70-fold molar excess of NaBH.sub.3CN with respect to the aldehydic groups. The reactions were carried out for 3 days at 37° C. Fourteen volumes of 10 mM sodium phosphate buffer, pH 7.2, were then added, followed by a 25-fold molar excess of NaBH.sub.4 (relative to the relative to the number of moles of aldehydic groups). The pH was controlled at 8.5 and the mixtures were stirred for 2 hrs at room temperature in order to quench any residual aldehydic groups. At the end of the quenching step, the pH was corrected again to 7.2, and the solutions filtered through a 0.2 μm-pore membrane.

(42) Purification of Conjugates

(43) The conjugates were purified from the excess of reagents and residual, unreacted oligosaccharides by ultrafiltration on a 30 KDa membrane. The reaction mixtures were diafiltered against 100 volumes of 0.01 M sodium phosphate buffer, pH 7.2, followed by 50 volumes of 10 mM histidine, pH 7.2. The solutions containing the purified conjugates were then filtered through a 0.2 μm-pore membrane and stored at 2-8° C.

(44) Confirmation of Conjugation to CRM.sub.197

(45) Conjugation of the MenA oligosaccharides to CRM.sub.197 was demonstrated by SDS-Page (FIG. 7). SDS-Page was carried out according to reference 267 using 7.5% acrylamide for stacking and 7.5% acrylamide for the separating gel. Before electrophoresis, samples were treated 1:4 with sample buffer and boiled for 10 min. Electrophoresis was carried out at 200 V constant voltage for about 40 min. Gels were developed with a Coomassie stain solution for approximately 20 min and destained in acetic acid/EtOH solution (7/40%) for approximately 4 hrs.

(46) The profile of the conjugates in FIG. 7 is shifted towards higher molecular weights compared to CRM.sub.197, and is markedly different from CRM.sub.197. The SDS-Page analysis also demonstrates the presence of high molecular weight material. This material may be formed during the conjugation reaction, which allows multiple attachment points of the CRM.sub.197 per oligosaccharide molecule.

(47) The conjugates were also analyzed for saccharide and protein content. Saccharide/protein ratios ranging from 0.20 to 0.32 (wt/wt) were observed.

(48) Stability of CRM.sub.197-MenA Conjugates

(49) The stability of the CRM.sub.197-MenA conjugates was determined by measuring the release of unconjugated saccharide over the time, which results from hydrolysis of the phosphodiester bonds.

(50) Centricon 30 devices (2 ml capacity) were conditioned by rinsing with 1 ml distilled water and spinning twice. 60 μl saline was added to 940 μl sample (CRM-MenA10/90 or CRM-MenA10/0) containing about 0.3 mg/ml of saccharide. Total phosphorus content was measured as described above before adding the mixtures to the devices. The devices were spun at 1942 g until 100-200 μl of solution was left in the retentate chamber, and then washed with 2×1 ml of saline and spun again. The solution in the permeate chamber was recovered and the sample volume adjusted with saline to 3 ml. The permeate derived from each sample was analyzed for total phosphorus content as described above.

(51) The value (P2/P1)×100, where P1 is the total phosphorus before centricon treatment and P2 is the total phosphorus after centricon treatment, represents the percentage of free saccharide. Spiking experiments to demonstrate the recovery of the free oligosaccharide through the membrane were conducted by adding 60 μl of about 2 mg/ml oligosaccharide to 940 μl of sample or saline and then applying the separation procedure described above. Recovery was consistently above 80%.

(52) FIG. 8 shows that the conjugate CRM-MenA10/90 showed a reduced tendency to release free saccharide compared to CRM-MenA10/0. FS (Free saccharide) is calculated as FS % (t)−FS % (0) where FS % (t) and FS % (0) are the free saccharide percentages at time t and 0 respectively.

(53) The stability of the CRM.sub.197-MenA conjugates was also determined by measuring phosphomonoester generation during storage. Briefly, solutions of the conjugates, in a concentration range from 157 to 253 μg/ml, were incubated at 37° C. in 10 mM histidine buffer, pH 7.2. At different time points over a period of 42 days, the conjugates were analysed for the amount of phosphomonoester generated during storage.

(54) FIG. 9 shows the increment of phosphomonoester groups during storage at 37° C. for the two conjugates mentioned above. The percentage of phosphomonoester was calculated as described above. The conjugate CRM-MenA10/90 showed a reduced tendency to generate phosphomonoester compared to CRM-MenA10/0.

(55) Immunogenicity of CRM-MenA Conjugates

(56) In order to assess the ability of the MenA conjugates to elicit antibodies recognizing the native MenA capsular polysaccharide, immunogenicity experiments were conducted in mice.

(57) Vaccine Formulation

(58) CRM-MenA10/90 and CRM MenA10/0 conjugates were mixed with sodium phosphate buffer and a AlPO.sub.4 suspension to obtain final concentrations of 20 μg/ml saccharide and 0.6 mg/ml Al.sup.3+ in 10 mM sodium phosphate buffer, pH 7.2. For non-adjuvanted formulations, the AlPO.sub.4 suspension was replaced with sodium phosphate buffer. Before immunization, the resultant vaccines were diluted 1:5 with saline.

(59) Immunization of Mice

(60) Groups of 8 Balb/c mice, females of 6-8 weeks, were immunized two or three times s.c. with 0.5 ml of conjugate vaccines containing 2 μg of saccharide. In the case of the two-injection schedule, the interval between the first and the second dose was four weeks. Bleedings were performed before the immunization and two weeks after the second dose. In the case of the three doses schedule, vaccines were given at 0, 14 and 28 days and bleedings were performed at time zero, one day before (post 2 doses sera) and 14 days after (post 3 doses sera) the third immunization.

(61) Immunogenicity

(62) The sera from the immunized mice were analyzed for specific anti-MenA capsular polysaccharide total IgG antibodies and for complement mediated serum bactericidal activity (SBA) against Neisseria meningitidis serogroup A.

(63) Specific anti-MenA capsular polysaccharide total IgG antibodies were determined essentially according the method of reference 268, adapted for animal sera analysis. Each individual mouse serum was analyzed in duplicate by a titration curve. Anti-MenA polysaccharide titers were calculated as Mouse Elisa Unit (MEU)/ml using software based on the Reference Line Assay Method. Geometric mean titers (GMT) were calculated for each immunization groups.

(64) SBA was measured on post II and post III (where appropriate) sera pools for each immunization group. The standard SBA protocol was based on the inoculum of the test bacterial strain (MenA F8238) in Mueller Hinton Broth with the addition of 0.25% glucose. The bacterial culture was incubated at 37° C. in the presence of 5% CO.sub.2 and growth stopped when the bacteria reached the early exponential phase of growth, around 0.220-0.240 OD.sub.600. The bacteria were then diluted to 10.sup.−4 with 1% BSA in GBBS buffer and incubated for 1 hour at 37° C. with 5% CO.sub.2 in the presence of heat inactivated sera pools (30 minutes at 56° C.) and 25% baby rabbit serum as a source of complement. The reaction mixtures were then plated on Mueller Hinton agar and incubated overnight at 37° C. Bactericidal titres were expressed as the reciprocal serum dilution yielding 50% killing of the bacteria.

(65) Table II shows the anti-MenA capsular polysaccharide total IgG titers expressed as GMT (+/−95 confidence limits) as measured by ELISA and the SBA titers induced by CRM-MenA10/90, and CRM-MenA10/0. Both conjugates were capable of inducing in mice specific anti-MenA polysaccharide antibodies with bactericidal functional activity.

(66) TABLE-US-00002 TABLE II Post 2 ELISA Titre Vaccine GMT (+/−95% CI) Post 2 SBA Titre CRM-MenA 10/0 lot 5/AlPO4 346 (230; 520) >4096 < 8192 CRM-MenA 10/90 lot 5/AlPO4 270 (217; 336) 4096

(67) In a second experiment, the immunogenicity in mice of CRM-MenA10/90 was tested with and without AlPO.sub.4. The immunogenicity of the CRM-MenA10/90 is confirmed in Table III, which shows the specific anti-MenA IgG antibody titers induced after two and three immunizations and the complement mediated bactericidal activity of these antibodies. Pre-immunization titres were found to be negative (SBA<4). These data suggest that the presence of the adjuvant enhances the antibody response. The immunogenicity observed in the conjugate is clearly a consequence of the chemical conjugation of the oligosaccharide to the protein carrier, as a physical mixture of MenA oligosaccharide, CRM.sub.197 and AlPO.sub.4 was not immunogenic.

(68) TABLE-US-00003 TABLE III Post 2 Post3 Post 2 ELISA Post 3 ELISA Titre SBA SBA Vaccine Titre GMT (+/−95% CI) GMT (+/−95% CI) Titre Titre CRM-MenA10/90 lot11 867 (585; 1285) 1299 (1008; 1675) 2048 4096 AlPO.sub.4 CRM-MenA 10/90 lot 11 388 (249; 604)  426 (241; 751)  1024 2048 OligoMenA10/90 lot 11 + 2 2 <4 <4 CRM.sub.197 + AlPO.sub.4 (physical mix of unconjugated antigens)

Example 2

Modification of Men A Polysaccharide

(69) Chemical Modification of MenA Polysaccharide

(70) 20 mg of native MenA capsular polysaccharide (0.072 mmol) was added to 170 mg (2.5 mmol) of imidazole and 1 mL of CH.sub.3CN. Stirring with a magnetic bar, 163 μL (1.59 mmol) of acetic anhydride was added and the reaction was incubated at 55° C. for 21 h. The imidazole:acetic anhydride molar ratio was 2:4. A diafiltration step using a Centricon cellulose membrane (1 kDa molecular weight cut-off) against Milli-Q water (1:7 vol/vol) was used to purify the reaction product. The material was finally dried under vacuum (SpeedVac).

(71) Confirmation of Chemical Modifications by .sup.1H and .sup.13C NMR

(72) To establish the degree of acetylation, a complete structural characterisation of the modified MenA capsular polysaccharide was carried out by .sup.1H and .sup.13C NMR spectroscopy.

(73) Quantitative NMR analysis was used to quantify the level of O-acetylation of the saccharide chains. The O-acetylation percentage was estimated by integration of H.sub.2.sup.3OAc peak (proton at position C-2 of the N-acetyl-mannosamine residues O-acetylated at C-3), H.sub.2.sup.4OAc peak (proton at position C-2 of the N-acetyl-mannosamine residues O-acetylated at C-4) and H.sub.2.sup.deOAc peak (proton at position C-2 of the N-acetyl-mannosamine residues without O-acetylation), in comparison to H.sub.1 (proton at position C-1 of the N-acetyl-mannosamine residues). The total O-acetylation level was obtained by the sum of H.sub.2.sup.3OAc and H.sub.2.sup.4OAc peak integrations.
% O-Acetylation=[H.sub.2.sup.3OAc+H.sub.2.sup.4OAc]/[H.sub.1.sup.deOAc]

(74) Moreover, the O-acetylation percentage was estimated by integration of H.sub.2.sup.3OAc/H.sub.2.sup.4OAc peak (proton at position C-3 of the N-acetyl-mannosamine residues O-acetylated at C-3 and proton at position C-4 of the N-acetyl-mannosamine residues O-acetylated at C-4), in comparison to H.sub.1 (proton at position C-1 of the N-acetyl-mannosamine residues).
% O-Acetylation=[H.sub.2.sup.3OAc/H.sub.2.sup.4OAc]/[H.sub.1.sup.OAc+H.sub.1.sup.deOAc]
Stability of MenA Polysaccharides

(75) .sup.31P NMR analysis was used to evaluate the stability of the fully acetylated modified MenA capsular polysaccharide in comparison to the native polysaccharide and corresponding oligosaccharide at 37° C. for 42 days in 10 mM histidine buffer pH 7.2, as described above.

(76) The fully O-acetylated modified MenA polysaccharide was much more stable than the native capsular polysaccharide and corresponding oligosaccharide.

(77) TABLE-US-00004 TABLE IV avDP Sample 0 d 7 d 14 d 21 d 28 d 35 d 42 d Poly MenA Native >50 >50 44.6 29.6 26.9 20.8 18.3 Oligo MenA Native 17.3 15.5 13.0 12.0 11.0 10.4 9.6 Poly MenA Fully Ac >50 >50 >50 >50 >50 >50 >50

(78) These results confirm that the stability of the MenA oligosaccharide can be enhanced by blocking the hydroxyl groups in position 4 and 3 of N-acetylmannosamine with a blocking group according to the present invention.

(79) 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.

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