Nucleic acids delivered by HD-map

Abstract

The present invention relates to stable nucleic acid formulations for administration via a microprojection array in which the microprojections are densely packed and in which the formulations are rapidly sprayed or layered on to the microprojections in relatively small amounts such that the formulations dry rapidly. The present invention relates in particular to nucleic acid cancer vaccines delivered by high density micro-array MAPs (HD-MAP).

Description

NUCLEIC ACIDS DELIVERED BY HD-MAPCross-Reference to Related Application

[0001] This application claims priority to US Provisional Patent Application No. 63 / 674,458 filed on 23 July 2024 and US Provisional Patent Application No. 63 / 754,149 filed on 5 February 2025.Background of the Invention

[0002] The present invention relates to stable nucleic acid formulations for administration via a microprojection array in which the microprojections are densely packed and in which the formulations are rapidly deposited on to the microprojections in relatively small amounts such that the formulations dry rapidly. The present invention relates in particular to nucleic acid cancer vaccines delivered by high-density microprojection array patch (HD-MAP).Description of the Prior Art

[0003] In recent years, attempts have been made to devise new methods of delivering drugs and other bioactive materials, for vaccination and other purposes, which provide alternatives that are more convenient and / or enhanced in performance to the customary routes of administration such as intramuscular and intradermal injection. Limitations of intradermal injection include: cross-contamination through needle-stick injuries in health workers; the need for training in this precise technique; injection phobia from a needle and syringe; and most importantly, as a result of its comparatively large scale and method of administration, the needle and syringe cannot target key cells in the outer skin layers. This is a serious limitation to many existing and emerging strategies for the prevention, treatment and monitoring of a range of untreatable diseases. There is also a need to reduce the amount of material delivered due to toxicity of the material or due to the need to conserve the material because it is difficult or expensive to produce.

[0004] Cancer vaccines help the body to acquire immunity to attacks by cancer cells. These vaccines can treat existing cancer (therapeutic cancer vaccines) or prevent the development of cancer (prophylactic cancer vaccines). Cancer vaccines also often contain adjuvant substances,which are helpful in strengthening the immune response. Cancer DNA vaccines are engineered DNA molecules that encode one or several predetermined tumor antigens, with or without other immunomodulatory molecules. DNA vaccines must pass through the cell membrane of antigen-presenting cells (APCs) to the cytoplasm and migrate to the nucleus to initiate transcription. The resulting mRNAs translocate to the cytoplasm, where they are translated to tumor antigen proteins. These proteins can be degraded by proteasomes and processed through the endoplasmic reticulum as intracellular antigens, which are presented as peptides bound to MHC I. Alternatively, the proteins can be degraded in endosomes as extracellular antigens, producing peptides that are bound to MHC II. APCs can present the epitopes to CD4+(helper) or CD8+(cytotoxic) T cells and also to B cells. The final destinations for encoded antigens are lymphatic organs, such as spleen and lymph nodes, where resident B and T cells are activated by APCs. RNA vaccines also deliver genetic information encoding tumor antigens; these vaccines are produced by in vitro transcription of template DNA using RNA polymerase. mRNA vaccines may be a pre-defined sequence used for many patients, or can be patient specific; mRNA collected from tumor samples can be amplified by PCR, yielding a large amount of complementary DNA encoding patient-specific tumor antigens. Recent progress using mRNA vaccines in preclinical and clinical trials has resulted from improvements in mRNA stability, structure, transfection methods, and purification techniques, which remove impurities and double-stranded RNA (dsRNA). mRNA vaccines need only cross the cell membrane and the overall immunogenicity observed to date is slightly better than that achieved with DNA vaccines.

[0005] Despite cancer vaccines’ high degree of therapeutic efficacy, there are challenges associated with their effective delivery to the target organs and cells, which is key for inducing the immune recognition process. Poor transfection efficiency of nucleic acids is a major problem, along with challenges in vaccine formulation and storage, which can affect performance and safety. Nucleic acid vaccines have the potential to address issues of safety and effectiveness sometimes associated with vaccines based on live attenuated viruses and recombinant viral vectors. In addition, methods to manufacture nucleic acid vaccines are suitable as generic platforms and for rapid response.

[0006] There is a need for devices and methods of vaccination in which the vaccine is stable and which can be administered at a reduced dose with the same efficacy as a needle and syringe. In addition, there is a need for devices for vaccination which can be produced in large quantities under GMP conditions at a low cost which avoid reconstitution and increase the onset of immunogenicity. In addition to dose sparing and the ease of mass manufacture of MAPs at a very affordable cost, MAPs have further advantages. MAP dry-coated vaccines are generally more thermostable than liquid vaccines required for injection with the needle and syringe.

[0007] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.Summary of the Present Invention

[0008] The present invention relates to stable nucleic acid formulations for administration via a microprojection array in which the microprojections are densely packed and in which the formulations are rapidly sprayed or layered on to the microprojections in relatively small amounts such that the formulations dry rapidly. The present invention relates in particular to nucleic acid cancer vaccines delivered by high-density microprojection array patch (HD- MAP).

[0009] In one broad form an aspect of the present invention relates to methods of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition coated onto a microprojection array (HD-MAP) comprising a base and a plurality of solid microprojections by projecting the microprojection array into the human’s skin thereby penetrating the skin with the microprojections coated with the composition such that the vaccine composition is stripped from the microprojections and enters the skin.

[0010] In one embodiment the present invention relates to methods of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition where the nucleic acid vaccine composition is comprised of DNA or mRNA, and combinations thereof.

[0011] In one embodiment the present invention relates to methods of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition where the vaccine composition further comprises one or more excipients.

[0012] In one embodiment the present invention relates to methods of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition where the vaccine composition comprises mRNA.

[0013] In one embodiment the present invention relates to methods of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition where the vaccine composition further comprises lipid nanoparticles.

[0014] In one embodiment the present invention relates to methods of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition where the vaccine composition comprises naked DNA.

[0015] In one embodiment the present invention relates to methods of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition where the vaccine composition is selected from the group consisting of antigens or immunological epitopes related to human epidermal growth factor receptor 1 (HER1), human epidermal growth factor receptor 2 (HER2 / neu), human epidermal growth factor receptor 3 (HER3), human epidermal growth factor receptor 4 (HER4), prostate-specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, MART-1, MC1R, GplOO, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, Brachyury, Brachyury (TIVS7-2, polymorphism), Brachyury (IVS7 T / C polymorphism), T Brachyury, T, hTERT, hTRT, iCE, MUC1, MUC1 (VNTR polymorphism), MUClc, MUCln, MUC2, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, b-catenin / m, Caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HSP70- 2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPVmbcr-abl, ETV6 / AML, LDLR / FUT, Pml / RARa, HPV E6, HPV E7,and TEL / AML 1.

[0016] In another broad form an aspect of the present invention relates to devices for administering a nucleic acid vaccine composition into a patient comprising a microprojection array having a base and a plurality of solid microprojections wherein the microprojections are coated with the cancer vaccine and wherein the number of microprojections is from 1000 to 3000.

[0017] In one embodiment the present invention relates to devices where the cancer vaccine composition is comprised of nucleic acids.

[0018] In one embodiment the present invention relates to devices where the cancer vaccine composition further comprises one or more excipients.

[0019] In one embodiment the present invention relates to devices where the cancer vaccine composition comprises mRNA.

[0020] In one embodiment the present invention relates to devices where the cancer vaccine composition further comprises lipid nanoparticles.

[0021] In one embodiment the present invention relates to devices where the cancer vaccine composition comprises naked DNA.

[0022] In one embodiment the present invention relates to devices where the cancer vaccine composition is selected from the group consisting of antigens or immunological epitopes related to human epidermal growth factor receptor 1 (HER1), human epidermal growth factor receptor 2 (HER2 / neu), human epidermal growth factor receptor 3 (HER3), human epidermal growth factor receptor 4 (HER4), prostate-specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM- 10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, MART-1, MC1R, GplOO, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, Brachyury, Brachyury (TIVS7-2, polymorphism), Brachyury (IVS7 T / C polymorphism), T Brachyury, T, hTERT, hTRT, iCE, MUC1, MUC1 (VNTR polymorphism), MUClc, MUCln, MUC2, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, b-catenin / m, Caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HSP70- 2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE,SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPVmbcr-abl, ETV6 / AML, EDER / FUT, Pml / RARa, HPV E6, HPV E7,and TEL / AML 1.

[0023] It will be appreciated that the broad forms of the invention and their respective features can be used in conjunction, interchangeably and / or independently, and reference to separate broad forms is not intended to be limiting.Brief Description of the Drawings

[0024] Various examples and embodiments of the present invention will now be described with reference to the accompanying drawings, in which: -

[0025] Figure 1: (A). A schematic illustrating the vaccination schedule and sample collection.(B). IgG titers in vaccinated mice collected on day 42 tested against the rat and human Her-2 proteins as indicated. Data is presented as the mean of n = 8 mice with error bars representing the standard deviation. (C). Splenocytes were collected on day 42 and stimulated with either the Her-2 (negative control) or Neu peptides and analysed for interferon-y production via ELISpot. Data are presented as the mean of n = 5 mice with error bars representing the standard deviation, ns, not significant. *, p < 0.05, **, p < 0.01, as determined by one-way ANOVA with Sidak’s multiple comparisons test.

[0026] Figure 2: Splenocytes from mice vaccinated with the indicated doses of pRHuT via intramuscular (IM) or high-density microarray patch (HD-MAP) were stimulated with the Neu peptide and analyzed for production of (A), interferon-y (IFN-y), (B). interleukin-2 (IE-2) and(C). tumor necrosis factor alpha (TNFa) via intracellular cytokine staining. Data are presented as the geometric mean of n = 5 mice with error bars represented the standard deviation. *, p < 0.05 as determined by Welch’s ANOVA with Games-Howell post-hoc test (for the IFN-y data) or the Kruskal-Wallis H test for the remaining data.

[0027] Figure 3: (A). A timeline of the vaccination and tumor challenge. (B). IgG titers in serum from mice either pre — or post-immunization. Titers were measured against either the Rat or Human Her-2 proteins. Data presented as mean of n = 6 mice / group with error bars representing the standard deviation. (C). Mean tumor diameter for groups vaccinated via intramuscular (IM), HD-MAP or IM followed by electroporation as indicated. Each linerepresents a mouse from groups of n = 6 mice / group. ns, not significant, **, p < 0.01, ****, p < 0.001 as determined by one-way ANOVA with Sidak’s multiple comparisons test. IM, intramuscular. HD-MAP, high-density microarray patch. Elec., electroporation.Detailed Description of the Preferred Embodiments

[0028] The present invention relates to HD-MAPs and their use in the immunization of patients with respect to vaccines, in particular the nucleic acid based cancer vaccines. HD-MAPs are microprojection arrays having projections extending from the surface of a base. The projections and base may be formed from any suitable material, including but not limited to silicon and various polymers including liquid crystal polymers. The projections may be solid, non-porous and non-hollow.

[0029] The HD-MAP may have a square, rectangular, circular, or irregular shape depending on its use, and various arrangements of projections such as circular, staggered, or grid arrangements that are compatible with rapid coating methods may be used. In order to further improve or enhance the targeting accuracy, the substrate may be designed such that the features to be coated are located on radial lines from the center point of the rotation or located on concentric circles or on a continuous spiral. The substrate may be designed such that the feature spacing on each arc is designed to match an integer number of steps of the motor for a given radius. The substrate may be designed such that the rows facilitate an integer number of steps of the motor across rows. The substrate may be designed such that the columns facilitate an integer number of steps of the motor across columns. The substrate may be designed such that a repeating pattern is designed to match an integer number of steps of the motor in one or more directions. Each projection includes a tip for penetrating tissue of the biological subject and projections will typically have a profile which tapers from the base to the tip. The taper may be smooth (continuous) or irregular (stepped or staggered in one or more places).

[0030] The HD-MAP is applied to the biological subject by positioning the HD-MAP against a surface of a subject or by positioning the HD-MAP near the surface of the subject if an applicator that can propel the HD-MAP toward the skin is utilized. The tips of the projections penetrate the surface of the skin and may penetrate tissue beneath the surface of the skin to a given depth as the HD-MAP is applied. The HD-MAP may be used to deliver material orstimulus to internal tissues of a patient. The MAP may be delivered such that the projections pierce the Stratum Corneum (SC) and penetrate through the Viable Epidermis (VE) to penetrate the Dermis DE) by a dermal penetration depth. The HD-MAP may be used to deliver material or stimulus to any part or region in the subject. The HD-MAP can be provided in a variety of different configurations to suit different material or stimulus delivery requirements. Accordingly, the specific configuration of the HD-MAP can be selected to allow the delivery of material and stimulus to particular tissues, at a specific depth, to induce a desired response.

[0031] The microprojection arrays can be varied in size depending on its use. The area of the HD-MAP and the force applied will have an impact on the ability to penetrate the subject, but this must be balanced by the need to induce cell damage over a sufficiently large area to induce a response. Consequently the HD-MAPs typically have dimensions of between 0.5 x 0.5 mm and 20 x 20 mm, between 0.5 x 0.5 mm and 15 x 15 mm and more typically between 1 x 1 mm and 12 x 12 mm.

[0032] In one embodiment the microprojection array is 10.6x10.6mm. The microprojection arrays may have a density of projections of between from 1,000 to 5,000 per cm2, or from 2,500 to 20,000 per cm2or from 2,500 to 15,000 per cm2or from 2,500 to 10,000 per cm2or from 2,500 to 7,500 per cm2or from 2,500 to 5,000 per cm2or from 5,000 to 20,000 per cm2or from 5,000 to 15,000 per cm2or from 5,000 to 10,000 per cm2or from 5,000 to 9,000 per cm2or from 5,000 to 8,000 per cm2or from 5,000 to 7,000 per cm2or from 5,000 to 6,000 per cm2. The applicators of the present invention are often utilized to project high density microprojection arrays into the skin. Such high density arrays are microprojection arrays of sufficient size and density such that forces that can be applied manually will be insufficient to overcome the elasticity of the skin.

[0033] The microprojection array may be made of any suitable materials including but not limited to silicon, polymers, and plastic. In silicon embodiments the base thickness is about 60 pm or silicon with a thin (1mm) polymer backing. The overall mass of some embodiments of the microprojection array is about 0.8 grams. The microprojection array may have bevelled edges to reduce peak stresses on the edge of the array. The HD-MAP can be quartered or subdivided by other ratios to reduce the stress load on the HD-MAP and mitigate HD-MAP breakage. Polymer embodiments may have reduced mass. The microprojection array may alsohave an overall weakly convex shape of the HD-MAP to improve the mechanical engagement with skin and mitigate the effect of high-speed rippling application: a ‘high velocity / low mass’ system. The microprojection array may have a mass of less than 1 gram, or less than 0.9 grams or less than 0.8 grams or less than 0.7 grams, or less than 0.6 grams or less than 0.5 grams or less than 0.4 grams. The microprojection array may have a mass of from about 0.1 grams to about 1.0 grams or from about 0.1 grams to about 0.9 grams, or from about 0.1 grams to about 0.8 grams or from about 0.1 grams to about 0.7 grams, or from about 0.1 grams to about 0.6 grams or from about 0.1 grams to about 0.5 grams or from about 0.1 grams to about 0.4 grams, or from about 0.1 grams to about 0.3 grams or from about 0.1 grams to about 0.2 grams. In one embodiment of the applicator / microprojection system the mass of the array is about 0.3 grams, the array is projected at a velocity of about 20-26 m / s by the applicator.

[0034] The amount of antigen used in the devices and methods of the present invention include amounts necessary to provide an immune response. At least one dose selected from the group consisting of a Ipg dose, 2pg dose, 3pg dose, 4pg dose, 5pg dose, 6pg dose, 7pg dose, 8pg dose, 9pg dose, lOpg dose, 15pg dose, 20pg dose, 25pg dose and a 30pg dose may be sufficient to induce an immune response in humans. The dose of the antigen may be administered to the human within a range of doses including from about Ipg to about 50pg, from about Ipg to about 30pg, from about Ipg to about 25 g, from about Ipg to about 20pg, from about Ipg to about 15 pg, from about Ipg to about 10 pg, from about 2 pg to about 10 pg, from about 2 pg to about 8pg, from about 3 pg to about lOpg, from about 3 pg to about 8pg, from about 3 pg to about 5pg, from about 4pg to about lOpg, from about 4pg to about 8pg, from about 5pg to about lOpg, from about 5pg to about 9pg, and from about 5pg to about 8pg. More than one dose may be appropriate for some cancer vaccines.

[0035] Excipients may be included in the vaccine and include but are not limited to Histidine, Sodium acetate, Sodium chloride, Sodium citrate, Sodium phosphate, Sodium sulfate, Sodium succinate, Gelatin, Hydrolysed Gelatin, Protamine sulfate, Arginine, Aspartic acid, Glutamic acid, Glycine, Isoleucine, Lactic acid, Lysine, Maleic acid, Malic acid, Methionine, Urea, EDTA, Magnesium chloride, Benzalkonium chloride, Brij 35, Poloxamer 188 (Pluronic F-68), Polysorbate 20, Polysorbate 80, Sodium docusate, Triton X-100, Lactose, Sucrose, Trehalose, Glycerol, Mannitol, Sorbitol, Gamma-Cyclodextrin, 2-hydroxypropyl-beta-cyclodextran,Sulfobutyl ether beta-cyclodextrin, Carboxymethyl cellulose, Dextran sulfate, Dextran 40, PEG-3350, Sodium Hyaluronate, Sodium thioglycolate, Cysteine, and Glutathione, saponins (such as QS-21), serum albumin and combinations thereof.

[0036] Stability of vaccine compositions and components can be measured by a loss in antigen potency. This loss in potency can be determined under a variety of conditions, such as storage temperature and storage humidity at various time points. Typically, vaccines which are in solution are stored at 4°C or at room temperature (about 25°C); for nucleic acid vaccines, storage conditions are frequently at freezing temperatures, such as -80°C or -20°C. It would be preferable to be able to store vaccine at at least room temperature or higher temperatures (35 °C - 45°C) such that cold storage would be unnecessary; for nucleic acid vaccines, storage at 4°C would be preferable over frozen conditions.

[0037] The methods and compositions of the present invention provide microprojection arrays that can be coated with multiple incompatible vaccine antigens that are stable over time. The vaccine compositions of the present invention are stable at at least 2-8 °C for at least 1 or at least 2 or at least 3 or at least 4 or at least 5 or at least 6 or at least 7 or at least 8 or at least 9 or at least 10 or at least 12 or at least 13 or at least 14 or at least 15 or at least 16 or at least 17 or at least 18 or at least 19 or at least 20 or at least 21 or at least 22 or at least 23 or at least 24 or at least 30 or at least 36 months at various temperatures and conditions. The stability of the vaccine formulations may be measured by a variety of techniques including but not limited to ELISA and SDS-PAGE silver stain.

[0038] Plasmids used for cancer gene therapy or DNA vaccination must contain at least one expression cassette that directs the expression of a protein that will induce the therapeutic effect. After DNA uptake by the cell, it needs to reach the nucleus, where the gene will direct the therapeutic protein expression in the same way the cell produces its own proteins.

[0039] For therapy to be effective, the correct design and optimization of the plasmid are important. For example, if more than one gene of interest needs to be expressed using a single plasmid, they can be expressed independently (each gene with its own promoter), in a multicistronic system (two or more genes under the control of the same promoter), or as a fusion protein (a linker sequence between both sequences may be added). For the multicistronicsystem, an internal ribosome entry site (IRES) or a virus-derived T2A sequence must be placed between the different genes.

[0040] Codon optimization of the gene of interest may be important, since the richness of guanines and cytosines increases messenger RNA (mRNA) levels. Furthermore, the DNA molecule per se may stimulate the immune system through its unmethylated cytosine- phosphate-guanine (CpG) motifs and double- stranded structure. CpG sequences in DNA vaccines have been shown to increase immunogenicity, acting as immunostimulatory sequences (ISS) through recognition by the Toll-like receptor 9 (TLR9) present in antigen- presenting cells (APCs); however, they may decrease gene expression.

[0041] Depending on the strategy intended for the plasmid, the gene of interest or the mRNA may encode a therapeutic protein to kill cancer cells directly, for example, a proapoptotic protein, an enzyme that activates a prodrug, a cytotoxic peptide, or a bacterial toxin. Plasmids encoding specific small interfering RNA (siRNA) molecules may be used for cancer gene therapy. In this case, a tumor- specific promoter can direct the transgene expression in cancer cells.

[0042] The target antigens or immunological epitopes coated onto the HD-MAP are tumorspecific antigens, a tumor-associated antigens, bacterial antigens, viral antigens, yeast antigens, fungal antigens, protozoan antigens, parasite antigens, mitogens, or a combinations thereof. In some aspects, the target antigens or immunological epitopes thereof is human epidermal growth factor receptor 1 (HER1), human epidermal growth factor receptor 2 (HER2 / neu), human epidermal growth factor receptor 3 (HER3), human epidermal growth factor receptor 4 (HER4), prostate-specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE- A10, MAGE-A12, BAGE, DAM- 6, DAM- 10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE- 7B, NA88-A, NY-ESO-1, MART-1, MC1R, GplOO, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, Brachyury, Brachyury (TIVS7-2, polymorphism), Brachyury (IVS7 T / C polymorphism), T Brachyury, T, hTERT, hTRT, iCE, MUC1, MUC1 (VNTR polymorphism), MUClc, MUCln, MUC2, PRAME, P15, RU1, RU2, SART-1, SART- 3, AFP, b-catenin / m, Caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HSP70- 2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP,Annexin II, CDC27 / m, TPEmbcr-abl, ETV6 / AML, LDLR / FUT, Pml / RARa, HPV E6, HPV E7,and TEL / AML 1.

[0043] The nucleic acid sequence encoding the antigen has at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to the wild type antigen.

[0044] Illustrative stimuli or material that can be delivered with the device of the present invention include any or more of: small chemical or biochemical compounds including drugs, metabolites, amino acids, sugars, lipids, saponins, and hormones; macromolecules such as complex carbohydrates, phospholipids, peptides, polypeptides, peptidomimetics, and nucleic acids; or other organic (carbon containing) or inorganic molecules; and particulate matter including whole cells, bacteria, viruses, virus-like particles, cell membranes, dendrimers and liposomes.

[0045] In some embodiments, the stimulus or material is selected from nucleic acids, illustrative examples of which include DNA, RNA, sense oligonucleotides, antisense oligonucleotides, ribozymes, small interfering oligonucleotides (siRNAs), micro RNAs (miRNAs), repeat associated RNAs (rasiRNA), effector RNAs (eRNAs), and any other oligonucleotides known in the art, which inhibit transcription and / or translation of a mutated or other detrimental protein. In illustrative examples of this type, the nucleic acid is in the form of an expression vector from which a polynucleotide of interest is expressible. The polynucleotide of interest may encode a polypeptide or an effector nucleic acid molecule such as sense or antisense oligonucleotides, siRNAs, miRNAs and eRNAs.

[0046] In other embodiments, the stimulus or material is selected from peptides or polypeptides, illustrative examples of which include insulin, proinsulin, follicle stimulating hormone, insulin like growth factor- 1, insulin like growth factor-2, platelet derived growth factor, epidermal growth factor, fibroblast growth factors, nerve growth factor, colony stimulating factors, transforming growth factors, tumor necrosis factor, calcitonin, parathyroid hormone, growth hormone, bone morphogenic protein, erythropoietin, hemopoietic growth factors, luteinizing hormone, glucagon, glucagon likepeptide-1, anti-angiogenic proteins, clotting factors, anti-clotting factors, atrial natriuretic factor, plasminogen activators,bombesin, thrombin, enkephalinase, vascular endothelial growth factor, interleukins, viral antigens, non-viral antigens, transport proteins, and antibodies.

[0047] In still other embodiments, the stimulus or material is selected from receptor ligands. Illustrative examples of receptors include Fc receptor, heparin sulfate receptor, vitronectin receptor, Vcam-1 receptor, hemaglutinin receptor, Pvr receptor, Icam-1 receptor, decayaccelerating protein (CD55) receptor, Car (coxsackievirus-adenovirus) receptor, integrin receptor, sialic acid receptor, HAVCr-1 receptor, low-density lipoprotein receptor, BGP (biliary glycoprotien) receptor, aminopeptidease N receptor, MHC class- 1 receptor, laminin receptor, nicotinic acetylcholine receptor, CD56 receptor, nerve growth factor receptor, CD46 receptor, asialoglycoprotein receptor Gp-2, alpha-dystroglycan receptor, galactosylceramide receptor, Cxcr4 receptor, Glvrl receptor, Ram-1 receptor, Cat receptor, Tva receptor, BLVRcpl receptor, MHC class-2 receptor, toll-like receptors (such as TLR-1 to -6) and complement receptors.

[0048] In specific embodiments, the stimuli or material are selected from antigens including endogenous antigens produced by a host that is the subject of the stimulus or material delivery or exogenous antigens that are foreign to that host. The antigens may be in the form of soluble peptides or polypeptides or polynucleotides from which an expression product (e.g., protein or RNA) is producible. Suitable endogenous antigens include, but are not restricted to, cancer or tumor antigens. Non-limiting examples of cancer or tumor antigens include antigens from a cancer or tumor selected from ABL1 proto-oncogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, oesophageal cancer, Ewing's Sarcoma, Extra-Hepatic Bile Duct Cancer, Eye Cancer, Eye: Melanoma, Retinoblastoma,Fallopian Tube cancer, Fanconi anemia, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestational-trophoblastic-disease, glioma, gynecological cancers, haematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhan's cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid tumor of kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer (NSCLC), ocular cancers, esophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal tumours, pituitary cancer, polycythemia vera, prostate cancer, rare cancers and associated disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell- cancer-(bladder), transitional-cell-cancer-(renal-pelvis- / -ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom's macroglobulinemia, Wilms' tumor. In certain embodiments, the cancer or tumor relates to melanoma. Illustrative examples of melanoma-related antigens include melanocyte differentiation antigen (e.g., gplOO, MART, Melan-A / MART-1, TRP-1, Tyros, TRP2, MC1R, MUC1F, MUC1R or a combination thereof) and melanoma-specific antigens (e.g., BAGE, GAGE-1, gpl00In4, MAGE-1 (e.g., GenBank Accession No. X54156 and AA494311), MAGE-3, MAGE4, PRAME, TRP2IN2, NYNSOla, NYNSOlb, LAGE1, p97 melanoma antigen (e.g., GenBank Accession No. M12154) p5 protein, gp75, oncofetalantigen, GM2 and GD2 gangliosides, cdc27, p21ras, gpl00Pmel117or a combination thereof. Other tumour- specific antigens include, but are not limited to: etv6, amll, cyclophilin b (acute lymphoblastic leukemia); Ig-idiotype (B cell lymphoma); E-cadherin, a-catenin, P-catenin, y- catenin, pl20ctn (glioma); p21ras (bladder cancer); p21 ras (biliary cancer); MUC family, HER2 / neu, c-erbB-2 (breast cancer); p53, p21ras (cervical carcinoma); p21ras, HER2 / neu, c- erbB-2, MUC family, Cripto-lprotein, Pim-1 protein (colon carcinoma); Colorectal associated antigen (CRC)-CO17-1A / GA733, APC (colorectal cancer); carcinoembryonic antigen (CEA) (colorectal cancer; choriocarcinoma); cyclophilin b (epithelial cell cancer); HER2 / neu, c-erbB- 2, ga733 glycoprotein (gastric cancer); a-fetoprotein (hepatocellular cancer); Imp-1, EBNA-1 (Hodgkin's lymphoma); CEA, MAGE-3, NY-ESO-1 (lung cancer); cyclophilin b (lymphoid cell-derived leukemia); MUC family, p21ras (myeloma); HER2 / neu, c-erbB-2 (non-small cell lung carcinoma); Imp-1, EBNA-1 (nasopharyngeal cancer); MUC family, HER2 / neu, c-erbB- 2, MAGE-A4, NY-ESO-1 (ovarian cancer); Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2 / neu, c-erbB-2, ga733 glycoprotein (prostate cancer); HER2 / neu, c-erbB-2 (renal cancer); viral products such as human papillomavirus proteins (squamous cell cancers of the cervix and esophagus); NY-ESO-1 (testicular cancer); and HTLV-1 epitopes (T cell leukemia).

[0049] Foreign antigens are suitably selected from transplantation antigens, allergens as well as antigens from pathogenic organisms. Transplantation antigens can be derived from donor cells or tissues from e.g., heart, lung, liver, pancreas, kidney, neural graft components, or from the donor antigen-presenting cells bearing MHC loaded with self antigen in the absence of exogenous antigen.

[0050] Non-limiting examples of allergens include Fel d 1 (i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91 / 06571), Der p I, Der p II, Der fl or Der fTI (i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94 / 24281). Other allergens may be derived, for example from the following: grass, tree and weed (including ragweed) pollens; fungi and moulds; foods such as fish, shellfish, crab, lobster, peanuts, nuts, wheat gluten, eggs and milk; stinging insects such as bee, wasp, and hornet and the chimomidae (non-bitingmidges); other insects such as the housefly, fruitfly, sheep blow fly, screw worm fly, grain weevil, silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle; spiders and mites, including the house dust mite; allergens found in the dander, urine, saliva, blood or other bodily fluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil; airborne particulates in general; latex; and protein detergent additives.

[0051] Exemplary pathogenic organisms include, but are not limited to, viruses, bacteria, fungi parasites, algae and protozoa and amoebae. Illustrative viruses include viruses responsible for diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Epstein-Barr virus and other herpesviruses such as papillomavirus, Ebola virus, influenza virus, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No. M24444), hantavirus, Sendai virus, respiratory syncytial virus, othromyxoviruses, vesicular stomatitis virus, visna virus, cytomegalovirus and human immunodeficiency virus (HIV) (e.g., GenBank Accession No. U18552). Any suitable antigen derived from such viruses are useful in the practice of the present invention. For example, illustrative retroviral antigens derived from HIV include, but are not limited to, antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components. Illustrative examples of hepatitis viral antigens include, but are not limited to, antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA. Illustrative examples of influenza viral antigens include; but are not limited to, antigens such as hemagglutinin and neuraminidase and other influenza viral components. Illustrative examples of measles viral antigens include, but are not limited to, antigens such as the measles virus fusion protein and other measles virus components. Illustrative examples of rubella viral antigens include, but are not limited to, antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components. Illustrative examples of cytomegaloviral antigens include, but are not limited to, antigens such as envelope glycoprotein B and other cytomegaloviral antigen components. Non-limiting examples of respiratory syncytial viralantigens include antigens such as the RS V fusion protein, the M2 protein and other respiratory syncytial viral antigen components. Illustrative examples of herpes simplex viral antigens include, but are not limited to, antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components. Non-limiting examples of varicella zoster viral antigens include antigens such as 9PI, gpll, and other varicella zoster viral antigen components. Non-limiting examples of Japanese encephalitis viral antigens include antigens such as proteins E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80% E, and other Japanese encephalitis viral antigen components. Representative examples of rabies viral antigens include, but are not limited to, antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. Illustrative examples of papillomavirus antigens include, but are not limited to, the LI and L2 capsid proteins as well as the E6 / E7 antigens associated with cervical cancers, See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M., 1991, Raven Press, New York, for additional examples of viral antigens.

[0052] Illustrative examples of fungi include Acremonium spp., Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces dermatidis, Candida spp., Cladophialophora carrionii, Coccoidiodes immitis, Conidiobolus spp., Cryptococcus spp., Curvularia spp., Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp., Fonsecaea compacta, Fonsecaea pedrosoi, Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Hortaea werneckii, Lacazia loboi, Lasiodiplodia theobromae, Leptosphaeria senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia furfur, Microsporum spp., Neotestudina rosatii, Onychocola canadensis, Paracoccidioides brasiliensis, Phialophora verrucosa, Piedraia hortae, Piedra iahortae, Pityriasis versicolor, Pseudallesheria boydii, Pyrenochaeta romeroi, Rhizopus arrhizus, Scopulariopsis brevicaulis, Scytalidium dimidiatum, Sporothrix schenckii, Trichophyton spp., Trichosporon spp., Zygomcete fungi, Absidia corymbifera, Rhizomucor pusillus and Rhizopus arrhizus. Thus, representative fungal antigens that can be used in the compositions and methods of the present invention include, but are not limited to, Candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components;and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.

[0053] Illustrative examples of bacteria include bacteria that are responsible for diseases including, but not restricted to, diphtheria (e.g., Corynebacterium diphtheria), pertussis (e.g., Bordetella pertussis, GenBank Accession No. M35274), tetanus (e.g., Clostridium tetani, GenBank Accession No. M64353), tuberculosis (e.g., Mycobacterium tuberculosis), bacterial pneumonias (e.g., Haemophilus influenzae.), cholera (e.g., Vibrio cholerae), anthrax (e.g., Bacillus anthracis), typhoid, plague, shigellosis (e.g., Shigella dysenteriae), botulism (e.g., Clostridium botulinum), salmonellosis (e.g., GenBank Accession No. LO3833), peptic ulcers (e.g., Helicobacter pylori), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No. U59487), Other pathogenic bacteria include Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. Thus, bacterial antigens which can be used in the compositions and methods of the invention include, but are not limited to: pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, F M2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diphtheria bacterial antigens such as diphtheria toxin or toxoid and other diphtheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components, streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components, pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pnermiococcal bacterial antigen components; Haemophilus influenza bacterial antigens such as capsular polysaccharides and other Haemophilus influenza bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as romp A and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.

[0054] Illustrative examples of protozoa include protozoa that are responsible for diseases including, but not limited to, malaria (e.g., GenBank Accession. No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. LOS 198), toxoplasmosis, trypanosomiasis, leishmaniasis, giardiasis (GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinosis. Thus, protozoal antigens which can be used in the compositions and methods of the invention include, but are not limited to: Plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, blood-stage antigen pf 155 / RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; Leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and Trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components.

[0055] The present invention also contemplates toxin components as antigens. Illustrative examples of toxins include, but are not restricted to, staphylococcal enterotoxins, toxic shock syndrome toxin; retroviral antigens (e.g., antigens derived from HIV), streptococcal antigens, staphylococcal enterotoxin-A (SEA), staphylococcal enterotoxin-B (SEB), staphylococcal enterotoxin 1-3 (SE1-3), staphylococcal enterotoxin-D (SED), staphylococcal enterotoxin-E (SEE) as well as toxins derived from mycoplasma, mycobacterium, and herpes viruses.

[0056] In specific embodiments, the antigen is delivered to antigen-presenting cells. Such antigen-presenting cells include professional or facultative antigen-presenting cells. Professional antigen-presenting cells function physiologically to present antigen in a form that is recognised by specific T cell receptors so as to stimulate or anergise a T lymphocyte or B lymphocyte mediated immune response. Professional antigen-presenting cells not only process and present antigens in the context of the major histocompatability complex (MHC), but also possess the additional immunoregulatory molecules required to complete T cell activation or induce a tolerogenic response. Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells. Nonprofessional or facultative antigen-presenting cells typically lack one or more of the immunoregulatory molecules required to complete T lymphocyte activation or anergy. Examples of non-professional or facultative antigen-presenting cells include, but are not limited to, activated T lymphocytes, eosinophils, keratinocytes, astrocytes, follicular cells, microglial cells, thymic cortical cells, endothelial cells, Schwann cells, retinal pigment epithelial cells, myoblasts, vascular smooth muscle cells, chondrocytes, enterocytes, thymocytes, kidney tubule cells and fibroblasts. In some embodiments, the antigen-presenting cell is selected from monocytes, macrophages, B lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells. In certain advantageous embodiments, the antigen- presenting cell expresses CD 11c and includes a dendritic cell or Langerhans cell. In some embodiments the antigen-presenting cell stimulates an immune response. In other embodiments, the antigen-presenting cell induces a tolerogenic response.

[0057] The delivery of exogenous antigen to an antigen-presenting cell can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen- presenting cells, especially dendritic cells. These methods include insertion of antigen into pH- sensitive liposomes (Zhou and Huang, 1994, Immunomethods, 4:229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et al., 1988, Cell, 54:777-785), coupling of antigens to potent adjuvants (Aichele et al., 1990, J. Exp. Med., 171: 1815-1820; Gao et al., 1991, J. Immunol, 147: 3268-3273; Schulz et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 991-993; Kuzu et al., 1993, Euro. J. Immunol., 23: 1397-1400; and Jondal et al., 1996, Immunity 5: 295-302) and apoptotic cell delivery of antigen (Albert et al. 1998, Nature 392:86- 89; Albert et al. 1998, Nature Med. 4:1321-1324; and in International Publications WO 99 / 42564 and WO 01 / 85207). Recombinant bacteria (eg. E. coll) or transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery. Recombinant chimeric virus-like particles (VLPs) have also been used as vehicles for delivery of exogenous heterologous antigen to the MHC class I processing pathway of a dendritic cell line (Bachmann et al., 1996, Eur. J. Immunol., 26(11): 2595-2600).

[0058] Alternatively, or in addition, an antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the MHC class I pathway. Exemplary cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997, Vaccine 15(3): 248-256 and U.S. Pat. No. 6,352,697), phospholipases (see, e.g., Camilli et al., 1991, J. Exp. Med. 173: 751-754), pore-forming toxins (e.g., an a-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin O (LLO, e.g., Mengaud et al., 1988, Infect. Immun. 56: 766-772 and Portnoy et al., 1992, Infect. Immun. 60: 2710-2717), streptolysin O (SLO, e.g., Palmer et al., 1998, Biochemistry 37(8): 2378-2383) and perfringolysin O (PFO, e.g., Rossjohn et al., Cell 89(5): 685-692). Where the antigen- presenting cell is phagosomal, acid activated cytolysins may be advantageously used. For example, listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al., Infect. Immun. 1992, 60: 2710-2717).

[0059] The cytolysin may be provided together with a pre-selected antigen in the form of a single composition or may be provided as a separate composition, for contacting the antigen- presenting cells. In one embodiment, the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits the delivery of the antigen to the cytosol of the target cell. In another embodiment, the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast. Suitably, when the delivery vehicle is a microbial delivery vehicle, the delivery vehicle is non-virulent. In a preferred embodiment of this type, the delivery vehicle is a non-virulent bacterium, as for example described by Portnoy et al. in U.S. Pat. No. 6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding one or more pre-selected antigens. Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc. A wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterisedstrains, particularly laboratory strains of E. coli, such as MC4100, MCI 061, DH5a, etc. Other bacteria that can be engineered for the invention include well-characterised, nonvirulent, non- pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc. In a particular embodiment, the bacteria are attenuated to be non- replicative, non-integrative into the host cell genome, and / or non-motile inter- or intracellularly.

[0060] One target of interest is the ErbB2 receptor as it plays key roles in numerous physiological processes such as embryogenesis, proliferation, differentiation, adhesion and cell motility, while in adult life it is expressed at low levels and by few cells. By contrast, its overexpression and dysregulation occur in 15% of invasive breast cancers, 54-100% of colorectal cancers, 25% of ovarian cancers, 17-82% of pancreatic cancers and 34% of prostate cancers. These aberrations are associated with greater tumor aggressiveness, increased risk of recurrence, and poor prognosis. In addition, its expression on the cell surface of tumor cells makes ErbB2 a target for both antibodies and cell-mediated immunity. Two plasmids (RHuT and HuRT) code for chimeric rat / human ErbB2 proteins. RHuT is a hybrid plasmid coding for a chimeric rat / human protein in which the 410-NH2-terminal amino acids are from neu (rat Her2 ortholog) and the remaining residues from Her2.

[0061] The delivery vehicles described above can be used to deliver one or more antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells. In embodiments when the delivery vehicle is a microbe, the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen-presenting cell vacuole (including phagosomes and endosomes).

[0062] In other embodiments, the antigen is produced inside the antigen-presenting cell by introduction of a suitable expression vector as for example described above. The antigenencoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques. In one example of a variant, the codon composition of an antigen-encoding polynucleotide is modified to permit enhanced expression of the antigen in a target cell or tissue of choice using methods as set forth in detail in International Publications WO 99 / 02694 and WO 00 / 42215. Briefly, these methodsare based on the observation that translational efficiencies of different codons vary between different cells or tissues and that these differences can be exploited, together with codon composition of a gene, to regulate expression of a protein in a particular cell or tissue type. Thus, for the construction of codon-optimised polynucleotides, at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational efficiency in a target cell or tissue than the existing codon it replaces. Although it is preferable to replace all the existing codons of a parent nucleic acid molecule with synonymous codons which have that higher translational efficiency, this is not necessary because increased expression can be accomplished even with partial replacement. Suitably, the replacement step affects 5, 10, 15, 20, 25, 30%, more preferably 35, 40, 50, 60, 70% or more of the existing codons of a parent polynucleotide.

[0063] The expression vector for introduction into the antigen-presenting cell will be compatible therewith such that the antigen-encoding polynucleotide is expressible by the cell. For example, expression vectors of this type can be derived from viral DNA sequences including, but not limited to, adenovirus, adeno-associated viruses, herpes- simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses. Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000, Curr. Opin. Biotechnol. l l(2):205-208), Vigna and Naldini (2000, J. Gene Med. 2(5):308-316), Kay, et al. (2001, Nat. Med. 7(l):33-40), Athanasopoulos, et al. (2000, Int. J. Mol. Med. 6(4):363-375) and Walther and Stein (2000, Drugs 60(2):249-271).

[0064] LNPs can be used as a delivery vehicle for messenger RNA (mRNA) vaccines, which deliver mRNA encoding for antigen proteins into the cytoplasm of host cells; the mRNA is translated into the protein, which acts as an antigen and leads to development of an immune response to the pathogen. The compositions of the lipid nanoparticles may contain an ionizable lipid which is positively charged at low pH (enabling RNA complexation) and neutral at physiological pH (reducing the potential toxic effects and facilitating payload release). They also contain a PEGylated lipid to reduce antibody association (opsonization) by serum proteins and clearance by phagocytes thus conferring longer systemic circulation. The phospholipid distearoylphosphatidylcholine (DSPC) and cholesterol help to pack the cargo into the LNPs. Proprietary cationic lipids ALC-0315 (Pfizer) and SM-102 (Modema) are used in COVID-19vaccine nanoparticles; both lipids are tertiary amines which are protonated (and thus positively charged) at low pH. Their hydrocarbon chains are connected through biodegradable ester groups, enabling safe clearance after mRNA delivery. The cationic lipids used in the mRNA vaccines contain branched hydrocarbon chains, which optimize the formation of nonlamellar phases and the mRNA delivery efficiency. The PEG-lipids are both PEG-2000 conjugates. The LNPs are prepared at low pH (pH 4.0), at which the ionizable lipid is positively charged, so that it can easily form complexes with mRNA. A microfluidic device is used to mix a stream containing mRNA in water with a stream containing a lipid mixture in ethanol. When rapidly mixed, the constituents of these two streams form nanoparticles which entrap the negatively charged mRNA.

[0065] The vaccines of the present invention may be spray coated onto the HD-MAPs which minimizes the inefficiencies associated with spray coating or dip coating that may overcoat or undercoat the microprojections. Moreover, dip coating or spray coating is less accurate than ink jet coating. US 11254126 describes the process for coating microprojections of the HD- MAP. Once the vaccine is coated onto the microprojections of the HD-MAP it dries rapidly to form a stable coating on the microprojection arrays. When the HD-MAP is projected into the skin the vaccine coating is stripped from the microprojections and enters the skin. The efficiency of the stripping of the coating from the microprojections is about 40% or about 50% or about 60% or about 70% or about 80% or about 90% or about 95%. The efficiency of the stripping of the coating from the microprojections is from about 40 to 95% or from about 40 to 90% or form about 40 to 80% or from about 40 to 70% or from about 40 to 60% or from about 40 to 50% or from about 50 to 95% or from about 50 to 90% or form about 50 to 80% or from about 50 to 70% or from about 50 to 60% or from about 60 to 95% or from about 60 to 90% or form about 60 to 80% or from about 60 to 70% or from about 70% to 95% or from about 70 to 90% or form about 70 to 80%.ExamplesExample 1Coating ofpRHUT on HD-MAP

[0066] Using a formulation containing 1% methylcellulose to add viscosity and aid in coating morphology, HD-MAPs were coated with vaccine. A subset of HD-MAPs were platinum coated prior to vaccine coating to enhance visualization of vaccine removal via scanning electron microscopy (SEM). SEM analysis of these HD-MAPs clearly illustrated removal of vaccine from the tips of the microprojections. The amount of plasmid remaining on the HD- MAPs after delivery was measured and compared to undelivered HD-MAPs. Measurement of this delivery efficiency showed that coating with 25 pg delivered just under 5 pg of plasmid, and coating with 125 pg plasmid resulted in delivery of ~15 pg of plasmid.Example 2Breast Cancer Vaccine RHuT

[0067] This study was conducted to determine the immunogenicity in BALB / c mice of the hybrid plasmid RHuT, that code for chimeric rat / human extracellular and transmembrane domains of Her2. The RHuT plasmid was delivered either with Directjet-coated high density polymer micro-projection array patches (HD-MAPs) or via intramuscular (IM) injection. The formulation comprised RHuT plasmid in 1% methylcellulose in water. With respect to the HD- MAP the dose was coated onto the HD-MAP at 125 pg to deliver 15 pg of vaccine and 25 pg to deliver 5 pg. The dose was delivered as 5 pg and 30 pg across 2 patches which means that each mouse got 2x patches. The HD-MAP wear time was 2 minutes. The HD-MAP had 5000 conical projections and was delivered to the mice skin at about 20 m / s. Long term stability at room temp at 37 °C = 100% recovery measured via nanodrop and PicoGreen assay, on patches (25 pg coated). Female BALB / c mice (6-8 weeks old) were divided into nine groups of six mice each: with immunisations via HD-MAP or IM injections with 5 pg, 15 pg, 30 pg pRHuT or a vehicle only control along with a 100 pg pRHuT IM control. All mice were immunised twice at 14-day intervals. Blood was collected before each immunisation and 14 days post final vaccination via retro-orbital bleed or cardiac puncture. The serum fraction of the blood samplewas recovered by allowing the blood to clot overnight at 4 °C before centrifugation at 10,000 xg for 10 minutes at 4 °C and storing them at -20 °C until analysis. (Figure 1A). Analysis of the IgG titer in serum from day 28 against both the rat and human Erbb2 proteins showed that HD-MAP-delivered pRHuT was immunogenic (Figure IB).Table 1 - Listing of experimental groups, dosing and delivery mechanism

[0068] At all doses, the HD-MAP immunized mice tended to have higher IgG titers compared to their IM counterparts, though this only reached statistical significance (p < 0.05) in the 15 pg group for both the rat and human assays. Notably, IgG titers between mice vaccinated with 100 pg IM and 15 and 30 pg via the HD-MAP were similar. To assess cellular immunity, splenocytes collected on day 28 were stimulated with either the immunodominant neu peptide (TYVPANASL) or the Her-2 peptide (TYLPTNASL) as a negative control and analyzed via interferon-y (IFNy) ELISpot (Fig. 1C). No responses were seen when splenocytes were stimulated with the Her-2 peptide. When stimulated with the neu peptide, all HD-MAP groups showed IFNy responses that were higher than their IM counterparts. This reached statistical significance when comparing the 30 pg groups (p < 0.01). Notably the 30 pg dose, when delivered via the HD-MAP, induced more spot forming units compared to the 100 pg IM dose (p < 0.01) indicating at least 3 -fold dose sparing is achieved by using HD-MAP delivery compared to standard intramuscular injection.

[0069] Various ELISA Assays for rat and human anti Her2 antibodies pre-bleed and finalbleed for HD-MAP and IM delivery were conducted. In addition, 500k splenocytes were stimulated with 15 pg / ml peptide at 37C for 16h.

[0070] Intracellular cytokine staining was conducted on the splenocytes. Splenocytes were pulsed with either the neu or Her-2 peptides as before, and stained for expression of IFNy, interleukin (IL)-2, and tumour necrosis factor alpha (TNFa) (Figures 3A-C). The ICS analysis observed markedly higher responses of IFNy (5pg: p > 0.05; 15pg and 30pg: ns) and TNFa (5pg and 30pg: p > 0.05; 15pg: ns) from the HD-MAP group. Significant differences of IFNy and TNFa responses between the two different delivery methods are observed in the 5pg group, indicating that dose sparing is achieved again by the HD-MAP delivery. However, IL- 2 responses were minimal, with only the 30pg pRHuT groups observing a slight increase when compared to the unvaccinated group.Example 3Tumor growth assessment

[0071] The highest and the lowest doses from Example 2 (5 and 30pg) were studied to determined its protective efficacy when mice are subjected to tumour challenge post vaccination. Ten groups of mice (n=6) were vaccinated twice at 14 day intervals with 5 or 30 pg of pRHuT or 30pg of pVAXl via HD-MAP application, IM injection or IM injection followed by electroporation (IM+elec). As a positive control, one group of mice received 100 pg of pRHuT via IM injection. Sera was also collected before each vaccination and tumor challenge and analysed for neu and HER2 specific IgG responses. IM vaccinated mice did not elicit detectable anti-neu or anti-HER2 antibodies even after two immunisations for either dosage. Interestingly, there were also no detectable antibodies (both anti-neu and anti-HER2) raised against the lOOpg pRHuT IM control. Following the confirmation of antibodies elicited, 14 days post final vaccination, mice were challenged with a lethal dose (IxlO5) rat-ErbB2 positive mammary tumor cells (TUBO) subcutaneously into their mammary fat pad, (Fig. 3A). All mice were bled twice: before the immunizations and the day before the TUBO cell challenge. Mice were then monitored weekly by palpation for tumour growth for 4 weeks following challenge. Untreated control mice had onset of tumour growth following challenge and rapidly increased in tumour diameter across the 4 weeks (Fig. 3C). Similarly, the pVAXl control groups regardless of delivery methods, observed similar tumour growth trends as the untreated control group. Tumor growth was monitored twice a week. Spleens were collectedat the time of cull and stored for later analysis. Collected serum was analyzed for anti-rat and anti-human erbB2 protein (ELISA assay).Table 2 - Listing of experimental groups, dosing and delivery mechanismTable 3 - Listing of experimental groups, dosing and delivery mechanismTable 4 - Tumor size measurements per mouse at given listed day for TUBO challenge with 30 pg pVAXl delivered IMTable 5 - Tumor size measurements per mouse at given listed day for TUBO challenge with30 pg pVAXl delivered HD-MAPTable 6 - Tumor size measurements per mouse at given listed day for TUBO challenge with30 pg pVAXl delivered by electroporationTable 7 - Tumor size measurements per mouse at given listed day for TUBO challenge with 5 pg pRHuT delivered IMTable 8 - Tumor size measurements per mouse at given listed day for TUBO challenge with30 pg pRHuT delivered by HD-MAPTable 9 - Tumor size measurements per mouse at given listed day for TUBO challenge with30 pg pRHuT delivered by electroporationTable 10 - Tumor size measurements per mouse at given listed day for TUBO challenge with 30 pg pRHuT delivered IMTable 11 - Tumor size measurements per mouse at given listed day for TUBO challenge with 30 pg pRHuT delivered by HD -MAPTable 12 - Tumor size measurements per mouse at given listed day for TUBO challenge with 30 pg pRHuT delivered by electroporationTable 13 - Tumor size measurements per mouse at given listed day for TUBO challenge with 100 pg pRHuT delivered IMTable 14 - Tumor size measurements per mouse at given listed day for TUBO challenge with untreated mice

[0072] There were no detectable antibodies observed in the 100 pg IM pRHuT group, two out of six mice had delayed onset of tumour growth, with growth only observed day 18 post challenge. This was also observed in the 5 pg pRHuT IM groups, with no detectable antibodies post vaccination but observed two mice having reduced tumour growth which maintained in diameter after day 14 post challenge while three mice in the 30 pg pRHuT IM group had no onset of tumour growth. Mice vaccinated with 5 pg or 30 pg pRHUT via HD-MAP or IM+elec observed complete protection from tumour challenge, indicating that these were the most effective group in terms of preventing tumour growth.

[0073] Throughout this specification and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers. As used herein and unless otherwise stated, the term "approximately" means ±20%.

[0074] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0075] It will of course be realised that whilst the above has been given by way of an illustrative example of this invention, all such and other modifications and variations hereto, as would be apparent to persons skilled in the art, are deemed to fall within the broad scope and ambit of this invention as is herein set forth.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:1) A method of immunizing a human against cancer by administering to the human a nucleic acid vaccine composition coated onto a microprojection array (HD-MAP) comprising a base and a plurality of solid microprojections by projecting the microprojection array into the human’s skin thereby penetrating the skin with the microprojections coated with the composition such that the vaccine composition is stripped from the microprojections and enters the skin.2) The method of claim 1 wherein the nucleic acid vaccine composition is comprised of DNA or mRNA, and combinations thereof.3) The method of claim 1 wherein the vaccine composition further comprises one or more excipients.4) The method of claim 1 wherein the vaccine composition comprises mRNA.5) The method of claim 4 wherein the vaccine composition further comprises lipid nanoparticles.6) The method of claim 1 wherein the vaccine composition comprises naked DNA.7) The method of claim 1 wherein the vaccine composition is selected from the group consisting of antigens or immunological epitopes human epidermal growth factor receptor 1 (HER1), human epidermal growth factor receptor 2 (HER2 / neu), human epidermal growth factor receptor 3 (HER3), human epidermal growth factor receptor 4 (HER4), prostate-specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, MART-1, MC1R, GplOO, PSA, PSM, Tyrosinase, TRP- 1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, Brachyury, Brachyury (TIVS7-2, polymorphism), Brachyury (IVS7 T / C polymorphism), T Brachyury, T, hTERT, hTRT, iCE, MUC1, MUC1 (VNTR polymorphism), MUClc, MUCln, MUC2, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, b-catenin / m, Caspase-8 / m, CDK-4 / m, ELF2M, GnT- V, G250, HSP70- 2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPVmbcr-abl, ETV6 / AML, LDLR / FUT, Pml / RARa, HPV E6, HPV E7,and TEL / AML 1.8) A device for administering a cancer vaccine composition into a patient comprising a microprojection array having a base and a plurality of solid microprojections wherein the microprojections are coated with the cancer vaccine and wherein the number of microprojections is from 1000 to 3000.9) The device of claim 8 wherein the cancer vaccine composition is comprised of nucleic acids.10) The device of claim 8 wherein the cancer vaccine composition further comprises one or more excipients.11) The device of claim 8 wherein the cancer vaccine composition comprises mRNA.12) The device of claim 11 wherein the cancer vaccine composition further comprises lipid nanoparticles.13) The device of claim 8 wherein the cancer vaccine composition comprises naked DNA.14) The device of claim 1 wherein the cancer vaccine composition is selected from the group consisting of antigens or immunological epitopes human epidermal growth factor receptor 1 (HER1), human epidermal growth factor receptor 2 (HER2 / neu), human epidermal growth factor receptor 3 (HER3), human epidermal growth factor receptor 4 (HER4), prostate-specific antigen (PSA), PSMA, folate receptor alpha, WT1, p53, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, MART-1, MC1R, GplOO, PSA, PSM, Tyrosinase, TRP- 1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, BRCA1, Brachyury, Brachyury (TIVS7-2, polymorphism), Brachyury (IVS7 T / C polymorphism), T Brachyury, T, hTERT, hTRT, iCE, MUC1, MUC1 (VNTR polymorphism), MUClc, MUCln, MUC2, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, b-catenin / m, Caspase-8 / m, CDK-4 / m, ELF2M, GnT- V, G250, HSP70- 2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPVmbcr-abl, ETV6 / AML, LDLR / FUT, Pml / RARa, HPV E6, HPV E7, and TEL / AML 1.

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