Capsular polysaccharide solubilisation and combination vaccines

Abstract

Precipitated bacterial capsular polysaccharides can be efficiently re-solubilised using alcohols as solvents. The invention provides a process for purifying a bacterial capsular polysaccharide, comprising the steps of (a) precipitation of said polysaccharide, followed by (b) solubilisation of the precipitated polysaccharide using ethanol. CTAB can be used for step (a). The material obtained, preferably following hydrolysis and sizing, can be conjugated to a carrier protein and formulated as a vaccine. Also, in vaccines comprising saccharides from both serogroups A and C, the invention provides that the ratio (w/w) of Men A saccharide:MenC saccharide is >1.

Claims

1. A liquid composition comprising a Neisseria meningitidis serogroup A capsular saccharide conjugate and a Neisseria meningitidis serogroup C capsular saccharide conjugate, and further comprising (i) an aluminum hydroxide adjuvant and (ii) a histidine buffer, and where each conjugate has a saccharide:protein ratio (w/w) of between 0.5:1 and 5:1.

2. The composition of claim 1, wherein each conjugate comprises capsular saccharides that are oligosaccharides with an average degree of polymerization of less than 30 conjugated to the carrier protein selected from bacterial toxins and toxoids.

3. The composition of claim 2, wherein the bacterial toxins and toxoids are diphtheria toxoids or tetanus toxoids.

4. The composition of claim 1, wherein each conjugate comprises a linker.

5. The composition of claim 1, wherein the capsular saccharide in each conjugate was activated or functionalized prior to conjugation.

6. The composition of claim 5, wherein the activation was with a cyanylating reagent.

7. The composition of claim 1, wherein the composition comprises Neisseria meningitidis serogroup A capsular saccharides, Neisseria meningitidis serogroup C capsular saccharides, Neisseria meningitidis serogroup W135 capsular saccharides, and Neisseria meningitidis serogroup Y capsular saccharides.

8. The composition of claim 1, wherein the composition further comprises proteins from serogroup B of Neisseria meningitidis.

9. The composition of claim 1, wherein the composition further comprises antigens from pneumococcus, hepatitis A virus, hepatitis B virus, Bordetella pertussis, diphtheria, tetanus, Helicobacter pylori, polio and/or Haemophilus influenzae.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the effect of varying ethanol:water ratios on polysaccharide solubilisation.

(2) FIG. 2 shows IgG titres obtained in mice against serogroup A oligosaccharide antigen.

(3) FIG. 3 shows IgG titres obtained in mice against for serogroup Y oligosaccharide antigen.

(4) FIG. 4 shows IgG titres obtained in mice against for serogroup W135 oligosaccharide antigen.

(5) FIGS. 5A-B shows post-II IgG titres obtained in mice with a mixture of oligosaccharide conjugates for serogroups A and C: FIG. 5A shows the anti-serogroup A responses; and FIG. 5B shows anti-serogroup C responses.

(6) FIG. 6 shows anti-serogroup W135 IgG titres obtained in mice with a mixture of oligosaccharide conjugates for serogroups C, W135 and Y.

(7) FIG. 7 shows anti-serogroup Y IgG titres obtained in mice with a mixture of oligosaccharide conjugates for serogroups C, W135 and Y.

(8) FIG. 8 shows anti-serogroup C IgG titres obtained in mice with a mixture of oligosaccharide conjugates for serogroups C, W135 and Y.

(9) FIG. 9 shows the anti-serogroup W135 post-II IgG titres obtained in mice with a mixture of oligosaccharide conjugates for serogroups A, C, W13S and Y.

(10) FIG. 10 shows anti-serogroup Y post-II IgG titres obtained in mice with a mixture of oligosaccharide conjugates for serogroups A, C, W13S and Y.

(11) FIG. 11 shows anti-serogroup A post-II IgG titres obtained in mice with a mixture of oligosaccharide conjugates for serogroups A, C, W13S and Y.

(12) FIG. 12 is a calibration curve obtained using test MenA polysaccharide samples at different hydrolysis times. The curve shows the linear relationship between the reciprocal of the degree of polymerisation and optical rotatory power.

(13) FIG. 13 is a calibration curve obtained using test Men Y polysaccharide samples at different hydrolysis times. The curve shows the linear relationship between the log of the degree of polymerisation and KD (distribution coefficient).

(14) FIGS. 14 to 16 show post-II IgG titres, split by IgG subclass, obtained in mice after immunisation with oligosaccharide conjugates for serogroups: (14) A; (15) C; (16) W135 and (17) Y.

(15) FIGS. 17A-D shows post-II IgG titres, split by IgG subclass, obtained in mice after immunisation with a tetravalent mixture of oligosaccharide conjugates. FIG. 17A shows MenA. FIG. 17B shows MenC. FIG. 17C shows MenW135. FIG. 17D shows MenY.

(16) FIG. 18 illustrates the preparation of an oligosaccharide conjugate.

(17) FIGS. 19A-B show (FIG. 19A) anti-MenA and (FIG. 19B) anti-MenC GMT(95% confidence intervals) obtained in a guinea pig model. Values above bars are serum bactericidal assay (SBA) titres i.e. the reciprocal of the sera dilution yielding the 50% of killing.

MODES FOR CARRYING OUT THE INVENTION

(18) A. Production and Purification of Meningococcal Polysaccharides

(19) Meningococci of serogroups A, W135 and Y were grown in 500 ml flasks containing 150 ml of Franz A as medium, for 12 hours at 351 C. Agitation was set at 150 rpm using a 35 mm throw Shaker. 85 ml of the culture was then inoculated in 20 L fermentor containing Watson as medium.

(20) After 18.5 hours (W135 and Y) or 16.5 hours (A), when OD10 was reached, the fermentation was interrupted by adding 300 ml of formalin and then, after 2 hours of incubation, and the fermentor was cooled to 10 C. The supernatant was collected by centrifugation followed by filtration (0.22 m), and ultrafiltration with a 30 kDa membrane.

(21) The crude concentrated polysaccharide was then precipitated by addition of CTAB as a 100 mg/ml water solution. The volumes added are shown in the following table. After 12 hours at room temperature, the CT AB complexes were recovered by centrifugation. The CT AB complex was extracted by adding a 95% ethanol solution at room temperature for 16-20 hrs under vigorous stirring. The volume of ethanol added is shown in the following table:

(22) TABLE-US-00003 Serogroup CTAB volume (ml) Volume of 95% EtOH (l/kg wet paste) A 475 3.5 to 6 W135 200 4 to 6 Y 650 3.4

(23) The resulting suspensions were filtered through a CUNO 10 SP depth filter. The filtrate was recirculated through a CUNO ZETACARBON cartridge until OD.sub.275nm<0.2. The Z carbon filtrate was then collected and filtered through a 0.22 m filter. The polysaccharide was eventually precipitated from the ethanol phase by addition of a CaCh 2M water solution (10-12 ml/l of EtOH final solution). The purified polysaccharide was then collected by centrifugation, washed with 95% ethanol and dried under vacuum.

(24) In other experiments, the final concentration of ethanol used for extraction was varied (FIG. 1). For serogroup A polysaccharide, a range of between 80 and 95% ethanol was most effective, with extraction efficiency decreasing at lower percentages. For serogroup W135, good extraction was achieved with between 75% and 90% ethanol, with 95% being less effective. For serogroup Y, the best results were achieved with between 75% and 85% ethanol, with higher percentages (e.g. 90%, 95%) being less effective. In general, it was noted that ethanol percentages below those reported here tended to increase the co-extraction of contaminants such as proteins. Ethanol percentages given in this paragraph are expressed as a final concentration (ethanol as percentage of total volume of ethanol water) and are based on a water content in the CTAB-poJysaccharide pastes recovered by centrifugation of about 50% (i.e. 500 g H.sub.2O per kg wet paste). This value was determined empirically in small scale-up experiments.

(25) B. Conjugation of Serogroup A Polysaccharides

(26) a) Hydrolysis

(27) The serogroup A meningococcal polysaccharide was hydrolysed in 50 mM sodium acetate buffer, pH 4.7 for about 3 hrs at 73 C. The hydrolysis was controlled in order to obtain oligosaccharides with an average degree of polymerisation (DP) of approximately 10, as determined by the (w/w) ratio between the total organic phosphorus and the monoester phosphate.

(28) The DP ratio of (total organic phosphorus) to (phosphorus monoester) is inversely proportional to optical rotatory power (a), as shown in FIG. 12. This relationship can be used to monitor the extent of hydrolysis more conveniently than direct phosphorus measurements.

(29) b) Sizing

(30) This step removes short-length oligosaccharides generated during the hydrolysis process. The hydrolysate obtained above was ultrafiltered through a 30 kDa cut-off membrane (12 diafiltration volumes of 5 mM acetate buffer, pH 6.5). The retentate, containing the high Mw species, was discarded; the permeate was loaded onto a onto a Q-Sepharose Fast Flow column equilibrated in acetate buffer 5 mM, pH 6.5. The column was then washed with 5 column volumes (CV) of equilibrating buffer, then with 10 CV of 5 mM acetate buffer/125 mM NaCl pH 6.5 in order to remove oligosaccharides with DP6. The sized oligosaccharide was then eluted with 5 CV of 5 mM acetate buffer/0.5 M NaCl pH 6.5.

(31) The eluted oligosaccharide population has an average DP of about 15.

(32) c) Introduction of a Primary Amino Group at the Reducing Terminus

(33) Ammonium salt (acetate or chloride) was added to the sized oligosaccharide solution for a final concentration ranging from 49-300 g/L, then sodium-cyano-borohydride was added to a final concentration ranging from 12-73 g/L. After adjusting the pH to between 6-7.3, the mixture was incubated at 37 C. for 5 days.

(34) The amino-oligosaccharides 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 diafihration volumes of 20 mM NaCl. The purified amino-oligosaccharide solution was analysed for phosphorus content (one chemical activity of the antigen) by the procedure of ref. 146 and the amount of introduced amino groups by the procedure of ref. 147.

(35) The purified oligosaccharides were then dried with rotary evaporator to remove water.

(36) d) Perivatisation to Active Ester

(37) The 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.

(38) 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.

(39) The amount of active ester groups introduced into the oligosaccharide structure was determined by a colorimetric method as described in ref. 148.

(40) e) Conjugation to CRM.sub.197

(41) The dried activated oligosaccharide was added to a 45 mg/ml solution of CRM.sub.197 in 0.01 M 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 hydrophobic chromatography or tangential flow ultrafiltration. The purified MenA-CRM.sub.197 conjugate was sterile filtered and stored at 20 C. or 60 C. until vaccine formulation.

(42) The conjugate was analysed for: protein content (microBCA Protein Assay), MenA saccharide content (colorimetric analysis of phosphorus), free saccharide content, MPLC profile (on TSKgel G4000SW 7.5 mm ID30 cm), and SDS-PAGE. Characteristics of typical preparations are shown in the following table:

(43) TABLE-US-00004 Saccharide Lot Code (mg/ml) protein (mg/ml) Glycosylation KD 210201/A 0.257 0.864 0.3 0.489 210201/BS 0.308 1.354 0.23 0.503 210201/BL 0.28 1.482 0.19 0.501 351230595 0.138 0.3 0.46 010900 0.092 0.337 0.27 DP29 0.105 0.245 0.43 A1 (UNSIZED) 0.08 0.291 0.27 A2 (SIZED) 0.446 2.421 0.18
C. Conjugation of Serogroup W135 Polysaccharides
a) Hydrolsis

(44) The group W meningococcal polysaccharide was 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.

(45) The DP ratio of (total SA) to (reduced terminal SA) is related to the KD of the as determined by HPLC-SEC, as shown in FIG. 13. This relationship can be used to monitor the extent of hydrolysis more conveniently than direct SA measurements.

(46) b) Sizing

(47) 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.

(48) c) Introduction of a Primary Amino Group at the Reducing Terminus

(49) 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.

(50) The amino-oligosaccharides were then purified by tangential How ultrafiltration as described for serogroup A. The purified material was analysed for its content of sialic acid (colorimetric method according 10 ref. 149 and/or galactose (HPLC) (chemical activities of the MenW135 antigen). The purified oligosaccharides were then dried with rotary evaporator to remove water.

(51) d) Derivatisation to Active Ester

(52) The dried amino-oligosaccharides were derivatised as described above for serogroup A.

(53) e) Conjugation to CRM.sub.197

(54) Conjugation was performed as described above for serogroup A but, to purify the conjugate, diafiltration with a 30 kDa membrane was used (50 diafiltration volumes of 10 mM phosphate buffer, pH 7.2). The purified conjugate was sterile filtered and stored at 20 C. of 60 C. until vaccine formulation.

(55) The conjugate was analysed for the same parameters as described above for serogroup A. MenW saccharide content was assayed by colorimetric sialic acid determination:

(56) TABLE-US-00005 saccharide Lot code (mg/ml) protein (mg/ml) Glycosylation KD lot 1 5.73 3.52 1.63 0.296 lot 2/4, 5 3.51 2.88 1.22 0.308 lot 3S 2.49 2.25 1.11 0.380 lot 3Sd 2.03 2.24 0.91 0.394 lot 3L 2.32 2.3 1.01 0.391 lot 3Ld 1.94 2.29 0.85 0.383 Lot 3S/pr. Glic6 0.363 0.82 0.44 0.498 Lot 3S/pr. Glic9 0.424 0.739 0.57 0.447 Lot 3S/pr. Glic12 0.479 0.714 0.671 0.414
D. Conjugation of Serogroup Y Polysaccharides
a) Hydrolysis

(57) The group Y meningococcal polysaccharide was hvdrolysed as described above for serogroup W135. This gave oligosaccharides with an average DP of about 15 to 20 as determined by ratio between SA and reduced terminal SA (conveniently measured indirectly as described under C(a) above).

(58) b) Sizing c) Introduction of Amino Group, d) Derivatisation to Active Ester and e) Conjugation

(59) These steps were performed as described above for serogroup W135. The purified conjugate was sterile filtered and stored at 20 C. or 60 C. until vaccine formulation.

(60) The conjugate was analysed in the same way as described above for serogroup W135:

(61) TABLE-US-00006 Lot Code saccharide (mg/ml) protein (mg/ml) Glycosylation KD lot 1A 1.16 0.92 1.26 0.303 lot 1B 4.57 3.55 1.29 0.339 Lot 2/4, 5 2.32 6.1 0.38 0.467 lot 2/6 1.75 5.73 0.3 0.498
E. Immunogenicity of Individual Conjugates

(62) The frozen bulk conjugates were thawed. Each was diluted, under stirring, to a final concentration of 20 g 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 g saccharide/mf for mouse immunisation.

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

(64) 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.

(65) Specific anti-poly saccharide IgG antibodies were determined in the sera of immunised animals essentially as described in ref. 150. Each individual mouse serum was analysed in duplicate by a titration curve and GMT was calculated for each immunisation group. Titles 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.

(66) As shown in FIG. 2, the MenA 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. 3) and MenW135 (FIG. 4).

(67) The IgG subclass of the post-II immune responses was measured for various groups. Specific subclasses were determined using the same ELISA method as used for the determination of the total IgG titer in section E above, but using alkaline phosphatase-anti mouse-IgG1, -IgG2a, -IgG2b or -IgG3 (Zymed) as the secondary antibody. Titres were expressed as OD.sub.405nm obtained after 30 minutes of substrate development using serum diluted 1:3200, and are shown in FIGS. 14 (MenA), 15 (MenW135) and 16 (MenY). Responses are primarily in subclass IgG1, which is the subclass predominantly induced in mice by T-dependent antigens. Because polysaccharides are inherently T-independent antigens which are not able to induce immunological memory, these data show that conjugation has had the desired effect.

(68) Post-II sera were also 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: MenA G8238, A1, F6124; MenW135 5554(OAc+) and 242317(OAc); MenY 242975(OAc) and 240539(OAc+).

(69) Results for MenA included:

(70) TABLE-US-00007 Poly/oligo Approx. Aluminium Carrier saccharide DP adjuvant GMT Bactericidal activity CRM.sub.197 O 15 461 F8238: 2048-4096; F6124: 2048-4096 CRM.sub.197 O 15 phosphate 920 F8238: 4096; F6124: 4096 P phosphate 3 F8238: 8; F6124: 128 CRM.sub.197 O 15 290 F8238: 512-1024 P 2 F8238: <4 CRM.sub.197 O 15 155 F8238: 512-1024 CRM.sub.197 O 15 393 F8238: 1024 CRM.sub.197 O 15 396 CRM.sub.197 O 15 phosphate 1396 F8238: 4096 CRM.sub.197 O 15 phosphate 1461 F8238: 2048-4096 CRM.sub.197 O 15 phosphate 1654 F8238: 2048 CRM.sub.197 O 29 phosphate 1053 F8238: 2048 CRM.sub.197 unsized O 10 phosphate 1449 F8238: 2048 CRM.sub.197 O 15 phosphate 626 F8238: 2048-4096 CRM.sub.197 O 15 742 CRM.sub.197 O 15 2207 CRM.sub.197 O 29 1363 CRM.sub.197 unsized O 10 615 CRM.sub.197 O 15 phosphate 1515 CRM.sub.197 O 15 phosphate 876 CRM.sub.197 O 15 phosphate 1232 CRM.sub.197 O 15 phosphate 852 CRM.sub.197 O 15 phosphate 863 F8238: 2048; A1: 2048; F6124: >2048 CRM.sub.197 O 27 phosphate 1733 F8238: 4096-8192; F6124: 4096-8192 CRM.sub.197 O 15 phosphate 172 F8238: 1024; A1: 1024-2048; F6124: 2048 CRM.sub.197 O 15 hydroxide 619 F8238: 1024; A1: 2048; F6124: 2048

(71) Results for MenW135 included:

(72) TABLE-US-00008 Poly/oligo Aluminium Carrier saccharide OAc adjuvant GMT Bactericidal activity CRM.sub.197 O + 14 5554: 256-512 CRM.sub.197 O + phosphate 23 5554: 256-512 P 5554: 4 CRM.sub.197 O + 45 5554: 1024 CRM.sub.197 O + 101 5554: 64-128 CRM.sub.197 O + 80 5554: 256-512 CRM.sub.197 O + phosphate 221 5554: 1024-2048; 242317: 1024-2048 CRM.sub.197 O 52 5554: 512-1024 CRM.sub.197 O phosphate 329 5554: 1024-2048; 242317: 1024-2048 CRM.sub.197 O + 41 5554: 256-512 CRM.sub.197 O + phosphate 24 5554: 1024; 242317: 128-256 CRM.sub.197 O 116 5554: 256-512 CRM.sub.197 O phosphate 185 5554: 1024; 242317: 512-1024 CRM.sub.197 O + phosphate 565 5554: 2048 CRM.sub.197 O + phosphate 328 5554: 512-1024 CRM.sub.197 O + phosphate 490 5554: 1024-2048 CRM.sub.197 O + hydroxide 189 5554: 512-1024; 242317: 512-1024 CRM.sub.197 O + phosphate 80 5554: 512-1024; 242317: 512-1024 CRM.sub.197 O + hydroxide 277 5554: 512-1024; 242317: 1024-2048

(73) Results for MenY included:

(74) TABLE-US-00009 Poly/oligo Aluminium Carrier saccharide DP adjuvant GMT Bactericidal activity CRM.sub.197 O >15 751 242975: 8192 CRM.sub.197 O >15 phosphate 1190 242975: 8192-16384; 240539: 8192-16384 CRM.sub.197 O >15 284 242975: 2048-4096 CRM.sub.197 O >15 phosphate 775 242975: 2048-4096 P 242975: 256 CRM.sub.197 O >15 1618 242975: 4096-8192 CRM.sub.197 O >15 2123 242975: 2048 CRM.sub.197 O <10 253 242975: 512-1024 CRM.sub.197 O <10 1060 242975: 256-512 CRM.sub.197 O >15 hydroxide 1167 242975: 8192; 240539: 8192-16384 CRM.sub.197 O >15 phosphate 665 242975: 8192; 240539: 8192-16384 CRM.sub.197 O >15 phosphate 328 242975: 4096; 240539: 2048-4096 CRM.sub.197 O >15 hydroxide 452 242975: 2048; 240539: 1024-2048
F. Immunogenicity of MenA Conjugate in Combination with MenC Conjugate

(75) CRM-MenC concentrated bulk (from Chiron Vaccines, Italy) was mixed with CRM-MenA concentrated bulk (obtained as described above) were diluted and mixed by stirring. Three different preparations were made. Each contained 20 g saccharide/m) for MenA, but different amounts of MenC conjugate were included: (i) 20 g saccharide/m) (ii) 10 g saccharide/ml; (iii) 5 g saccharide/ml. Ratios of MenA:MenC (w/w) were thus; (i) 1:1; (ii) 2:1; (iii) 4.1.

(76) Each preparation also contained 5 mM sodium phosphate, 9 mg/ml NaCl, aluminium phosphate (to give an Al.sup.3+ concentration of 0.6 mg/ml), pH 7.2. Each mixture was then kept, without stirring, at 2-8 C. overnight and further diluted 1:5 with saline before mice immunisation.

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

(78) For each of the six vaccines, ten Balb/c mice were immunised as described above. Control groups received saline or MenA conjugate alone.

(79) Anti-poly saccharide antibodies for MenA and MenC were determined as described above.

(80) The results obtained with the mixture of MenA+MenC conjugates clearly indicate that the ratio (w/w) between A and C components plays a crucial role for MenA immunogenicity.

(81) The specific anti-MenApS titre obtained with the MenA conjugate control was higher (with or without alum adjuvant) than for the MenA+MenC combination at the same dosage (FIG. 5a). When a lower amount of MenC conjugate is used in the combination, a better anti-MenApS titre is induced by the MenA conjugate component. At the same time, the anti-MenC titre remains acceptable (FIG. 5b).

(82) Experiments were also performed using a guinea pig model. Three different preparations were made, using the same aluminium phosphate adjuvant as before (amorphous hydroxyphosphate, PO.sub.4/Al molar ratio between 0.84 and 0.92, 0.6 mg Al.sup.3+/ml):

(83) TABLE-US-00010 Preparation Men A* MenC* MenA:MenC ratio A 20 g/ml 20 g/ml 1:1 B 40 g/ml 20 g/ml 2:1 C 20 g/ml 10 g/ml 1: *Expressed as saccharide

(84) These preparations were diluted 1:2 with saline and used to immunise guinea pigs. Five guinea pigs (Hartelley strain, female, 450-500 grams) for each immunisation group were injected s.c. twice with 0.5 ml vaccine at days 0 and 28, Bleedings were performed before the first immunisation and then at day 42, Sera were stored at 70 C. prior to analysis by ELISA and serum bactericidal assay (against MenA strain MK 83/94 or .MenC strain C11). Results are shown in FIG. 19.

(85) G. Combination Vaccine for Serogroups C, W135 and Y

(86) Conjugates of polysaccharides from serogroups C, W135 and Y were mixed as described above to give a final concentration of 20 g saccharide/ml for each conjugate. The vaccine contained a final concentration of 5 mM sodium phosphate and 9 mg/ml NaCl, pH 7.2. After overnight storage, the mixture was diluted to contain 4 g saccharide/ml for each conjugate for immunisation.

(87) Immunisations and analysis took place as before.

(88) The results show that the immunogenicity of MenW135 conjugate is enhanced when administered in combination with MenC and MenY conjugates, when compared to that obtained with the MenW135 conjugate alone (FIG. 6). MenY immunogenicity was comparable in the combination to that obtained with the individual conjugate (FIG. 7) and was also comparable to the immunogenicity of the MenC conjugate (FIG. 8).

(89) H. Combination Vaccine for Serogroups A, C, W135 and Y

(90) Conjugates of polysaccharides from serogroups A, C, W135 and Y were mixed as described above to give a final concentration of 20 g saccharide/ml for the serogroup A, W135 and Y conjugates and 5 g saccharide/ml for the serogroup C conjugate. The vaccine contained a final concentration of 5 mM sodium phosphate, 9 mg/ml NaCl, aluminium phosphate (to give an Al.sup.3+ concentration of 0.6 mg/ml), pH 7.2. The mixture was then kept, without stirring, at 2-8 C. overnight and further diluted with saline to give 4 g saccharide/ml for the A, W135 and Y conjugates and Jpg saccharide/ml for the C conjugate. This diluted mixture was used for immunisation.

(91) Immunisations and analysis took place as before, with controls including the individual conjugates except for serogroup C.

(92) FIG. 9 shows that, as before, the immunogenicity of the MenW135 conjugate was enhanced when administered in combination with the MenA, MenC and MenY conjugates. FIG. 10 shows that the immunogenicity of the MenY conjugate is not significantly different when delivered in combination with the MenA, MenC and MenW135 conjugates. FIG. 11 shows that the immunogenicity of the MenA conjugate decreases markedly in the combination, even with the MenC conjugate administered at a lower dosage (). This antigenic competition is not seen in the non-conjugated tetravalent (ACWY) polysaccharide vaccine [5].

(93) I. Lyophilised Serogroup A Antigen

(94) The capsular polysaccharide of serogroup A N. meningitidis is particularly susceptible to hydrolysis. Conjugates of MenA capsular oligosaccharide were therefore prepared in lyophilised form, ready for re-constitution at the time of administration. The lyophilised form was prepared to have components which give the following composition after reconstitution into a unit dose:

(95) TABLE-US-00011 Component Concentration CRM-MenA 20 g saccharide/ml Potassium phosphate buffer 5 mM Mannitol 15 mg/ml

(96) This composition has no adjuvant. Two adjuvants were prepared for its reconstitution:

(97) TABLE-US-00012 Component Concentration Concentration Aluminium hydroxide 0.68 mg Al.sup.3+/ml Aluminium phosphate* 0.6 mg Al.sup.3+/ml Sodium phosphate buffer 10 mM Histidine buffer 10 mM Sodium chloride 9 mg/ml 9 mg/ml Tween 80 0.005% 0.005% PH 7.2 0.05 7.2 0.05 *amorphous hydroxyphosphate, PO.sub.4/Al molar ratio between 0.84 and 0.92

(98) When reconstituted with water for injection, stability of the saccharide component was as follows:

(99) TABLE-US-00013 Stored at 2-8 C. Stored at 36-38 C. Free Free Free Free Total saccharide saccharide saccharide Total saccharide saccharide saccharide Time (days) (g/ml) (g/ml) % (g/ml) (g/ml) % 0 17.72 1.04 5.9 17.72 1.04 5.9 15 17.01 0.88 5.2 16.52 2.26 13.7 30 17.82 0.89 5.0 17.29 2.64 15.3

(100) Over the same 4 week time scale, pH was stable at 12 both at 2-8 C. and at 36-38 C., protein content was stable at around 24.5 g/ml, and moisture content was below 2.5%.

(101) When reconstituted with the aluminium phosphate adjuvant solution at and stored at 2-8 C., stability was as follows:

(102) TABLE-US-00014 Time Total saccharide Free saccharide (hours) (g/ml) (g/ml) Free saccharide % 0 16.62 1.09 6.6 24 16.51 0.98 5.9 48 16.83 0.99 5.9
J. Combination Vaccine for Serogroups A, C, W135 and Y (Lyophilised Serogroup A Conjugate)

(103) A trivalent mixture of the MenC, W135 and Y components either adsorbed onto an aluminium hydroxide adjuvant (2 mg/ml) or mixed with an aluminium phosphate adjuvant (amorphous hydroxyphosphate, PO.sub.4/Al molar ratio between 0.84 and 0.92, 0.6 mg/ml Al.sup.3+, in presence of 10 mM phosphate buffer) was prepared. The compositions of the two trivalent mixtures were as follows:

(104) TABLE-US-00015 Component Concentration Concentration Aluminium hydroxide 0.68 mg Al.sup.3+/ml Aluminium phosphate* 0.6 mg Al.sup.3+/ml CRM-MenC 20 g saccharide/ml 20 g saccharide/ml CRM-MenY 20 g saccharide/ml 20 g saccharide/ml CRM-MenW135 20 g saccharide/ml 20 g saccharide/ml Sodium phosphate buffer 10 mM Histidine buffer 10 mM Sodium chloride 9 mg/ml 9 mg/ml Tween 80 0.005% 0.005% *amorphous hydroxyphosphate, PO.sub.4/Al molar ratio between 0.84 and 0.92

(105) For the hydroxide mixture, stability of the saccharide components were as follows:

(106) TABLE-US-00016 Stored at 2-8 C. Stored at 36-38 C. Time Free saccharide Free Free saccharide Free (days) (g/ml) saccharide % (g/ml) saccharide % MenC bulk 0 <1.2 <6 <1.2 <6 15 <1.2 <6 <1.2 <6 30 <1.2 <6 <1.2 <6 MenC vials 0 <1.2 <6 <1.2 <6 15 <1.2 <6 <1.2 <6 30 <1.2 <6 1.3 6.6 MenW 135 bulk 0 2.5 12.5 2.5 12.5 15 2.3 11.4 3.4 16.8 30 2.3 11.5 3.5 17.3 MenW 135 vials 0 2.1 10.6 2.1 10.6 15 2.3 11.7 2.7 13.3 30 20. 10.2 3.3 16.3 MenY bulk 0 1.7 8.3 1.7 8.3 15 <1.3 <6.3 2.0 10.2 30 1.3 6.3 2.4 12.2 MenY vials 0 1.4 7.1 1.4 7.1 15 1.5 7.6 2.1 10.7 30 1.3 6.3 2.9 14.3

(107) Over the same 4 week time scale, pH was stable at 7.150.05 both at 2-8 C. and at 36-38 C.

(108) For the phosphate mixture, stability of the saccharide components were as follows:

(109) TABLE-US-00017 Stored at 2-8 C. Stored at 36-38 C. Free Free Free Free Total saccharide saccharide saccharide Total saccharide saccharide saccharide Time (days) (g/ml) (g/ml) % (g/ml) (g/ml) % MenC bulk 0 22.8 <1.0 <5 22.8 <1.0 <5 15 17.2 <1.0 <5 18.6 <1.0 <5 30 18.9 <1.0 <5 20.5 <1.0 <5 MenC vials 0 20.5 <1.0 <5 20.5 <1.0 <5 15 18.3 <1.0 <5 23.4 <1.0 <5 30 18.0 <1.0 <5 20.5 <1.0 <5 MenW135 bulk 0 20.7 2.0 10.4 20.7 2.0 10.4 15 21.9 2.3 11.6 21.2 2.1 10.3 30 19.6 2.1 10.6 21.0 2.4 11.8 MenW135 vials 0 23.4 1.7 8.4 23.4 1.7 8.4 15 21.2 1.9 9.5 20.1 2.2 11.1 30 20.1 2.2 11.2 21.3 3.2 16.1 MenY bulk 0 19.1 <1.1 <5.3 19.1 <1.1 <5.3 15 20.1 1.4 6.8 18.7 1.3 6.4 30 18.6 1.4 7.6 19.2 1.7 8.3 MenY vials 0 21.4 <1.1 <5.3 21.4 <1.1 <5.3 15 19.6 1.4 6.8 19.0 1.5 7.4 30 17.7 1.2 6.2 18.4 1.9 9.4

(110) Over the same 4 week time scale, pH was stable at 7.050.05 both at 2-8 C. and at 36-38 C.

(111) The trivalent liquid compositions were diluted and 0.5 ml used to reconstitute the lyophilised MenA conjugate. The resulting tetravalent mixture was administered to ten Balb/c mice (female 6-8 weeks old) per group by subcutaneous injection at day 0 and 28. The mixture contained 2 g of each saccharide conjugate per dose, which represents of the single human dose (SHD). Controls were saline or unconjugated homologous polysaccharides. Bleedings were performed before immunization and then at day 42, with sera stored at 70 C. IgG was determined as described above.

(112) All the conjugates used were safe and immunogenic in the animals. GMT post-II ELISA titres (with 95% confidence intervals) were as follows:

(113) TABLE-US-00018 Vaccine Adjuvant A Y W135 C MenA (lyophilised and Aluminium 172 resuspended) phosphate (69-439) Aluminium 619 hydroxide (419-906) MenY Aluminium 328 phosphate (147-731) Aluminium 452 hydroxide (344-593) MenW Aluminium 80 phosphate (28-225) Aluminium 277 hydroxide (185-411) MenC Aluminium 317 phosphate (152-659) Aluminium 723 hydroxide (615-851) MenA (lyophilized) + Aluminium 32 397 99 114 MenC, W135, Y phosphate (15-68) (252-627) (35-288) (53-246) Aluminium 206 141 139 163 hydroxide (112-372) (97-205) (76-251) (122-218)

(114) FIG. 17 shows the results of IgG subclass analysis for: (17A) MenA; (17B) MenC; (17C MenW135; and (17D) MenY. IgG1 is clearly the most prominent subclass. Serum bactericidal titres were as follows:

(115) TABLE-US-00019 Anti- Anti- Anti-MenA Anti-MenY MenW135 MenC Vaccine Adjuvant F8238 A1 F6124 242975 240539 5554 242317 C11 MenA Aluminium 512- 1024- 2048 (lyophilised) phosphate 1024 2048 Aluminium 1024- 1024- 2048 hydroxide 2048 2048 MenY Aluminium 4096 2048- phosphate 4096 Aluminium 2048 1024- hydroxide 2048 MenW Aluminium 512 512- phosphate 1024 Aluminum 1024 1024- hydroxide 2048 MenC Aluminum 2048- phosphate 4096 Aluminum 4096 hydroxide MenA Aluminium 128- 1024 1024- 2048 256- 1024 512 (lyophilized + phosphate 256 2048 512 MenC, W135, Aluminum 512 1024- 1024- 2048- 256- 1024 512- Y hydroxide 2048 2048 4096 512 1024
K. Combination Vaccine for Serogroups A, C, W135 and Y (Different Dosages)

(116) Mice were immunised as described above, but the vaccine compositions contained different ratios of the various oligosaccharide conjugates. Doses were variously 0.5, 1, 2 or 4 g/dose. Lyophilised MenA oligo-conjugate was used in all experiments.

(117) ELISA titres were as follows:

(118) TABLE-US-00020 Antigen quality (g/dose) Aluminum GMT ELISA (95% confidence interval) A C W135 Y adjuvant A C W135 Y 4 2 2 2 Phosphate 177 367 239 239 (107-291) (263-510) (135-424) (184-311) 4 2 2 2 Hydroxide 390 494 338 158 (313-486) (345-706) (266-430) (96-260) 2 2 2 2 Phosphate 132 582 143 247 (59-296) (268-1155) (75-272) (152-400) 2 2 2 2 Hydroxide 337 569 171 100 (239-476) (462-679) (117-251) (59-169) 4 2 1 1 Phosphate 137 192 18 315 (47-397) (88-421) (4-75) (174-571) 4 2 1 0.5 Phosphate 152 207 51 220 (85-271) (100-428) (21-125) (125-388) 4 2 1 2 Phosphate 113 230 23 267 (49-263) (98-540) (6-91) (81-877) 4 2 0.5 1 Phosphate 267 504 46 583 (109-656) (300-847) (15-134) (330-1030) 4 2 2 1 Phosphate 87 118 24 214 (49-155) (51-278) (8-72) (140-326) 2 2 1 1 Phosphate 217 514 110 206 (132-355) (332-796) (66-183) (141-300) 2 2 1 0.5 Phosphate 105 381 90 206 (40-279) (180-808) (34-236) (96-445) 2 2 1 2 Phosphate 155 374 53 502 (71-339) (196-713) (28-100) (335-752) 2 2 0.5 1 Phosphate 224 358 43 624 (125-400) (223-577) (14-128) (426-914) 2 2 2 1 Phosphate 180 306 70 423 (113-288) (190-492) (34-146) (258-696)

(119) Serum bactericidal titres were as follows:

(120) TABLE-US-00021 Antigen quality (g/dose) Aluminum Bactericidal antibody titre A C W135 Y adjuvant A C W135 Y 4 2 2 2 Phosphate 256- 1024- 1024- 4096- 512 2048 2048 8192 4 2 2 2 Hydroxide 1024- 256- 1024- 1024- 2048 512 2048 2048 2 2 2 2 Phosphate 512- 1024- 128- 8192- 1024 2048 256 16384 2 2 2 2 Hydroxide 256 1024- 256 512- 2048 1024 4 2 1 1 Phosphate 512- 2048 128 2048- 1024 4096 4 2 1 0.5 Phosphate 512- 1024- 128 2048- 1024 2048 4096 4 2 1 2 Phosphate 512- 2048- 128 8192- 1024 4096 16384 4 2 0.5 1 Phosphate 1024- 8192 256- 8192- 2048 512 16384 4 2 2 1 Phosphate 2048- 128 4096- 4096 8192 2 2 1 1 Phosphate 1024- 1024- 256 4096- 2048 2048 8192 2 2 1 0.5 Phosphate 1024- 2048- 256- 2048- 2048 4096 512 4096 2 2 1 2 Phosphate 512- 1024- 128 8192- 1024 2048 16384 2 2 0.5 1 Phosphate 1024- 2048 256- 4096- 2048 512 8192 2 2 2 1 Phosphate 128- 512- 64- 1024- 256 1024 128 2048

(121) A second set of experiments was performed using a dosage of 2 g/ml saccharide for MenA and MenC, half that dosage for MenY, and a quarter dosage for MenW135. ELISA titres were as follows:

(122) TABLE-US-00022 Antigen quality (g/dose) Aluminum GMT ELISA (95% confidence interval) A C W135 Y adjuvant A C W135 Y 2 2 2 2 Phosphate 32 114 99 397 (15-68) (53-246) (35-288) (252-627) Hydroxide 206 163 139 141 (112-372) (122-218) (76-251) (97-205) 2 2 0.5 1 Phosphate 96 238 42 315 (49-187) (101-561) (20-89) (114-867) Hydroxide 293 267 83 244 (144-597) (158-451) (43-163) (152-392)

(123) Serum bactericidal titres were as follows:

(124) TABLE-US-00023 Antigen quantity (g/dose) Aluminum A C W135 Y A C W Y adjuvant F8238 A1 F6124 C11 5554 242317 242975 2 2 2 2 Phosphate 128- 1024 1024- 512 256- 1024 2048 256 2048 512 Hydroxide 512 1024- 1024- 512- 256- 1024 2048- 2048 2048 1024 512 4096 2 2 0.5 1 Phosphate 256 1024- 512 256- 1024 2048- 2048 512 4096 Hydroxide 128 512- 512- 512- 1024 1024 1024 1024 1024
L. MenA, W135 and Y Oligosaccharide Conjugates

(125) The following table shows data relating to MenA, MenW135 and MenY conjugates suitable for making combination compositions of the invention:

(126) TABLE-US-00024 A W135 Y DP after sizing 16.6 21.9 21.1 Saccharide/protein ratio 0.5 1.1 0.7 KD 0.44 0.36 0.41 Free saccharide 5% 10% 5% Free protein <2% <2% <2%

(127) 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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