Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture

Abstract

An immunogenic composition comprising: (i) a non-virion influenza virus antigen, prepared from a virus grown in cell culture; and (ii) an adjuvant. Preferred adjuvants comprise oil-in-water emulsions.

Claims

1. A method of raising an immune response in a human comprising administering an effective amount of a composition to the human, wherein the composition comprises: (i) at least one non-virion influenza virus antigen, prepared from a cell culture-grown human influenza virus, and (ii) an adjuvant comprising an oil-in-water emulsion.

2. The method of claim 1, wherein the at least one non-virion influenza virus antigen comprises one or more purified surface antigens.

3. The method of claim 1, wherein the at least one non-virion influenza virus antigen comprises one or more split virions.

4. The method of claim 1, wherein the composition further comprises non-virion influenza virus antigens from more than one human influenza virus strain.

5. The method of claim 4, wherein the non-virion antigens are prepared from human influenza A virus and human influenza B virus.

6. The method of claim 1, wherein the at least one non-virion influenza virus antigen is from an H1, H2, H3, H5, H7, or H9 influenza A virus subtype.

7. The method of claim 1, wherein the composition contains from 0.1 to 20 μg of haemagglutinin per viral strain.

8. The method of claim 1, wherein the composition is free of chicken DNA, ovalbumin, and ovomucoid.

9. The method of claim 1, wherein the composition contains less than 10 ng of cellular DNA from cell culture host.

10. The method of claim 1, wherein the oil-in-water emulsion has sub-micron droplets.

11. The method of claim 1, wherein the oil-in-water emulsion comprises a terpenoid.

12. The method of claim 1, wherein the oil-in-water emulsion comprises a tocopherol.

13. The method of claim 1, wherein the oil-in-water emulsion comprises a polyoxyethylene sorbitan esters surfactant, an octoxynol surfactant, and/or a sorbitan ester.

14. The method of claim 1, wherein the oil-in-water emulsion comprises polysorbate 80.

15. The method of claim 1, wherein the oil-in-water emulsion comprises sorbitan trioleate.

16. The method of claim 1, wherein the oil-in-water emulsion comprises squalene.

17. The method of claim 1, wherein the oil-in-water emulsion comprises about 4.3% squalene by weight, about 0.5% polysorbate 80 by weight, and about 0.48% sorbitan trioleate by weight.

18. The method of claim 1, wherein the composition comprises 3-O-deacylated monophosphoryl lipid A.

19. The method of claim 1, wherein the composition is substantially free of thiomersal.

20. The method of claim 1, wherein the composition is substantially free of mercurial material.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the percentage of CD4.sup.+ T cells that gave an antigen-specific cytokine response when stimulated by HA.

(2) FIGS. 2 to 4 show the Log 10 serum antibody titers (ELISA) for mice immunized with different compositions. Arrows show compositions adjuvanted by the MF59 emulsion. FIG. 2 shows the H1N1 results; FIG. 3 shows H3N2; FIG. 4 shows influenza B.

(3) FIG. 5 shows serum HI titers with different adjuvants.

(4) FIG. 6 is similar to FIG. 1, and shows the effect of adding CpG to various adjuvants. The left bar of each pair shows the % of cells with an antigen-specific cytokine response; the right bar shows the % of cells which show an antigen-specific interferon-γ response.

(5) FIG. 7 is similar to FIG. 5, and shows HI titers for adjuvants (1) to (4), with (plain, foreground) and without (hatched, background) CpG when using 0.1 μg antigen.

(6) FIG. 8 shows GMTs (AU/ml) for IgG against the H3N2 strain with different adjuvants and combinations. The left bar in each pair shows IgG1; the right shows IgG2a. The scale is logarithmic.

Modes for Carrying Out the Invention

(7) Influenza virus strains Wyoming H3N2 (A), New-Caledonia H1N1 (A) and Jiangsu (B) were separately grown on MDCK cells, thereby avoiding the presence of any egg-derived proteins (specifically ovalbumin) in the final vaccines. A trivalent surface glycoprotein vaccine was prepared and was used to immunize immune-nave Balb/C mice at two doses (0.1 and 1 μg HA per strain) at days 0 and 28. Animals were bled at day 42 and various assays were performed with the blood: HI titers; anti-HA responses, measured by ELISA; and the level of CD4.sup.+ T cells that release cytokines in an antigen-specific manner, including a separate measurement of those that release γ-interferon. IgG responses were measured specifically in respect of IgG1 and IgG2a.

(8) In contrast to the reports in reference 1 of enhanced T cell responses when using antigens purified from influenza grown in mammalian cell culture, only a modest number of CD4.sup.+ T cells released cytokines in an antigen-specific manner. To improve these results, vaccines were adjuvanted with one of the following: (1) an aluminum hydroxide, used at 1 mg/ml and including a 5 mM histidine buffer; (2) MF59 oil-in-water emulsion with citrate buffer mixed at a 1:1 volume ratio with the antigen solution; (3) calcium phosphate, used at 1 mg/ml and including a 5 mM histidine buffer; (4) microparticles formed from poly(lactide co-glycolide) 50:50 co-polymer composition, intrinsic viscosity 0.4 (‘PLG’), with adsorbed antigen; (5) a CpG immunostimulatory oligonucleotide with a phosphorothioate backbone; (6) resiquimod; or (7) a negative control with no adjuvant.

(9) FIG. 1 shows the number of T cells that release cytokine(s) in an antigen-specific manner after immunization with one of the seven compositions. Each of the six adjuvants increased the T cell responses, but the emulsion-based composition (arrow) gave by far the best enhancement.

(10) FIGS. 2 to 4 show anti-HA ELISA responses. From these Figures, and from similar data in FIG. 5, it is again apparent that the emulsion-based composition gave the best responses. The data in FIG. 5 also show a good anti-HA response when using an immunostimulatory oligonucleotide.

(11) FIG. 6 shows the effect on T cells of adding the CpG oligonucleotide (5) to adjuvants (1) to (4). With the emulsion (2), there is little effect on the overall T cell response, but the proportion of interferon-γ secreting cells is much greater, indicating a more TH1-like response. A similar effect is seen when CpG is added to calcium phosphate (3), but with an increased overall T cell response. Adding CpG to the aluminum hydroxide adjuvant had no beneficial effect on T cell responses.

(12) The same shift towards a TH1-like response is seen in FIG. 8. Adjuvants (1) to (4) all showed a dominant IgG1 response (TH2) on their own, as did CpG (5) alone. Adding CpG to adjuvants (1) to (4) increased the levels of IgG2a (TH1) in all cases, including the production of an IgG2a response for the aluminum hydroxide and PLG adjuvants which was not seen in the absence of CpG. Moreover, the addition of CpG to the oil-in-water emulsion (2) and to calcium phosphate (3) shifted the IgG response such that IgG2a was dominant. Adding CpG to the adjuvants generally enhanced HI titers (FIG. 7). Thus addition of CpG enhances both T cell responses and B cell responses for all adjuvants, except for the aluminum salt.

(13) Thus, in contrast to the findings in reference 1 using whole virion antigens derived from viruses grown in mammalian cell culture, antigen-specific T cell responses against purified influenza antigens were found to be weak in the absence of adjuvant. By adding adjuvants, however, T cell responses could be enhanced. In particular, oil-in-water emulsions are excellent adjuvants, both in terms of T cell responses and anti-HA antibodies. By both of these criteria the MF59 emulsion is superior to an aluminum salt adjuvant.

(14) 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.

REFERENCES (THE CONTENTS OF WHICH ARE HEREBY INCORPORATED BY REFERENCE)

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