CONJUGATION OF STAPHYLOCOCCUS AUREUS TYPE 5 AND TYPE 8 CAPSULAR POLYSACCHARIDES

Abstract

The invention provides a process for preparing a conjugate of a S.aureus type 5 or type 8 capsular polysaccharide and a carrier molecule, comprising the steps of: (a) depolymerising the capsular polysaccharide, to give a polysaccharide fragment; (b) oxidising the fragment in order to introduce an aldehyde group into at least one saccharide residue in the fragment, to give an oxidised saccharide residue; and (c) coupling the oxidised saccharide residue to a carrier molecule via the aldehyde group, thereby giving the conjugate. The coupling in step (c) may be direct, or may be via a linker molecule. The invention also provides a conjugate obtained or obtainable by this process.

Claims

1. A process for preparing a conjugate of a S.aureus type 5 or type 8 capsular polysaccharide and a carrier molecule, comprising the steps of: (a) depolymerising the capsular polysaccharide, to give a polysaccharide fragment; (b) oxidising the fragment in order to introduce an aldehyde group into at least one saccharide residue in the fragment, to give an oxidised saccharide residue; and (c) coupling the oxidised saccharide residue to a carrier molecule via the aldehyde group, thereby giving the conjugate.

2. A process for treating a S.aureus type 5 capsular polysaccharide comprising the step of depolymerising the capsular polysaccharide, to give a polysaccharide fragment having a -D-FucNAc-(1.fwdarw.moiety at its non-reducing terminus.

3. A process for treating a S.aureus type 8 capsular polysaccharide comprising the step of depolymerising the capsular polysaccharide, to give a polysaccharide fragment having an -D-FucNAc-(1.fwdarw.moiety at is non-reducing terminus.

4. The process according to any preceding claim, wherein the depolymerising is carried out by acid hydrolysis using acetic acid.

5. The process according to any preceding claim, wherein the average molecular mass of the fragment is between 5 and 100 kDa.

6. The process according to any preceding claim, wherein the degree of O-acetylation of the fragment is 10-90%.

7. The process of any of claims 1 and 4-6, wherein step (a) is the step defined in claim 2 or claim 3.

8. A process for providing a S.aureus type 5 capsular polysaccharide derivative comprising the step of oxidising a S.aureus type 5 capsular polysaccharide having a -D-FucNAc-(1.fwdarw.moiety at its non-reducing terminus to convert two vicinal hydroxyl groups in the -D-FucNAc-(1.fwdarw.moiety into two aldehyde groups.

9. A process for providing a S.aureus type 8 capsular polysaccharide derivative comprising the step of oxidising a S.aureus type 8 capsular polysaccharide having an -D-FucNAc-(1.fwdarw.moiety at is non-reducing terminus to convert two vicinal hydroxyl groups in the -D-FucNAc-(1.fwdarw.moiety into two aldehyde groups.

10. The process of claim 7, wherein step (b) is the step defined in claim 8 or claim 9.

11. A process for providing a coupled S.aureus type 5 capsular polysaccharide comprising the step of coupling to a carrier molecule a S.aureus type 5 capsular polysaccharide having a -D-FucNAc-1.fwdarw.moiety at its non-reducing terminus that has been oxidised to convert two vicinal hydroxyl groups into two aldehyde groups, wherein the coupling is via one of the aldehyde groups.

12. A provides a process for providing a coupled S.aureus type 8 capsular polysaccharide comprising the step of coupling to a carrier molecule a S.aureus type 8 capsular polysaccharide having an -D-FucNAc-(1.fwdarw.moiety at its non-reducing terminus that has been oxidised to convert two vicinal hydroxyl groups into two aldehyde groups, wherein the coupling is via one of the aldehyde groups.

13. The process of claim 10, wherein the process is for preparing a conjugate of a S.aureus type 5 capsular polysaccharide and a carrier molecule, comprising the steps of: (a) depolymerising the capsular polysaccharide, to give a polysaccharide fragment having a -D-FucNAc-(1.fwdarw.moiety at its non-reducing terminus; (b) oxidising the fragment in order to convert two vicinal hydroxyl groups in the -D-FucNAc-(1.fwdarw.moiety into two aldehyde groups; and (c) coupling the oxidised fragment to a carrier molecule via one of the aldehyde groups, thereby giving the conjugate.

14. The process of claim 10, wherein the process is for preparing a conjugate of a S.aureus type 8 capsular polysaccharide and a carrier molecule, comprising the steps of: (a) depolymerising the capsular polysaccharide, to give a polysaccharide fragment having a -D-FucNAc-(1.fwdarw.moiety at its non-reducing terminus; (b) oxidising the fragment in order to convert two vicinal hydroxyl groups in the -D-FucNAc-(1.fwdarw.moiety into two aldehyde groups; and (c) coupling the oxidised fragment to a carrier molecule via one of the aldehyde groups, thereby giving the conjugate.

15. The process of any of claims 1 and 4-7 and 10-14, wherein the coupling is direct coupling by reacting the aldehyde group with an amine group in the carrier by reductive amination.

16. The process of any of claims 1 and 4-7 and 10-14, wherein the coupling is via a linker by reacting the aldehyde group with an amine group in the linker by reductive amination.

17. The process of claim 16, wherein the linker is attached to the carrier molecule.

18. The process of any of claims 1 and 4-7 and 10-17, wherein the coupling results in a polysaccharide:protein ratio (w/w) of between 1:5 and 1:2.

19. A conjugate, fragment, derivative or coupled polysaccharide obtained or obtainable by the process of any preceding claim.

20. An immunogenic composition comprising a conjugate or coupled polysaccharide according to claim 19.

21. The composition of claim 20 further comprising one or more S.aureus protein antigen(s) selected from the group consisting of a clfA antigen; a clfB antigen; a sdrE2 antigen; a sdrC antigen; a sasF antigen; a emp antigen; a sdrD antigen; a spa antigen; a esaC antigen; a esxA antigen; a esxB antigen; a sta006 antigen; a isdC antigen; a Hla antigen; a sta011 antigen; a isdA antigen; a isdB antigen; and a sta073 antigen.

22. The composition of claim 21, wherein the one or more S.aureus protein antigen(s) are selected from the group consisting of a esxA antigen; a esxB antigen; a sta006 antigen; a Hla antigen; a sta011 antigen; and a sta073 antigen.

23. The composition of claim 22, wherein the composition comprises S.aureus protein antigens according to one of combinations (1) to (10) below: (1) a esxA antigen, a esxB antigen, a sta006 antigen and a Hla antigen; (2) a esxA antigen, a esxB antigen, a sta006 antigen and a sta011 antigen; (3) a esxA antigen, a esxB antigen and a sta011 antigen; (4) a esxA antigen, a esxB antigen, a Hla antigen, a sta006 antigen and a sta011 antigen; (5) a esxA antigen, a esxB antigen and a Hla antigen; (6) a Hla antigen, a sta006 antigen and a sta011 antigen; (7) a esxA antigen and a esxB antigen; (8) a esxA antigen, a esxB antigen and a sta006 antigen; (9) a esxA antigen, a esxB antigen, a sta011 antigen and a sta073 antigen; and (10) a sta006 antigen and a sta011 antigen.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0281] FIG. 1 shows a scheme for making an S.aureus type 5 capsular polysaccharide-CRM197 conjugate using an adipic acid dihydrazine linker and carbodiimide chemistry.

[0282] FIG. 2a shows an SDS-PAGE analysis of the S.aureus type 5 capsular polysaccharide-CRM197 conjugate made using an adipic acid dihydrazine linker and carbodiimide chemistry. FIG. 2b shows an S300 Sephacryl chromatogram of the S.aureus type 5 capsular polysaccharide-CRM197 conjugate made using an adipic acid dihydrazine linker and carbodiimide chemistry.

[0283] FIG. 3 shows an S300 Sephacryl chromatogram of depolymerised type 5 capsular polysaccharide.

[0284] FIG. 4A compares 1D .sup.1H signals in the .sup.1H anomeric region for depolymerised and native type 5 capsular polysaccharide. Some notable differences are marked. FIGS. 4B and 4C compare the anomeric and Methyl-Fucose regions respectively of 2D (.sup.1H, .sup.1H) scalar coupling spectra for these polysaccharides. Some notable differences are marked.

[0285] FIG. 5a shows an SDS-PAGE analysis of a S.aureus type 5 capsular polysaccharide-CRM197 conjugate made using a method of the invention. FIG. 5b shows an S300 Sephacryl chromatogram of a S.aureus type 5 capsular polysaccharide-CRM197 conjugate made using a method of the invention.

[0286] FIG. 6 shows an S300 Sephacryl chromatogram of S.aureus type 5 capsular polysaccharides depolymerised under various conditions.

[0287] FIG. 7 shows SDS-PAGE analyses of S.aureus type 5 capsular polysaccharide-CRM197 conjugates made using methods of the invention.

[0288] FIG. 8 shows the IgG response to various antigens in a mouse kidney abscess model of S.aureus infection.

[0289] FIG. 9 compares IgG and IgM responses to various antigens in the mouse kidney abscess model.

[0290] FIG. 10 shows protective responses to various antigens in the mouse kidney abscess model.

[0291] FIG. 11 compares the IgG response to different conjugates in the mouse kidney abscess model.

[0292] FIG. 12 compares protective responses to different conjugates in the mouse kidney abscess model.

[0293] FIGS. 13A and 13B compare protective responses to further conjugates in the mouse kidney abscess model.

[0294] FIG. 14 compares IgG and IgM responses to further conjugates in the mouse kidney abscess model.

[0295] FIG. 15 compares protective responses to a further conjugate when adjuvanted with different agents in the mouse kidney abscess model.

[0296] FIG. 16 compares responses to various antigens in a mouse lethal model of S.aureus infection.

[0297] FIG. 17 shows a SEC-HPLC chromatogram of S.aureus type 8 capsular polysaccharide depolymerised with 2M hydrochloric acid.

[0298] FIG. 18 shows an NMR spectrum of S.aureus type 8 capsular polysaccharide depolymerised with 2M hydrochloric acid.

MODES FOR CARRYING OUT THE INVENTION

[0299] Conjugate Production and Characterisation

[0300] A purified S.aureus type 5 capsular polysaccharide was conjugated to CRM197 using carbodiimide chemistry and an adipic acid dihydrazine linker, similar to the method used in reference 2 (see below). In this method, the capsular polysaccharide is conjugated to derivatised CRM197 using EDC (FIG. 1). The reaction involves the carboxyl groups of the capsular polysaccharide. The carbodiimide (EDC) activates the carboxyl groups to bind to the NH.sub.2 group from the derivatised carrier protein (CRMadh), forming an amide linkage. The derivatised CRMadh is prepared using the same carbodiimide chemistry.

[0301] CRMadh Preparation:

[0302] To a solution of CRM197 was added 100 mM MES pH6.0 buffer in order to reach a final concentration of 10-12 mg/ml. Then 3.5 mg/ml of ADH (adipic acid dihydrazide) and 0.15 (EDC/CRM, w/w) was added, and the reaction kept under mild stirring for 1 h at RT. The mixture was then dialyzed against first 200 mM NaCl, 10 mM MES pH7.3 buffer and then against 5 mM MES pH7.0 buffer, using a 6-8 kDa membrane (SpectraPor). The product was characterized by MicroBCA, SDS-Page (3-8%), HPLC and MS. The CRMadh was found to be derivatised with 6-8 linker of ADH).

[0303] Conjugation Reaction:

[0304] The conjugation reaction was performed at capsular polysaccharide concentration of 2 mg/mL in 50 mM MES buffer pH6.04. The derivatised carrier protein, CRMadh, was added to the solution of capsular polysaccharide to a final concentration of 4.0 mg/ml. The solution was kept at RT for 3 h. The polysaccharide:protein ratio in the reaction mixture was 1:2 (weight/weight), the polysaccharide:EDC ratio was 1:6.66 (equivalent/equivalent) and the polysaccharide:SulfoNHS ratio was 1:0.53 (equivalent/equivalent).

[0305] After 3 h, formation of the conjugate was verified by SDS-PAGE using a NuPAGE 3-8% Tris-Acetate Gel (Invitrogen) (FIG. 2a). After conjugation, the conjugate was purified by gel-filtration chromatography (performed on an Akta system (G&E Healthcare) using a S300 Sephacryl resin (G&E Healthcare), with a 10 mM NaPi, 10 mM NaCl, pH7.2 mobile phase buffer). The conjugate was detected at 215 nm, 254 nm and 280 nm (FIG. 2b). The conjugate solution was stored at 20 C. until further use. Total saccharide in the conjugate was determined by HPAEC-PAD analysis and protein content by MicroBCA assay, as described in reference 281 (Table 1).

TABLE-US-00001 TABLE 1 Conjugate Protein Saccharide Saccharide/protein (lot) (g/ml) (g/ml) (w/w) 1 26.00 12.10 0.47 2 33.90 11.00 0.32 3 62.21 29.40 0.47 4 45.21 9.30 0.21

[0306] Purified S.aureus type 5 and type 8 capsular polysaccharides were separately conjugated to CRM197 using a method of the invention (see below).

[0307] Depolymerisation Purified capsular polysaccharide was dissolved in distilled water at 2 mg/mL. Acetic acid was added to a final concentration of 2% (v/v) and the reaction kept at 90 C. for 3 hours (or overnight in the case of Lot B). The solution was then neutralized with 1M NaOH and the depolymerised polysaccharide purified on a gel-filtration column (performed on an Akta system (G&E Healthcare) using a S300 Sephacryl resin (G&E Healthcare), with a 10 mM NaPi, 10 mM NaCl, pH7.2 mobile phase buffer). The saccharide was detected at 215 nm (FIG. 3). Pooled fractions were dialyzed against distilled water using a 1 kDa membrane (SpectraPor) and lyophilized.

[0308] The site of cleavage was verified as being at (1.fwdarw.3) glycosidic linkages within the type 5 polysaccharide using .sup.1H NMR. Briefly, samples of native and depolymerised type 5 capsular polysaccharide were freeze-dried to eliminate protonated water solvent and dissolved in deuterium oxide (99.9% deuterium, Sigma-Aldrich). All NMR spectra were recoded at 50 C. on a Bruker Avance III 400 MHz spectrometer using a 5-mm broadband probe and the TopSpin 2.1 software package (Bruker) for data acquisition and processing. 1D .sup.1H spectra were collected using a standard one-pulse experiment over a spectral width of 4,000 Hz and collecting 32 k data points. The transmitter was set at the residual HDO frequency (4.79 ppm). The spectra were obtained in a quantitative manner using a total recycle time to ensure a full recovering of each signal (5 Longitudinal Relaxation Time T1). Spectra were Fourier transformed after applying a 0.2 Hz line broadening function. 2D (.sup.1H, .sup.1H) scalar correlation spectra were recorded by DQF-COSY pulse sequence. 4096 data points were collected in the F2 domain and 256 in the F1 domain.

[0309] 1D .sup.1H signals for the native polysaccharide were compared with published values and found to be in agreement (Table 2).

TABLE-US-00002 TABLE 2 Measured Pubd. Pubd. Signal (ppm)* (ppm)** (ppm)*** H.sub.3.sup.L-FucNAc-OAc 4.958 5.005 5.005 H.sub.1.sup.L-FucNAc-OAc 4.929 4.981 4.975 H.sub.3.sup.L-FucNAc-deOAc 4.864 4.935 4.911 H.sub.1.sup.ManNAc-deOAc 4.801 4.860 4.847 H.sub.1.sup.ManNAc-OAc 4.638 4.698 4.683 H.sub.2.sup.ManNAc-deOAc 4.625 4.680 4.670 H.sub.2.sup.ManNAc-deOAc 4.584 4.645 4.629 H.sub.1.sup.D-FucNAc-OAc/deOAc 4.405 4.461 4.452 H.sub.4.sup.L-FucNAc-OAc 4.320 4.382 4.367 H.sub.2.sup.L-FucNAc-OAc 4.292 4.368 4.338 H.sub.5.sup.L-FucNAc-OAc 4.121 4.175 4.168 H.sub.5.sup.L-FucNAc-deOAc 4.080 4.142 4.126 H.sub.2.sup.L-FucNAc-deOAc 4.033 4.105 4.077 H.sub.4.sup.L-FucNAc-deOAc 4.005 4.060 4.051 NAc.sup.D-FucNAc-OAc 2.083 2.149 2.131 NAc.sup.D-FucNAc-deOAc 2.067 2.126 2.115 OAc.sup.L-FucNAc-OAc 2.004 2.070 2.051 NAc.sup.L-FucNAc-deOAc 1.995 2..057 2.002 NAc.sup.L-FucNAc-OAc 1.955 2.023 2.043 NAc.sup.ManNAc-deOAc 1.948 2.018 1.996 NAc.sup.ManNAc-OAc 1.943 2.011 1.992 H.sub.6.sup.D-FucNAc-OAc 1.238 1.300 1.287 H.sub.6.sup.D-FucNAc-deOAc 1.298 H.sub.6.sup.L-FucNAc-OAc/deOAc 1.183 1.242 1.231 *HDO signal at 4.484 ppm **Jones C. Carbohydr. Res. 2005, 340(6), 1097-1106 - HDO signal at 4.484 ppm ***Jones C. Carbohydr. Res. 2005, 340(6), 1097-1106 - H1L-FucNAc = 5.005 ppm, therefore HDO signal at 4.532 ppm instead of 4.484 ppm

[0310] FIG. 4A compares 1D .sup.1H signals in the .sup.1H anomeric region for the depolymerised and native polysaccharides. FIGS. 4B and 4C compare the anomeric and Methyl-Fucose regions respectively of 2D (.sup.1H, .sup.1H) scalar coupling spectra for these polysaccharides. The data show that the acetic acid treatment resulted in cleavage of (1.fwdarw.3) glycosidic linkages between the -L-FucNAc(3OAc) and -D-FucNAc residues in the type 5 capsular polysaccharide.

[0311] Oxidation

[0312] The depolymerised capsular polysaccharide was dissolved in distilled water at 2 mg/mL. NaIO.sub.4 was added at a polysaccharide:NaIO.sub.4 ratio of 1:1 (weight/weight) and the reaction kept at room temperature for 1-2 hours in the dark. The solution was then dialyzed against distilled water using a 1 kDa membrane (SpectraPor) and lyophilized once again.

[0313] Conjugation

[0314] The oxidised capsular polysaccharide was dissolved in a 200 mM NaPi, 1M NaCl, pH7.2 buffer at a concentration of 10 mg/mL. CRM197 was added to the solution at a polysaccharide:protein ratio of 4:1 (weight/weight) and NaBH.sub.3CN (Aldrich) added at a sacchaaride:NaBCNH.sub.3 ratio of 2:1 (weight/weight). The solution was kept at 37 C. for 2 days. SDS-PAGE was used to confirm formation of the conjugate (see FIG. 5a for the type 5 conjugate). After conjugation, the conjugate was purified by gel-filtration chromatography (performed on an Akta system (G&E Healthcare) using a S300 Sephacryl resin (G&E Healthcare), with a 10 mM NaPi, 10 mM NaCl, pH7.2 mobile phase buffer). The conjugate was detected at 215 nm, 254 nm and 280 nm (see FIG. 5b for the type 5 conjugate). The conjugate solution was stored at 20 C. until further use. Total saccharide in the conjugate was determined by HPAEC-PAD analysis and protein content by MicroBCA assay (see Table 3a for the type 5 conjugate and Table 3b for type 8 conjugate).

TABLE-US-00003 TABLE 3a Conjugate Protein Saccharide Saccharide/protein (lot) (g/ml) (g/ml) (w/w) A 51.52 1.72 0.03 B 161.80 17.10 0.11 C 34.42 4.22 0.12 D 40.56 12.70 0.31 E 196.00 55.17 0.28

TABLE-US-00004 TABLE 3b Conjugate Protein Saccharide Saccharide/protein (lot) (g/ml) (g/ml) (w/w) 518.00 82.30 0.16 11.00 7.94 0.72 23.22 5.57 0.24 22.87 5.08 0.22

[0315] Purified S.aureus type 5 was conjugated to CRM197 using another method of the invention. In this method, the depolymerisation, oxidation and conjugation steps were carried out as described above, except that the conjugation step was carried out with the derivatised carrier protein described above (CRMadh) instead of CRM197. Total saccharide in the conjugate was determined by HPAEC-PAD analysis and protein content by MicroBCA assay (Table 4).

TABLE-US-00005 TABLE 4 Conjugate Protein Saccharide Saccharide/protein (lot) (g/ml) (g/ml) (w/w) A 58.25 2.49 0.043

[0316] Alternative Depolymerisation Methods

[0317] In other studies, different conditions were tested for depolymerisation of the purified capsular polysaccharide. The polysaccharide was dissolved in distilled water at 2 mg/mL. Acetic acid was added to a final concentration of 2% or 5% (v/v) and the reaction kept at 90 C. for 30 minutes, 3 hours, 5 hours or 6 hours. The solution was then neutralized and purified on a gel-filtration column as described above. The saccharide was detected at 215 nm and pooled (FIG. 6).

[0318] The pooled fractions were then oxidised and dialyzed against water as described above. The fractions were conjugated to CRM197 or CRMadh as described above and the resultant conjugates purified by gel-filtration chromatography also as described above (FIG. 7).

[0319] In another study, hydrochloric acid at 0.5M was used instead of acetic acid for type 8 capsular polysaccharide, and the reaction kept at 90 C. for 2.5 hours, with the reaction being sampled every 30 minutes. Samples were analysed by NMR and SEC-HPLC. The polysaccharide did not hydrolyse and the level of O-acetylation remained almost unchanged. In contrast, when hydrochloric acid at 2M was used, and the reaction kept at 100 C., hydrolysis was observed even after only 30 minutes. The level of O-acetylation gradually fell over the 2.5 hours (FIGS. 17 and 18 (with acetyl peak circled)).

[0320] Immunisation StudyAbscess Model (1)

[0321] General assay protocol: Mice were immunized according to the schedule described below and challenged by intravenous injection of a bacterial suspension of S.aureus. The culture of S.aureus was centrifuged, washed twice and diluted in PBS before challenge. Further dilutions were needed for the desired inoculum, which was experimentally verified by agar plating and colony formation. For organ harvest, mice were euthanized and their kidneys removed and homogenized in 1% Triton X-100. Aliquots were then diluted and plated on agar media for triplicate determination of CFU. For histology, kidney tissue was incubated at room temperature in 10% formalin for 24 hours. Tissues were embedded in paraffin, thin sectioned, hematoxylin/eosin stained and examined by microscopy.

[0322] CD1 mice at 3 weeks old were immunised at days 0 and 11 by intraperitoneal injection with a 5 g dose of antigen in an injection volume of 200 l. The mice were bled on days 0 and 20 and challenged with S.aureus on day 21. Organs were harvested at day 25 Immunisations were carried out in groups of eight mice according to the following scheme: [0323] Group 1Alum alone [0324] Group 2Type 5 capsular polysaccharide alone [0325] Group 3Type 5 capsular polysaccharide plus alum [0326] Group 4Type 5 capsular polysaccharide-CRMadh conjugate (Lot 1) [0327] Group 5Type 5 capsular polysaccharide-CRMadh conjugate (Lot 1) plus alum

[0328] The conjugate induced a specific IgG response against type 5 polysaccharide. The alum formulation gave an improved response (FIG. 8). The conjugate also induced a specific IgM response against type 5 polysaccharide (FIG. 9). The alum conjugate formulation also gave the best protection from kidney infection (FIG. 10).

[0329] Immunisation StudyAbscess Model (2)

[0330] CD1 mice at 3 weeks old were immunised at days 1, 14 and 28 by intraperitoneal injection with a 5 g dose of antigen in an injection volume of 200 l. The mice were bled on days 0, 27 and 37 and challenged with S.aureus on day 38. Organs were harvested at day 42 Immunisations were carried out in groups of eight mice according to the following scheme: [0331] Group 1Alum alone [0332] Group 2Type 5 capsular polysaccharide plus alum [0333] Group 3Type 5 capsular polysaccharide-CRMadh conjugate (Lot 2) plus alum [0334] Group 4Type 5 capsular polysaccharide-CRMadh conjugate (Lot A) plus alum

[0335] The conjugates induced a specific IgG response against type 5 polysaccharide. The conjugates of the invention (represented by lot A) gave a particularly high titre (FIG. 11). The conjugates of the invention gave the best protection from kidney infection (FIG. 12).

[0336] Immunisation StudyAbscess Model (3)

[0337] CD1 mice at 3 weeks old were immunised at days 1, 14 and 28 by intraperitoneal injection with a 5 g dose (or 0.5 g dose in the case of lot A) of antigen in an injection volume of 200 l. The mice were bled on days 0, 27 and 37 and challenged with S.aureus (grown in liquid or solid medium) on day 38. Organs were harvested at day 42. Immunisations were carried out in groups of eight mice according to the following scheme: [0338] Group 1Alum alone [0339] Group 2Type 5 capsular polysaccharide plus alum [0340] Group 3Type 5 capsular polysaccharide-CRMadh conjugate (Lot 2) plus alum [0341] Group 4Type 5 capsular polysaccharide-CRMadh conjugate (Lot 3) plus alum [0342] Group 5Type 5 capsular polysaccharide-CRMadh conjugate (Lot A) plus alum [0343] Group 6Type 5 capsular polysaccharide-CRM conjugate (Lot A) plus alum [0344] Group 7Type 5 capsular polysaccharide-CRM conjugate (Lot B) plus alum

[0345] The conjugates of the invention (represented by lots A, A and B) gave protection from kidney infection (FIGS. 13A and 13B). The conjugates of the invention gave high titres of specific IgG antibodies with low tites of IgM antibodies (FIG. 14).

[0346] Immunisation StudyAbscess Model (4)

[0347] CD1 mice at 3 weeks old were immunised at days 1 and 14 by intraperitoneal injection with a 1 g dose of antigen in an injection volume of 200 l. The mice were bled on days 0, 13 and 27 and challenged with S.aureus on day 28. Organs were harvested at day 32 Immunisations were carried out in groups of eight or nine mice according to the following scheme: [0348] Group 1Type 8 capsular polysaccharide-CRM conjugate (lot a) plus alum [0349] Group 2Type 8 capsular polysaccharide-CRM conjugate (lot a) plus MF59 [0350] Group 3Alum alone [0351] Group 4MF59 alone

[0352] The conjugates of the invention gave protection from kidney infection (FIG. 15). The alum formulation gave better protection than the MF59 formulation.

[0353] Immunisation StudyLethal Model (1)

[0354] General assay protocol: Mice were immunized according to the schedule described below and challenged by intraperitoneal injection of a bacterial suspension of S.aureus. Cultures of S.aureus were centrifuged, washed twice and diluted in PBS before challenge. Further dilutions were needed for the desired inoculum, which was experimentally verified by agar plating and colony formation. Animals were monitored for 14 days and lethal disease recorded.

[0355] CD1 mice were immunised by intraperitoneal injection with a 5 g dose of antigen in an injection volume of 200 l. Immunisations were carried out in groups of twelve mice according to the following scheme, prior to challenge with 510.sup.8 CFU type 5 S.aureus: [0356] Group 1PBS plus alum [0357] Group 2Type 5 capsular polysaccharide-CRM conjugate (Lot C) plus alum [0358] Group 3Type 5 capsular polysaccharide-CRMadh conjugate (Lot 3) plus alum

[0359] The conjugates of the invention (represented by lot C) gave higher survival (FIG. 16).

[0360] Immunisation StudyLethal Model (2)

[0361] CD1 mice were immunised by intraperitoneal injection with a 2 g (saccharide) and 10(protein, where present) doses of antigen in an injection volume of 200 l. Immunisations were carried out in groups of twelve mice according to the following scheme, prior to challenge with 510.sup.8 CFU type 5 S.aureus: [0362] Group 1PBS plus alum [0363] Group 2Type 5 capsular polysaccharide-CRM conjugate (Lot D) plus alum [0364] Group 3Type 5 capsular polysaccharide-CRMadh conjugate (Lot 4) plus alum [0365] Group 4Type 5 capsular polysaccharide-CRM conjugate (Lot D) plus EsxAB, Sta006 and Sta011 proteins and alum [0366] Group 5Type 5 capsular polysaccharide-CRM conjugate (Lot D) plus HlaH35L, Sta006 and Sta011 proteins and alum

[0367] Survival data is presented in Table 5.

TABLE-US-00006 TABLE 5 Time (days) Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 100 25 17 17 17 17 17 17 17 17 8 0 0 0 2 100 50 50 50 50 50 50 50 50 42 42 42 42 42 3 100 50 42 42 42 42 42 42 42 33 33 33 33 33 4 100 67 67 67 67 67 67 67 67 67 67 67 67 67 5 100 100 100 100 100 100 83 83 75 75 75 75 75 75

[0368] The conjugates of the invention (represented by lot D) gave higher survival. Survival was enhanced by addition of S.aureus protein antigens.

[0369] Immunisation StudyLethal Model (3)

[0370] CD1 mice were immunised by intraperitoneal injection with a 2 g (type 5 polysaccharide) 1 g (type 8 polysaccharide, where present) and 10 (protein, where present) doses of antigen in an injection volume of 200 l. Immunisations were carried out in groups of twelve mice according to the following scheme, prior to challenge with 510.sup.8 CFU type 5 S.aureus: [0371] Group 1PBS plus alum [0372] Group 2Type 5 capsular polysaccharide-CRM conjugate (lot E) plus EsxAB, Sta006 and Sta011 proteins and alum [0373] Group 3Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot ) plus EsxAB, Sta006 and Sta011 proteins and alum [0374] Group 4Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot ) plus EsxAB, Sta011 and Sta073 proteins and alum [0375] Survival data is presented in Table 7.

TABLE-US-00007 TABLE 7 Time (days) Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 100 50 42 42 42 42 42 42 42 42 33 33 33 33 2 100 42 42 42 42 42 42 42 42 42 33 33 33 33 3 100 75 75 75 75 75 75 75 75 75 58 50 50 50 4 100 92 92 83 83 83 83 83 83 83 75 75 75 75

[0376] Immunisation StudyLethal Model (4)

[0377] CD1 mice were immunised by intraperitoneal injection with a 2 g (type 5 capsular polysaccharide) 1 g (type 8 capsular polysaccharide, where present) and 10 (protein, where present) doses of antigen in an injection volume of 200 l. Immunisations were carried out in groups of twelve mice according to the following scheme, prior to challenge with 510.sup.8 CFU type 5 S.aureus: [0378] Group 1PBS plus alum [0379] Group 2Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot first dose, lot second dose) plus EsxAB, Sta006 and Sta011 proteins and alum [0380] Group 3Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot first dose, lot second dose) plus alum [0381] Group 4Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot first dose, lot second dose) plus EsxAB protein and alum [0382] Group 5Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot first dose, lot second dose) plus Sta006 protein and alum [0383] Group 6Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot first dose, lot second dose) plus Sta011 protein and alum [0384] Group 7Type 5 capsular polysaccharide-CRM conjugate (lot E) and Type 8 capsular polysaccharide-CRM conjugate (lot first dose, lot second dose) plus Sta006 and Sta011 proteins and alum [0385] Group 8Type 5 capsular polysaccharide-CRM conjugate (lot E) plus HlaH35L, Sta006 and Sta011 proteins and alum [0386] Group 9Type 5 capsular polysaccharide-CRM conjugate (lot E) plus HlaH35L protein and alum [0387] Survival data is presented in Table 8.

TABLE-US-00008 TABLE 8 Time (days) Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 100 100 13 13 13 13 13 13 13 13 13 13 13 13 2 100 88 75 75 63 63 63 50 50 50 50 50 50 50 3 100 100 63 63 38 38 38 38 38 38 38 38 38 38 4 100 100 75 75 75 75 75 75 63 50 50 25 25 25 5 100 100 50 50 50 50 50 38 38 38 38 38 38 38 6 100 100 25 25 25 25 25 25 25 25 25 13 13 13 7 100 88 63 63 63 63 63 50 50 50 50 50 50 50 8 100 100 100 100 88 88 88 88 88 75 75 75 75 75 9 100 88 88 63 38 38 38 38 38 13 13 13 13 13

[0388] 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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