SOLUBLE NEEDLE ARRAYS FOR DELIVERY OF INFLUENZA VACCINES

Abstract

Influenza vaccines are administered using solid biodegradable microneedles. The microneedles are lubricated from the influenza vaccine in combination with solid excipient(s) and, after penetrating the skin, they dissolve in situ and release the vaccine to the immune system. The influenza vaccine is (i) a purified influenza virus surface antigen vaccine, rather than a live vaccine or a whole-virus or split inactivated vaccine (ii) an influenza vaccine prepared from viruses grown in cell culture, not eggs, (iii) a monovalent influenza vaccine e.g. for immunising against a pandemic strain, (iv) a bivalent vaccine, (v) a tetravalent or >4 -valent vaccine, (vi) mercury-free vaccine, or (vii) a gelatin-free vaccine.

Claims

1. A skin patch comprising a plurality of solid biodegradable microneedles, wherein the microneedles comprise a mixture of (i) a biosoluble and biodegradable matrix material and (ii) influenza vaccine selected from the group consisting of a purified influenza virus surface antigen vaccine, art influenza vaccine prepared from viruses grown in cell culture, a monovalent influenza vaccine, a bivalent vaccine, a tetravalent or >4-valent vaccine, a mercury-free vaccine, and a gelatin-free vaccine.

2. A process for preparing a skin patch comprising a plurality of solid biodegradable microneedles, comprising steps of: (i) mixing a biosoluble and biodegradable matrix material with an influenza vaccine selected from the group consisting of a purified influenza virus surface antigen vaccine, art influenza vaccine prepared from viruses grown in cell culture, a monovalent influenza vaccine, a bivalent vaccine, a tetravalent or >-4-valent vaccine, a mercury-free vaccine, and a gelatin-free vaccine; and (ii) adding the mixture from step (i) to a mold containing cavities for forming microneedles.

3. An aqueous liquid or solid material comprising (i) a biosoluble and biodegradable matrix material and (ii) an influenza vaccine selected from the group consisting of a purified influenza. virus surface antigen vaccine, an influenza vaccine prepared from viruses grown in cell culture, a monovalent influenza vaccine, a bivalent vaccine, a tetravalent or >4-valent vaccine, a mercury-free vaccine, and a gelatin-free vaccine.

4. The patch, process or material of any one of claims 1-3, wherein the influenza vaccine is a purified influenza virus surface antigen vaccine.

5. A skin patch comprising a plurality of solid biodegradable microneedles, wherein the microneedles comprise a mixture of (i) a biosoluble and biodegradable matrix material and (ii) an influenza virus hemagglutinin, wherein the amount of influenza virus hemagglutinin per patch is ≤16 μg per strain.

6. The patch of claim 5, wherein the patch comprises a whole virus inactivated influenza vaccine, a split virus influenza vaccine, or a purified influenza virus surface antigen vaccine.

7. The patch, process or material of any one of claims 1-6, wherein the influenza vaccine is an influenza vaccine prepared from viruses grown in cell culture.

8. The patch, process or material of any one of claims 1-6, wherein the influenza vaccine is a monovalent influenza vaccine.

9. The patch, process or material of any one of claims 1-6, wherein the influenza vaccine is a bivalent influenza vaccine.

10. The patch, process or material of any one of claims 1-6, wherein the influenza vaccine is a tetravalent influenza vaccine.

11. The patch, process or material of any one of claims 1-6, wherein the influenza vaccine is a >4-valent influenza vaccine.

12. The patch, process or material of any one of claims 1-6, wherein the influenza vaccine is a mercury-free influenza vaccine.

13. The patch, process or material of any one of claims 1-6, wherein the influenza vaccine is a gelatin-free influenza vaccine.

14. The patch, process or material of any preceding claim, wherein the matrix material comprises one or more carbohydrates.

15. The patch, process or material of claim 14, wherein the matrix material comprises a cellulose and/or a dextrin and/or a disaccharide.

16. The patch or process of any preceding claim, wherein the microneedles are 100-2500 μm long and are tapered with a skin-facing point.

17. The patch or process of any preceding claim, wherein a single patch has >20 microneedles.

18. The patch or process of any preceding claim, wherein the patch has an area of ≤2 cm.sup.2.

19. The patch or process of any preceding claim, wherein a skin-facing area of the patch includes an adhesive to facilitate adherence to a subject's skin.

20. The patch, process or material of any preceding claim, comprising between 0.5-50 μg or detergent per μg of hemagglutinin.

21. The patch, process or material of any preceding claim, containing 1-15 μg, of hemagglutinin per influenza virus strain.

22. A method of raising an immune response in a subject, comprising the step of applying a patch of any preceding claim to the subject's skin, such that the patch's microneedles penetrate the skin's dermis.

23. A process for determining the amount of influenza hemagglutinin in a skin patch, wherein (a) the patch comprises a biosoluble & biodegradable matrix material and an influenza vaccine, and (b) the process comprises steps of (i) dissolving the patch in a solvent to provide a dissolved patch solution; and (ii) assaying hemagglutinin in the dissolved patch solution by enzyme-linked immunosorbent assay (ELISA).

24. The process of claim 23, wherein the ELISA is a capture ELISA using immobilised anti-hemagglutinin antibodies.

25. The process of claim 23 or claim 24, wherein the influenza vaccine is a multivalent influenza vaccine, and wherein the ELISA separately uses strain-specific anti-hernagglutinin antibody for each strain.

Description

BRIEF DESCRIPTION THE DRAWINGS

[0104] FIG. 1 shows scanning electron micrograph images of a patch of the invention, Panels B, C & D show individual needles from the patch shown in panel A.

[0105] FIG. 2 shows SDS-PAGE analysis of antigens, either in solution or after formulation into a patch. Lanes are: (1) markers; (2) 3-valent antigen at 30 μg HA per strain; (3) 3-valent antigen at 15 μg HA/strain; (4) 3-valent antigen at 7.5 μg HA/strain; (5-7) monovalent HAs at 15 μg; (8) empty patch after TCA treatment; (9) patch after TCA treatment.

[0106] FIG. 3 shows ELISA results for antigen from two different strains. The circles show data for a trivalent vaccine. The triangles show data with a dummy patch spiked with trivalent vaccine. The squares show data for a patch with integral trivalent vaccine. The crosses show a dummy patch.

[0107] FIG. 4 shows strain-specific IgG titers after immunisations. Each of the seven triplets of bars shows titers for the three strains in the trivalent vaccine. The triplets are, from left to right: unadjuvanted injected vaccine at 0.1 μg dose; patch-administered vaccine at 0.1 μg dose; adjuvanted injected vaccine at 0.1 μg dose; unadjuvanted injected vaccine at 0.01 μg dose; patch-administered vaccine at 0.01 μg dose; adjuvanted injected vaccine at 0.01 μg dose; naïve mice.

[0108] FIG. 5 shows serum H1 titers. The bars are grouped as in FIG. 4.

[0109] FIG. 6 shows strain-specific IgG titers. The five pairs of bars show titers after 1 dose or 2 doses. The pairs are, from left to right: unadjuvanted injected vaccine at 0.1 μg dose; patch-administered vaccine at 0.1 μg dose; unadjuvanted injected vaccine at 1 μg dose; patch-administered vaccine at 1 μg dose; mice receiving PBS alone.

[0110] FIG. 7 shows serum H1 titers against one vaccine strain. The five groups are as in FIG. 6.

[0111] FIG. 8 shows % weight loss in mice after challenge. Diamonds show data for unadjuvanted injected vaccine at 0.1 μg (empty) or 1 μg (filled). Squares show data for patch-administered vaccine at 0.1 μg (empty) or 1 μg (filled). Crosses show data for mice receiving PBS alone.

[0112] FIG. 9 shows microneutralization titers (IC80). The five groups are as in FIG. 6.

MODES FOR CARRYING OUT THE INVENTION

Vaccine Patch Fabrication

[0113] An influenza virus vaccine was prepared using the MDCK cell culture and antigen purification techniques used for manufacturing the OPTAFLU™ product [69]. This provides a surface antigen inactivated vaccine free from mercury, antibiotics, formaldehyde, and egg-derived materials.

[0114] Bulk monovalent antigens from each of A/H1N1, A/H3N2 and B strains included a high HA concentration (200-600 μg/ml) with about 0.5% w/v Tween 80. These three bulks were mixed to give a trivalent bulk at high HA concentration. This hulk was mixed with trehalose and sodium carboxymethylcellulose, and a microneedle patch was prepared by filling a micromold with the mixture then centrifuging at 4000 rpm for 5 minutes. The centrifuged material was then dried to give the patch. Antigens were incorporated to give a final concentration per patch of 0.01 μg, 0.1 μg, 1 μg or 15 μg of HA per strain.

[0115] FIG. 1 shows scanning electron micrographs of a patch after sputter coating with gold palladium alloy for two minutes.

Assays for Antigen in Fabricated Patches

[0116] To confirm that vaccine antigens were properly incorporated and stable, patches were characterized qualitatively by SDS-PAGE and quantitatively by capture ELISA.

[0117] Patches containing trivalent antigen at 15 μg per strain were dissolved in 1 ml sterile water. Vials were vortexed for 10 minutes to ensure the entire patch was in solution. 100 μl of 0.5% deoxycholate was added to the samples. Samples were allowed to sit at room temperature for 10 minutes. After incubation 80 μl of 60% TCA was added to the sample. Samples were placed on microcentrifuge for minutes at room temperature at 12k RPM. The supernatant was removed and the pellet was dried. 60 μl of 4× reducing loading buffer and 20 μl of 1M Tris-HCl pH 8 was added to the pellet. The sample was vortexed and placed on a heating block set at 90° C. for 10 minutes. Samples were allowed to cool to room temperature and were 9 μl was added to each well in a 4-20% SDS-PAGE gel. Gels were stained overnight, de-stained in distilled water, and imaged. An antigen-free patch was treated in the same way for comparison.

[0118] FIG. 2 shows results. Lanes 2-4 contain non-patch trivalent antigen in lanes 2-4 at 2×, 1× and 0.5× the concentration in the patch. Lanes 5-7 show non-patch monovalent antigens. Lane 8 shows an antigen-free patch, and lane 9 shows the TCA-precipitated patch. The three individual antigens are clearly visible in the patch.

[0119] Antigen content of the patches was analyzed by capture ELISA. In this technique ELISA plates were coated to capture the antigen. The dissolved patches were added to the plates and incubated, followed by biotinylated IgG antibody for 30 min. Subsequently, unbound IgG and antigen was washed off and a streptavidin antibody conjugated to alkaline phosphatase was added. Antigen content was then determined by enzymatic reaction with a pNPP substrate. Absorbance was measured at 405 nm and antigen concentration was extrapolated from antigen-specific standard curves.

[0120] Results are shown in FIG. 3. The capture ELISA was able to recover the full antigen content from patches, confirming that the matrix excipients from the patch do not interfere with the assay.

[0121] In contrast, mass spectrometry methods were able to recover around 50% of the HA content. Recovery was calculated by comparing the area of the peak in the patch sample with the area of the peak in a standard mix sample, repeated with five different peptides for each strain. This process was performed on patches which had been treated with or without TCA to precipitate their proteins. Recovery for one strain was 17% without TCA or 43% with TCA; for another strain it was 24% without TCA or 49% with TCA. Spiking studies were also used, and recovery was again poor (ranging from 41-55% across three different strains). Thus mass spectrometry was not useful for quantifying HA in the patches, presumably due to some interference from the patch excipients.

Immunization and Challenge Studies

[0122] Patches for immunization studies had a much lower antigen content (1, 0.1 or 0.01 μg HA per strain than the patches which were used for antigen assays (15 μg per strain).

[0123] In a first series of experiments patches were loaded at 0.1 or 0.01 μg HA per strain per patch. Patches were applied to shaved mice (female Balb/C mice, 8-10 weeks old) with pressure for 3 minutes, and then removed 15 minutes later, by which time the tips of the needles were completely dissolved. Two immunizations were carried out 30 days apart and serum samples were collected before the first immunization and two weeks after each immunization. Individual serum samples were analyzed for IgG titers by ELISA (FIG. 4) and hemagglutination inhibition (HI) titers (FIG. 5). The results of the ELISA indicate comparable IgG titers upon intramuscular injection of trivalent influenza vaccine or upon patch administration at the 0.1 μg dose.

[0124] In a second series of experiments patches were loaded at 0.1 or 1 μg HA per strain per dose. Mice were immunised and assayed in the same way as before. FIG. 6 shows strain-specific IgG titers, and FIG. 7 shows H1 results. In addition to these assays, two weeks after the second immunization the animals were challenged with one of the wild-type vaccine strains at 10 MLD.sub.50 (300,000 TCID.sub.50/mice). Animals were monitored every two days for weight loss after challenge, and after 14 days neutralization titers were determined to confirm protection.

[0125] FIG. 8 shows body weight. About 10-15% weight loss was observed in the first three days after viral challenge, but mice in the treated groups recovered within a week. In contrast, untreated control group suffered a ˜20% weight loss and recovered only to 97% of original weight after two weeks.

[0126] FIG. 9 shows neutralization titers, calculated as the sera dilution at which 80% of the cells are protected against virus infection. The titer is expressed as IC80 and calculated using a 4 parameter curve fitting. Administration of the vaccine via the patch at 0.1 μg dose resulted in neutralization titers slightly lower than non-adjuvanted vaccine administered intramuscularly.

[0127] In conclusion, intradermal administration of influenza vaccine by the patch induced HI titers for all three influenza strains which were comparable to those achieved by intramuscular administration of non-adjuvanted vaccine. This effect was seen with HA doses as low as 0.1 μg/strain. Additionally, ELISA results indicated comparable IgG titers. In the challenge study, both microneedle patches and non-adjuvanted influenza antigen at 0.1 and 1 μg doses resulted in positive neutralization titers.

[0128] 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

[0129] [1] Vaccines. (eds. Plotkin & Orenstein). 4th edition. 2004, ISBN: 0-7216-9688-0.

[0130] [2] Koutsonanos et al. (2009) PLoS ONE 4(e): e4773.

[0131] [3] Quan et at. (2009) PLoS ONE 4(9):e7152.

[0132] [4] WO2007/030477.

[0133] [5] U.S. Pat. No. 6,945,952.

[0134] [6] U.S. Pat. No. 7,211,062.

[0135] [7] Sullivan et al. (2008) Adv Mater 20:933-8.

[0136] [8] U.S. Pat. No. 7,182,747.

[0137] [9] Oh et al. (2006) American Association of Pharmaceutical Scientists, 2006 Annual Meeting and Exposition. The AAPS Journal. 8(S2). (Annual Meeting).

[0138] [10] Intradermal Delivery of Vaccines: A review of the literature and the potential for development for use in low- and middle-income countries. (2009) Program for Appropriate Technology in Health.

[0139] [11] WO02/28422.

[0140] [12] WO02/067983.

[0141] [13] WO02/074336.

[0142] [14] WO01/21151.

[0143] [15] WO02/097072.

[0144] [16] WO2005/113756.

[0145] [17] Huckriede et al. (2003) Methods Enzymol 373:74-91.

[0146] [18] WO96/37624.

[0147] [19] WO98/46262.

[0148] [20] WO95/18861.

[0149] [21] Bright et at. (2008) PLoS ONE 3:e1501.

[0150] [22] Crevar & Ross (2008) Virology Journal 5:131.

[0151] [23] WO97/37000.

[0152] [24] Brands et al. (1999) Dev Biol. Stand 98:93-100.

[0153] [25] Halperin et al. (2002) Vaccine 20:1240-7.

[0154] [26] Tree et al. (2001) Vaccine 19:3444-50.

[0155] [27] Kistner et al. (1998) Vaccine 16:960-8.

[0156] [28] Kistner et al. (1999) Dev Biol Stand 98:101-110.

[0157] [29] Brutal et al. (2000) Vaccine 19:1149-58.

[0158] [30] Pau et al. (2001) Vaccine 19:2716-21.

[0159] [31] WO03/076601.

[0160] [32] WO2005/042728.

[0161] [33] WO03/043415.

[0162] [34] WO01/85938.

[0163] [35] WO2006/108846.

[0164] [36] EP-A-1260581 (WO01/64846).

[0165] [37] WO2006/071563.

[0166] [38] WO2005/113758.

[0167] [39] WO2006/027698.

[0168] [40] WO03/023021

[0169] [41] WO03/023025

[0170] [42] WO97/37001.

[0171] [43] EP-B-0870508.

[0172] [44] U.S. Pat. No. 5,948,410.

[0173] [45] WO2007/052163.

[0174] [46] WO2008/068631.

[0175] [47] Rota et al. (1992) J Gen Viral 73:2737-42.

[0176] [48] GenBank sequence G1:325176.

[0177] [49] Holmes et al. (2005) PLoS Biol. 3(9):e300.

[0178] [50] McCullers et al. (1999) J Virol 73:7343-8.

[0179] [51] GenBank sequence G1:325237.

[0180] [52] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.

[0181] [53] Le et al. (2005) Nature 437(7062):1108.

[0182] [54] WO2009/001217

[0183] [55] Banzhoff (2000) Immunology Letters 71:91-96.

[0184] [56] Lasley (2007) Pediatric Asthma Allergy & Immunology 20(3): 201-5.

[0185] [57] Coop et al. (2008) Int Arch Allergy Immunol. 146(1):85-8.

[0186] [58] Treanor et al. (1996) J Infect Dis 173:1467-70.

[0187] [59] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10.

[0188] [60] U.S. Pat. No. 4,680,338.

[0189] [61] U.S. Pat. No. 4,988,815.

[0190] [62] WO92/15582.

[0191] [63] Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-2278.

[0192] [64] Evans et al. (2003) Expert Rev Vaccines 2:219-229.

[0193] [65] WO03/011223.

[0194] [66] Wong et al. (2003) J Clin Pharmacol 43(7):735-42.

[0195] [67] US2005/0215517.

[0196] [68] Potter & Oxford (1979) Br Med Bull 35: 69-75.

[0197] [69] Doroshenko & Halperin (2009) Expert Rev Vaccines 8:679-88.