Hypo- and hyper-acetylated meningococcal capsular saccharides

Abstract

Capsular saccharides derived from serogroups W135 and Y of Neisseria meningitidis have altered levels of O-acetylation at the 7 and 9 positions of their sialic acid residues, and can be used to make immunogenic compositions. Relative to unmodified native saccharides, derivatives of the invention are preferentially selected during conjugation to carrier proteins, and conjugates of the derivatives show improved immunogenicity compared to native polysaccharides.

Claims

1. A modified serogroup Y meningococcal capsular saccharide, conjugated to a carrier protein, wherein (a) between 2-9% of the sialic acid residues in the saccharide are O-acetylated at the 7 position; and/or (b) between 35-55% of the sialic acid residues in the saccharide are O-acetylated at the 9 position.

2. The modified meningococcal capsular saccharide of claim 1 wherein between 4-8% of the sialic acid residues in the saccharide are O-acetylated at the 7 position.

3. The modified meningococcal capsular saccharide of claim 1, wherein between 40-50% of the sialic acid residues in the saccharide are O-acetylated at the 9 position.

4. A modified meningococcal capsular saccharide conjugated to a carrier protein, wherein the saccharide comprises n or more repeating units of the disaccharide unit:
[sialic acid]-[hexose] wherein the hexose is glucose and n is an integer from 1 to 100, and wherein: (a) x % of the sialic acid residues in said n or more repeating units are O-acetylated at the 7 position; and/or (b) y % of the sialic acid residues in said n or more repeating units are O-acetylated at the 9 position, wherein x is between 2-9 and y is between 35-55.

5. The saccharide of claim 4, wherein 6%±0.6% of the sialic acid residues in said n or more repeating units are O-acetylated at the 7 position, and 45%±4.5% of the sialic acid residues in said n or more repeating units are O-acetylated at the 9 position.

6. A composition comprising a molecules of serogroup Y meningococcal capsular saccharide, wherein (i) the average number of sialic acid residues per capsular saccharide molecule is b, and wherein: (a) between 2-9% of the a.Math.b serogroup Y sialic acid residues in the composition are O-acetylated at the 7 position; and/or (b) between 35-55% of the a.Math.b serogroup Y sialic acid residues in the composition are O-acetylated at the 9 position, (ii) the saccharide is conjugated to a carrier protein.

7. A saccharide comprising n or more repeats of the following disaccharide unit: ##STR00002## n is an integer from 1 to 100, X and Y are different groups selected from —H and —OH, R.sub.1 is independently selected from —H and —COCH.sub.3 and may be the same or different in each disaccharide unit, R.sub.2 is independently selected from —H and —COCH.sub.3 and may be the same or different in each disaccharide unit, and, X is —H and Y is —OH, (a) 2-9% of R.sub.1 are —COCH.sub.3 and/or (b) 35-55% of R.sub.2 are —COCH.sub.3, and wherein the saccharide is conjugated to a carrier protein.

8. The saccharide of claim 1, wherein the saccharide has an average degree of polymerisation of less than 30.

9. The saccharide of claim 1, wherein the carrier protein is selected from the group consisting of: diphtheria toxoid, tetanus toxoid, H. influenzae protein D, and CRM.sub.197.

10. An immunogenic composition comprising (a) a modified capsular saccharide conjugate of claim 1, and (b) a pharmaceutically acceptable carrier.

11. The composition of claim 10, in aqueous form.

12. The composition of claim 10, in lyophilised form.

13. The composition of claim 10, further comprising a capsular saccharide antigen from serogroup C of N. meningitidis.

14. The composition of claim 10, further comprising a capsular saccharide antigen from serogroup A of N. meningitidis.

15. The composition of claim 10, further comprising a capsular saccharide antigen from serogroup W135 of N. meningitidis.

16. The composition of claim 14, wherein the serogroup A antigen is a modified saccharide in which one or more of the hydroxyl groups on the native saccharide has/have been replaced by a blocking group.

17. The composition of claim 10, further comprising an antigen from serogroup B of N. meningitidis.

18. The composition of claim 10, further comprising a saccharide antigen from Haemophilus influenzae type B.

19. The composition of claim 10, further comprising an antigen from Streptococcus pneumoniae.

20. The composition of claim 10, further comprising one or more of: an antigen from hepatitis A virus; an antigen from hepatitis B virus; an antigen from Bordetella pertussis; a diphtheria toxoid; a tetanus toxoid; or a poliovirus antigen.

21. A method for raising an antibody response in a mammal, comprising administering a composition of claim 10 to the mammal.

22. A process for preparing a modified serogroup Y meningococcal capsular saccharide conjugated to a carrier protein comprising the steps of: (1) providing a starting serogroup Y meningococcal capsular saccharide and the carrier protein, either or both of which is/are optionally modified to render it/them reactive towards the other; (2) forming a covalent bond between the saccharide and the carrier protein; and (3) purifying the resulting serogroup Y meningococcal capsular saccharide conjugated to a carrier protein, wherein, between steps (1) and (3), the degree of O-acetylation at the 9 position of sialic acid residues in the starting saccharide increases to 35-55% and/or the degree of O-acetylation at the 7-position of the sialic acid residues decreases to 2-9% to generate the modified serogroup Y meningococcal capsular saccharide.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIGS. 1 and 2 show annotated NMR spectra of hydrolysed MenW135 and MenY, respectively.

(2) FIGS. 3 and 4 show the O-acetylation status of the sialic acid residues at the 7 and 9 positions during the preparation of MenW135-CRM.sub.197 and MenY-CRM.sub.197 conjugates, respectively.

(3) FIGS. 5 and 6 show IgG titres obtained in mice against oligosaccharide antigens using MenW135 and MenY, respectively.

MODES FOR CARRYING OUT THE INVENTION

(4) A. Production and Purification of Meningococcal Polysaccharides

(5) Capsular polysaccharides were purified from MenW135 and MenY as described in ref. 14.

(6) B. Preparation of Modified Serogroup W135 and Y Polysaccharides Conjugates

(7) The purified polysaccharides were hydrolysed in acetic 50 mM sodium acetate buffer, pH 4.7 for about 3 hours at 80° C. This resulted in oligosaccharides with an average DP of about 15 to 20 as determined by ratio between sialic acid (SA) and reduced terminal SA.

(8) The hydrolysate was ultrafiltered through a 30 kDa cut-off membrane (12 to 20 diafiltration volumes of 5 mM acetate buffer/15-30 mM NaCl pH 6.5). The retentate, containing the high MW species, was discarded while the permeate was loaded onto a Q-Sepharose Fast Flow column equilibrated in 5 mM acetate buffer/15 mM NaCl pH 6.5. The column was then washed with 10 CV equilibrating buffer, in order to remove oligosaccharides with DP≦3-4 and eluted with 3 CV 5 mM acetate buffer/500 mM NaCl pH 6.5.

(9) Ammonium chloride or ammonium acetate was added to the sized oligosaccharide solution to a final concentration of 300 g/L, then sodium-cyano-borohydride was added to 49 g/L or 73 g/L final concentration. The mixture was incubated at 50° C. for 3 days to produce amino-oligosaccharides, which were then purified by tangential flow ultrafiltration with a 1 kDa or 3 kDa cut-off membrane using 13 diafiltration volumes of 0.5 M NaCl followed by 7 diafiltration volumes of 20 mM NaCl. The purified oligosaccharides were then dried with rotary evaporator to remove water.

(10) Dried amino-oligosaccharides were solubilised in distilled water at a 40 mM amino group concentration, then 9 volumes of DMSO were added followed by triethyl-amine at a final concentration of 200 mM. To the resulting solution, adipic acid N-hydroxysuccinimido diester was added for a final concentration of 480 mM. The reaction was maintained under stirring at room temperature for 2 hours, then the activated oligosaccharide was precipitated with acetone (80% v/v final concentration). The precipitate was collected by centrifugation and washed several times with acetone to remove unreacted adipic acid N-hydroxysuccinimido diester and by-products. Finally the activated oligosaccharide was dried under vacuum. The amount of active ester groups introduced into the oligosaccharide structure was determined by a colorimetric method as described in ref. 231.

(11) The dried activated oligosaccharide was added to a 45 mg/ml solution of CRM.sub.197 in 0.01M phosphate buffer pH 7.2 for an active ester/protein (mole/mole) ratio of 12:1. The reaction was maintained under stirring at room temperature overnight. After this period, the conjugate was purified by diafiltration with a 30 kDa membrane (50 diafiltration volumes of 10 mM phosphate buffer, pH 7.2). The purified conjugate was sterile filtered and stored at −20° C. or −60° C. until vaccine formulation.

(12) C. O-Acetylation Status of Polysaccharides During Conjugation

(13) O-acetylation status of the C7 and C9 positions of the sialic acid residues in the population of modified saccharides derived from MenW135 and Men Y was measured by NMR analysis.

(14) The intermediate poly- and oligo-saccharides of the conjugation process (native polysaccharide, after hydrolysis, prior to amination, after activation and after conjugation) were characterised using 1D and 2D proton NMR experiments. .sup.1H NMR samples were prepared by dissolving lyophilized oligosaccharides in 0.75 mL of 99.9% deuterated .sup.2H.sub.2O (Aldrich™) to give 10-15 mM concentrated solutions. In all experiments, 5 mm Wilmad NMR tubes were used.

(15) .sup.1H NMR spectra were recorded at 298 K on a Bruker NMR Spectrometer Avance DRX 600 MHz equipped with a BGU unit and using standard Bruker pulse programs. A 5 mm TBI triple resonance probe with self shielded z-gradients was used. For processing data the Bruker XWINNMR 3.0 software was used.

(16) Proton standard spectral acquisition conditions are to collect 32 k data points over a spectral window of 6000 Hz with 4 scans. .sup.1H NMR spectra were Fourier-transformed after applying a 0.1 Hz line broadening function and referenced relative to the mono-deuterated water (HDO) at 4.72 ppm.

(17) The assignment of the resonances and hence determination of molecular structure were made based on literature data [232,4].

(18) To show the peak assignment, annotated NMR spectra of hydrolysed MenW135 and hydrolysed MenY are presented in FIG. 1 and FIG. 2 respectively.

(19) The following table gives the proportion of all sialic acid (N-acetyl-neuraminic acid) C7 and C9 positions in the population of saccharides derived from MenW135 that were found to be O-acetylated during conjugate preparation:

(20) TABLE-US-00003 % O- % O- acetylation at 7 acetylation at 9 Preparation step position position Native polysaccharide 30.1 25.0 After hydrolysis 16.9 26.4 Prior to amination 15.0 26.2 After activation 5.1 26.3 After conjugation 6.3 43.1

(21) Thus, the overall percentage of sialic acid O-acetylation at the 7 position fell during preparation of the conjugate, from about 30% to about 6%. At the same time, the percentage of O-acetylation at the 9 position increased from about 25% to about 43% (FIG. 3). The dramatic change seen at the 9 position in the final step shows that conjugation preferentially selects those saccharides that are O-acetylated at the 9 position.

(22) Similarly, the O-acetylation status of the sialic acid residues in the population of modified saccharides derived from MenY after each step of the conjugation process is given in the following table:

(23) TABLE-US-00004 % O-acetylation % O-acetylation at Preparation step at the 7 position the 9 position Native polysaccharide 10.3 28.0 After hydrolysis 3.3 24.1 Prior to amination 5.1 25.1 After activation 2.4 20.9 After conjugation 6.1 45.1

(24) Thus, the percentage of sialic acid O-acetylation at the 7 position fell during preparation of the conjugate of the present invention from about 10% to about 2%, before finally rising to about 6% during the conjugation reaction. At the same time, the percentage of O-acetylation at the 9 position fell from about 28% to about 21%, before finally rising to about 45% during the conjugation reaction (FIG. 4). The dramatic change seen at the 9 position in the final step shows that conjugation preferentially selects those saccharides that are O-acetylated at the 9 position.

(25) D. Immunogenicity of Conjugates

(26) The frozen bulk conjugates were thawed. Each was diluted, under stirring, to a final concentration of 20 μg saccharide/ml, 5 mM phosphate, 9 mg/ml NaCl, aluminium phosphate (to give an Al.sup.3+ concentration of 0.6 mg/ml), pH 7.2. The mixtures were then kept, without stirring, at 2-8° C. overnight and further diluted with saline to 4 μg saccharide/ml for mouse immunisation.

(27) A second set of vaccines was prepared for both serogroups in the same way, but the addition of aluminium phosphate was replaced with same volume of water.

(28) Ten Balb/c mice for each immunisation group were injected s.c. twice with 0.5 ml vaccine at weeks 0 and 4. Bleedings were performed before immunisation, the day before the second dose and 2 weeks after the second dose. Immunisations were performed with (a) the conjugate vaccine with or without alum, (b) saline control and (c) unconjugated polysaccharide control.

(29) Specific anti-polysaccharide IgG antibodies were determined in the sera of immunised animals essentially as described in ref. 233. Each individual mouse serum was analysed in duplicate by a titration curve and GMT was calculated for each immunisation group. Titres were calculated in Mouse Elisa Units (MEU) using ‘Titerun’ software (FDA). Anti-polysaccharide titre specificity was determined by competitive ELISA with the relevant polysaccharide as competitor.

(30) As shown in FIG. 5, the MenW135 conjugate induced high antibody titres in animals. As expected, the unconjugated polysaccharide was not immunogenic. The conjugate formulation with an aluminium phosphate as adjuvant induced a higher level of antibodies compared to the titre obtained by the conjugate alone. Similar results were seen for MenY (FIG. 6).

(31) Post-II sera were tested for bactericidal activity using an in vitro assay to measure complement-mediated lysis of bacteria. Post-II sera were inactivated for 30 minutes at 56° C. before the use in the assay, and 25% baby rabbit complement was used as source of complement. Bactericidal titre was expressed as the reciprocal serum dilution yielding 50% killing of bacteria against the following strains: MenW135, 5554 (OAc+); MenY, 242975 (OAc+).

(32) A capsular polysaccharide derived from MenW135 did not yield a GMT value and gave a bactericidal activity of only 4. In contrast, de-O-acetylated conjugates of the invention gave GMT values between 14 and 565, with bactericidal titres between 64 and 2048.

(33) A capsular polysaccharide derived from MenY did not yield a GMT value and gave a bactericidal activity of only 256. In contrast, de-O-acetylated conjugates of the invention gave GMT values between 253 and 1618, with bactericidal titres between 256 and 16384.

(34) 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. In particular, minor modifications that do not affect the immunogenicity of the modified capsular saccharide of the present invention are also encompassed.

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