AU2012244074B2 - Methods and compositions for intranasal delivery - Google Patents

Abstract

Provided herein are methods for generating dry vaccine powder formulations. Dry vaccine powder formulations can be used for intranasal delivery. Also provided are methods for stimulating local mucosal and systemic immunity by intranasal vaccine delivery. See also Figure 17B.

Description

AUSTRALIA Regulation 3.2 Patents Act 1990 Complete Specification Standard Patent APPLICANT: Shin Nippon Biomedical Laboratories, Ltd., and The Chemo-Sero Therapeutic Research Institute Invention Title: METHODS AND COMPOSITIONS FOR INTRANASAL DELIVERY The following statement is a full description of this invention, including the best method of performing it known to me: WO 2011/129120 PCT/JP2011/002225 Description Title of Invention: METHODS AND COMPOSITIONS FOR IN TRANASAL DELIVERY Technical Field [0001] CROSS-REFERENCE This application claims the benefit of U.S. provisional application Ser. No. 61/324,542, filed April 15, 2010, which is incorporated herein by reference in its entirety. Background Art [00021 Influenza vaccines formulated as liquids can be subject to chemical degradation, e.g., aggregation, denaturation, hydrolysis, and oxidation that can result in their inac tivation. Liquid vaccine formulations can also be sensitive to temperature: high tem peratures can increase inactivation, and freezing temperatures can result in ice that can damage antigen in the vaccine. Thus, to prevent inactivation, liquid vaccines are often stored and distributed in a temperature range between 2 and 8 degrees C. Such storage can be costly, both for long-term storage and transportation of vaccines, and from loss of vaccine due to expiration. Generation of vaccines that are stable at room tem perature would result in savings with respect to storage and would facilitate stockpiling. There is a need for means of generating vaccine formulations that are stable at room temperature, such as dry powder vaccines. [0003] Several methods of freeze-drying vaccines have been described. For example, lyophilization (freeze-drying) of influenza vaccine solution can be used to generate a vaccine powder. However, the influenza vaccine powder produced by this method can be a hard cake, which does not facilitate consistent and reliable administration. Spray freeze-drying (SFD) of an influenza vaccine solution can provide fine particles of influenza vaccine powder; however, SFD is a high-cost method. Thus, there is a need for low-cost methods of generating fine powder vaccines with relatively high flowability and relatively low hygroscopicity. [0004] The mode of administration of a vaccine can play a role in its efficacy. One mode of administration, nonparental administration (e.g., nasal), can induce and promote mucosal and systemic humoral and cell mediated immune responses. Mucosal vac cination can result in induction of secretory IgA (sIgA) responses in the respiratory tract and oropharyngeal region. One feature of mucosal sIgA antibodies is that they can provide cross-protection against antigenically distinct viruses; thus, mucosal sIgA responses have the potential to provide protection against a viral strain that has drifted from the strain used to generate the vaccine (for example, influenza virus HINI can 2 WO 2011/129120 PCT/JP2011/002225 drift to H2N1 or H1N2). Furthermore, sIgA can help bind a virus or other pathogen at the mucosal surface, preventing access of the pathogen to deeper tissues and/or de creasing the likelihood of full-blown infection. Described herein are novel methods for generating an sIgA inducing vaccine, for example, a powder vaccine formulation for nonparental administration. Summary of Invention [0005] Disclosed herein is a dry vaccine powder formulation comprising: one or more antigens, one or more saccharides, one or more buffers; and microcrystalline cellulose. An antigen in a vaccine powder formulation described herein can be a viral antigen. A viral antigen can be live attenuated virus, whole inactivated virus, split-inactivated virus, subunit antigens, virosome, or cold-adapted live influenza virus. A viral antigen can be influenza virus; for example, an antigen could HiN1; or H5N1; or a mixture of H INI, H3N2 and Influenza type B. An antigen in a vaccine powder formulation described herein can be a bacterial antigen. A bacterial antigen can be killed whole bacteria, attenuated bacteria, toxoids, purified surface protein, or purified recombinant surface protein. A bacterial antigen can be tetanus toxoid or diphtheria toxoid. An antigen in the dry vaccine powder formulation can also be a protist. An antigen could also be protein. The saccbaride used can be trehalose, mannitol, or lactose. The saccharide used can be trehalose. The buffer used can be a phosphate buffer. A vaccine powder formulation described herein can be stable at room temperature and 60% relative humidity for at least 12 months. [00061 Also provided herein is a method for generating a dry vaccine powder formulation comprising: preparing a liquid formulation comprising an antigen; quick freezing said liquid formulation, wherein the quick freezing does not comprise spray freezing; blending the freeze-dried sample with one or more excipients to generate the dry vaccine powder formulation. A viral antigen can be live attenuated virus, whole in activated virus, split-inactivated virus, subunit antigens, virosome, or cold-adapted live influenza virus. A viral antigen can be influenza virus; for example, an antigen could HIN1; or H5Nl; or a mixture of HIN1, H3N2 and Influenza type B. An antigen in a vaccine powder formulation described herein can be a bacterial antigen. A bacterial antigen can be killed whole bacteria, attenuated bacteria, toxoids, purified surface protein, or purified recombinant surface protein. A bacterial antigen can be tetanus toxoid or diphtheria toxoid. An antigen in the dry vaccine powder formulation can also be a protist. An antigen could also be protein. The preparation of a liquid formulation can comprise addition of a saccharide, for example trehalose, mannitol, or lactose. Preparation of a liquid formulation can also comprise addition of a buffer, such as a phosphate buffer. The powder can comprise fine particles. The powder can be stable at 3 WO 2011/129120 PCT/JP2011/002225 room temperature and 60% relative humidity for at least 12 months. Excipients useful in methods described herein can comprise one or more nasal carriers, such as micro crystalline cellulose and tribasic calcium phosphate. An excipient can improve flowability of the powder and/or reduce hygroscopicity of the powder. Some vaccine powders produced by a method herein do not comprise an adjuvant. Quick freezing can comprise the use of liquid nitrogen. [0007] Another method provided herein is a method of stimulating an sIgA response in a subject to an antigen comprising administering a dry vaccine powder formulation to a subject, wherein the dry powder formulation comprises the antigen and wherein the dry powder formulation is generated by quick freezing a liquid vaccine formulation, wherein the quick freezing does not comprise spray-freezing. In some instances, an IgG response is also stimulated. sIgA production can be stimulated at the site of ad ministration and/or at a mucosal site other than the site of administration. Admin istration can be intranasal. An antigen in a vaccine powder formulation described herein can be a viral antigen. A viral antigen can be live attenuated virus, whole in activated virus, split-inactivated virus, subunit antigens, virosome, or cold-adapted live influenza virus. A viral antigen can be influenza virus; for example, an antigen could HINI; or H5NI: or a mixture of HINI, H3N2 and Influenza type B. An antigen in a vaccine powder formulation described herein can be a bacterial antigen. A bacterial antigen can be killed whole bacteria, attenuated bacteria, toxoids, purified surface protein, or purified recombinant surface protein. A bacterial antigen can be tetanus toxoid or diphtheria toxoid. An antigen in the dry vaccine powder formulation can also be a protist. An antigen could also be protein. The preparation of a liquid formulation can comprise addition of a saccharide, for example trehalose, mannitol or lactose. Preparation of a liquid formulation can also comprise addition of a buffer, such as a phosphate buffer. The powder can comprise fine particles. The powder can be stable at room temperature and 60% relative humidity for at least 12 months. Excipients useful in methods described herein can comprise one or more nasal carriers, such as micro crystalline cellulose and tribasic calcium phosphate. An excipient can improve flowability of the powder and/or reduce hygroscopicity of the powder. [0008] Also provided herein is a device for administration of a vaccine powder formulation disclosed herein. Such a device can be configured for a single use. [00091 INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be in corporated by reference.

4 WO 2011/129120 PCT/JP2011/002225 Brief Description of Drawings [0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth il lustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [fig. I ]Figure 1 illustrates properties of influenza vaccine powders generated using con ventional slow freezing and freeze-drying processing with trehalose, mannitol and lactose. [fig.2]Figure 2 illustrates a process for preparing a dry nasal vaccine powder for mulation by quick freezing with liquid nitrogen. Exemplary properties of powders before and after addition of nasal carriers are also described. [fig.3]Figure 3 illustrates an embodiment of a manufacturing process of the provided invention. [fig.4]Figure 4 illustrates a study design for testing a HINI nasal influenza vaccine powder formulation. [fig.5A]Figure 5A tabulates HI titers measured in serum samples collected during a test of a HINI nasal influenza vaccine powder formulation. [fig.5B]Figure 5B tabulates HI titers measured in nasal wash samples collected during a test of a H1NI nasal influenza vaccine powder formulation. [fig.6A]Figure 6A tabulates serum IgG antibody titers measured in samples collected during a test of a Hi NI nasal influenza vaccine powder formulation. [fig.6B]Figure 6B tabulates nasal wash sIGA antibody titers measured in samples collected during a test of a H IN 1 nasal influenza vaccine powder formulation. [fig.7]Figure 7 graphically illustrates IgG and sIgA antibody titers measured during a test of a HIN1 nasal influenza vaccine powder formulation. [fig.8]Figure 8 tabulates HI titers measured in serum and nasal wash samples collected during a test of a H IN I nasal influenza vaccine powder formulation. (fig.9]Figure 9 tabulates serum IgG and nasal wash sIgA antibody titers measured in samples collected during a test of a HIN1 nasal influenza vaccine powder formulation. [fig. 10]Figure 10 illustrates a study design for testing a H5N1 nasal influenza vaccine powder formulation. [fig. 1 lAlFigure 11 A tabulates serum IgG antibody titers measured in samples collected during a test of a H5N1 nasal influenza vaccine powder formulation. [fig. 11B]Figure 11 B tabulates nasal wash sIgA antibody titers measured in samples collected during a test of a H5N 1 nasal influenza vaccine powder formulation. [fig. 12]Figure 12 graphically illustrates IgG and sIgA antibody titers measured during 5 WO 2011/129120 PCT/JP2011/002225 a test of a H5N 1 nasal influenza vaccine powder formulation. [fig. 13]Figure 13 illustrates a study design for testing a Tetanus toxoid nasal vaccine powder formulation. [fig. 14A]Figure 14A tabulates the absorbance ratio of serum IgG measured in samples collected during a test of a Tetanus toxoid nasal vaccine powder formulation [fig. 14B]Figure 14B graphically illustrates the absorbance ratio of serum IgG measured in samples collected during a test of a Tetanus toxoid nasal vaccine powder formulation [fig. 15]Figure 15 tabulates IFN gamma levels measured in samples collected during a test of a Tetanus toxoid nasal vaccine powder formulation. [fig. 16]Figure 16 illustrates a study design for testing a Diphtheria toxoid nasal vaccine powder formulation. [fig. 17A]Figure 17A tabulates serum IgG antibody titers measured in samples collected during a test of a Diphtheria toxoid nasal vaccine powder formulation. [fig. 17B]Figure 17B graphically illustrates serum IgG antibody titers measured in samples collected during a test of a Diphtheria toxoid nasal vaccine powder for mulation. [fig. 18]Figure 18 illustrates a study design for testing a homogenized ovalbumin nasal vaccine powder formulation. [fig. 19A]Figure 19A tabulates serum IgG antibody titers measured in samples collected during a test of a homogenized ovalbumin nasal vaccine powder formulation. [fig. 19B]Figure 19B graphically illustrates serum IgG antibody titers measured in samples collected during a test of a homogenized ovalbumin nasal vaccine powder for mulation. [fig.20A]Figure 20A tabulates nasal wash sIgA antibody titers measured in samples collected during a test of a homogenized ovalbumin nasal vaccine powder formulation. [fig.20B]Figure 20B graphically illustrates nasal wash sIgA antibody titers measured in samples collected during a test of a homogenized ovalbumin nasal vaccine powder formulation. Description of Embodiments [0011] DETAILED DESCRIPTION OF THE INVENTION I. OVERVIEW Conventional freeze-drying processes for liquid influenza vaccine formulations, such as cooling from room temperature to -40 degrees C over 24 hr, can lead to suboptimal particle properties or loss of antigenic (e.g. influenza hemagglutinin (HA)) potency (Figure 1). For example, liquid influenza vaccine formulations with trehalose that are subjected to a conventional freeze-drying process can form a partially caked powder 6 WO 2011/129120 PCT/JP2011/002225 (Figure 1). Liquid influenza vaccine formulations with mannitol that are subjected to a conventional freeze-drying process can have reduced HA potency (Figure 1). Liquid influenza vaccine formulations with lactose that are subjected to a conventional freeze drying process can form a partially caked powder and can have reduced HA potency (Figure 1). [0012] The present disclosure provides methods comprising a quick freezing step for generating a dry vaccine powder formulation (see e.g., Figures 2 and 3) which overcomes the limitations of previous freeze drying methods, resulting in high potency powdered vaccines with high flowability. The methods can comprise a step of generating a liquid formulation containing one or more antigens, such as a pathogen or a component thereof (e.g., a whole inactivated influenza virus) with one or more agents (e.g., a saccharide and/or buffer, e.g., phosphate buffer). A liquid vaccine formulation can be freeze-dried (e.g., comprising quick freezing in liquid nitrogen) to generate a powder (e.g., a vaccine powder). The powder can comprise fine particles and can be stable at room temperature. If the antigen is an influenza virus, the powder can have high HA potency (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). After freeze drying, the powder can be blended (e.g., by vortexing) with one or more excipients (e.g., nasal carriers and/or flowability agents) to form a dry vaccine powder formulation. [0013] A dry vaccine powder formulation described herein can be stable at room tem perature. This is an advance over liquid influenza vaccines, which are unstable at room temperature and can require expensive storage and distribution under refrigerated conditions (e.g., cold-chain distribution). In some vaccine preparations, a liquid for mulation containing disaccharides, for example, trehalose or lactose, is prepared. Such additives generally allow for maintenance of HA potency of a dry influenza vaccine powder formulation. Although the use of such component saccharides is known, the methods described herein can provide a dry vaccine form which does not form hard cakes using these saccharide components. Hard caking can be avoided using the buffers and quick freezing techniques described herein. The powders produced from quick frozen and dried antigen preparations can then be combined with one or more excipients, such as a nasal carrier (e.g., microcrystalline cellulose) and/or a flowability agent (e.g., tribasic calcium phosphate). The present formulations can result in dry powder vaccines suitable for intranasal delivery which can be stable at room tem perature and under accelerated conditions. A dry vaccine powder formulation provided herein can afford complete and consistent delivery from a nasal delivery device and result in stimulation of the recipient's immune response to the antigen/pathogen to which the vaccine is directed [0014] The methods provided herein can allow for reducing hygroscopicity and improving 7 WO 2011/129120 PCT/JP2011/002225 the flowability of a dry vaccine powder formulation provided herein. The methods can include addition of a physiologically acceptable agent (e.g., microcrystalline cellulose) to a powder formulation to reduce hygroscopicity and improve flowability of a dry vaccine powder formulation. [0015] Methods provided herein can allow for improving the efficacy of a vaccine. The methods can comprise steps for generating a dry vaccine powder compositions that can stimulate a local immune response, for example, a mucosal immune response (e.g., involving mucosal sIgA). sIgA can provide cross-protection against mutated influenza viruses (e.g., a dry vaccine powder formulation can be used as a pandemic influenza vaccine) and/or viruses which have undergone genetic drift. A dry vaccine powder for mulation, e.g., a dry nasal influenza powder formulation, can induce protection in distal mucosal sites. For example, introduction of a vaccine of the present disclosure at the nasal mucosa can lead to protection (e.g., sIgA production in the upper respiratory tract, the lower respiratory tract, the gastrointestinal tract, and vagina). A dry vaccine powder formulation can stimulate a systemic immune response (e.g., producing serum IgG). Dry vaccine powder compositions can comprise microcrystalline cellulose. In some embodiments, a dry vaccine powder formulation does not comprise adjuvant. [0016] II, LIQUID FORMULATIONS FOR USE IN GENERATING A POWDER FOR MULATION To generate a dry vaccine powder formulation, a liquid formulation can be first generated. The liquid formulation can comprise one or more antigens (e.g., one or more pathogens or components of pathogens), one or more saccharides, one or more buffers, and one or more other components. Typically, the liquid formulation is subjected to quick freezing (e.g., by immersion in liquid nitrogen) and freeze-drying prior to producing the dry vaccine powder formulation. [0017] The volume of the liquid formulation can be about 0.1 mL, 1.0 mL, 10 mL, 25 mL, 50 mL, 100 mL, 250 mL, 500 mL, 1 L, 10 L, 50 L, 100 L, 250 L, 500 L, or 1000 L. The volume of the liquid formulation can be more than about 0.1 mL, 1.0 mL, 10 mL, 25 mL, 50 mL, 100 mL, 250 mL, 500 mL, 1 L, 10 L, 50 L, 100 L, 250 L, 500 L, or 1000 L. The volume of the liquid formulation can be about 0.01 -1 mL, about 1- 10 mL, about 10-50 mL, about 50-100 mL, about 1-1000 mL, about 100-1000 mL, about 1 -10 L, about 10-50 L, about 50-100 L, about 100-500 L, about 100-1000 L, or about 1-1000 L. Following freeze drying, the amount of dry vaccine produced can be between about 0.05 mg to 500 mg, about 0.0.05 mg to I mg, about 1 mg to about 100 mg, or about 100 mg to about 500 mg. [0018] A. VIRAL VACCINE COMPONENTS The methods of generating a dry vaccine powder formulation described herein can be used to produce a vaccine with a live attenuated virus, whole inactivated virus, split 8 WO 2011/129120 PCT/JP2011/002225 virus, subunit antigen, virosome, or cold-adapted live influenza virus. [0019] The methods of generating a dry vaccine powder formulation described herein can be used to produce a vaccine with a live attenuated virus. Live attenuated vaccines can be derived from serial passage in cultured cells, including, for example, human diploid cells (e.g. fetal lung tissue, other fibroblasts), monkey kidney cells, and chick embryos. Adaptation of a virus to growth in the cultured cells can be accompanied by a gradual loss of virulence for the natural host. Avirulence can be conferred, e.g., by accu mulation of point mutations. Genetic engineering can be used to achieve viral at tenuation by, e.g., generating temperature sensitive mutants, generating deletion mutants, site-directed mutagenesis, or generating live recombinant viruses. [0020] The methods of generating a dry vaccine powder formulation described herein can be used to produce a vaccine with a whole inactivated virus. Inactivated viruses can be generated, for example, by using ultraviolet light, low pH (e.g., acid, e.g., caprylic acid), pasteurization, solvents/detergents, sodium thiocyanate, formalin, beta propiolactone, or ethylenimines. UV rays can damage DNA through by creating nucleic acid diners, which can inactivate viruses by preventing the replication of genetic material. Some viruses denature upon exposure to low pH solutions. This method can be particularly effective when employed verses enveloped viruses. Pas teurization can inactivate viruses by means of temperature induced denaturation. Solvent/detergent inactivation is only effective against viruses enveloped in a lipid coat. The detergent used is typically Triton-X 100. Sodium thiocyanate can denature the protein coat of viruses, rendering the virus inactive. Formalin can chemically modify the surface proteins of the viral coat, which can prevent infection. Ethylenimines and beta-propiolactone can act on the nucleic acids of the virus while leaving the protein coat mostly unmodified. Inactivation can destroy infectivity of the virus while maintaining its immunogenicity. Multiple applications of inactivated virus can be administered to a subject. [0021] The methods of generating a dry vaccine powder formulation described herein can be used to produce a vaccine with one or more antigenic proteins (vaccine proteins) from one or more pathogens. An antigenic protein can be from any pathogen to which a vaccine is to be produced. For example, where the vaccine is to target influenza virus, an antigenic protein can be hemagglutinin (HA) and/or neuraminidase (NA). Hemag glutinin is an antigenic glycoprotein and a major surface protein of the influenza A virus. It mediates the biding between an influenza virus and the cell to be infected by binding to sialic acid-containing receptors on the surface of the cell. Viral particles bound to the surface of the cell are engulfed into endosomes. Inside the endosome, HA mediates a fusion of the viral membrane and the endosomal membrane, releasing the viral genome into the cell. Structurally, HA consists of three identical monomers 9 WO 2011/129120 PCT/JP2011/002225 organized into a helical coil. A function blocking antibody could inhibit either the cell binding or membrane fusing functions of HA. Neuraminidase is another glycoprotein found on the surface of an influenza virus. NAs are enzymes that function by cleaving sialic acid groups from glycoproteins. This cleavage seems to serve two functions: to prevent viral clumping and to release progeny viruses from the surface of a cell. [0022] There are at least 16 known HA subtypes. A vaccine antigen can be HAL, HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9, HA10, HA11, HA12, HA13, HA14, HA15, or HA 16. There are 9 known NA subtypes. A vaccine antigen can be NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8, or NA9. A vaccine prepared from a HA and/or NA subtype can be used individually or in any combination. For example, two or more of the various HA and NA antigens can be mixed during manufacture of a dry vaccine powder formulation, or dry powder formulations of individual HA and NA antigens can be combined. An antigenic protein can be surface proteins from the pathogen. An antigenic protein can be produced recombinantly. For example, nucleic acid encoding an antigen of interest can be introduced in a prokaryotic cell (e.g. bacteria), eukaryotic cells (e.g., yeast cells and insect cells), and the protein can be expressed and purified from the cells. Where the pathogen is a virus, nonessential components of a vision can be removed (e.g., using ether and detergents). [0023] The methods of generating a dry vaccine powder formulation described herein can be used to produce a virosomal vaccine. A virosomal vaccine comprises virus-like particles of reconstituted virus envelopes with no genetic material of the native virus. Influenza virosomes are vesicles consisting of a unilamellar phospholipid bilayer with intercalated HA and NA proteins. Because they have no genetic material, virosomes are not infectious. [0024] The concentration of a vaccine protein (e.g., antigen or antigen containing component) in a liquid vaccine formulation can be from about 0.05 mg/mL to 10 mg/ mL, about 0.1 mg/mL to 10 mg/mL, about 0.1 mg/mL to 5 mg/mL, about 0.1 mg/mL to 2.5 mg/mL, about 0.1 mg/mL to 1 mg/mL, about 0.1 mg/mL to 0.5 mg/ML, about 0.5 mg/mL to 1 mg/mL, about 0.05 mg/mL to 1 mg/mL, or about 0.05 mg/mL to 2.5 mg/mL. The concentration of a vaccine protein (e.g., antigen or antigen containing component) in a liquid vaccine formulation can be about 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3 mg/mL, 3.5 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 5.5 mg/mL, 6 mg/mL, 6.5 mg/mL, 7.0 mg/ mL, 8.0 mg/mL, 8.5 mg/mL, 9 mg/mL, or 10 mg/mL. The concentration of a vaccine protein (e.g. antigen or antigen-containing component) in a liquid vaccine formulation can be more than about 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 10 WO 2011/129120 PCT/JP2011/002225 0.5 mg/mL, 0.6 ng/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/nL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3 mg/mL, 3.5 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 5.5 mg/mL, 6 mg/mL, 6.5 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 8.5 mg/mL, 9 mg/ mL, or 10 mg/mL. [0025] A dry vaccine powder formulation can be used to prevent and/or treat infection by one or more influenza viruses. Influenza viruses belong to the Orthomyxoviridae family of viruses, which includes five genera: Influenzavirus A, Influenzavirus B, In fluenzavirus C, Isavirus, and Thogotovirus. Dhori virus is a species of the genus Thogotovirus. An influenza virus can infect humans and other species. Influenza type A viruses can infect humans, birds, pigs, horses, seals and other animals. Wild birds can be natural hosts for these viruses. Influenza type A viruses can be divided into subtypes and named on the basis of two proteins on the surface of the virus: hemag glutinin (HA) and neuraminidase (NA). For example, an "H7N2 virus" designates an influenza A subtype that has an HA7 protein and an NA2 protein. Similarly an "H5N1" virus has an HA 5 protein and an NA 1 protein. There are 16 known HA subtypes and 9 known NA subtypes. Many different combinations of HA and NA proteins are possible. Any number of the known HA subtypes (HA 1, HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9, HA10, HA11, HA12, HA13, HA14, HA15, and HA16) can be combined with any number of the known NA subtypes (NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8, and NA9) to produce a vaccine to prevent or treat an infection. The HA and NA subtypes can also be used individually in a vaccine to prevent or treat infection. Different subtype vaccines can be combined at the point of use, either se quentially or simultaneously, to prevent or treat an infection. Some influenza A subtypes (e.g., HlN1, H1N2, and H3N2) are currently in general circulation among people. Other subtypes can be found in other animal species. For example, H7N7 and H3N8 viruses can cause illness in horses, and H3N8 also has recently been shown to cause illness in dogs (http://www.cdc.gov/flu/avian/gen-info/flu-viruses). [0026] Antiviral agents can be used to protect high-risk groups (e.g., individuals in a hospital unit, individuals at an institute caring for the elderly, or immuno-suppressed individuals). A potential use for an antiviral agent is to limit the spread and severity of the future pandemics whether caused by, e.g. avian H5N1 or another strains of influenza virus (e.g., H1N1). Avian influenza A viruses of the subtypes H5 and H7, including H5N1, H7N7, and H7N3 viruses, have been associated with high pathogenicity, and human infection with these viruses have ranged from mild (e.g., H7N3, H7N7) to severe and fatal disease (e.g., H7N7, H5Nl). Human illness due to infection with low pathogenicity viruses has been documented, including very mild symptoms (e.g., conjunctivitis) to influenza-like illness. Examples of low 11 WO 2011/129120 PCT/JP2011/002225 pathogenicity viruses that have infected humans include H7N7, H9N2, and H7N2. (http://www.cdc.gov/flu/avian/gen-info/flu-viruses). [0027] Influenza B viruses can be found in humans and can also infect seals. Unlike influenza A viruses, these viruses are not classified according to subtype. Influenza B viruses can cause morbidity and mortality among humans, but in general are associated with less severe epidemics than influenza A viruses. Although influenza type B viruses can cause human epidemics, they have not caused pandemics. (http://www.cdc.gov/flu/avian/gen-info/flu-viruses). [0028] Influenza type C viruses can cause mild illness in humans and do not cause epidemics or pandemics. These viruses can also infect dogs and pigs. These viruses are not classified according to subtype. (http://www.cdc.gov/flu/avian/gen-info/flu-viruses). [0029] The methods and compositions described herein can be useful for the prevention and/ or treatment of infection by any virus, including, for example, Abelson leukemia virus, Abelson murine leukemia virus, Abelson's virus, Acute laryngotracheobronchitis virus, Adelaide River virus, Adeno associated virus group, Adenovirus, African horse sickness virus, African swine fever virus, AIDS virus, Aleutian mink disease parvovirus, Alpharetrovirus, Alphavirus, ALV related virus, Amapari virus, Aph thovirus, Aquareovirus, Arbovirus, Arbovirus C, arbovirus group A, arbovirus group B, Arenavirus group, Argentine hemorrhagic fever virus, Argentine hemorrhagic fever virus, Arterivirus, Astrovirus, Ateline herpesvirus group, Aujezky's disease virus, Aura virus, Ausduk disease virus, Australian bat lyssavirus, Aviadenovirus, avian erythrob lastosis virus, avian infectious bronchitis virus , avian leukemia virus, avian leukosis virus, avian lymphomatosis virus, avian myeloblastosis virus, avian paramyxovirus, avian pneumoencephalitis virus, avian reticuloendotheliosis virus, avian sarcoma virus, avian type C retrovirus group, Avihepadnavirus, Avipoxvirus, B virus, B19 virus, Babanki virus, baboon herpesvirus, baculovirus, Barmah Forest virus, Bebaru virus, Berrimah virus, Betaretrovirus, Birnavirus, Bittner virus, BK virus, Black Creek Canal virus, bluetongue virus, Bolivian hemorrhagic fever virus, Boma disease virus, border disease of sheep virus, borna virus, bovine alphaherpesvirus 1, bovine alphaher pesvirus 2, bovine coronavirus, bovine ephemeral fever virus, bovine immunode ficiency virus, bovine leukemia virus, bovine leukosis virus, bovine mammillitis virus, bovine papillomavirus, bovine papular stomatitis virus, bovine parvovirus, bovine syncytial virus, bovine type C oncovirus, bovine viral diarrhea virus, Buggy Creek virus, bullet shaped virus group, Bunyamwera virus supergroup, Bunyavirus, Burkitt's lymphoma virus, Bwamba Fever, CA virus, Calicivirus, California encephalitis virus, camelpox virus, canarypox virus, canid herpesvirus, canine coronavirus, canine distemper virus, canine herpesvirus , canine minute virus, canine parvovirus, Cano 12 WO 2011/129120 PCT/JP2011/002225 Delgadito virus, caprine arthritis virus, caprine encephalitis virus, Caprine Herpes Virus, Capripox virus, Cardiovirus, caviid herpesvirus 1, Cercopithecid herpesvirus 1, cercopithecine herpesvirus 1, Cercopithecine herpesvirus 2, Chandipura virus, Changuinola virus, channel catfish virus, Charleville virus, chickenpox virus, Chikungunya virus, chimpanzee herpesvirus, chub reovirus, chum salmon virus, Cocal virus, Coho salmon reovirus, coital exanthema virus, Colorado tick fever virus, Coltivirus, Columbia SK virus, common cold virus, contagious ecthyma virus, contagious pustular dermatitis virus, Coronavirus, Corriparta virus, coryza virus, cowpox virus, coxsackie virus, CPV (cytoplasmic polyhedrosis virus), cricket paralysis virus, Crimean-Congo hemorrhagic fever virus, croup associated virus, Cryptovirus, Cypovirus, Cytomegalovirus, cytomegalovirus group, cytoplasmic polyhedrosis virus, deer papillomavirus, deltaretrovirus, dengue virus, Densovirus, Dependovirus, Dhori virus, diploma virus, Drosophila C virus, duck hepatitis B virus, duck hepatitis virus 1, duck hepatitis virus 2, duovirus, Duvenhage virus, Deformed wing virus DWV, eastern equine encephalitis virus, eastern equine encephalomyelitis virus, EB virus, Ebola virus, Ebola-like virus, echo virus, echovirus, echovirus 10, echovirus 28, echovirus 9, ectromelia virus, EEE virus, EIA virus, EIA virus, encephalitis virus, encephalomy ocarditis group virus, encephalomyocarditis virus, Enterovirus, enzyme elevating virus, enzyme elevating virus (LDH), epidemic hemorrhagic fever virus, epizootic hem orrhagic disease virus, Epstein-Barr virus, equid alphaherpesvirus 1, equid alphaher pesvirus 4, equid herpesvirus 2, equine abortion virus, equine arteritis virus, equine en cephalosis virus, equine infectious anemia virus, equine morbillivirus, equine rhinop neumonitis virus, equine rhinovirus, Eubenangu virus, European elk papillomavirus, European swine fever virus, Everglades virus, Eyach virus, felid herpesvirus 1, feline calicivirus, feline fibrosarcoma virus, feline herpesvirus, feline immunodeficiency virus, feline infectious peritonitis virus, feline leukemia /sarcoma virus, feline leukemia virus, feline panleukopenia virus, feline parvovirus, feline sarcoma virus, feline syncytial virus, Filovirus, Flanders virus, Flavivirus, foot and mouth disease virus, Fort Morgan virus, Four Corners hantavirus, fowl adenovirus 1, fowlpox virus, Friend virus, Gammaretrovirus, GB hepatitis virus, GB virus, German measles virus, Getah virus, gibbon ape leukemia virus, glandular fever virus, goatpox virus, golden shinner virus, Gonometa virus, goose parvovirus, granulosis virus, Gross' virus, ground squirrel hepatitis B virus, group A arbovirus, Guanarito virus, guinea pig cy tomegalovirus, guinea pig type C virus, Hantaan virus, Hantavirus, hard clam reovirus, hare fibroma virus, HCMV (human cytomegalovirus), hemadsorption virus 2, hemag glutinating virus of Japan, hemorrhagic fever virus, hendra virus, Henipaviruses, Hep adnavirus, hepatitis A virus, hepatitis B virus group, hepatitis C virus, hepatitis D virus, hepatitis delta virus, hepatitis E virus, hepatitis F virus, hepatitis G virus, 13 WO 2011/129120 PCT/JP2011/002225 hepatitis nonA nonB virus, hepatitis virus, hepatitis virus (nonhuman), hepatoen cephalomyelitis reovirus 3, Hepatovirus, heron hepatitis B virus, herpes B virus, herpes simplex virus, herpes simplex virus 1, herpes simplex virus 2, herpesvirus, herpesvirus 7, Herpesvirus ateles, Herpesvirus hominis, Herpesvirus infection, Herpesvirus saimiri, Herpesvirus suis, Herpesvirus varicellae, Highlands J virus, Hirame rhabdovirus, hog cholera virus, human adenovirus 2, human alphaherpesvirus 1, human alphaher pesvirus 2, human alphaherpesvirus 3, human B lymphotropic virus, human betaher pesvirus 5, human coronavirus, human cytomegalovirus group, human foamy virus, human gammaherpesvirus 4, human gammaherpesvirus 6, human hepatitis A virus, human herpesvirus I group, human herpesvirus 2 group, human herpesvirus 3 group, human herpesvirus 4 group, human herpesvirus 6, human herpesvirus 8, human im munodeficiency virus, human immunodeficiency virus 1, human immunodeficiency virus 2, human papillomavirus, human T cell leukemia virus, human T cell leukemia virus I, human T cell leukemia virus II, human T cell leukemia virus III, human T cell lymphoma virus I, human T cell lymphoma virus I, human T cell lymphotropic virus type 1, human T cell lymphotropic virus type 2, human T lymphotropic virus I, human T lymphotropic virus II, human T lymphotropic virus III, Ichnovirus, infantile gas troenteritis virus, infectious bovine rhinotracheitis virus, infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus, influenza virus A, influenza virus B, influenza virus C, influenza virus D, influenza virus pr8, insect iridescent virus, insect virus, iridovirus, Japanese B virus , Japanese encephalitis virus, JC virus, Junin virus, Kaposi's sarcoma-associated herpesvirus, Kemerovo virus, Kilham's rat virus, Klamath virus, Kolongo virus, Korean hemorrhagic fever virus, kumba virus, Kysanur forest disease virus, Kyzylagach virus, La Crosse virus, lactic dehydrogenase elevating virus, lactic dehydrogenase virus, Lagos bat virus, Langur virus, lapine parvovirus, Lassa fever virus, Lassa virus, latent rat virus, LCM virus, Leaky virus, Lentivirus, Leporipoxvirus, leukemia virus, leukovirus, lumpy skin disease virus, lym phadenopathy associated virus, Lymphocryptovirus, lymphocytic choriomeningitis virus, lymphoproliferative virus group, Machupo virus, mad itch virus, mammalian type B oncovirus group, mammalian type B retroviruses, mammalian type C retrovirus group, mammalian type D retroviruses, mammary tumor virus, Mapuera virus, Marburg virus, Marburg-like virus, Mason Pfizer monkey virus, Mastadenovirus, Mayaro virus, ME virus, measles virus, Menangle virus, Mengo virus, Mengovirus, Middelburg virus, milkers nodule virus, mink enteritis virus, minute virus of mice, MLV related virus, MM virus, Mokola virus, Molluscipoxvirus, Molluscum con tagiosum virus, monkey B virus, monkeypox virus, Mononegavirales, Morbillivirus, Mount Elgon bat virus, mouse cytomegalovirus, mouse encephalomyelitis virus, mouse hepatitis virus, mouse K virus, mouse leukemia virus, mouse mammary tumor 14 WO 2011/129120 PCT/JP2011/002225 virus, mouse minute virus, mouse pneumonia virus, mouse poliomyelitis virus, mouse polyomavirus, mouse sarcoma virus, mousepox virus, Mozambique virus, Mucambo virus, mucosal disease virus, mumps virus, murid betaherpesvirus 1, murid cy tomegalovirus 2, murine cytomegalovirus group, murine encephalomyelitis virus, murine hepatitis virus, murine leukemia virus, murine nodule inducing virus, murine polyomavirus, murine sarcoma virus, Muromegalovirus, Murray Valley encephalitis virus, myxoma virus, Myxovirus, Myxovirus multiforme, Myxovirus parotitidis, Nairobi sheep disease virus, Nairovirus, Nanirnavirus, Nariva virus, Ndumo virus, Neethling virus, Nelson Bay virus, neurotropic virus, New World Arenavirus, newborn pneumonitis virus, Newcastle disease virus, Nipah virus, noncytopathogenic virus, Norwalk virus, nuclear polyhedrosis virus (NPV), nipple neck virus, O'nyong'nyong virus, Ockelbo virus, oncogenic virus, oncogenic viruslike particle, oncornavirus, Orbivirus, Orf virus, Oropouche virus, Orthohepadnavirus, Orthomyxovirus, Or thopoxvirus, Orthoreovirus, Orungo, ovine papillomavirus, ovine catarrhal fever virus, owl monkey herpesvirus, Palyam virus, Papillomavirus, Papillomavirus sylvilagi, Pa povavirus, parainfluenza virus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, Paramyxovirus, Parapoxvirus, paravaccinia virus, Parvovirus, Parvovirus B 19, parvovirus group, Pestivirus, Phlebovirus, phocine distemper virus, Picodnavirus, Picornavirus, pig cytomegalovirus - pigeonpox virus, Piry virus, Pixuna virus, pneumonia virus of mice, Pneumovirus, poliomyelitis virus, poliovirus, Polydnavirus, polyhedral virus, polyoma virus, Poly omavirus, Polyomavirus bovis, Polyomavirus cercopi theci, Polyomavirus hominis 2, Polyomavirus maccacae 1, Polyomavirus muris 1, Polyomavirus muris 2, Polyomavirus papionis 1, Polyomavirus papionis 2, Poly omavirus sylvilagi, Pongine herpesvirus 1, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine parvovirus, porcine transmissible gastroenteritis virus, porcine type C virus, pox virus, poxvirus, poxvirus variolae, Prospect Hill virus, Provirus, pseudocowpox virus, pseudorabies virus, psittacinepox virus, quailpox virus, rabbit fibroma virus, rabbit kidney vaculolating virus, rabbit pa pillomavirus, rabies virus, raccoon parvovirus, raccoonpox virus, Ranikhet virus, rat cytomegalovirus, rat parvovirus, rat virus, Rauscher's virus, recombinant vaccinia virus, recombinant virus, reovirus, reovirus 1, reovirus 2, reovirus 3, reptilian type C virus, respiratory infection virus, respiratory syncytial virus, respiratory virus, reticu loendotheliosis virus, Rhabdovirus, Rhabdovirus carpia, Rhadinovirus, Rhinovirus, Rhizidiovirus, Rift Valley fever virus, Riley's virus, rinderpest virus, RNA tumor virus, Ross River virus, Rotavirus, rougeole virus, Rous sarcoma virus, rubella virus, rubeola virus, Rubivirus, Russian autumn encephalitis virus, SA 11 simian virus, SA2 virus, Sabia virus, Sagiyaina virus, Saimirine herpesvirus 1, salivary gland virus, sandfly 15 WO 2011/129120 PCT/JP2011/002225 fever virus group, Sandjimba virus, SARS virus, SDAV (sialodacryoadenitis virus), sealpox virus, Semliki Forest Virus, Seoul virus, sheeppox virus, Shope fibroma virus, Shope papilloma virus, simian foamy virus, simian hepatitis A virus, simian human immunodeficiency virus, simian immunodeficiency virus, simian parainfluenza virus, simian T cell lymphotrophic virus, simian virus, simian virus 40, Simplexvirus, Sin Nombre virus, Sindbis virus, smallpox virus, South American hemorrhagic fever viruses, sparrowpox virus, Spumavirus, squirrel fibroma virus, squirrel monkey retrovirus, SSV I virus group, STLV (simian T lymphotropic virus) type I, STLV (simian T lymphotropic virus) type II, STLV (simian T lymphotropic virus) type III, stomatitis papulosa virus, submaxillary virus, suid alphaherpesvirus 1, suid herpesvirus 2, Suipoxvirus, swamp fever virus, swinepox virus, Swiss mouse leukemia virus, TAC virus, Tacaribe complex virus, Tacaribe virus, Tanapox virus, Taterapox virus, Tench reovirus, Theiler's encephalomyelitis virus, Theiler's virus, Thogoto virus, Thot tapalayam virus, Tick borne encephalitis virus, Tioman virus, Togavirus, Torovirus, tumor virus, Tupaia virus, turkey rhinotracheitis virus, turkeypox virus, type C retroviruses, type D oncovirus, type D retrovirus group, ulcerative disease rhabdovirus, Una virus, Uukuniemi virus group, vaccinia virus, vacuolating virus, varicella zoster virus, Varicellovirus, Varicola virus, variola major virus, variola virus, Vasin Gishu disease virus, VEE virus, Venezuelan equine encephalitis virus, Venezuelan equine en cephalomyelitis virus, Venezuelan hemorrhagic fever virus, vesicular stomatitis virus, Vesiculovirus, Vilyuisk virus, viper retrovirus, viral haemorrhagic septicemia virus, Visna Maedi virus, Visna virus, volepox virus, VSV (vesicular stomatitis virus), Wallal virus, Warrego virus, wart virus, WEE virus, West Nile virus, western equine encephalitis virus, western equine encephalomyelitis virus, Whataroa virus, Winter Vomiting Virus, woodchuck hepatitis B virus, woolly monkey sarcoma virus, wound tumor virus, WRSV virus, Yaba monkey tumor virus, Yaba virus, Yatapoxvirus, yellow fever virus, and the Yug Bogdanovac virus. [0030] B. NON-VIRAL PATHOGEN VACCINE COMPONENTS A vaccine described herein can comprise bacterial, fungal, or protist cells or components thereof. For example, a vaccine to a bacterial pathogen can comprise a killed bacterium or a purified antigenic determinant thereof. Attenuated bacteria can also be used as an antigen. Tn some instances, a vaccine to a toxin produced by a cellular pathogen (e.g., cholera toxin) can be produced by combining the inactivated toxin (toxoid) with one or more of the vaccine components described herein. An antigenic peptide from a target pathogen can be purified from a source pathogen and/or produced recombinantly prior to combining with the one or more of the components of the vaccine. Conjugate antigens can also be used. In a conjugate antigen, the poorly antigenic polysaccharide outer coat of a bacterial pathogen is attached to toxic protein 16 WO 2011/129120 PCT/JP2011/002225 that can stimulate an immune response. Typically, vaccines to non-viral pathogens will be designed to produce immune responses (e.g., sIgA production) to pathogens which affect mucosal surfaces, or gain access to the body via mucosal surfaces. Non-limiting examples of such pathogens include Cryptococcus neoformans, Shigella spp., Salmonella typhi, Sa. paratyphi, enterotoxigenic Escherischia coli, Yersinia pestis, My cobacterium tubercolosis, Ureaplasma urealyticum, Cryptosporidium spp., Clostridium tetani, Corynebacterium diphtheriae, Neisseria meningitidis, Bordetella pertussis, Streptococcus pneumoniae, Bacillus anthracis, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira alexanderi, Leptospira weilii, Leptospira borgpetersenii, Leptospira santarosai, Leptospira kmetyi, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Enterococcus faccalis, Enterococcus faecium, Francisella tularensis, Haemophilus influenzae, Heli cobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria mono cytogenes, Mycobacterium leprae, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiac, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Candida albicans, Aspergillus fumigatus, Aspergillus flavus, Cryp tococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii, Stachybotrys chartarum, Plasmodium falciparum, etc. [0031] C. PREPARATION OF ANTIGENIC COMPONENTS In order to preserve the antigenic function of the proteins or other cellular components of the pathogen, the present disclosure provides methods for preparing a vaccine which can preserve some or all of a three-dimensional configuration of the antigenic component (e.g., virus, protein). Thus, the methods provided herein can allow for the production of vaccines in which the antigenic determinants on the pathogen or component thereof are preserved in an intact state. For example, retaining three-dimensional structure of a protein in a vaccine can allow for retention of "confor mational" epitopes to which an immune response can be triggered. "Conformational" epitopes are those which rely upon protein folding and generally are not comprised entirely of amino acids in linear form (e.g., a digested or linearized protein). Fur thermore, the methods provided herein to produce vaccines can result in retention of antigenic potency (i.e., the ability to induce an immune response), such that the level of immune response in a reaction to a given amount of vaccine is at least about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 17 WO 2011/129120 PCT/JP2011/002225 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%. 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% as compared to exposure to the pathogen or other naturally-occurring antigenic source. Additionally, the methods provided herein can allow for the production of a vaccine in which a particular antigen retains high levels of antigenic capability (e.g., at least about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50%) of the total antigenic protein subjected to the quick freezing methods described herein. [0032] One embodiment of this methodology is shown in Figure 2. In this example, the antigen (shown as an open circle) is combined with a stabilizer (trehalose) and a buffer (phosphate buffer). The components are mixed and freeze-dried (e.g., by immersion in liquid nitrogen). The dried vaccine component produced comprises fine particles in which the antigen or antigens are still capable of eliciting an immune response and is stable at room temperature. The vaccine component can then be combined with a carrier suitable for nasal administration (e.g., microcrystalline cellulose). Non-limiting examples of components of vaccines disclosed herein are provided below. [0033] Components of a liquid formulation can be chosen to perform certain functions. For example, one component can be utilized to provide stability to the antigen for which the vaccine is being developed. Primarily, such components can prevent antigenic degradation during the subsequent freezing process. These components can comprise any stabilizing molecule or compound, for example sugars, amino acids and/or polymers. One or more such antigenic stabilizing agents can be used in a formulation. Typically, an antigenic stabilizing agent will be wholly or partially water soluble. Preferred antigenic stabilizers will not produce hard cakes in the processes described herein. Exemplary sugars which can be utilized to produce a liquid vaccine for mulations include, but are not limited to trehalose, mannitol, sucrose, lactose, inulin, sorbose, melezitose, raffinose, mannitol, xylitol, erythritol, threitol, stachyose, sorbitol, glycerol, fructose, mannose, maltose, arabinose, xylose, ribose, rhamnose, galactose, glucose, L-gluconate, and/or the like.. Exemplary amino acids which can be utilized, include, but are not limited to isoleucine, valine, leucine, arginine, asparagine, glutamine, glycine, histidine, glutamate and lysine. An exemplary polymer is polyethylene glycol (PEG), but other polymers that can be utilized can include dextran, human serum albumin (HSA), nonhydrolyzed gelatin, metbylcellulose, xanthan gum, carrageenan, collagen, chondroitin sulfate, a sialated polysaccharide, actin, myosin, microtubules, dynein, kinetin, polyvinyl pyrrolidone, hydrolyzed gelatin, and/or the like. A surfactant can be, e.g., a polyethylene glycol, sorbitan monolaurate (Tween 20), 18 WO 20111129120 PCT/JP2011/002225 a polyoxyethylenesorbitan monooleate (Tween 80), a block copolymer of polyethylene and polypropylene glycol (Pluronic), and/or the like. [0034] Although such antigenic stabilizers have been utilized in vaccine preparations, the use of one or more of these stabilizers in the present methods can result in a frozen vaccine formulation that does not form a hard cake upon drying. For example, the use of trehalose is known to provide protection to proteins when frozen, but leads to caking if the substance is not spray frozen (see, e.g., Chefson et al.. J Biotechnol. 2007 Jul 15;130(4):436-40). However, one embodiment provided herein is the combination of trehalose with a proteinaceous antigen, a phosphate buffer and a quick freeze method (e.g., exposure to liquid nitrogen). Such methods can lead to the production of fine powders in which the protein retains activity (e.g., antigenic capability) without forming a hard cake. This is an advance as the grinding of hard cakes is an extra step in the vaccine preparation process and can result in low recovery rate and degradation of the antigenic protein in the hard cake, through heat and/or mechanical forces. [0035] The ratio of antigen to stabilizer can be, for example, about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, or 1:100. The ratio of antigen to stabilizer can be, for example, about 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, 1:250, 1:260, 1:270, 1:280, 1:290, 1:300, 1:310, 1:320, 1:330, 1:340, 1:350, 1:360, 1:370, 1:380, 1:390, 1:400, 1:410, 1:420, 1:430, 1:440, 1:450, 1:460, 1:470, 1:480, 1:490, 1:500, 1:510, 1:520, 1:530, 1:540, 1:550, 1:560, 1:570, 1:580, 1:590, 1:600, 1:610, 1:620, 1:630, 1:640, 1:650, 1:660, 1:670, 1:680, 1:690, 1:700, 1:710, 1:720, 1:730, 1:740, 1:750, 1:760, 1:770, 1:780, 1:790, 1:800, 1:810, 1:820, 1:830, 1:840, 1:850, 1:860, 1:870, 1:880, 1:890, 1:900, 1:910, 1:920, 1:930, 1:940, 1:950, 1:960, 1:970, 1:980, 1:990, or 1:1000. A vaccine liquid formulation to be used in the freeze-drying step can comprise one or more pH buffers (Figure 2 and 3). The pH buffer can be, e.g., potassium phosphate, sodium phosphate, disodium hydrogen phosphate, potassium dihydrogenphosphate, sodium hydroxide, sodium acetate, histidine, HEPES, ACES, ADA, ADA, disodium salt, ADA monosodium salt, AMPSO, 2-aminoethanol, 2-amino-2-methyl-1,3-propanediol, 2-amino-2-methyl-1-propanol, 3-amino-1-propanesulfonic acid sodium salt, BES, bicine, Bis-Tris, Bis-Tris HCI, Bis-Tris propane, CAPS, CAPSO, CHES, DIPSO, DIPSO sodium salt, glycinamide HCI, glycine, HEPPS, HEPPSO, MES, MOPS, 19 WO 2011/129120 PCT/JP2011/002225 MOPSO, PIPES, TAPS, TAPSO, TES, tricine, triethanolamine, imidazole, sodium citrate, sodium succinate, ammonium bicarbonate, and/or a carbonate. A buffer can be phosphate buffered saline. The pH can be maintained at between about pH 3 to about pH 8, about pH 4 to 8, about pH 5 to 8, about pH 6 to 8, or about pH 6.0, 61, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. A liquid formulation can comprise, consist essentially of, or consist of one or more antigens and one or more buffers. A liquid formulation can comprise, consist essentially of, or consist of one or more antigens, one or more stabilizers, and one or more buffers. [0036] A liquid formulation used to generate a powder formulation by the methods described herein can contain one or more other drugs, bulking agents, and/or sustained release polymers. Other drugs useful in the compositions of the invention, can include, e.g., aids to penetration, decongestants, bronchiole relaxers, expectorants, analgesics, and the like. Bulking agents can include, e.g., lactose, mannitol, and/or hydroxyethyl starch (HES). Sustained release semi-permeable polymer matrix of the compositions can include, e.g., polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate, or liposomes. [0037] A vaccine described herein can be made without including an adjuvant. Thus, the final vaccine can be produced using only the pathogen/antigen, a stabilizer, and a buffer which is then freeze-dried. Following freeze-drying, the vaccine can be combined with a carrier without the need to add an adjuvant prior to producing the final vaccine product. Alternately, the formulation can comprise adjuvant, a substance added to a vaccine to improve the immune response of the vaccine. An adjuvant can be added prior to, or after, freeze drying. Examples of adjuvant include mineral salts, e.g., aluminum hydroxide and aluminum or calcium phosphate gels, oil emulsions and surfactant based formulations, e.g., MF59 (microfluidised detergent stabilized oil in-water emulsion), QS21 (purified saponin), ASO2 ([SBAS2J (oil-in-water + MPL + WS-21)), Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion); par ticulate adjuvants, (e.g, virsomes (unilamellar liposomal vehicles incorporating influenza hemagglutinin), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins and lipids), polylactide co-glycolide (PLG); microbial derivatives (natural and synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC-5291 (synthetic acylated monosaccharide), DCChol (lipoidal immunostimulators able to self organise into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects); endogenous human immunomodulators, e.g., hGM-CSF or hIL- 12 (cytokines that can be administered either as protein or plasmid encoded), Im mudaptin (C3d tandem array); inert vehicles, such as gold particles; and squalene. The 20 WO 2011/129120 PCT/JP2011/002225 liquid formulation and the final dry vaccine powder formulation can have no adjuvant. [00381 III. FREEZE DRYING A liquid formulation can be converted to a powder by freeze drying. Freeze drying is a process by which material is frozen and subsequently dried by removal of water by sublimation. Quick freezing can be accomplished, e.g., by immediate immersion of spray droplets (spray-freeze drying) in liquid nitrogen or a stream of cold gas. Quick freezing can also be accomplished by a process that does not comprise a spray-freezing step. Quick freezing can be accomplished by contacting a liquid vaccine formulation with liquid nitrogen (-196 degrees C). Quick freezing can be accomplished by contacting a liquid vaccine formulation with liquid nitrogen combined with another chemical, e.g., hexane/liquid nitrogen (-94 degrees C), methanol/liquid nitrogen (-98 degrees C), and pentane/liquid nitrogen (-131 degrees C) (Gordon AJ and Ford RA "The Chemist's Companion. Wiley. New York 1972). Quick freezing can be ac complished by contacting a liquid vaccine formulation with a dry ice/organic solvent (e.g., ethanol, methanol, ethylene glycol, carbon tetracholride, acetonitrile, isopropyl alcohol, or acetone) bath, e.g., carbon tetrachloride/dry ice (-23 degrees C), ace tonitrile/dry ice (-42 degrees C), or acetone or isopropyl alcohol/dry ice bath (-78 degrees C). (Gordon, supra). Quick freezing can be accomplished by immersing a liquid vaccine formulation in a slurry of ice and inorganic salt (e.g., NaCl or CaCl 2 ), which can reach -40 degrees C. The temperature at which a liquid vaccine formulation can be frozen can be less than about 0 degrees C, -5 degrees C, -10 degrees C, -15 degrees C, -20 degrees C, -25 degrees C, -30 degrees C, -35 degrees C, -40 degrees C, -45 degrees C, -50 degrees C, -55 degrees C, -60 degrees C, -65 degrees C, -70 degrees C, -75 degrees C, -80 degrees C, -85 degrees C, -90 degrees C, -95 degrees C, -100 degrees C, -105 degrees C, -110 degrees C, -115 degrees C, -120 degrees C, -125 degrees C, -130 degrees C, -135 degrees C, -140 degrees C, -145 degrees C, -150 degrees C, -155 degrees C, -160 degrees C, -165 degrees C, -170 degrees C, -175 degrees C, -180 degrees C, -185 degrees C, -90 degrees C, -195 degrees C, -200 degrees C, -205 degrees C, or -210 degrees C. The temperature at which at which a liquid vaccine formulation can be frozen can be about 0 degrees C to -210 degrees C, 50 degrees C to about -210 degrees C, -100 degrees C to about -210 degrees C, or -150 degrees C to about -200 degrees C. The temperature at which a liquid vaccine for mulation can be frozen can be about 0 degrees C, -5 degrees C, -10 degrees C, -15 degrees C, -20 degrees C, -25 degrees C, -30 degrees C, -35 degrees C, -40 degrees C, -45 degrees C, -50 degrees C, -55 degrees C, -60 degrees C, -65 degrees C, -70 degrees C, -75 degrees C, -80 degrees C, -85 degrees C, -90 degrees C, -95 degrees C, -100 degrees C, -105 degrees C, -110 degrees C, -115 degrees C, -120 degrees C, -125 degrees C, -130 degrees C, -135 degrees C, -140 degrees C, -145 degrees C, -150 21 WO 2011/129120 PCT/JP2011/002225 degrees C, - 155 degrees C, -160 degrees C, -165 degrees C, -170 degrees C, -175 degrees C, -180 degrees C, -185 degrees C, -190 degrees C, -195 degrees C, -200 degrees C, -205 degrees C, or -210 degrees C. The method of freezing can prevent loss of the three-dimensional shape of an antigen in the liquid vaccine formulation. [0039] Some antigen-containing solutions disclosed herein can contain carbohydrates. For example an antigen-containing solution can contain a sugar, including, but not limited to trehalose, mannitol, sucrose, lactose or inulin. Such sugars are utilized for various purposes, for example to protect proteinaceous components of a solution from losing or decreasing antigenic capability upon freezing. For example, the addition of trehalose to a solution can prevent loss of antigenicity of proteins, such as hemagglutinin (HA) of influenza, in liquid formulations containing proteins (e.g., liquid vaccine liquid for mulations). However, the addition of trehalose and other sugars can result in the formation of a hard cake in vaccine preparations, unless spray freezing is utilized. The novel methods disclosed herein allow for the use of such sugars in a quick freezing method which does not require spray freezing, and does not result in the formation of a hard cake. This is an advance over prior approaches which require milling of hard cakes, as such treatments can result in the loss of antigenicity of component biological molecules. The combinations of buffers and sugars disclosed herein in the antigen containing solutions allows for such results. Quick freezing of a sugar-containing solution disclosed herein can result in the generation of a powder. [0040] A liquid vaccine formulation can be exposed to a cold liquid, e.g., liquid nitrogen, for about 30 seconds to 5 min, 1 min to 60 min, 1 min to 50 min, 1 to 40 min, 1 to 30 min, 1 to 20 min, 1 to 10 min, or 1 to 5 min. A liquid vaccine formulation can be exposed to the cold liquid, e.g., liquid nitrogen, for about 30 seconds, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min. A liquid vaccine formulation can be exposed to the cold liquid, e.g., liquid nitrogen for more than about 30 seconds, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min. A liquid vaccine formulation can be exposed to the cold liquid by putting the liquid vaccine formulation in a container and immersing the containing in the cold liquid (e.g., liquid nitrogen). A liquid vaccine formulation can be exposed to the cold liquid by directly introducing a liquid vaccine formulation into the cold liquid (e.g., liquid nitrogen). A liquid vaccine formulation can be exposed to the cold liquid by pouring the cold liquid (e.g., liquid nitrogen) over the liquid vaccine formulation. [0041] DRYING After quick freezing, e.g., in liquid nitrogen, the frozen formulation can be freeze 22 WO 2011/129120 PCT/JP2011/002225 dried in a freeze-dryer. Freeze drying can occur in one or more steps (e.g., different temperatures at the same pressure). The freeze drying can occur at, for example, at about -210 degrees C, -205 degrees C, -200 degrees C, -195 degrees C, -190 degrees C, -185 degrees C, -180 degrees C, -175 degrees C, -170 degrees C, -165 degrees C, -160 degrees C, -155 degrees C, -150 degrees C, -145 degrees C, -140 degrees C, -135 degrees C, -130 degrees C, -125 degrees C, -120 degrees C, -115 degrees C, -110 degrees C, -105 degrees C, -100 degrees C, -95 degrees C, -90 degrees C, -85 degrees C, -80degrees C, -75 degrees C, -70 degrees C, -65 degrees C, -60 degrees C, -55 degrees C, -50 degrees C, -45 degrees C, -40 degrees C, -35 degrees C, -30 degrees C, -25 degrees C, -20 degrees C, -15 degrees C, -10 degrees C, -5 degrees C, 0 degrees C, 5 degrees C, 10 degrees C, 15 degrees C, 20 degrees C, 25 degrees C, or 30 degrees C. Freeze drying can occur at, for example, at more than about -210 degrees C, -205 degrees C, -200 degrees C, -195 degrees C, -190 degrees C, -185 degrees C, -180 degrees C, -175 degrees C, -170 degrees C, -165 degrees C, -160 degrees C, -155 degrees C, -150 degrees C, -145 degrees C, -140 degrees C, -135 degrees C, -130 degrees C, -125 degrees C, -120 degrees C, -115 degrees C, -110 degrees C, -105 degrees C, -100 degrees C, -95 degrees C, -90 degrees C, -85 degrees C, -80degrees C, -75 degrees C, -70 degrees C, -65 degrees C, -60 degrees C, -55 degrees C, -50 degrees C, -45 degrees C, -40 degrees C, -35 degrees C, -30 degrees C, -25 degrees C, -20 degrees C, -15 degrees C, -10 degrees C, -5 degrees C, 0 degrees C, 5 degrees C, 10 degrees C, 15 degrees C, 20 degrees C, 25 degrees C, or 30 degrees C. Freeze drying can occur at, e.g., at about -80degrees C to 30 degrees C, about -50 degrees C to 25 degrees C, or about -40 degrees C to 20 degrees C. Freeze-drying can occur at one temperature, two different temperatures, three different temperatures, four different temperatures, five different temperatures, six different temperatures, seven different temperatures, eight different temperatures, nine different temperatures, or ten different temperatures. [0042] The freeze-drying can occur at one or more different pressures. The pressure can be, for example, about 10 mtorr to 300 mtorr, about 25 mtorr to 300 mtorr, about 50 mtorr to 250 mtorr, or about 50 mtorr to 200 mtorr. The freeze-drying can occur at about 10 mtorr, 20 mtorr, 30 mtorr, 40 mtorr, 50 mtorr, 60 mtorr, 70 mtorr, 80 mtorr, 90 mtorr, 100, mtorr, 110 mtorr, 120 mtorr, 130 mtorr, 140 mtorr, 150 mtorr, 160 mtorr, 170 mtorr, 180 mtorr, 190 mtorr, 200 mtorr, 210 mtorr, 220 mtorr, 230 mtorr, 240 mtorr, 250 mtorr, 260 mtorr, 270 mtorr, 280 mtorr, 290 mtorr, or 300 mtorr. Freeze-drying can occur at more than about 10 mtorr, 20 mtorr, 30 mtorr, 40 mtorr, 50 mtorr, 60 mtorr, 70 mtorr, 80 mtorr, 90 mtorr, 100, mtorr, 110 mtorr, 120 mtorr, 130 mtorr, 140 mtorr, 150 mtorr, 160 mtorr, 170 mtorr, 180 mtorr, 190 mtorr, 200 mtorr, 210 mtorr, 220 mtorr, 230 mtorr, 240 mtorr, 250 mtorr, 260 mtorr, 270 mtorr, 280 mtorr, 290 23 WO 2011/129120 PCT/JP2011/002225 mtorr, or 300 imtorr. [0043] The duration of each freeze-drying step can be from about 1 hr to 48 hr, about 1 hr to 36 hr, about 1 hr to 24 hr, about 4 hr to 24 hr, about 6 hr to 24 hr, or about 8 hr to 24 hr. The duration of each freeze-drying step can be about 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 11 hr, 12 hr, 13 hr, 14 hr, 15 hr, 16 hr, 17 hr, 18 hr, 19 hr, 20 hr, 21 hr, 22 hr, 23 hr, 24 hr, 25 hr, 26 hr, 27 hr, 28 hr, 29 hr, 30 hr, 31 hr, 32 hr, 33 hr, 34 hr, 35 hr, 36 hr, 37 hr, 38 hr, 39 hr, 40 hr, 41 hr, 42 hr, 43 hr, 44 hr, 45 hr, 46 hr, 47 hr, or 48 hr. The duration of each freeze-drying step can be more than about 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 1 Ihr, 12 hr, 13 hr, 14 hr, 15 hr, 16 hr, 17 hr, 18 hr, 19 hr, 20 hr, 21 hr, 22 hr, 23 hr, 24 hr, 25 hr, 26 hr, 27 hr, 28 hr, 29 hr, 30 hr, 31 hr, 32 hr, 33 hr, 34 hr, 35 hr, 36 hr, 37 hr, 38 hr, 39 hr, 40 hr, 41 hr, 42 hr, 43 hr, 44 hr, 45 hr, 46 hr, 47 hr, or 48 hr. [0044] One or more drying steps can be utilized in the methods disclosed herein. Primary drying of a frozen sample can be performed by any relevant methodology, e.g., by lyophilization. Secondary drying can be performed by, e.g., continued freeze drying at a higher temperature in a vacuum chamber, contact exposure to temperature controlled surfaces, or by suspension of particles in a vortex or fluidized bed of temperature/ humidity controlled gas. A dried powder particle product can be recovered, e.g., from process containers, or by sizing and settling of particles from process gas streams. [0045] Other drying processes include, for example, air drying, desiccation under nitrogen purge (including grinding and sieving), freeze-drying (including milling and sieving), and supercritical fluid drying (SCF). The drying process can preserve the three dimensional structure of an antigen. For example, the process can preserve the structure of an influenza HA antigen, providing a high HA potency. [0046] After freeze drying, the powder can be stored (preserved) at a temperature of about 4 to 25 degrees C. The relative humidity of the preservation condition can be about 0% to 70%, about 0% to 60%, about 0% to 50%, about 0% to 40%, about 0% to 30%, about 0% to 20%, about 0% to 10%, or about 0% to 5%. The relative humidity of the preservation can be less than about 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 24%, 23%, 22%, 21%, 60%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. [0047] The water content of the powder after freeze drying can be about 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. The water content of the powder after freeze drying can be less than 12 %, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01%. [0048] Average particle diameter size of the powder generated after freeze drying can be about 5 to 100 micro m, about 5 to 60 micro m, or about 5 to 30 micro m. Average particle diameter size of the powder generated after freeze drying can be less than 24 WO 2011/129120 PCT/JP2011/002225 about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 micro m. [0049] IV. CARRIERS A powder produced by the freeze drying methods described herein can be blended with one or more additional components to generate a dry vaccine powder formulation. Such components include pharmaceutically acceptable carriers, for example, carriers appropriate for mucosal administration. Carriers suitable for mucosal administration can be physiologically acceptable substances such as microcrystalline cellulose. Micro crystalline cellulose can be a specific microcrystalline cellulose that has a larger specific surface area. Although any microcrystalline cellulose can be utilized, in some embodiments, the microcrystalline cellulose used to produce the vaccines of the present application can be Ceolus (registered trademark) PH-F20JP or Avicel(registered trademark) PH- 105. [0050] One way of defining powdered particles of a carrier, or the complete vaccine is based on average particle size. The average particle size of the microcrystalline cellulose and/ or vaccine particles can be measured by any means known in the art, for example, sifting, sieving or laser diffraction. The average particle size of the carrier (e.g., micro crystalline cellulose) and/or vaccine can be, e.g., about 10 micro m, 11 micro m, 12 micro m, 13 micro m, 14 micro m, 15 micro m, 16 micro m, 17 micro m, 18 micro m, 19 micro m, 20 micro m, 21 micro m, 22 micro m, 23 micro m, 24 micro m, 25 micro m, 26 micro m, 27 micro m, 28 micro m, 29 micro m, 30 micro m, 31 micro m, 32 micro m, 33 micro m, 34 micro m, 35 micro m, 36 micro m, 37 micro m, 38 micro m, 39 micro m, 40 micro m, 41 micro m, 42 micro m, 43 micro m, 44 micro m, 45 micro m, 46 micro m, 47 micro m, 48 micro m, 49 micro m, 50 micro m, 51 micro m, 52 micro m, 53 micro m, 54 micro m, 55 micro m, 56 micro m, 57 micro m, 58 micro m, 59 micro m, 60 micro m, 61 micro m, 62 micro m, 63 micro m, 64 micro m, 65 micro m, 66 micro m, 67 micro m, 68 micro m, 69 micro m, 70 micro m, 71 micro m, 72 micro m, 73 micro m, 74 micro m, 75 micro m, 76 micro m, 77 micro m, 78 micro m, 79 micro m, 80 micro m, 81 micro m, 82 micro m, 83 micro m, 84 micro m, 85 micro m, 86 micro m, 87 micro m, 88 micro m, 89 micro m, 90 micro m, 91 micro m, 92 micro m, 93 micro m, 94 micro m, 95 micro m, 96 micro m, 97 micro m, 98 micro m, 99 micro m, 100 micro m, 110 micro m, 120 micro m, 130 micro m, 140 micro m, 150 micro m, 160 micro m, 170 micro m, 180 micro m, 190 micro m, or 200 micro m. In some embodiments, microcrystalline cellulose used as a carrier for the vaccine com positions described herein can have an average particle size of 25 micro m, 39 micro m, or 57 micro m, measured by, for example, laser diffraction, sieving or sifting. [0051] A carrier (e.g., microcrystalline cellulose) and/or vaccine powder can be prepared to be of useful particle size distribution. Preparations of carrier and/or vaccine can have a particle size distribution of, for example 10 - 200 micro m, 20 - 200 micro m, 30 - 200 25 WO 2011/129120 PCT/JP2011/002225 micro in, 40 - 200 micro in, 50 - 200 micro m, 60 - 200 micro n, 70 - 200 micro m, 80 - 200 micro m, 90 - 200 micro m, 100 - 200 micro m, 110 - 200 micro m, 120 - 200 micro m, 130 - 200 micro m, 140 - 200 micro m, 150 - 200 micro m, 160 - 200 micro m, 170 - 200 micro m, 180 - 200 micro m, 190 - 200 micro m, or any included sub ranges of particle size distribution. Powders described herein can have a particle size additional particle size distributions, for example 10 - 100 micro m, 20 - 100 micro m, 30 - 100 micro m, 40 - 100 micro m, 50 - 100 micro in, 60 - 100 micro m, 70 - 100 micro m, 80 - 100 micro m, 90 - 100 micro m, 10 - 50 micro m, 10 - 60 micro M, 20 60 micro m, 30 - 70 micro m, 40 - 80 micro m, 50 - 90 micro m, 60 - 100 micro m, 70 110 micro m, 80 - 120 micro m, 90 - 130 micro m, 100 - 140 micro m, 110 - 150 micro m, 120 - 160 micro m, 130 - 170 micro m, 140 - 180 micro m, 150 - 190 micro m, 160 - 200 micro m, or any included sub-range of particle sizes. The carrier and/or vaccine can have a particle size distribution of, for example, 10 - 50 micro m, 11 - 50 micro m, 12- 50 micro m, 13 - 50 micro m, 14 - 50 micro m, 15 - 50 micro m, 16 - 50 micro m, 17- 50 micro m, 18 - 50 micro m, 19 - 50 micro m, 20 - 50 micro in., 21 - 50 micro m, 22 - 50 micro m, 23 - 50 micro in, 24 - 50 micro m, 25 - 50 micro m, 26 - 50 micro m, 27 - 50 micro m, 28 - 50 micro m, 29 - 50 micro m, 30 - 50 micro m, or any included sub-range of particle sizes. In a particular embodiment, the carrier and/or vaccine can have a particle size distribution of 19 - 60 micro m, or a particle size distribution of 19 - 50 micro m. [0052] A microcrystalline cellulose powder, or other carrier compound, useful for the preparation of vaccines described herein can be specified or unspecified, with regards to a particular physical aspect. For example microcrystalline cellulose powder can be specified to have larger particles, which can protect the lungs. A microcrystalline cellulose powder can be specified to have smaller particles, which can enhance the immune response. Physical characteristics of powders can be specified by sieving or otherwise processed to minimize the presence of particles that are, for example less than about 10 micro m, less than about 20 micro m, less than about 30 micro in, less than about 40 micro in, less than about 50 micro m, less than about 60 micro m, less than about 70 micro in, less than about 80 micro in, less than about 90 micro in, less than about 100 micro m and/or minimize particles that are greater than about 20 micro m, greater than about 30 micro m, greater than about 40 micro m, greater than about 50 micro in, greater than about 60 micro m, greater than about 70 micro m, greater than about 80 micro m, greater than about 90 micro m, greater than about 100 micro m, greater than about 110 micro m, greater than about 120 micro m, greater than about 130 micro m, greater than about 140 micro m, greater than about 150 micro m, greater than about 160 micro m, greater than about 170 micro m, greater than about 180 micro in, greater than about 190 micro m, or greater than about 200 micro in.

26 WO 2011/129120 PCT/JP2011/002225 [0053] An additional parameter of the powder compositions that can be varied to achieve desired results (e.g., enhanced immunogenicity) described herein is the specific surface area of the powder. For example, powder compositions can be prepared such that the specific surface area of the carrier (e.g., microcrystalline cellulose) and/or vaccine is, 1.0 m 2 /g, 1.1 m 2 /g, 1.2 m 2 /g, 1.3 m/g, 1.4 m/g, 1.5 m 2 /g, 1.6 M/g, 1.7 m 2 /g, 1.8 m 2 /g, 1.9 m 2 /g, 2.0 m 2 /g, 2.1 m 2 /g, 2.2 m 2 /g, 2.3 m 2 /g, 2.4 m 2 /g, 2.5 m 2 /g, 2.6 m 2 /g, 2.7 m 2 /g, 2.8 m/g, 2.9 m 2 /g, 3.0 m 2 /g, 3.1 m 2 /g, 3.2 m 2 /g, 3.3 m2/g, 3.4 M 2 /g, 3.5 m 2 /g, 3.6 m 2 /g, 3.7 m 2 /g, 3.8 m 2 /g, 3.9 m 2 /g, 4.0 m/g, 4.1 m 2 /g, 4.2 m 2 /g, 4.3 m 2 /g, 4.4 m 2 /g, 4.5 m 2 /g, 4.6 m 2 /g, 4.7 m 2 /g, 4.8 m 2 /g, 4.9 m 2 /g, 5.0 m/g, 5.1 m/g, 5.2 m 2 /g, 5.3 m 2 /g, 5.4 m 2 /g, 5.5 m 2 /g, 5.6 m 2 /g, 5.7 m 2 /g, 5.8 m 2 /g, 5.9 m 2 /g, 6.0 m 2 /g, 6.1 m 2 /g, 6.2 m 2 /g, 6.3 m 2 /g, 6.4 m 2 /g, 6.5 m 2 /g, 6.6 m 2 /g, 6.7 m 2 /g, 6.8 m 2 /g, 6.9 m 2 /g, 7.0 m 2 /g, 7.1 m 2 /g, 7.2 m 2 /g, 7.3 m 2 /g, 7.4 m 2 /g, 7.5 m 2 /g, 7.6 m 2 /g, 7.7 m/g, 7.8 m 2 /g, 7.9 m 2 /g, 8.0 m 2 /g, 8.1 M 2 /g, 8.2 m 2 /g, 8.3 m 2 /g, 8.4 m 2 /g, 8.5 m 2 /g, 8.6 m/g, 8.7 m 2 /g, 8.8 m 2 /g, 8.9 m 2 /g, 9.0 m 2 /g, 9.1 M/g, 9.2 m 2 /g, 9.3 m 2 /g, 9.4 m 2 /g, 9.5 m 2 /g, 9.6 m 2 /g, 9.7 m 2 /g, 9.8 m 2 /g, 9.9 m 2 /g, 10.0 m 2 /g, 10.1 m 2 /g, 10.2 m 2 /g, 10.3 m 2 /g, 10.4 m 2 /g, 10.5 M/g, 10.6 M 2 /g, 10.7 m2/g, 10.8 m2/g, 10.9 m 2 /g, 11.0 m 2 /g, 11.1 m 2 /g, 11.2 m 2 /g, 11.3 m 2 /g, 11.4 m/g, 11.5 m 2 /g, 11.6 M 2 /g 11.7 m 2 /g, 11.8 M/g, 11.9 M 2 /g, 12.0 m/g, 12.1 m 2 /g, 12.2 m 2 /g, 12.3 m 2 /g, 12.4 m 2 /g, 12.5 m 2 /g, 12.6 m 2 /g, 12.7 m/g, 12.8 m/g, 12.9 m 2 /g, 13.0 m 2 /g, 13.1 m 2 /g, 13.2 m 2 /g, 13.3 m 2 /g, 13.4 m 2 /g, 13.5 m 2 /g, 13.6 m 2 /g, 13.7 M 2 /g, 13.8 m 2 /g, 13.9 M 2 /g, 14.0 m 2 /g, 14.1 m 2 /g, 14.2 m 2 /g, 14.3 m 2 /g, 14.4 m/g, 14.5 m 2 /g, 14.6 m 2 /g, 14.7 M 2 /g, 14.8 m 2 /g, 14.9 m 2 /g, 15.0 m 2 /g, 15.1 m 2 /g, 15.2 m 2 /g, 15.3 m 2 /g, 15.4 m 2 /g, 15.5 M 2 /g, 15.6 m 2 /g, 15.7 m 2 /g, 15.8 m 2 /g, 15.9 m 2 /g, 16.0 m/g, 16.1 m 2 /g, 16.2 m 2 /g, 16.3 m 2 /g, 16.4 m 2 /g, 16.5 m 2 /g, 16.6 m 2 /g, 16.7 m 2 /g, 16.8 m/g, 16.9 m 2 /g, 17.0 mn/g, 17.1 m 2 /g, 17.2 m 2 /g, 17.3 m 2 /g, 17.4 m 2 /g, 17.5 m 2 /g, 17.6 m/g, 17.7 m 2 /g, 17.8 m 2 /g, 17.9 M 2 /g, 18.0 M 2 /g 18.1 m 2 /g, 18.2 m 2 /g, 18.3 m 2 /g, 18.4 m 2 /g, 18.5 m 2 /g, 18.6 m 2 /g, 18.7 mn 2 /g, 18.8 M/g, 18.9 m 2 /g, 19.0 m 2 /g, 19.1 m 2 /g, 19.2 m 2 /g, 19.3 m 2 /g, 19.4 m 2 /g, 19.5 M/g, 19.6 m 2 /g, 19.7 m 2 /g, 19.8 m 2 /g, 19.9 m 2 /g, or 20.0 m 2 /g. The specific surface are of the powder can be, for example, about 21 m 2 /g, 22 m 2 /g, 23 m 2 /g, 24 m 2 /g, 25 m 2 /g, 26 M 2 g, 27 m 2 /g, 28 m/g, 29 m 2 /g, 30 m 2 /g, 31 m 2 /g, 32 m 2 /g, 33 m 2 /g, 34 m 2 /g, 35 M 2 /g, 36 m 2 /g, 37 m 2 /g, 38 m 2 /g, 39 m 2 /g, 40 m 2 /g, 41 m 2 /g, 42 m 2 /g, 43 m 2 /g, 44 m 2 /g, 45 m/g, 46 m 2 /g, 47 m 2 /g, 48 m 2 /g, 49 m 2 /g, or 50 m 2 /g. In particular embodiments, the specific surface area of a carrier (e.g., microcrystalline cellulose) and/or vaccine powder can be equal to or less than 1.3 M/g, equal to or greater than 1.3 m 2 /g or can be about 2.3 m 2 / g. [00541 Still another parameter which can describe a powdered composition (carrier and/or vaccine) is by bulk density. In some embodiments, a powder used can have a bulk density range. A powder of the present invention can have a bulk density of, for example, 0.10 - 1.00 g/cM 3 , 0.10 - 0.90 g/cM, 0.10 - 0.80 g/cm 3 , 0.10 - 0.70 g/cm 3

,

27 WO 2011/129120 PCT/JP2011/002225 0.10 - 0.60 g/cm, 0.10 - 0.50 g/cm 3 , 0.10 - 0.40 g/cn 3 , 0.10 - 0.30 g/cr 3 , 0.20 - 1.00 g/ cml, 0.20 - 0.90 g/cm 3 , 0.20 - 0.80 g/cm 3 , 0.20 - 0.70 g/cm 3 , 0.20 - 0.60 g/cm 3 , 0.20 0.50 g/cm 3 , 0.20 - 0.40 g/cm 3 , 0.20 - 0.30 g/cm 3 , 0.30 - 1.00 g/cm 3 , 0.30 - 0.90 g/cm 3 , 0.30 - 0.80 g/cm 3 , 0.30 - 0.70 g/cm 3 , 0.30 - 0.60 g/cm 3 , 0.30 - 0.50 g/cm 3 , 0.30 - 0.40 g/ cm 3 , 0.40 - 1.00 g/cM 3 , 0.40 - 0.90 g/cm 3 , 0.40 - 0.80 g/cm 3 , 0.40 - 0.70 g/cm 3 , 0.40 0.60 glcm, 0.40 - 0.50 g/cm 3 , 0.50 - 1.00 g/cm 3 , 0.50 - 0.90 g/cm 3 , 0.50 - 0.80 g/cm 3 , 0.50 - 0.70 g/cm 3 , 0.50 - 0.60 g/cm 3 , 0.60 - 1.00 g/cm, 0.60 - 0.90 g/cM 3 , 0.60 - 0.80 g/ cm 3 , 0.60 - 0.70 g/cM 3 , 0.70 - 1.00 g/cm 3 , 0.70 - 0.90 g/cm 3 , 0.70 - 0.80 g/cm 3 , 0.80 1.00 g/cm 3 , 0.80 - 0.90 g/cm 3 , 0.9 - 1.0 g/cm3, or any included sub range. In particular embodiments, a carrier (e.g., microcrystalline cellulose) and/or vaccine powder with a bulk density of 0.13 - 2.9 g/cm 3 or 0.26 - 0.48 g/cm 3 can be used. In other em bodiments, a powder can have a particular bulk density of, for example 0.10 g/cm 3 , 0.11 g/cm 3 , 0.12 g/cm 3 , 0.13 g/cm 3 , 0.14 g/cm 3 , 0.15 g/cm 3 , 0.16 g/cm 3 , 0.17 g/cm 3 , 0.18 g/cm 3 , 0.19 g/cm 3 , 0.20 g/cm 3 , 0.21 g/cM 3 , 0.22 g/cm 3 , 0.23 g/cm 3 , 0.24 g/cm 3 , 0.25 g/cm 3 , 0.26 g/cm 3 , 0.27 g/cm 3 , 0.28 g/cm 3 , 0.29 g/cm 3 , 0.30 g/cm, 0.31 g/cm 3 , 0.32 g/cm 3 , 0.33 g/cm 3 , 0.34 g/cm 3 , 0.35 g/cm 3 , 0.36 g/cm 3 . 0.37 g/cm 3 , 0.38 g/cm 3 , 0.39 g/cm 3 , 0.40 g/cm 3 , 0.41 g/cm 3 , 0.42 g/cm 3 , 0.43 g/cm 3 , 0.44 g/cm 3 , 0.45 g/cm 3 , 0.46 g/cm 3 , 0.47 g/cm 3 , 0.48 g/cm 3 , 0.49 g/cM 3 , 0.50 g/cm 3 , 0.51 g/cm 3 , 0.52 g/cm 3 , 0.53 g/cm 3 , 0.54 g/cm 3 , 0.55 g/cm 3 , 0.56 g/cm 3 , 0.57 g/cm 3 , 0.58 g/cm 3 , 0.59 g/cm 3 , 0.60 g/cm3, 0.61 g/cm 3 , 0.62 g/cM 3 , 0.63 g/cm, 0.64 g/cm, 0.65 g/cm 3 , 0.66 g/cm 3 , 0.67 g/cm 3 , 0.68 g/cm 3 , 0.69 g/CM 3 , 0.70 g/cm 3 , 0.71 g/cm 3 , 0.72 g/cm 3 , 0.73 g/cm 3 , 0.74 g/cm 3 , 0.75 g/cm 3 , 0.76 g/cm 3 , 0.77 g/cm3, 0.78 g/cm 3 . 0.79 g/cmn, 0.80 g/cm', 0.81 g/cm , 0.82 g/cm 3 , 0.83 g/cm 3 , 0.84 g/cm 3 , 0.85 g/cm 3 , 0.86 g/cm 3 , 0.87 g/cm 3 , 0.88 g/cm 3 , 0.89 g/cm 3 , 0.90 g/cm 3 , 0.91 g/cm 3 , 0.92 g/cm 3 . 0.93 g/cm 3 , 0.94 g/cm 3 , 0.95 g/cm 3 , 0.96 g/cm 3 , 0.97 g/cm 3 , 0.98 g/cM 3 , 0.99 g/cm 3 , or 1.00 g/cm 3 . In some em bodiments, a carrier (e.g., microcrystalline cellulose) and/or vaccine powder can have a bulk density of 0.23 g/cm or 0.41 g/cm 3 . 10055] A carrier, such as microcrystalline cellulose, can comprise about 25% to about 98% of the mass of the dry vaccine powder formulation. In some embodiments, the carrier can comprise no more than about 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%. 30%, or 25% of a dry vaccine powder formulation. [0056] Another carrier useful in the vaccines of the present invention can be tribasic calcium phosphate (TCP). TCP can comprise about 0.5% to about 5% of the dry vaccine powder formulation. TCP can comprise no more than about 0.5%, 0.8%, 0.9%, 1%, 1.2%, 1.4%, 1.6%, 1.8%. 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 4%, or 5% of the dry vaccine powder formulation. [0057] A carrier can be added by blending, e.g., by vortexing. The duration of the blending, e.g., vortexing, can be about 30 secs to 120 min, about 30 secs to 2 min, about 30 secs 28 WO 2011/129120 PCT/JP2011/002225 to 7.5 min, about 20 sees to 15 min, about 30 secs to 30 min, about 30 sees to 45 min, about 30 secs to 60 min, about 30 secs to 75 min, about 30 sees to 90 min. about 30 sees to 120 min. The duration of the blending, e.g., vortexing, can be more than about 30 sees, 1 min, 2 min, 4 min, 8 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, or 120 min. The duration of the blending, e.g., vortexing, can be about 30 sees, 1 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, or 120 min. [0058] Upon blending the particle size of a dry vaccine formulation comprising freeze dried antigen-containing powder and a carrier can be any appropriate size for delivery of the dry powder vaccine to an anatomical site of interest. Additionally, size of the particles can be adjusted for different delivery devices. Thus, average particle diameter size of a dry powder vaccine formulation containing freeze dried antigen (e.g., influenza) and a carrier (e.g., microcrystalline cellulose) generated by the methods herein can be less than about 10 micro m, 11 micro in, 12 micro m, 13 micro m, 14 micro m, 15 micro m, 16 micro in, 17 micro m, 18 micro m, 19 micro m, 20 micro m, 21 micro m, 22 micro m, 23 micro m, 24 micro m, 25 micro in, 26 micro m, 27 micro m, 28 micro m, 29 micro m, 30 micro m, 31 micro in, 32 micro m, 33 micro m, 34 micro in, 35 micro m, 36 micro in, 37 micro m, 38 micro in, 39 micro m, 40 micro m, 41 micro m, 42 micro m, 43 micro m, 44 micro m, 45 micro m, 46 micro m, 47 micro m, 48 micro m, 49 micro m, 50 micro m, 51 micro m, 52 micro m, 53 micro m, 54 micro m, 55 micro m, 56 micro m, 57 micro m, 58 micro m, 59 micro m, 60 micro m, 61 micro m, 62 micro m, 63 micro in, 64 micro m, 65 micro in, 66 micro in, 67 micro m, 68 micro m, 69 micro m, 70 micro m, 71 micro in, 72 micro m, 73 micro m, 74 micro m, 75 micro m, 76 micro in, 77 micro m, 78 micro m, 79 micro m, 80 micro m, 81 micro m, 82 micro m, 83 micro m, 84 micro m, 85 micro in, 86 micro m, 87 micro in, 88 micro m, 89 micro m, 90 micro m, 91 micro m, 92 micro m, 93 micro m, 94 micro m, 95 micro m, 96 micro in, 97 micro m, 98 micro in, 99 micro m, 100 micro m, 110 micro m, 120 micro m, 130 micro m, 140 micro m, 150 micro in, 160 micro m, 170 micro m, 180 micro m, 190 micro m, or 200 micro m. [0059] The dry vaccine formulation comprising freeze dried antigen-containing powder and a carrier can have a range of particle sizes, for example 10 - 200 micro m, 20 - 200 micro m, 30 - 200 micro m, 40 - 200 micro m, 50 - 200 micro in, 60 - 200 micro m, 70 - 200 micro m, 80 - 200 micro m, 90 - 200 micro m, 100 - 200 micro m, 110 - 200 micro m, 120 - 200 micro m, 130 - 200 micro m, 140 - 200 micro m, 150 - 200 micro m, 160 - 200 micro m, 170 - 200 micro m, 180 - 200 micro m, 190 - 200 micro m, or any included sub-ranges of particle size. The dry vaccine formulation comprising freeze dried antigen-containing powder and a carrier can have a range of particle sizes, for example 10 - 100 micro m, 20 - 100 micro m, 30 - 100 micro m, 40 - 100 micro m, 50 - 100 micro in, 60 - 100 micro m, 70 - 100 micro m, 80 - 100 micro m, 90 - 100 29 WO 2011/129120 PCT/JP2011/002225 micro m, 10 - 50 micro m, 20 - 60 micro m, 30 - 70 micro in, 40 - 80 micro m, 50 - 90 micro m, 60 - 100 micro m, 70 - 110 micro m, 80 - 120 micro m, 90 - 130 micro m, 100 - 140 micro m, 110 - 150 micro m, 120 - 160 micro m, 130 - 170 micro m, 140 180 micro m, 150 - 190 micro m, 160 - 200 micro m, or any included sub-range of particle sizes. [0060] Dry vaccine formulation comprising freeze dried antigen-containing powder and a carrier can be specified by sieving or otherwise processed to minimize particles that are, for example less than about 10 micro m, less than about 20 micro m, less than about 30 micro m, less than about 40 micro m, less than about 50 micro m, less than about 60 micro m, less than about 70 micro m, less than about 80 micro m, less than about 90 micro m, less than about 100 micro m. and/or minimize particles that are greater than about 20 micro m, greater than about 30 micro m, greater than about 40 micro m, greater than about 50 micro m, greater than about 60 micro m, greater than about 70 micro m, greater than about 80 micro m, greater than about 90 micro m, greater than about 100 micro m, greater than about 110 micro m, greater than about 120 micro m, greater than about 130 micro m, greater than about 140 micro m, greater than about 150 micro m, greater than about 160 micro m, greater than about 170 micro m, greater than about 180 micro m, greater than about 190 micro m, or greater than about 200 micro m. [0061] V. STABILTY AND HYGROSCOPICITY A dry vaccine powder formulation prepared as described herein can be stable at room temperature (25 degrees C and 60% relative humidity) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. The stability of the dry vaccine powder formulation can also be stable under accelerated conditions (45degrees C and 75% relative humidity) for extended time periods. Under accelerated conditions, a dry vaccine powder formulation can be stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. A dry vaccine powder formulation prepared as described herein can be stable at other temperatures (e.g., -20 degrees C to 55 degrees C) and relative hu midities (0% to 100%). [0062] Stability, as used herein, can refer to several aspects of the dry vaccine powder under storage conditions. One such aspect is vaccine potency, i.e., retention of antigenicity of the antigenic component of the vaccine. This aspect of stability, for example, of a dry influenza vaccine powder formulation comprising HA, can be determined by measuring HA antigenicity. A vaccine powder is considered stable if it retains greater the 50% antigenicity (compared to initial potency) after a particular time under particular conditions (e.g., 18 months under accelerated conditions).

30 WO 2011/129120 PCT/JP2011/002225 [0063] Alternately, stability can refer to the ability of the dry powder to resist uptake of en vironmental water under storage conditions. Such uptake of water can lead to increased clumping, which, in turn can lead to undesirable properties such as decreased flowability and decreased bioavailability. [0064] A dry vaccine powder formulation described herein can have low hygroscopicity. Hygroscopicity of a dry vaccine powder formulation can be measured over time by weighing the dry vaccine powder formulation. An increase in weight indicates ac quisition of water. Hygroscopicity can be determined by the amount of water absorbed by the dry vaccine powders of the present invention when the powder is stored in an air-tight container, a non-air tight container or in an open system. [0065] VI. ROUTES AND MEANS OF ADMINSTRATION In some embodiments, a device can be configured to deliver a substantial fraction of a single dose of a dry vaccine powder therapeutic formulation into the nostril of a subject. In some cases, a device may be configured to deliver a substantial fraction of an amount of a dry vaccine powder therapeutic formulation residing within the device into the nostril of a subject. In some cases, a dry vaccine powder therapeutic for mulation or a substantial fraction thereof may be delivered after a single engagement of the device. In some cases, a powdered therapeutic formulation or a substantial fraction thereof can be delivered after multiple engagements of the device, such as for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 engagements. In some cases, multiple en gagements of a device may constitute a single use of a device. According to the methods, devices, and compositions described herein a substantial fraction of the dry vaccine powder therapeutic formulation delivered by the device encompasses at least 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9%, 99.95%, or 100% of the amount of dry powdered medicine therapeutic such as the amount in a single dose or the amount residing in the device. [0066] Nasal applicators appropriate for use with dry vaccine powder formulations generated by the methods of the provided invention are described in pending U.S. Ap plication Serial No. 61/260,367, which is herein incorporated by reference in its entirety. [0067] VII. EFFECTS OF DRY POWDER FORMULATION ON IMMUNITY The methods and compositions of the provided invention can be used to stimulate a local immune response. A local immune response can be in peripheral lymphoid tissue. For example, a vaccine dry powder formulation can be administered intranasally to stimulate mucosa-associated lymphoid tissue (MALT), which can play a role in mucosal immunity. Examples of mucosa include buccal mucosa, esophageal mucosa, gastric mucosa, intestinal mucosa, nasal mucosa, olfactory mucosa, oral mucosa, 31 WO 2011/129120 PCT/JP2011/002225 bronchial mucosa, uterine mucosa, endometrium (mucosa of the uterus), and penile mucosa. In particular, nasopharynx-associated lymphoid tissue (NALT) can be targeted. NALT can play a role in the generation of T helper 1 and T helper 2 cells, and IgA-committed B cells. Intranasal immunization can lead to the induction of antigen specific protective immunity in both the mucosal and systemic immune compartments. [0068] The methods and compositions of the provided invention can be used to stimulate production of the principal antibody of the mucosal immune system, secretory IgA (sIgA) (Figures 6, 7, 9, 11, 12 and 20). sIgA is a dimer or tetramer composed of two or four monomers, a J-chain polypeptide, and a polypeptide chain called secretory component. The J-chain polypeptide can facilitate the polymerization of both serum and secretory IgA. The secretory component is a 70 kDa polypeptide produced by ep ithelial cells of mucous membranes and can protect sIgA by making it less susceptible to proteolytic enzymes in mucous secretions. sIgA can be produced locally by mucosal plasma cells that are descended from precursors initially stimulated in organized, mucosal lymphoid organs designed for antigen sampling. After an initial trigger, the precursor cells can pass via regional lymph nodes, lymph, and blood to disseminate widely among mucosal sites, thus leading to protection at mucosal sites other than the site of administration (e.g., nasal administration). After secretion from a local plasma cell, sIgA can bind to an epithelial cell surface receptor, and the complex can pass through the epithelial cell into the secretions where it can serves as a nonphlogistic im munologic barrier to inhibit uptake of antigens. [0069] In addition to stimulating a mucosal (i.e., sIgA) response, the dry powder for mulations disclosed herein can also stimulate an IgG response (Figures 6, 7, 9, 11, 12, 14, 17 and 19). Such stimulation can lead to an additional layer of protection, for example by priming the humoral response to react to a pathogen which eludes or evades the protection provided by the sigA induced by a vaccine disclosed herein. Thus, in one embodiment, the vaccines disclosed herein can induce both mucosal and humoral antibody responses. Examples [0070] Example 1: Preparation and testing of whole inactive HIN dry vaccine powder for mulation In this example, various dry powder formulations of the seasonal flu vaccine (H1NI) are generated and tested. A preferred embodiment of the invention is also tested verses traditional liquid nasal and injection formulations of the seasonal flu vaccine. [0071] Example 1A: Preparation of influenza vaccine (H1N1) powders using non-quick freezing techniques In this experiment, various antigen stabilizers are used in a conventional freeze- 32 WO 2011/129120 PCT/JP2011/002225 drying process to generate vaccine powders, which are then examined for consistency and stability. In a 10 mL bottle, 0.4 mL of a 1.6 mg/mL solution of whole inactive influenza (HiNi, strain A/Brisbane/59/2007, The Chemo-Sero-Therapeutic Research Institute) is combined with a stabilizer (13.6 mg) in 0.4 mL of Phosphate Buffered Saline (PBS or phosphate buffer), pH 7.4, to give a final antigen to stabilizer ratio of 1:21. The mixture is slowly frozen at -40 degrees C for over 5 hours. The frozen com position is then freeze dried in four steps: -40 degrees C, less than 140 mtorr for 24 hr; 30 degrees C, less than130 mtorr for 24 hr; -10 degrees C, less thanl0O mtorr for 4 hr; and 20C, less than50 mtorr for 4 hr. The resulting lyophilized powder contains 29 micro g of influenza vaccine protein per 1 mg of influenza vaccine powder. The influenza vaccine powder is combined (blended) with nasal carriers (e.g., micro crystalline cellulose) with specific surface area of greater than 1.3 square meter per gram and tribasic calcium phosphate (TCP) (Ca 3 (PO4) 2 ). Influenza vaccine powder (49.3 mg, including 1.44 mg of influenza vaccine protein, is combined with 309.1 mg of Ceolus(registered trademark) PH-F20JP microcrystalline cellulose (mean particle size: 57 micro m; bulk density: 0.23 g/cm3; specific surface area: 2.3 m2/g), 40.0 mg of Ceolus(registered trademark)PH-301 microcrystalline cellulose (mean particle size: 39 micro m; bulk density: 0.41 g/cm 3 ), and 1.6 mg of TCP in a 10 mL glass bottle, and the components are blended using a vortex mixer for one minute. The resulting dry influenza vaccine powder formulation contains 90 micro g of influenza vaccine protein per 25 mg of dry influenza vaccine powder formulation. In one case, trehalose is used as the stabilizer to generate an influenza vaccine powder that is partially caked and stable HA potency. In another case, mannitol is used as the stabilizer to generate an influenza vaccine powder that comprises fine particles and has unstable HA potency. In yet another case, lactose is used as the stabilizer to generate an influenza vaccine powder that is a partially caked and has stable HA potency (Figure 1). In this example, stability is defined as retaining greater than 50 % HA potency after freeze drying; unstable is equal to, or less than, 50 % HA potency after freeze drying; results are summarized in Table 1. Because the formulations lack both full HA potency and good flowability, such approaches require improvements to produce effective and fully de liverable intranasal vaccines. [0072] [Table 1] Influenza (HINI) vaccine powder generated by non-quick freezing technique Antigen stabilizer Total protein of antigen ! Powder HA potency stabilizer ratio (by weight) Property Stable, >50%; Jnstabie 50% Trehalose 1: 21 Cake Stable Mannitol 1: 21 Fine Unstable Lactose 1: 21 Cake Stable [0073] Example 1B: Preparation of a nasal influenza (H1N1) vaccine powder using a quick 33 WO 2011/129120 PCT/JP2011/002225 freezing process In this experiment, various stabilizers are used in a quick freezing and drying process to generate vaccine powders, which are then examined for consistency and stability. The general manufacturing process is outlined in Figures 2 and 3; specific details relating to the generation of a H1N1 nasal vaccine formulation are provided infra. In a 10 mL bottle, 0.4 mL of a 1.6 mg/mL solution of whole inactive influenza (HIN1, strain A/Brisbane/59/2007) is combined with a stabilizer (13.6 mg) in 0.4 mL of Phosphate Buffered Saline (PBS or phosphate buffer), pH 7.4, to give a final antigen to stabilizer ratio of 1:21. The mixture is quickly frozen in liquid nitrogen for 10 minutes and an influenza powder is generated by a four step freeze-drying process: -40 degrees C, less thanl40 mtorr for 24 hr; -30 degrees C, less than130 mtorr for 24 hr; -10 degrees C, less than100 mtorr for 4 hr; and 20C, less than50 mtorr for 4 hr. The powder, which contains 29 micro g of influenza vaccine protein per 1 mg of influenza vaccine powder, is comprised of fine particles and is stable at room temperature, stability being defined as retaining greater than 50 % HA potency (Table 2). The influenza vaccine powder is combined (blended) with nasal carriers (e.g., micro crystalline cellulose) with specific surface area of greater than 1.3 square meter per gram and tribasic calcium phosphate (TCP) (Ca 3

(PO

4

)

2 ). Influenza vaccine powder (49.3 mg, including 1.44 mg of influenza vaccine protein and 30.60 mg of trehalose), is combined with 309.1 mg of Ceolus(registered trademark) PH-F20JP micro crystalline cellulose (mean particle size: 57 micro m; bulk density: 0.23 g/cm 3 ; specific surface area: 2.3 m 2 /g), 40.0 mg of Ceolus(registered trademark)PH-301 micro crystalline cellulose (mean particle size: 39 micro m; bulk density: 0.41 g/cm3), and 1.6 mg of TCP in a 10 mL glass bottle, and the components are blended using a vortex mixer for one minute. The resulting dry influenza vaccine powder formulation contains 90 micro g of influenza vaccine protein per 25 mg of dry influenza vaccine powder formulation. In one case, trehalose was used as the antigen stabilizer resulting in a for mulation with stable HA potency and fine particle size. In another case, lactose was used as the antigen stabilizer, which also produced a stable formulation consisting of fine particle size. Mannitol was not tested as an antigen stabilizer for HINi vaccine powders. [0074] [Table 2] Influenza (HINI) vaccine powder generated by quick freezing technique Antigen stabilizer Total protein of antigen / Powder HA potency stabilizer ratio (by weight) Property Stable, >50%; Unstable, _150% Trehalose 121 Fine Stable Mannitol not tested not tested not tested Lactose 1: 21 Fine Stable [0075] Example IC: Study design and results of nasal influenza vaccine powder formulation 34 WO 2011/129120 PCT/JP2011/002225 In this experiment, the ability of a dry powder HI NIvaccine to elicit an immune response is tested and compared to conventional nasal and injected liquid formulations. The vaccine is prepared using a quick freezing process and blended with micro crystalline cellulose carriers, as explained supra. In each condition, 0.09 mg of influenza vaccine protein (HiNi, strain A/Brisbane/59/2007, inactive whole influenza vaccine) was administered to 4 groups of cynomolgus monkeys. Cynomolgus monkeys have similar anatomy of nasal cavity and similar immune response as humans. Group 1 was administered 25 mg of nasal influenza (HiNi) vaccine powder formulation, prepared by the quick freezing process outlined supra, containing 0.09 mg influenza vaccine protein, 1.91 mg trehalose, 19.28 mg Ceolus(registered trademark) PH-F20JP, 2.50 mg Ceolus(registered trademark) PH-301, and 0.10 mg TCP; Group 2 was ad ministered 0.1 ml of nasal influenza vaccine solution containing 0.09 mg influenza vaccine protein; Group 3 was administered 0.1 ml of nasal influenza vaccine solution containing 0.09 mg influenza vaccine protein, 0.5 microL Tween 80 with 0.02 mg adjuvant alpha-galactosylceramide; and Group 4 was administered 0.5 mL of SC influenza vaccine solution containing 0.09 mg influenza vaccine protein. Vaccines were administered and samples were collected as described in Figure 4. Antibody levels were determined by hemagglutination inhibition (HI) and enzyme-linked im munosorbent assay (ELISA). [0076] Hemagglutination Inhibition (HI) antibody titers in serum and nasal wash samples were determined as follows. Samples were treated with Receptor Destroying Enzyme (RDE, Denka Seiken Co Ltd., Tokyo, Japan) for 15 to 18 hours at 37 degrees C and then heat inactivated for 1 hour at 56 degrees C. Two-fold serial dilution series of the samples were prepared, mixed with HIN1 (strain A/Brisbane/59/2007) HA antigen (Denka Seiken) at a concentration of 4 hemagglutination units per well, and incubated for 1 hour at room temperature. To each well, 50 micro L of a 0.5 % suspension of chicken red blood cells was added and hemagglutination was assessed an hour later. The highest dilution of sample that inhibits hemagglutination is the HI title of the sample. [0077] The results of HI testing of samples collected in this study are shown in Figures 5A and B, which contain tables of the HI titers produced by monkeys exposed to the different formulations of the whole inactive HI NI virus (strain A/Brisbane/59/2007) vaccine. The HI titers measured in serum samples are found in 5A; the HI titers measured in nasal wash samples are found in 5B. The SC injection vaccine (Group 4) produced the highest HI titers in the serum samples; however, no increase in HI titer was detected in nasal wash samples. Of the nasal preparations, the whole inactive nasal influenza (H1N1, strain A/Brisbane/59/2007) vaccine powder formulation produced the highest titers in both the serum and nasal wash samples, demonstrating a clear im- 35 WO 2011/129120 PCT/JP2011/002225 provement over the liquid formulations. Taken together, these results demonstrate that both serum and nasal wash HI titers were elevated in test group 1. [0078] Enzyme-linked immunosorbent assay (ELISA) antibody titers in serum and nasal wash samples were determined as follows. ELISA plates were coated with an antigen for 17 hours at 4 degrees C, washed, and blocked in 100 micro L of blocking solution (0.5 % bovine serum albumin in phosphate buffer) for 1 hour at room temperature. After washing, 2-fold serial dilutions of the test samples were made in 0.5% BSA and PBS and the dilutions were added to the wells of the ELISA plate. After an incubation at 37 degrees C for 1 hour, the plates were washed and incubated with a horseradish peroxidase (HRP)-conjugated goat anti-monkey IgG or HRP-conjugated sheep anti monkey secretory component detection antiserum for 1 hour at 37 degrees C. The plates were then washed, incubated with o-Phenylenediamine (OPD) for 15 minutes at 37 degrees C, and the color reaction stopped by the addition of 100 micro L of 1M sulfuric acid (HS0 4 ). The samples were measured by OD492 on an ELISA reader. [0079] The results of the ELISA antibody titers measured in the samples collected in this study are shown in Figures 6 and 7. Figure 6A and B are table of sIgA (5B) and IgG (5A) antibody titers produced by monkeys exposed to the different flu vaccine for mulations. Figure 7 provides a graphical representation of the data and indicating similar results from each animal tested (different animals indicated by different lines). SC Flu vaccine solution produced the most IgG among all test articles. Nasal influenza (HIN1, strain A/Brisbane/59/2007) vaccine powder formulation produced the most IgG among all nasal preparations. Nasal influenza vaccine powder formulation produced the most sIgA among all test articles. SC injected influenza vaccine produced the least sIgA among all test articles. Nasal influenza vaccine solution with adjuvant produced the least sIgA among all nasal preparations even though added the adjuvant. [0080] Example 1D: HI, IgG and sigA titers during the recovery period A subset of the animals from Example 1C was monitored following the end of the experiment to determine whether the elevated antibody titers were held. Serum and nasal wash samples were taken on day 80 (31 days after the last vaccination), day 101 (52 days post-vaccination) and day 115 (66 days post-vaccination). The results are found in Figures 8 and 9. Figure 8 contains a table of HI titers; Figure 9 contains a table of IgG and sigA titers. Antibody titer levels were held at high levels in the animal treated with the nasal powder formulation (Figures 8 and 9, Group 1). Antibody titer levels were held at a lower level in animals treaded with the nasal liquid formulation, without (Group 2) or with (Group 3) the addition of an adjuvant. IgG and HI titer levels in animals injected with a liquid formulation (Group 4) decreased remarkably throughout the recovery period; the levels of sIgA antibodies were not significantly raised in animals treated with the injected vaccine formulation.

36 WO 2011/129120 PCT/JP2011/002225 [0081] Example 1E: Survivability/challenge studies. In this example, the ability of the influenza vaccine to protect animals from subsequent challenge will be determined. Nasal challenge of the monkeys vaccinated in the previous experiment is performed 3 weeks after the final immunization. The animals are challenged with an embryonated-chicken-egg grown canine influenza (A/Brisbane/59/2007 IVR-148) virus. Each animal receives a total of approximately 10 7 TCID 5 0 of virus in a 2 ml volume. For mock challenge, the monkeys are challenged with 2 ml of virus-free allantoic fluid. As further controls, three un-vaccinated monkeys are exposed to 107 TCID 50 of virus or challenged with 2 ml of virus-free allantoic fluid. [0082] Animals from each group are monitored daily for body mass, hypothermia, general appearance and clinical symptoms. The monkeys are observed for flu-related clinical signs for 28 days post-challenge. All monkeys are fed with a standard diet and water is available ad libitum. For each group studied, nasal swabs and blood samples are taken at -7 days, 3 days, 7 days, 14 days and 28 days following the initial challenge. Antibody titers (sIgA and IgG) are determined for each animal. [0083] Example iF: Determining stability and hygroscopicity of a dry vaccine powder for mulation. In this example, the stability and hygroscopicity of a dry vaccine powder formulation will be examined. A dry, whole inactive Hi NI influenza vaccine powder formulation is generated by the methods of the provided invention. The stability of the vaccine powder formulation is tested at 45 degrees C and at 20 degrees C to 25 degrees C. The dry vaccine powder formulation to be tested is stored both in sealed bottles and in unsealed containers. Stability is measured by determining HA antigenicity. [0084] The hygroscopicity of a dry vaccine powder formulation is measured by determining the mass of a sample over time. To determine the effects of different environmental conditions on the hygroscopic stability of dry vaccine powders, 50 mg of vaccine powder is stored under various conditions. Samples of dry vaccine powder are stored under air-tight conditions, in a sealed container and an open container. The samples are weighed at monthly intervals for 6 months and weighed. An increase in weight indicates acquisition of water. [0085] Vaccine powder formulation stored for longer than 6 months are tested in a nasal delivery device. The percent of vaccine powder formulation delivered from the device is determined and compared to the percent of vaccine powder formulation freshly made. [0086] Example 2: Preparation and testing of whole inactive H5N1 dry vaccine powder for mulation In this example, various dry powder formulations of the avian flu vaccine (H5N 1) are 37 WO 2011/129120 PCT/JP2011/002225 generated and tested. A preferred embodiment of the invention is also tested verses tra ditional liquid nasal and injection formulations of the avian flu vaccine. [0087] Example 2A: Preparation of a nasal influenza (H5N 1) vaccine powder using a quick freezing process This example was performed to determine the optimal antigen stabilizer, and antigen to stabilizer ratio, for use in a quick freezing and drying process to generate a H5N I nasal vaccine powder. The general manufacturing process is outlined in Figures 2 and 3; specific details relating to the generation of a H5NI nasal vaccine formulation are provided infra. Four ratios of antigen to stabilizer were tested (1:11, 1:21, 1:49, and 1:101); the numbers cited infra correspond to the 1:49 ratio formulation. In a 10 mL bottle, 0.4 mL of a 0.526 mg/mL antigen solution containing whole inactive H5N1 virus (strain A/Vietnam/1 194/2004, Sinovac Biotech Ltd) is combined with 10.4 mg of a stabilizer (trehalose, mannitol, or lactose) in 0.4 mL phosphate buffer pH 7.2, to yield a final ratio of antigen to stabilizer of 1:49. The mixture is quickly frozen in liquid nitrogen for 10 minutes and an influenza powder is generated by a four step freeze drying process: -40 degrees C, less than140 mtorr for 24 hr; -30 degrees C, less than130 mtorr for 36 hr; -10 degrees C, less than100 mtorr for 4 hr; and 20 degrees C, less than50 mtorr for 4 hr. The resulting powder contains 11.2 micro g of antigen per 1 mg of powder. The influenza vaccine powder is combined (blended) with nasal carriers (e.g., microcrystalline cellulose) with specific surface area of greater than 1.3 square meter per gram and tribasic calcium phosphate (TCP) (Ca 3 (PO4) 2 ). Influenza vaccine powder (104 mg, including 1.2 mg of influenza vaccine protein, is combined with 254.4 mg of Ceolus (registered trademark) PH-F20JP microcrystalline cellulose (mean particle size: 57 micro m; bulk density: 0.23 g/cm3; specific surface area: 2.3 m2/g), 40.0 mg of Ceolus(registered trademark)PH-301 microcrystalline cellulose (mean particle size: 39 micro m; bulk density: 0.41 g/cm3), and 1.6 mg of TCP in a 10 mL glass bottle, and the components are blended using a vortex mixer for one minute. The resulting dry influenza vaccine powder formulation contains 58.9 micro g of influenza vaccine protein per 20 mg of dry influenza vaccine powder formulation. Use of trehalose, mannitol, and lactose as a stabilizer produce stable powders consisting of fine particles at the 1:21 and 1:49 antigen to stabilizer ratios. At the 1:101 antigen to stabilizer ratio, both trehalose and lactose containing formulations produced caked but stable powders; mannitol produced a stable powder consisting of fine particles at the 1:101 antigen to stabilizer ratio. Use of trehalose, mannitol, and lactose produced unstable formulations at antigen to stabilizer ratios of 1:11. The results are summarized in Table 3. [0088] 38 WO 2011/129120 PCT/JP2011/002225 [Table 3] Influenza (H5N1) vaccine powder generated by quick freezing technique. Total protein of antigen Trehalose Mannitol Lactose / stabilizer ratio Powder HA potency Powder HA potency Powder HA potency (by weight) Property stable:>so% Property stable:>50% Property table:>50% Unstable: 50% Unstable: 50% Unstable: 50% 1:11 Fine Unstable Fine Unstable Fine Unstable 1: 21 Fine Stable Fine Stable Fine Stable 1 : 49 Fine Stable Fine Stable Fine Stable 1 : 101 Cake Stable Fine Stable Cake Stable [0089] Example 2B: Study design and results of nasal influenza vaccine powder formulation In this experiment, the ability of a dry powder vaccine to elicit an immune response in cynomolgus monkeys, was tested and compared to conventional nasal and injected liquid formulations. Cynomolgus monkeys have similar anatomy of nasal cavity and similar immune response as humans. The dry powder vaccine was prepared from whole inactivated H5N1 (strain A/Vietnam/1 194/2004) antigen, using a quick freezing then freeze drying process, and blended with microcrystalline cellulose carriers as described supra. For every 20 mg of nasal influenza (H5N1) vaccine powder for mulation, 58.9 micro g whole inactive H5N 1 virus is delivered along with 2.9 mg trehalose, 12.7 mg Ceolus(registered trademark) PH-F2OJP, 2.0 mg Ceolus(registered trademark) PH-301, and 0.08 mg tribasic calcium phosphate. In each condition, 30 micro g of H5NI antigen was administered. Group 1 was administered 20 mg of nasal vaccine powder in each nostril (30 micro g total antigen); Group 2 was administered 0.15 mL nasal flu spray in each nostril (30 micro g total antigen); and Group 3 was ad ministered 0.3 mL of liquid vaccine by intramuscular injection (IM). Vaccines were administered and samples were collected according to the schedule in Figure 10. Samples were tested by enzyme-linked immunosorbent assay (ELISA) according to the methods outlined in Example 1. [0090] The results of the ELISA antibody titers measured in the samples collected in this study are shown in Figures I1 and 12. Figure 11 provides titers of sIgA (11B) and IgG (11 A) produced by monkeys exposed to the different flu vaccine formulations. Figure 12 provides a graphical representation of the data with different animals indicated by different lines. Animals vaccinated by injection with a liquid formulation (group 3) produced the highest IgG titers in the study; however, this same group produced levels of sIgA antibodies that were nearly undetectable. Animals vaccinated using a nasal liquid formulation (group 2) produced the lowest levels if IgG antibodies in this ex periment; this group also produced low levels of sIgA antibodies. Animals vaccinated with the nasal powder formulation (group 1) produced the highest levels of IgG an tibodies of the nasal vaccines; the nasal powder formulation also elicited the highest level of immune response as measured by sigA antibody levels. These results indicate that both sIgA and IgG antibody titers were successfully elevated in animals treated 39 WO 2011/129120 PCT/JP2011/002225 with the H5N1 nasal powder vaccine fornulation. [0091] Example 2C: Test method and results of stability test under stress conditions In this experiment, the stability of the dry powder H5N 1 vaccine formulation, prepared as described in Example 2A, is subjected to stress conditions and compared to a H5N1 nasal flu spray formulation. H5N1 influenza vaccine powder in encapsulated form was stored at 60 degrees C and 0% relative humidity and examined at two and three week time points. At two weeks, the powder consisted of fine particles; however, at three weeks, partial aggregation of the powder was observed. In another test, H5N1 influenza vaccine powder was loaded in a single use delivery device (Shin Nippon Biomedical Laboratory, LTD) and stored with an oxygen and moisture absorbing desiccant (PharmaKeep KC-20, Mitsubishi Gas Chemical Company, Inc.) in an aluminum canister at 60 degrees C and 75% relative humidity for two weeks, after which the powder still consisted of fine particles. In yet another test, H5N1 influenza vaccine powder was put in bottles and stored at 60 degrees C and 0% relative humidity and examined for HA potency at two and three week time points. At both times, the HA potency of the H5N1 nasal vaccine powder was stable. In another test of HA potency, H5N1 influenza vaccine powder was put in bottles and stored with an oxygen and moisture absorbing desiccant (PharmaKeep KC-20, Mitsubishi Gas Chemical Company, Inc.) at 60 degrees C and 75% relative humidity for two weeks, after which time the HA potency was determined to be stable. These results are summarized in Table 4. In contrast to the H5N1 nasal powder vaccine, H5N1 nasal spray vaccine, which was stored in a polypropylene microtube, lost all HA potency after two weeks at 60 degrees C. This demonstrates that increased stability at elevated temperature is achieved in the nasal powder formulation. [0092] [Table 4] H5N 1 influenza vaccine powder stress test results Powder Property HA Potency (Stable: > 50%, Unstable 5 50%) Time Encapsulated Loaded in Delivery Bottled Packaged t Device Initial Fine Particles Fine Particles Stable Stable 2 Weeks Fine Particles Fine Particles Stable Stable 3 Weeks Partially Aggregated Stable [0093] Example 3: Preparation and testing of a mixture of 3 HA split inactivated strain dry vaccine powder formulation In this example, various dry powder formulations of a nasal powder vaccine, containing a mixture of 3 split-inactivated strains (Hi N1 A/California/7/2009, H3N2 A/Victoria/210/2009, and B/Brisbane/60/2008 - collectively: "Trivalent HA influenza"), are generated and tested. [0094] Example 3A: Preparation of a Trivalent HA influenza vaccine powder using a quick 40 WO 2011/129120 PCT/JP2011/002225 freezing process This experiment was performed to determine the optimal antigen stabilizer, and antigen to stabilizer ratio, for use in a quick freezing and drying process to generate a Trivalent HA influenza nasal vaccine powder. The general manufacturing process is outlined in Figures 2 and 3; specific details relating to the generation of a Trivalent HA influenza nasal vaccine formulation are provided infra. Four ratios of antigen to stabilizer were tested (1:26, 1:56, 1:111, and 1:222); the numbers cited infra correspond to the 1:111 ratio formulation. In a 10 mL bottle, 0.6 mL of a >0.09 mg/mL antigen solution containing Trivalent HA influenza (H1N1 A/Califomia/7/2009, H3N2 A/Victoria/210/2009, and B/Brisbane/60/2008 , Denka Seiken Co Ltd) is combined with 6 mg of a stabilizer (trehalose, mannitol, or lactose) in 0.2 mL ultra pure water, to yield a final ratio of antigen to stabilizer of 1:111. The mixture is quickly frozen in liquid nitrogen for 10 minutes and an influenza powder is generated by a four step freeze-drying process: -40 degrees C, less than140 mtorr for 24 hr; -30 degrees C, less than130 mtorr for 36 hr; -10 degrees C, less than100 mtorr for 4 hr; and 20 degrees C, less than50 mtorr for 4 hr. The resulting powder contains >4.6 micro g of antigen per 1 mg of powder. The influenza vaccine powder is combined (blended) with nasal carriers (e.g., microcrystalline cellulose) with specific surface area of greater than 1.3 square meter per gram and tribasic calcium phosphate (TCP) (Ca 3 (P0 4

)

2 ). Influenza vaccine powder (97.75 mg, including 0.45 mg of influenza vaccine protein, is combined with 350.2 mg of Ceolus(registered trademark) PH-F20JP microcrystalline cellulose (mean particle size: 57 micro m; bulk density: 0.23 g/cm3; specific surface area: 2.3 m2/g), 50.0 mg of Ceolus(registered trademark)PH-301 microcrystalline cellulose (mean particle size: 39 micro m; bulk density: 0.41 g/cm3), and 2.0 mg of TCP in a 10 mL glass bottle, and the components are blended using a vortex mixer for one minute. The resulting dry influenza vaccine powder formulation contains >45 micro g of influenza vaccine protein per 25 mg of dry influenza vaccine powder formulation. Preparations using trehalose, mannitol, and lactose at an antigen to stabilizer ratio of 1:26 produced unstable powders consisting of fine particles. Both trehalose and lactose containing formulations produced stable powders with fine particle size at antigen to stabilizer ratios of 1:56 and 1:111; at these ratios, use of mannitol as the stabilizer yielded unstable HA potency with fine particle size. At an antigen to stabilizer ratio of 1:222, both trehalose and lactose containing formulations produced caked powders with stable HA potency; at the same ratio, the mannitol containing formulation produced a stable powder consisting of fine particles. The results are summarized in Table 5. [0095] 41 WO 2011/129120 PCT/JP2011/002225 [Table 5] Trivalent HA influenza vaccine powder generated by quick freezing technique. Conventional Trehalose Mannitol Lactose Freezing, and Particle HA potency Particle HA potency Particle HA potency DryingWeight ratio/ size Stable:>50% size Stable:>50% size Stable >50% Using excipients Unstable:o50% Unstable: o50% Unstable:<50% 1 :26 Fine Unstable Fine Unstable Fine Unstable 1 :56 Fine Stable Fine Unstable Fine Stable 1:111 Fine Stable Fine Unstable Fine Stable 1: 222 Cake Stable Fine Stable J Cake Stable [0096] Example 3B: Test method and results of stability test under stress conditions In this experiment, the stability of the dry powder Trivalent HA influenza vaccine formulation, prepared using a quick freezing process and blended with microcrystalline cellulose carriers, is tested under stress conditions and compared to a nasal spray Trivalent HA influenza vaccine formulation. Trivalent HA influenza vaccine powder in encapsulated form was stored at 60 degrees C and 0% relative humidity and examined at two and three week time points. At two weeks, the powder consisted of fine particles; however, at three weeks, partial aggregation of the powder was observed. In yet another test, Trivalent HA influenza vaccine powder was put in bottles and stored at 60 degrees C and 0% relative humidity and examined for HA potency at two and three week time points. At both times, the HA potency of the Trivalent HA nasal vaccine powder was stable. These results are summarized in Table 6. In contrast to the Trivalent HA nasal powder vaccine, nasal spray Trivalent HA nasal spray vaccine, which was stored in a polypropylene microtube, lost all HA potency after two weeks at 60 degrees C. This demonstrates that increased stability at elevated tem perature is achieved in the nasal powder formulation. [0097] [Table 6] Bottled Trivalent HA influenza nasal powder vaccine stress test results HA potency time Powder Consistency Stable:>50% Unstable: i50% Initial Fine Particles Stable 2 Weeks Fine Particles Stable 3 Weeks Partially Aggregated Stable [0098] Example 4: Preparation and testing of tetanus toxoid (TTx) dry vaccine powder for mulation In this example, various dry powder formulations of a tetanus toxoid (TTx) vaccine, are generated and tested. A preferred embodiment of the invention is also tested verses a traditional liquid injection formulation of the TTx vaccine. [0099] Example 4A: Preparation of a tetanus toxoid vaccine powder using a quick freezing process This experiment was performed to determine the optimal antigen stabilizer, and 42 WO 2011/129120 PCT/JP2011/002225 antigen to stabilizer ratio, for use in a quick freezing and drying process to generate a tetanus toxoid nasal vaccine powder. The general manufacturing process is outlined in Figures 2 and 3; specific details relating to the generation of a tetanus toxoid nasal vaccine formulation are provided infra. Five ratios of antigen to stabilizer were tested (1:26, 1:53, 1:111, 1:231, and 1: greater than 420); the numbers cited infra correspond to the 1:53 ratio formulation. In a 10 mL bottle, 0.5 mL of a less than0.08 mg/mL adsorbed tetanus toxoid antigen solution (Denka Seiken Co LTD) is combined with 2.1 mg of a stabilizer (trehalose, mannitol, or lactose) in 0.3 mL ultra pure water, to yield a final ratio of antigen to stabilizer of 1:53. The mixture is quickly frozen in liquid nitrogen for 10 minutes and an antigen powder is generated by a four step freeze drying process: -40 degrees C, less than140 mtorr for 24 hr; -30 degrees C, less than130 mtorr for 36 hr; -10 degrees C, less than100 mtorr for 4 hr; and 20 degrees C, less than50 mtorr for 4 hr. The resulting powder contains less than4.7 micro g of antigen per I mg of powder. The tetanus toxoid vaccine powder is combined (blended) with nasal carriers (e.g., microcrystalline cellulose) with specific surface area of greater than 1.3 square meter per gram and tribasic calcium phosphate (TCP) (Ca 3 (P0 4 )2). Tetanus toxoid vaccine powder (less than8.54 mg, including less than0.04 mg of antigen protein, is combined with 35.46 mg of Ceolus(registered trademark) PH-F20JP microcrystalline cellulose (mean particle size: 57 micro m; bulk density: 0.23 g/cm3; specific surface area: 2.3 m2/g), 5 mg of Ceolus(registered trademark)PH-301 micro crystalline cellulose (mean particle size: 39 micro m; bulk density: 0.41 g/cm3), and 0.2 mg of TCP in a 10 mL glass bottle, and the components are blended using a vortex mixer for one minute. The resulting dry tetanus toxoid vaccine powder formulation contains less than20 micro g of antigen protein per 25 mg of total powder. The use of trehalose, mannitol, and lactose produced antigen powders consisting of fine particles at antigen to stabilizer ratios of 1:26, 1:53, 1:105, and 1:210. At an antigen to stabilizer ratio of 1: 420, all three stabilizers (trehalose, mannitol, and lactose) produced caked powders. The results are summarized in Table 7. [0100] [Table 7] Tetanus toxoid vaccine powder generated by quick freezing technique. Trehalose Mannitol Lactose Total protein of antigen Powder Powder Powder / stabilizer ratio (by weight) Property Property Property 1: 26 Fine Fine Fine 1: 53 Fine Fine Fine 1:105 Fine Fine Fine 1: 210 Fine Fine Fine 1: 420 Cake Cake Cake [0101] Example 4B: Study design and results of nasal tetanus toxoid vaccine powder for mulation 43 WO 2011/129120 PCT/JP2011/002225 In this experiment, the ability of a tetanus toxoid nasal powder vaccine to elicit an immune response in cynomolgus monkeys, is tested and compared to a conventional injected liquid formulation. Cynomolgus monkeys have similar anatomy of nasal cavity and similar immune response as humans. The dry powder vaccine is prepared from adsorbed tetanus toxoid antigen, using a quick freezing then freeze drying process, and blended with microcrystalline cellulose carriers as described in Example 4A. For every 25 mg of nasal tetanus toxoid vaccine powder formulation, 2.5 Lf of adsorbed tetanus toxoid antigen is delivered along with 1.1 mg trehalose, 17.9 mg Ceolus(registered trademark) PH-F20JP, 2.6 mg Ceolus(registered trademark) PH-301, and 0.1 mg tribasic calcium phosphate. Multiple dosing levels are compared. Group I is administered 25 mg of nasal vaccine powder in each nostril (5 Lf dose); Group 2 is administered 25 mg of nasal vaccine powder two times in each nostril (10 Lf dose); Group 3 is administered 25 mg of nasal vaccine powder four times in each nostril (20 Lf dose); and, Group 4 is administered 2.0 mL liquid vaccine by subcutaneous injection (10 Lf dose). Vaccines are administered and samples are collected according to the schedule in Figure 13. Samples are tested by enzyme-linked immunosorbent assay (ELISA), according to the methods outlined in Example 1, and enzyme-linked immunosorbent spot (ELISpot). [0102] The ELISpot assay was performed as follows. Mouse anti-human/monkey interferon gamma (IFN gamma), monoclonal antibody, unconjugated, clone GZ-4 (15 micro g/ mL, MabTech, Sweden) was added to multiscreen plates (Millipore, USA) and incubated overnight at 4 degrees C. The next day, the plates were blocked with AIM-V (Life Technologies, USA) complete medium. 4X10 5 cells of peripheral blood mononuclear cells (PBMCs) separated from monkey whole blood and 25 mLf of Absorbed Tetanus Toxoid were added, and the plates incubated for 24 hour at 37 degrees C. The wells were then washed with PBS and 1 micro g/mL mouse anti-human IFN gamma, monoclonal antibody, biotinylated, clone 7-B6-1 (MabTech) was added. After 2 hour incubation at room temperature, the wells were washed with PBS. Strep tavidine-alkaline phosphatase diluted 1:1000 (MabTech) was added. After 1 hour in cubation at room temperature, the wells were washed with PBS. Staining was performed using 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium (BCTP/NBT-plus substrate) (Moss, USA). The plates were dried and numbers of spot in each well were evaluated on a scale of - to ++. The antibody titers measured in the samples collected in this study are shown in Figures 14 and 15. Figure 14A provides the absorbance ratio of serum IgG produced by monkeys exposed to the different flu vaccine formulations; Figure 14B displays those same results in graphical form. Figure 15 tabulates the results of ELISpot testing of the serum samples collected. The scoring in Figure 15 is as follows: (-) indicates 44 WO 2011/129120 PCT/JP2011/002225 negative control levels, (+/-) indicates low levels, (+) indicates medium levels, and (++) indicates high levels. In both the ELISA and ELISpot tests, the injected liquid for mulation of the TTx vaccine produced the greatest immune response. The 20 Lf dose of the nasal powder induced a detectable increase in IgG antibody titer during the study as measured by ELISA. The ELISpot measurements showed that all three doses of TTx Nasal powder vaccine were able to produce a dose-dependent immune response. [0103] Example 5: Preparation and testing of diphtheria toxoid (DTx) dry vaccine powder formulation In this example, various dry powder formulations of a diphtheria toxoid vaccine, are generated and tested. A preferred embodiment of the invention is also tested verses a traditional liquid injection formulation of the diphtheria toxoid vaccine. To test the stability of the diphtheria toxoid antigen during processing, the antigen powder produced by the quick freezing and freeze drying process detailed supra was re hydrated into a liquid formulation. This formulation will be referred to as reconstituted powder infra. [0104] Example 5A: Preparation of a diphtheria toxoid vaccine powder using a quick freezing process This experiment was performed to determine the optimal antigen stabilizer, and antigen to stabilizer ratio, for use in a quick freezing and drying process to generate a diphtheria toxoid nasal vaccine powder. The general manufacturing process is outlined in Figures 2 and 3; specific details relating to the generation of a diphtheria toxoid nasal vaccine formulation are provided infra. Five ratios of antigen to stabilizer were tested (2.5 Lf : 1.lmg, 2.5 Lf : 2.1 mg, 2.5 Lf : 4.2mg, 2.5 Lf : 8.4 mg and 2.5 Lf : 16.8 mg); the numbers cited infra correspond to the 2.5 Lf : 2.1 mg ratio formulation. In a 10 mL bottle, 0.5 mL of a less than 5 Lf/mL adsorbed diphtheria toxoid antigen solution (DTx, Research Institute for Microbial Disease, Osaka University) is combined with 2.1 mg of a stabilizer (trehalose, mannitol, or lactose) in 0.3 mL ultra pure water, to yield a final ratio of antigen to stabilizer of 2.5 Lf : 2.1 mg. The mixture is quickly frozen in liquid nitrogen for 10 minutes and an antigen powder is generated by a four step freeze-drying process: -40 degrees C, less than140 mtorr for 24 hr; -30 degrees C, less than130 mtorr for 36 hr; -10 degrees C, less thanl00 mtorr for 4 hr; and 20 degrees C, less than50 mtorr for 4 hr. The resulting powder contains less than 0.28 Lf of antigen per 1 mg of powder. The diphtheria toxoid vaccine powder is combined (blended) with nasal carriers (e.g., microcrystalline cellulose) with specific surface area of greater than 1.3 square meter per gram and tribasic calcium phosphate (TCP) (Ca 3

(PO

4

)

2 ). Diphtheria toxoid vaccine powder (1 mg, including less than0.28 Lf of antigen protein), is combined with 35.96 mg of Ceolus(registered trademark) PH-F20JP micro crystalline cellulose (mean particle size: 57 micro in; bulk density: 0.23 g/cm3; 45 WO 2011/129120 PCT/JP2011/002225 specific surface area: 2.3 m2/g), 5 mg of Ceolus(registered trademark)PH-301 micro crystalline cellulose (mean particle size: 39 micro m; bulk density: 0.41 g/cm3), and 0.2 mg of TCP in a 10 mL glass bottle, and the components are blended using a vortex mixer for one minute. The resulting dry diphtheria toxoid vaccine powder formulation contains less than 1.25 Lf of antigen protein per 25 mg of total powder. The results are summarized in Table 8. Use of trehalose, mannitol, or lactose generated powders consisting of fine particles at antigen to stabilizer ratios of 2.5 Lf : 1.1mg, 2.5 Lf: 2.1 mg, 2.5 Lf : 4.2mg and 2.5 Lf : 8.4 mg. At an antigen to stabilizer ratio of 2.5 Lf :16.8 mg, all three stabilizers used generated caked powders using this process. [0105] [Table 8] Diphtheria toxoid vaccine powder generated by quick freezing technique. Trehalose Mannitol Lactose Lf I Stabilizer Powder Powder Powder Property Property Property 2.5 LF / 1.1 mg Fine Fine Fine 2.5 LF /2.1 mg Fine Fine Fine 2.5 LF 14.2 mg Fine Fine Fine 2.5 LF / 8.4 mg Fine Fine Fine 2.5 LF / 16.8 mg Cake Cake Cake [01061 Example 5B: Study design and results of nasal diphtheria toxoid vaccine powder for mul ation In this experiment, the ability of a diphtheria toxoid nasal powder vaccine to elicit an immune response in cynomolgus monkeys, is tested and compared to a conventional injected liquid formulation and a reconstituted powder formulation. Cynomolgus monkeys have similar anatomy of nasal cavity and similar immune response as humans. The dry powder vaccine was prepared from adsorbed diphtheria toxoid antigen, using a quick freezing then freeze drying process, and blended with micro crystalline cellulose carriers as described supra. For every 25 mg of nasal diphtheria toxoid vaccine powder formulation, 1.25 Lf of diphtheria toxoid antigen is delivered along with 1.1 mg trehalose, 21.3 mg Ceolus(registered trademark) PH-F20JP, 3.0 mg Ceolus(registered trademark) PH-301, and 0.12 mg tribasic calcium phosphate. Group 1 was administered 25 mg of nasal vaccine powder in each nostril (2.5 Lf dose); Group 2 was administered 1.0 mL of liquid vaccine by subcutaneous injection (5 Lf dose); and, Group 3 was administered 1.0 mL of reconstituted powder vaccine by sub cutaneous injection (5 Lf dose). Vaccines were administered and samples were collected according to the schedule in Figure 16. Samples were tested by enzyme linked immunosorbent assay (ELISA), according to the methods outlined in Example 1. [0107] The antibody titers measured in this experiment are shown in Figures 17. Figure 17A is a table of the absorbance ratio of serum IgG; 17B is a bar graph (top) and line graph 46 WO 2011/129120 PCT/JP2011/002225 (bottom) of the data in 17A. The reconstituted powder fonnulation and the con ventional injected liquid formulations successfully induced an increase in serum IgG levels. The nasal powder formulation was also successful in increasing the IgG antibody titer, despite being administered at half the dose of the injected formulations. Taken together, these results indicate that the quick freeze drying methodology disclosed herein preserves diphtheria toxoid vaccine potency in animals. [0108] Example 6: Preparation and testing of ovalbumin dry vaccine powder formulation In this example, a dry powder formulation of ovalbumin (OVA, SIGMA A5503-IG) is generated and tested for the ability to elicit an immune response in cynomolgus monkeys. The nasally administered dry vaccine powder formulation is compared to traditional nasal and injected liquid formulations. The results demonstrate that nasal administration of an exemplary protein antigen using a formulation describe herein is capable of eliciting an immune response in animals. [01091 Example 6A: Preparation of an ovalbumin dry vaccine powder Three formulations of homogenized ovalbumin (hOVA) nasal powder are generated by blending different amounts of hOVA powder with nasal carriers (e.g., micro crystalline cellulose) with specific surface area of greater than 1.3 square meter per gram and tribasic calcium phosphate (TCP) (Ca 3

(PO

4

)

2 ). As hOVA is provided in powdered form, a quick freeze then freeze dry step was not needed. In formulation 1, 13.3 mg of hOVA powder is combined with 354.1 mg of Ceolus PH-F20JP, 40 mg Ceolus PH-301, and 1.6 mg of tribasic calcium phosphate (TCP), in a 10 mL bottle, and blended using a vortex mixer for one minute. The resulting mixture contains 1 mg antigen per 30 mg powder formulation. In formulation 2, 66.7 mg of hOVA powder is combined with 291.7 mg of Ceolus PH-F20JP, 40 mg Ceolus PH-301, and 1.6 mg of tribasic calcium phosphate (TCP), in a 10 mL bottle, and blended using a vortex mixer for one minute. The resulting mixture contains 5 mg antigen per 30 mg powder for mulation. In formulation 3, 200 mg of hOVA powder is combined with 158.4 mg of Ceolus PH-F20JP, 40 mg Ceolus PH-301, and 1.6 mg of tribasic calcium phosphate (TCP), in a 10 mL bottle, and blended using a vortex mixer for one minute. The resulting mixture contains 15 mg antigen per 30 mg powder formulation. [0110] Example 6B: Study design and results of nasal ovalbumin vaccine powder for mulation In this experiment, the ability of an ovalbumin nasal powder vaccine to elicit an immune response in cynomolgus monkeys, is tested and compared to conventional injected and nasal liquid formulations wherein hOVA was dissolved in phosphate buffer. Cynomolgus monkeys have similar anatomy of nasal cavity and similar immune response as humans. The dry powder vaccine was prepared from ho mogenized ovalbumin powder and blended with excipients as describe supra. Group 1 47 WO 2011/129120 PCT/JP2011/002225 was administered 30 ing of nasal vaccine powder formulation 1 in each nostril (2 mg dose); Group 2 was administered 30 mg of nasal vaccine powder formulation 2 in each nostril (10 mg dose); Group 3 was administered 30 mg of nasal vaccine powder for mulation 3 in each nostril (30 mg dose); Group 4 was administered 0.1 mL of liquid vaccine in each nostril (20 mg dose); Group 5 was administered 0.1 mL of liquid vaccine in each nostril (30 mg dose); Group 6 was administered 1.0 mL of liquid vaccine by subcutaneous injection (20 mg dose); and, Group 7 was administered 1.0 mL of liquid vaccine by subcutaneous injection (30 mg dose). Vaccines were ad ministered and samples were collected according to the schedule in Figure 18. Samples were tested by enzyme-linked immunosorbent assay (ELISA), according to the methods outlined in Example 1. [0111] The IgG antibody titers measured in the serum samples collected during this ex periment are shown in Figure 19. Figure 19A is a table of IgG antibody titers; 19B is a bar graph (top) and line graph (bottom) representation of the data in 19A. The nasal powder formulation and the injected liquid formulation were both capable of eliciting an immune response to similarly high levels; however, the highest titers were detected at an earlier time point in the animals treated with the nasal powder formulation. The nasal liquid formulation failed to elicit a detectable immune response as measured by IgG antibody titer. The sIgA antibody titers measured in this serum samples collected during this experiment are shown in Figure 20. Figure 20A is a table of sigA antibody titers; 20B is a bar graph (top) and line graph (bottom) representation of the data in 20A. Only the nasal powder formulation was capable of eliciting a detectable immune response as measured by sIgA antibody titer. No increase in sIgA titer was detected in animals vaccinated with either the nasal liquid or injected liquid formulations. Together, these results suggest that a nasal powder formulation described herein is capable of eliciting both mucosal and systemic immunogenicity in animals using an exemplary protein antigen. [0112 While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (18)

1. A method for generating an intranasal dry vaccine powder formulation comprising: a. preparing a liquid formulation comprising one or more antigens and one or more saccharides; b. quick freezing said liquid formulation to form a freeze-dried fine powder sample, wherein the quick freezing does not comprise spray freezing; and c. blending the freeze-dried fine powder sample with one or more excipients to generate the intranasal dry vaccine powder formulation.

2. The method of claim 1, wherein at least one of said one or more antigens is viral antigen.

3. The method of claim 1, wherein at least one of said one or more antigens is influenza virus.

4. The method of claim 1, wherein at least one of said one or more antigens is H1N1 influenza virus.

5. The method of claim 1, wherein at least one of said one or more antigens is H5N1 influenza virus.

6. The method of claim 1, wherein said one or more antigens comprises H1N1 influenza virus, H3N2 influenza virus, and Influenza B virus.

7. The method of claim 1, wherein at least one of said one or more antigens is live attenuated virus, whole inactivated virus, split virus, subunit antigen, virosome, or cold adapted live influenza virus.

8. The method of claim 1, wherein at least one of said one or more antigens is a bacterial antigen.

9. The method of claim 1, wherein at least one of said one or more antigens is killed whole bacteria, attenuated bacteria, toxoids, purified surface protein, or purified recombinant surface protein.

10. The method of claim 1, wherein at least one of said one or more antigens is tetanus toxoid. 49

11. The method of claim 1, wherein at least one of said one or more antigens is diphtheria toxoid.

12. The method of claim 1, wherein at least one of said one or more antigens is a protist antigen.

13. The method of claim 1, wherein at least one of said one or more antigens is a protein.

14. The method of claim 1, wherein at least one of said one or more saccharides is lactose.

15. The method of claim 1, wherein at least one of said one or more saccharides is trehalose.

16. The method of claim 1, wherein at least one of said one or more saccharides is mannitol.

17. The method of claim 1, wherein preparing said liquid formulation further comprises addition of one or more buffers.

18. The method of claim 17, wherein at least one of said one or more buffers is a phosphate buffer.