Vaccine

Abstract

The present invention relates to conjugates comprising polysaccharides comprising 3- deoxy-D-manno-actulosonic acid (KDO) moieties, particularly conjugates produced using random conjugation methods, methods for preparing such conjugates, immunogenic compositions and vaccines comprising the conjugates, and methods of treatment or medical uses using the compositions and vaccines.

Description

Abstract This invention relates to conjugates comprising polysaccharides containing a 3-deoxy-D-mannooctyl ketone acid (KDO) moiety, particularly conjugates produced using a random conjugation method, methods for preparing such conjugates, immunogenic compositions and vaccines comprising said conjugates, and therapeutic methods or medical uses using said compositions and vaccines.

Claims

CLAIMS 1. A conjugate comprising a polysaccharide comprising (a) a 3-deoxy-D-manno-octulosonic acid (KDO) moiety, and / or (b) an O-antigen conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide comprises more than one activated site.

2. A conjugate comprising a polysaccharide comprising (a) a KDO moiety, and / or (b) an O-antigen conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the polysaccharide by 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.

3. A method for producing a conjugate comprising a polysaccharide comprising (a) a KDO moiety, and / or (b) an O-antigen conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide.

4. A method for producing a conjugate comprising a polysaccharide comprising (a) a KDO moiety, and / or (b) an O-antigen conjugated to a carrier protein comprising a step of: (i) activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.

5. The conjugate or method of any one of the preceding claims, wherein: (a) the polysaccharide is an O-antigen;(b) the O-antigen is an O-antigen from Salmonella Paratyphi A (S. Paratyphi A); (c) the carrier protein is selected from the group consisting of CRM197, tetanus toxoid (TT) or diphtheria toxoid (DT); (d) the carrier protein is CRM197;(e) the conjugate further comprises a linker, optionally an adipic acid dihydrazide (ADH) linker; (f) the conjugate further comprises a linker between the saccharide or O- antigen and the carrier protein which is optionally an ADH linker; and / or (g) the polysaccharide or O-antigen comprises 1.5 or more, 2.0 or more, or 2.5 or more activated sites or the activating or oxidising step introduces 1.5 or more, 2.0 or more, or 2.5 or more activated sites.

6. The conjugate or method of any one of claims 2 or 4 to 5, wherein the activating or oxidising step introduces multiple activated sites into the polysaccharide or O- antigen.

7. The conjugate of any one of claims 1, 3 or 5 to 6, wherein the random conjugation method or introducing multiple activated sites comprises a step of: (i) activating the polysaccharide or the O-antigen by CDAP chemistry to provide an activated polysaccharide or O-antigen; or (ii) oxidising the polysaccharide or the O-antigen to provide an oxidised polysaccharide or O-antigen.

8. The conjugate or method of any one of claims 2 or 4 to 7, wherein: (a) activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises mixing the polysaccharide or the O-antigen with CDAP at a w / w ratio of between 0.05:1 and 5:1, between 0.1:1 and 5:1, between 0.2:1 and 2:1, or around 0.3:1 (CDAP to polysaccharide or O-antigen); (b) activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises mixing the O-antigen and / or the polysaccharide comprising a KDO moiety in a solution of NaCl or KCl at a concentration between 50 mM and 1 M,between 100 mM and 250 mM, between 125 mM and 200 mM, or around 150 mM; (c) activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises adjusting the pH to between 9 and 10 using triethylamine and optionally incubating the solution at room temperature for 1 to 5 minutes with stirring; (d) the method further comprises reacting the activated polysaccharide or O- antigen with hydrazide / amino groups on the carrier protein or a carrier protein- linker compound comprising the carrier protein, optionally wherein the carrier protein is CRM197and / or the carrier protein-linker compound is CRM197-ADH; and / or (e) the method further comprises reacting the activated polysaccharide or O- antigen with hydrazide / amino groups on the carrier protein or a carrier protein- linker compound comprising the carrier protein and reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on the carrier protein or the carrier protein-linker compound comprises mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound at a w / w ratio of between 0.1:1 and 5:1, between 0.2:1 and 3:1, between 0.5:1 and 2:1, or around 1:1 (polysaccharide or O-antigen to carrier protein or carrier protein-linker), optionally wherein the carrier protein is CRM197and / or the carrier protein-linker compound is CRM197-ADH.

9. The conjugate or method of any one of claims 2 or 4 to 8, wherein the conjugation method further comprises: (i) a step of adjusting the pH to between 9 and 10 using triethylamine and incubating the solution at room temperature for 1 to 10 hours with stirring after the step of mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound; and / or (ii) a step of adding glycine solution and adjusting the pH to pH 7 to 9 using triethylamine and incubating the solution at 2-8ºC for between 5 and 50, or between 10 and 20 hours; and / or (iii) a step of removing unreacted polysaccharide or O-antigen,optionally wherein the step of removing unreacted polysaccharide or O-antigen comprises a step of chromatography, optionally hydrophobic interaction chromatography or anion exchange chromatography.

10. The conjugate or method of any one of claims 2 or 4 to 7, wherein the step of oxidising the polysaccharide or O-antigen comprises mixing the polysaccharide or O- antigen with an oxidising agent, optionally at a pH between 4 and 6.

11. The conjugate or method of claim 10, wherein: (a) the oxidising agent is periodate, optionally sodium periodate; (b) mixing the polysaccharide or O-antigen with an oxidising agent comprises mixing the polysaccharide or O-antigen with an oxidising agent at a ratio of between 1 mg / mL: 10 mM and 100 mg / mL: 10 mM, between 1 mg / mL :10 mM and 50 mg / mL: 10 mM, between 1 mg / mL: 10 mM and 25 mg / mL: 10 mM, or around 10mg / mL:10 mM (polysaccharide or O-antigen to oxidising agent; and / or (c) the step of oxidising the polysaccharide or O-antigen comprises: (i) leaving a solution of the polysaccharide or O-antigen and the oxidising agent in the dark at a temperature between 20ºC and 30ºC for between 1 and 5 hours; and / or (ii) quenching excess oxidising agent, optionally using Na2SO3; and / or (iii) desalting the oxidised polysaccharide or O-antigen, optionally with a PD10 column.

12. The conjugate or method of any one of claims 2, 4 to 7, or 10 to 11, wherein the conjugation method further comprises a step of reacting the oxidised polysaccharide or O-antigen with the carrier protein.

13. The conjugate or method of claim 12, wherein: (a) the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen andthe carrier protein with a reducing agent, optionally wherein the reducing agent is sodium cyanoborohydride; (b) the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein at a w / w ratio of between 0.5: 1 and 20: 1, between 1:1 and 10: 1, between 1.5:1 and 5:1, or around 2:1 (polysaccharide or O-antigen to carrier protein); (c) the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the carrier protein and the reducing agent at a w / w ratio of between 0.25:1 and 20: 1, between 0.5:1 and 10: 1, between 0.75:1 and 5:1, or around 1:1 (carrier protein to reducing agent); and / or (d) the conjugation method further comprises: (i) incubating a reaction mixture comprising the oxidised polysaccharide or O-antigen and the carrier protein at a temperature between 35ºC and 39ºC for between 4 and 20 hours; (ii) quenching by adding sodium borohydride, optionally wherein the w / w ratio of oxidised polysaccharide or O-antigen to sodium borohydride is between 10:1 and 0.1: 1, between 5:1 and 0.5: 1, or around 1:1 (w / w polysaccharide or O-antigen to sodium borohydride).

14. A conjugate obtainable by the method of any one of claims 3 to 13.

15. A conjugate obtained by the method of any one of claims 3 to 13.

16. An immunogenic composition comprising the conjugate of any one of claims 1, 2 or 5 to 15, optionally further comprising a pharmaceutically acceptable excipient and / or an adjuvant.

17. The immunogenic composition of claim 16, wherein the immunogenic composition further comprises an antigen from Salmonella Typhi (S. Typhi), optionally a Vi polysaccharide.

18. The immunogenic composition of claim 17, wherein the Vi polysaccharide is a fragmented Vi polysaccharide (fVi).

19. The immunogenic composition of claim 18, wherein the fVi polysaccharide: (a) has an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; (b) has a target average molecular weight of between 51 kDa and 55 kDa; (c) is part of an fVi conjugate comprising fVi and a carrier protein; (d) is part of an fVi conjugate comprising fVi and a carrier protein which is CRM197or diphtheria toxoid; (e) is part of an fVi conjugate comprising fVi and a carrier protein which is CRM197; (f) is part of an fVi conjugate comprising fVi and a carrier protein, and wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, optionally via a linker; (g) is part of an fVi conjugate comprising fVi and a carrier protein, and wherein the fVi conjugate is obtained by or obtainable by a method comprising the steps of: (i) fragmenting Vi polysaccharide to obtain a fragmented Vi (fVi) polysaccharide having an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; (ii) activating the fVi polysaccharide by reacting the fVi polysaccharide obtained in step a. with a carbodiimide and N- hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative; and (iii) reacting the N-hydroxysuccinimide ester fVi derivative obtained in step b. with the carrier protein to produce the fVi conjugate; (h) is part of an fVi conjugate comprising fVi and a carrier protein, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry and the carbodiimide is EDC;(i) is part of an fVi conjugate comprising fVi and a carrier protein, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, the carbodiimide is EDC, and the carrier protein is derivatised by reacting it with a carbodiimide and a linker, optionally an adipic acid dihydrazide (ADH) linker; and / or (j) is part of an fVi conjugate comprising fVi and a carrier protein, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry and the carbodiimide is 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC) or the carbodiimide chemistry is EDAC chemistry.

20. The immunogenic composition of any one of claims 16 to 19, wherein: (a) the conjugate is stable in the immunogenic composition for at least 4 weeks, optionally wherein (i) the conjugate is stable in the immunogenic composition for at least 4 weeks if less than 20%, less than 18%, less than 17%, less than 15%, between 0% and 20%, or between 1% and 15% of the polysaccharide or O-antigen is released from the conjugate in 4 weeks; (ii) the conjugate is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is substantially less than the amount released in an equivalent immunogenic composition in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM197using direct reductive amination via an ADH-SIDEA linker, optionally wherein substantially less is at least 10% less, at least 15% less, at least 20% less, or at least 25% less; (iii) the conjugate is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is similar to the amount released in an equivalent reference immunogenic composition in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM197using CDAP chemistry, optionally wherein similar to is within 25%, within 15%, or within 10% of; and / or(iv) the amount of the polysaccharide or O-antigen released from the conjugate after 4 weeks is determined using a stability assay comprising the following steps: (A) incubate the immunogenic composition for 4 weeks at 37ºC; (B) prepare a post-incubation sample of the incubated immunogenic composition and remove the conjugated polysaccharide or O-antigen by deoxycholate precipitation; and (C) determine the amount of free polysaccharide or O-antigen in the post-incubation sample as a percentage of the amount of total polysaccharide or O-antigen in the post-incubation sample; and / or (b) the immunogenic composition induces a similar level of anti-S. Paratyphi O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises an S. Paratyphi O-antigen conjugated to CRM197using direct reductive amination via an ADH-SIDEA linker, optionally wherein: (i) a similar level is a level within 25%, within 15%, or within 10%; and / or (ii) the level of anti-S. Paratyphi O-antigen antibodies is measured using an immunogenic assay comprising the following steps: (A) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 μg (O-antigen); (B) measure the anti-S. Paratyphi O-antigen antibody level by ELISA at day 42.

21. The immunogenic composition of any one of claims 16 to 20 further comprising: (i) a Salmonella Typhimurium (S. Typhimurium) antigen, optionally wherein the S. Typhimurium antigen comprises or consists of S. Typhimurium GMMA; and / or (ii) a Salmonella Enteritidis (S. Enteritidis) antigen, optionally wherein the S. Enteritidis antigen comprises or consists of S. Enteritidis GMMA.

22. The immunogenic composition of claim 21, wherein: (a) the S. Typhimurium GMMA and / or the S. Enteritidis GMMA comprises detoxified lipid A; (b) the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from S. Typhimurium and / or S. Enteritidis that does not comprise: (i) a gene encoding a functional MsbB protein; and / or (ii) a gene encoding a functional PagP protein; and / or (c) the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from S. Typhimurium and / or S. Enteritidis that does not comprise gene encoding a functional tolR protein.

23. A vaccine comprising the immunogenic composition of any one of claims 16 to 22.

24. The immunogenic composition or vaccine of any one of claims 16 to 23, for use in a method of preventing an infection.

25. The immunogenic composition for use of claim 24, wherein the method of preventing an infection: (a) comprises administering an effective amount of the immunogenic composition or vaccine of any one of claims 16 to 23 to a subject; (b) is a method of preventing Salmonella infection; (c) is a method of preventing invasive non-typeable Salmonella infection; and / or (d) is a method of preventing infection by S. Typhimurium, S. Enteritidis, S. Typhi and / or S. Paratyphi A.

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