EMULSIONS WITH FREE AQUEOUS-PHASE SURFACTANT FOR ADJUVANTING SPLIT INFLUENZA VACCINES

Abstract

A split influenza virus vaccine is adjuvanted with an oil-in-water emulsion that contains free surfactant in its aqueous phase. The free surfactant can continue to exert a ‘splitting effect’ on the antigen, thereby disrupting any unsplit virions and/or virion aggregates that might be present.

Claims

1. An immunogenic composition comprising a split influenza virus antigen and an oil-in-water emulsion, wherein the emulsion includes free surfactant in its aqueous phase.

2. The method of claim 1, wherein the influenza virus antigen is from a H1, H2, H3, H5, H7 or H9 influenza A virus subtype.

3. The composition of claim 1 or claim 2, wherein the composition is grown in cell culture and is free from ovalbumin, ovomucoid and chicken DNA.

4. The composition claim 3, wherein the virus is grown on a cell culture of a cell line selected from the group consisting of: MDCK; Vero; and PER.C6.

5. The composition of claim 3 or claim 4, wherein the composition contains less than 10 ng of cellular DNA from the cell culture host.

6. The composition of any one of claims 3 to 5, wherein the composition contains less than 10 ng of DNA that is 100 nucleotides or longer.

7. The composition of any preceding claim, wherein the composition contains between 0.1 and 20 μg of haemagglutinin per viral strain.

8. The composition of any preceding claim, wherein the emulsion includes a squalene.

9. The composition of any preceding claim, wherein the emulsion includes a tocopherol.

10. The composition of claim 9, wherein the tocopherol is DL-α-tocopherol.

11. The composition of any preceding claim, wherein the emulsion has droplets with a sub-micron diameter.

12. The composition of any preceding claim, wherein the influenza virus antigen is prepared from an influenza virus having one or more RNA segments from an A/PR/8/34 influenza virus.

13. The composition of any preceding claim, wherein the influenza virus antigen is prepared from an influenza virus obtained by reverse genetics techniques.

14. The composition of any one of claims 3 to 13, wherein the cell culture is a microcarrier culture, an adherent culture, or a suspension culture.

15. The composition of any one of claims 3 to 14, wherein the cell culture is serum-free.

16. The composition of any preceding claim, wherein the emulsion comprises a 3-O-deacylated monophosphoryl lipid A (3dMPL).

17. The composition of claim 16, wherein at least 10% by weight of the 3dMPL is the hexaacyl chain form.

18. The composition of claim 16 or claim 17, wherein the 3dMPL is in the form of particles with a diameter <150 nm.

19. The composition of any preceding claim, being substantially free from mercurial material.

20. The composition of any preceding claim, including between 1 and 20 mg/ml sodium chloride.

21. The composition of any preceding claim, having an osmolality between 200 and 400 mOsm/kg.

22. The composition of any preceding claim, including one or more buffer(s).

23. The composition of claim 22, wherein the buffer(s) include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer.

24. The composition of any preceding claim, having a pH between 5.0 and 8.1.

25. The composition of any preceding claim, containing <1 endotoxin unit per dose.

26. The composition of any preceding claim, being gluten free.

27. The composition of any preceding claim, wherein the composition includes two influenza A strains and one influenza B strain.

28. The composition of any one of claims 1 to 26, wherein the composition is a monovalent vaccine against a pandemic influenza virus strain.

29. A kit comprising: (i) a first kit component comprising a split influenza virus antigen; and (ii) a second kit component comprising an oil-in-water emulsion adjuvant that includes free surfactant in its aqueous phase.

30. The kit of claim 29, wherein the first component and the second component are in separate containers.

31. The kit of claim 30, wherein the first and second components are in vials.

32. The kit of claim 30, wherein one of the first and second components is in a syringe, and wherein the other component is in a vial.

33. The kit of claim 31 or claim 32, wherein the vial is made of a glass or plastic material.

34. The kit of claim 31, claim 32 or claim 33, wherein the vial is sealed with a latex-free stopper.

Description

MODES FOR CARRYING OUT THE INVENTION

[0221] Analysis of Free Surfactant in a Squalene-In-Water Emulsion

[0222] A microfluidised squalene-in-water emulsion adjuvant comprising a Tween 80 surfactant was prepared as disclosed in chapter 10 of ref. 67. The emulsion was analysed to determine the level of Tween 80 in its aqueous phase. The oil phase of the adjuvant was removed, and the esters in the aqueous phase were saponified and fluorescently derivatised. After chromatographic separation, fluorescence detection was used to quantify the total amount of Tween 80 in the aqueous phase.

[0223] A RP-HPLC method was also used to quantify Tween 80 in the separated aqueous phase.

[0224] Both methods gave similar results, with 12±1% of the total Tween 80 in the emulsion being found in the aqueous phase.

[0225] Adjuvanting of Split Vaccines with MF59

[0226] Two commercially available unadjuvanted split virion trivalent influenza vaccines (“SPLIT (A)” and “SPLIT (B)”) were obtained and used to immunize mice. The vaccines were diluted to give a dose of 0.2 μg each HA. Dilution used either buffer alone, or buffer and the squalene-in-water emulsion. Groups of 8 female Balb/C mice, 8 weeks old, were immunized intramuscularly with the unadjuvanted and adjuvanted vaccines, with 50 μl doses on days 0 and 28. Sera were obtained on days 14 and 42, and were analysed for anti-HA titer (IgG), HI titer and T cells.

[0227] Serum IgG antibody titers (ELISA) were as follows, looking at each virus separately:

TABLE-US-00001 Day 14 Day 42 Plain O/W emulsion Plain O/W emulsion Anti-H1N1 SPLIT (A) 152 450 749 7690 SPLIT (B) 85 629 1175 7738 Anti-H3N2 SPLIT (A) 123 318 412 4583 SPLIT (B) 95 552 1111 6005 Anti-B SPLIT (A) 238 710 707 8716 SPLIT (B) 200 1063 1585 13682

[0228] HI serum antibody titers at day 42 were as follows:

TABLE-US-00002 Plain O/W emulsion Anti-H1N1 SPLIT (A) 140 800 SPLIT (B) 285 1300 Anti-H3N2 SPLIT (A) 290 510 SPLIT (B) 380 460 Anti-B SPLIT (A) 280 1560 SPLIT (B) 550 2280

[0229] Thus oil-in-water emulsions can enhance the immune responses achieved by split influenza vaccines. By including free surfactant in the aqueous phase, the emulsion can also continue to exert a ‘splitting effect’ on the virus, thereby disrupting any unsplit virions and/or virion aggregates that might otherwise be present.

[0230] 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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