Methods of influenza vaccination and compositions for the same

Abstract

The present invention relates to compositions and methods for inducing a robust cross-strain immune protection against influenza infection in a subject. The methods comprise administering a prime-boost combination of vaccines intranasally and intramuscularly, wherein at least one of the administered vaccines is a nanoemulsion influenza vaccine.

Description

Atty. Dkt. No.: 038491-0359METHODS OF INFLUENZA VACCINATION AND COMPOSITIONS FOR THE SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 716464, filed November 5, 2024, and US Provisional Application No. 63 / 763701, filed February 26, 2025, the entire disclosures of which are incorporated herein by reference.BACKGROUND[0002| Influenza has been established as a serious human affliction that can cause localized epidemics and global pandemics of acute respiratory infections. Each year the influenza virus is responsible for 20,000 to 40,000 deaths and up to 300,000 hospitalization cases in the U.S., primarily the elderly, young children and immune-compromised patients. Sandhu et al., “Influenza in the Older Adult, Indications for the Use of Vaccines and Antiviral Therapy,” Geriatrics 56:223-231 (2001). In the pandemic of 1918, it is widely believed that in excess of 40 million people died.

[0003] Influenza is caused by RNA viruses of the family Orthomyxoviridae , which includes three species that cause disease in vertebrates, including humans. Of these three species, influenza A virus and influenza B virus are the most common disease agents in humans. Influenza A virus is the pathogen associated with all known flu pandemics and is currently the most virulent form of the virus. A number of distinct serotypes have been isolated, including H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2 and H10N7. These serotypes are classified according to two viral surface proteins, hemagglutinin (H or HA) and neuroaminidase (N or NA). Within these serotypes, isolates are further characterized by a standard nomenclature specifying virus type, geographical location where first isolated, sequential number of isolation, year of isolation, and HA and NA subtype. For instance, one such isolate is A / Wisconsin / 67 / 2005 (H3N2).

[0004] Due to the highly variable and mutable nature of influenza antigens, developing a broadly-1-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 effective influenza vaccine has proven difficult. At present, no reliable treatment is available for influenza, and vaccination is the most proven method for protecting against the disease and its serious complications. The annual influenza vaccine must be reformulated and readministered each year in anticipation of the serotypes of the virus predicted to be prevalent in a population each flu season, and is therefore considered a “seasonal” vaccine. Typically, the most common human vaccine is a combination of two influenza A subtypes and one influenza B strain.

[0005] Presently, the most common influenza vaccines are administered by injection, these include Fluvirin®, Afluria®, FluLaval®, Fluarix®, Agrippal®, Influvac®, Mastaflu®, and Fluzone®. FluMist® is an intranasal influenza vaccine currently approved in the U.S. for use in patients between the ages of 2 and 50 years of age. For children between the ages of 2 and 8 years of age, two doses are required for vaccination, which requires two visits to a health care provider and incurs more costs. Further, no influenza vaccine is available for elderly patients that is not administered by injection. Finally, storage requirements can greatly affect both the cost and availability of influenza vaccines, as the recall of Fluvirin® in 2006 due to improper storage temperatures created shortages of the vaccine in New England.

[0006] As with most vaccines, greater immunogenicity is also sought as it correlates with greater efficacy in humans. Prior influenza vaccines have typically utilized recombinant proteins (e.g., U.S. Pat. Nos. 7,192,595; 6,194,546; 5,962,298), as well as the addition of adjuvants such as aluminum (U.S. Pat. No. 6,861,244) and muramyldipeptide (U.S. Pat. No. 4,826,687) to compositions to increase the immunogenicity. However, there still exists a need to develop highly effective influenza vaccines with improved storage stability and ease of administration, which are characteristics of the nanoemulsion (NE) vaccines of the present disclosure.

[0007] Prior teachings related to nanoemulsion vaccines include US 11,806,318 for “Nanoemulsion Compositions for Preventing, Suppressing or Eliminating Allergic and Inflammatory Disease”; US 11,173,207 for “Novel Adjuvant Compositions”; US 11,173,197 for “Stabilized Anthrax Vaccine”; US 11,147,960 for “Herpes Simplex Virus Nanoemulsion Vaccine”; US 11,083,788 for “Nanoemulsion Compositions for Preventing, Suppressing or Eliminating Allergic and Inflammatory Disease”; US 10,596,251, for “Nanoemulsion RSV-2-4924-5417-0743.1Atty. Dkt. No.: 038491-0359Subunit Vaccine”; US 10,525,121, for “Influenza Vaccine and Methods of Using the Same”; US 10,206,996 for “Herpes Simplex Virus Nanoemulsion Vaccine”; US 10,138,279 for “Compositions and Methods for Anthrax Vaccination”; US 9,561,271 for “Nanoemulsion RSV subunit vaccine”; US 9,492,525 for “Human Respiratory Syncytial Virus Vaccine”; and US 9,144,606 for “Influenza Vaccine and Methods of Using the Same”; and US 2020-0405846 Al for “Emulsion Adjuvant for Intramuscular, Intradermal and Subcutaneous Administration”. However, none of these references teach the methods, compositions and kits of the present invention.

[0008] There remains a need in the art for methods of vaccination for influenza which provide broad and persistent protection, and the present disclosure satisfies this need.SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure provides a method for inducing an immune response to influenza in a subject comprising: (a) intranasally (IN) administering to a subject at least one dose of a priming vaccine comprising (1) a nanoemulsion comprising: (i) droplets having an average diameter of less than about 1000 nm; (ii) an aqueous phase; (iii) at least one pharmaceutically acceptable oil; (iv) at least one non-ionic surfactant; (v) at least one cationic surfactant; and (vi) at least one organic solvent which is an alcohol; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) intramuscularly (IM) administering to the subject at least one dose of a boosting vaccine comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, wherein administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.[0010[ In another aspect, the present disclosure provides a method for inducing an immune response to influenza in a subject comprising: (a) intramuscularly (IM) administering to the subject at least one dose of a priming vaccine comprising one or more influenza immunogens or -3-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) intranasally (IN) administering to a subject at least one dose of a boosting vaccine comprising (1) a nanoemulsion comprising: (i) droplets having an average diameter of less than about 1000 nm; (ii) an aqueous phase; (iii) at least one pharmaceutically acceptable oil; (iv) at least one non-ionic surfactant; (v) at least one cationic surfactant; and (vi) at least one organic solvent which is an alcohol; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, wherein administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.

[0011] In some embodiments, the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are the same. In some embodiments, the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are different.

[0012] In some embodiments, (a) at least two doses of the priming vaccine are administered to the subject; (b) at least two doses of the boosting vaccine are administered to the subject; (c) only one dose of the priming vaccine is administered to the subject; (d) only one dose of the boosting vaccine is administered to the subject; or (e) any combination thereof.

[0013] In some embodiments, the vaccine administered IM comprises a nanoemulsion influenza vaccine, which can be the same or different from the IN-administered nanoemulsion influenza vaccine.[0014| In some embodiments, a first dose of the boosting vaccine is administered at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11-4-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months or at least 18 months or more following administration of at least one dose of the priming vaccine.

[0015] In some embodiments, (a) sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a protective immune response; and (b) administration of at least one dose of the priming vaccine alone or at least one dose of the boosting vaccine alone, both administered at the same dose as when administered sequentially, does not result in a protective immune response.

[0016] In some embodiments, the protective immune response resulting from sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine is greater than the magnitude of a protective immune response achieved following: (a) one or more administrations of the priming vaccine alone at the same dose; and / or one or more administrations of the boosting vaccine alone at the same dose; or (b) at least two sequential administrations of the priming vaccine alone at the same dose; or at least two sequential administrations of the boosting vaccine alone at the same dose; or (c) at least three sequential administrations of the priming vaccine alone at the same dose; or at least three sequential administrations of the boosting vaccine alone at the same dose.

[0017] In some embodiments, administration of the priming vaccine enhances the production of antibodies against the one or more influenza immunogens or recombinant influenza proteins comprised in or produced from the boosting vaccine relative to that achieved following administration of the boosting vaccine in the absence of the priming vaccine.

[0018] In some embodiments, the immune response and / or protective immune response is measured by increased levels of cellular immunity, increased levels of humoral immunity, increased antibody titers, increased seroconversion rates, increased neutralization titers, or any combination thereof. In some embodiments, the humoral immunity is assessed by levels of IgG, IgA, IgM, IgE, or any combination thereof. In some embodiments, the cellular immunity is assessed by levels of antigen-specific cytotoxic T-lymphocytes, macrophages and / or one or more cytokines released in response to the influenza immunogen or protein. In some embodiments, the-5-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 immune response and / or protective immune response comprises a Thl and / or Th2 response.

[0019] In some embodiments, a protective immune response is generated against an influenza strain, wherein an antigen of the influenza strain is not present in either the priming or boosting vaccine.

[0020] In some embodiments, each of the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine is selected from the group consisting of: (1) inactivated influenza virus, a recombinant immunogenic variant of an inactivated influenza virus, or an immunogenic fragment of an inactivated influenza virus; (2) H5N1, a recombinant immunogenic variant of H5N1, or an immunogenic fragment of H5N1; (3) H1N1, a recombinant immunogenic variant of H1N1, or an immunogenic fragment of H1N1; (4) H1N2, a recombinant immunogenic variant of H1N2, or an immunogenic fragment of H1N2; (5) H3N2, a recombinant immunogenic variant of H3N2, or an immunogenic fragment of H3N2; (6) H2N2, a recombinant immunogenic variant of H2N2, or an immunogenic fragment of H2N2; (7)H7N7, a recombinant immunogenic variant of H7N7, or an immunogenic fragment of H7N7; (8) H9N2, a recombinant immunogenic variant of H9N2, or an immunogenic fragment of H9N2; (9) H7N2, a recombinant immunogenic variant of H7N2, or an immunogenic fragment of H7N2; (10) H7N3, a recombinant immunogenic variant of H7N3, or an immunogenic fragment of H7N3; (11) H10N7, a recombinant immunogenic variant of H10N7, or an immunogenic fragment of H10N7; (12) Hl, a recombinant immunogenic variant of Hl, or an immunogenic fragment of Hl; (13) H2, a recombinant immunogenic variant of H2, or an immunogenic fragment of H2; (14) H3, a recombinant immunogenic variant of H3, or an immunogenic fragment of H3; (15) H5, a recombinant immunogenic variant of H5, or an immunogenic fragment of H5; (16) H7, a recombinant immunogenic variant of H7, or an immunogenic fragment of H7; (17) H9, a recombinant immunogenic variant of H9, or an immunogenic fragment of H9; (18) Nl, a recombinant immunogenic variant of Nl, or an immunogenic fragment of Nl; (19) N2, a recombinant immunogenic variant of N2, or an immunogenic fragment of N2; (20) N3, a recombinant immunogenic variant of N3, or an immunogenic fragment of N3; (21) N7, a-6-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 recombinant immunogenic variant of N7, or an immunogenic fragment of N7; (22) a seasonal influenza strain, a recombinant immunogenic variant of a seasonal influenza strain, or an immunogenic fragment of a seasonal influenza strain; (23) a pandemic influenza strain, a recombinant immunogenic variant of a pandemic influenza strain, or an immunogenic fragment of a pandemic influenza strain; (24) an influenza A virus strain, a recombinant immunogenic variant of an influenza A virus strain, or an immunogenic fragment of an influenza A virus strain; (25) an influenza B virus strain, a recombinant immunogenic variant of an influenza B virus strain, or an immunogenic fragment of an influenza B virus strain; (26) an influenza C virus strain, a recombinant immunogenic variant of an influenza C virus strain, or an immunogenic fragment of an influenza C virus strain; (27) A / New Caledonia / 20 / 99 lineage; (28) A / Fujian / 411 / 2002 lineage; (29) A / Kumamoto / 102 / 2002 lineage; (30) A / Wyoming / 3 / 2003 lineage; (31) A / Wellington / 1 / 2004 lineage; (32) A / California / 7 / 2004 lineage; (33) A / New York / 55 / 2004 lineage; (34) A / Solomon Islands / 3 / 2006 lineage; (35) A / Wisconsin / 67 / 2005 lineage; (36) A / Hiroshima / 52 / 2005 lineage; (37) A / Brisbane / 10 / 2007 lineage; (38) A / Indonesia; (39) A / Vietnam; (40) B / Hong Kong / 330 / 2001 lineage; (41) B / Shandong / 7 / 97 lineage; (42) B / Hong Kong / 1434 / 2002 lineage; (43) B / Brisbane / 32 / 2002 lineage; (44) B / Shanghai / 361 / 2002 lineage; (45) B / Jiangsu / 10 / 2003 lineage; (46) B / Jilin / 20 / 2003 lineage; (47) B / Malaysia / 2506 / 2004 lineage; (48) B / Florida / 4 / 2006 lineage, (49) B / Victoria / 2 / 87 lineage; (50) B / Yamagata / 16 / 88 lineage; (51) C / Aichi / 1 / 99 lineage; (52) C / Sao Paulo / 378 / 82 lineage; (53) C / Yamagata / 26 / 81 lineage; (54) C / Aichi / 1 / 81 lineage; (55) C / Aomori / 74 lineage; (56) C / Mississippi / 80 lineage; (57) recombinant protein or immunogenic fragment thereof derived from influenza virus HA, NA, Ml, NP, M2e, HA stalk, and / or M2; (58) any new strain or subtype that may arise due to antigenic drift and / or mutation; or (59) any combination thereof.[00211 In some embodiments, the total influenza immunogen and / or influenza protein present: (a) in the priming vaccine is from about 1 pg to about 300 pg, per dose, including any value inbetween these two numbers; (b) in the priming vaccine from about 25 pg to about 100 pg, including any value in-between these two numbers; (c) in the boosting vaccine from about 20 pg to about 150 pg, per dose, including any value in-between these two numbers; (d) in the boosting-7-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 vaccine at about 25 pg, about 50 pg, about 90 pg, or about 100 pg per dose; or (e) any combination thereof.

[0022] In some embodiments, the pharmaceutically acceptable oil is selected from the group consisting of mineral oil, squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyl adipate, C12-15 alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate, Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil, Coconut oil, com oil, Cottonseed oil, Flaxseed oil, Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine flower oil, lavender flower oil, manuka flower oil, Marhoram flower oil, orange flower oil, rose flower oil, ylang- ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil, sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl alcohol, semi-synthetic derivatives thereof, and any combinations thereof. In some embodiments, the pharmaceutically acceptable oil is soybean oil.-8-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[00231 In some embodiments, the organic solvent is a C1-C12 alcohol. In some embodiments, the C1-C12 alcohol is ethanol.

[0024] In some embodiments, the non-ionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 80, polysorbate 20, or any combination thereof.

[0025] In some embodiments, the cationic surfactant is cetylpyridinium chloride (CPC).

[0026] In some embodiments, the nanoemulsion comprises: (a) droplets having an average diameter of less than about 1000 nm; (b) an aqueous phase; (c) about 1% to about 80% (v / v) soybean oil; (d) about 0.001% to about 10% (v / v) of at least one non-ionic surfactant, which is Polysorbate 80, Polysorbate 20, or a combination thereof; (e) less than about 5% (v / v) of the cationic surfactant cetylpyridinium chloride (CPC); and (f) about 0.01% to about 50% (v / v) ethanol.[0027| In another aspect, the present disclosure provides a combination of vaccines, comprising: (a) at least one dose of a priming vaccine formulated for intranasal (IN) administration, wherein the vaccine comprises: (1) a nanoemulsion comprising: (i) droplets having an average diameter of less than about 1000 nm; (ii) an aqueous phase; (iii) at least one pharmaceutically acceptable oil; (iv) at least one non-ionic surfactant; (v) at least one cationic surfactant; (vi) at least one organic solvent which is an alcohol; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) at least one dose of a boosting vaccine formulated for intramuscular (IM) administration, comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins.

[0028] In a further aspect, the present disclosure provides a combination of vaccines, comprising: (a) at least one dose of a priming vaccine formulated for IM administration, comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) at least one dose of a boosting vaccine formulated for IN administration,-9-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 wherein the vaccine comprises: (1) a nanoemulsion comprising: (i) droplets having an average diameter of less than about 1000 nm; (ii) an aqueous phase; (iii) at least one pharmaceutically acceptable oil; (iv) at least one non-ionic surfactant; (v) at least one cationic surfactant; (vi) at least one organic solvent which is an alcohol; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins.

[0029] In some embodiments, the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are the same. In some embodiments, the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are different.10030] In one embodiment, each of the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine is selected from the list above (e.g., (1) inactivated influenza virus, a recombinant immunogenic variant of an inactivated influenza virus, or an immunogenic fragment of an inactivated influenza virus; . . . or (58) any combination thereof).

[0031] In another embodiment, the pharmaceutically acceptable oil is selected from the group listed above (e.g., “mineral oil, . . .”), and in one aspect the oil is soybean oil. The organic solvent can be a C1-C12 alcohol, such as but not limited to ethanol, and the non-ionic surfactant can be a polysorbate, such as but not limited to polysorbate 80, polysorbate 20, or any combination thereof. In another aspect, the cationic surfactant is cetylpyridinium chloride (CPC).

[0032] Finally, in a further aspect, the nanoemulsion can comprise: (a) droplets having an average diameter of less than about 1000 nm; (b) an aqueous phase; (c) about 1% to about 80% (v / v) soybean oil; (d) about 0.001% to about 10% (v / v) of at least one non-ionic surfactant, which is Polysorbate 80, Polysorbate 20, or a combination thereof; (e) less than about 5% (v / v) cetylpyridinium chloride (CPC); and (f) about 0.01% to about 50% (v / v) ethanol.-10-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[00331 Both the foregoing summary and the following description of the drawings and detailed description are exemplary and explanatory. They are intended to provide further details of the disclosure, but are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS[0034J FIG. 1 is a schematic illustrating the vaccine dosing timeline for the experiment described herein. In particular, the figure details two IN influenza vaccine administrations at Days 0 and 29 (NE adjuvant + recombinant H5 (A / Indonesia, clade 2.1) antigen), followed by an IM influenza vaccine (H5N1 IIV (Sanofi Pasteur Inactivated Influenza Virus Vaccine, H5N1)) administration at Day 197. Each ImL dose of the Sanofi vaccine is formulated to contain 90 micrograms (pg) hemagglutinin (HA) of the influenza virus strain A / Vietnam / 1203 / 2004 (H5N1, clade 1). Serum antibody titers were assessed at days 1, 57, 197, and 225 following the first administration of the IN vaccine composition.[0035J FIGs. 2A-2B are panels of graphs illustrating the geometric mean antibody titers (GMT) observed of serum samples obtained from subjects in the study: Study Groups A, B, C, D, and E, with 8 human subjects assigned to each study group (arm). Groups A-C comprised subjects who were IN-administered a NE influenza primer vaccine, with Groups A-C differing in the amount of influenza antigen (recombinant H5 (A / Indonesia, clade 2.1) antigen) present in the nanoemulsion (NE) adjuvant; Group A = 25 rH5 pg, Group B = 50 rH5 pg, and Group C = 100 rH5 pg. Group D was a control group where a NE primer influenza adjuvant was not administered, and Group E was a control group where only placebo was administered. FIG. 2A is a panel of graphs showing the GMT observed against the Indonesia strain antigen for Groups A- E, and FIG. 2B is a panel of graphs showing the GMT observed against the Vietnam strain antigen for Groups A-E.[0036| FIGs. 3A-3B are graphs depicting the seroconversion rate achieved in human subjects for Groups A-E using the various tested dosing protocols. FIG. 3A shows the seroconversion rate achieved against the Indonesia strain antigen for Groups A-E, and FIG. 3B shows the-11-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 seroconversion rate achieved against the Vietnam strain antigen for Groups A-E.

[0037] FIGs. 4A-4E are graphs showing the effect of IN-nanoemulsion influenza vaccine primeboost dosing protocol on neutralization titers against numerous H5N1 strains, including influenza strains that were not present in either the 1N-NE influenza vaccine or the IM influenza vaccine. FIG. 4A is a graph illustrating the neutralization titers against H5 A / Indonesia (Clade 2.1) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with Group A showing the highest level of neutralization titers at Day 225. FIG. 4B is a graph illustrating the neutralization titers against H5N1 A / Vietnam (Clade 1) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with Group A showing the highest level of neutralization titers at Day 225, although Group D showed modest levels. FIG. 4C is a graph illustrating the neutralization titers against H5 A / Turkey (Clade 2.2) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with all of Groups A-C showing positive levels of neutralization titers, with Group A being highest, next Group B, and finally Group C. FIG. 4D is a graph illustrating the neutralization titers against H5 AZEgypt (Clade 2.2.1) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with all of Groups A-C showing positive levels of neutralization titers, with Group A being highest, next Group B, and finally Group C. FIG. 4E is a graph illustrating the neutralization titers against H5 A / Anhui (Clade 2.3.4) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with all of Groups A-C showing positive levels of neutralization titers, with Group A being highest, next Group B, and finally Group C.[0038| FIGs. 5A-5B are graphs showing the production of H5-binding antibodies in serum samples as a function of dosing group (Group A-E), measured by surface plasmon resonance (SPR). FIG. 5A is a graph illustrating the amount of H5 Indonesia HA 1 -binding antibodies present in a serum sample as a function of dosing for Groups A-E, and FIG. 5B is a graph illustrating the amount of H5 Vietnam HA1 -binding antibodies present in a serum sample as a-12-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 function of dosing for Groups A-E.

[0039] FIGs. 6A-6B are graphs showing the correlation between antibody production and neutralization titers achieved against the Indonesia and Vietnam strains. FIG. 6A is a graph showing the correlation between the neutralization titer against the Indonesia strain virus and production of H5 Indo HA 1-320 D-S RU-specific antibodies. FIG. 6B is a graph showing the correlation between the neutralization titer against the Vietnam strain virus and production of Vietnam HA 1 -320 RU-specific antibodies.

[0040] FIGs. 7A-7B are graphs showing H5N1 clade 2.1 serum and nasal wash binding antibody production by group. FIG. 7A shows the H5 A / Indonesia (Clade 2. l)-specific serum IgG and IgA responses, as well as H5 stalk-specific IgG responses (EU / mL) as individual values and geometric mean concentration with 95% confidence intervals. FIG. 7B shows individual nasal wash responses (IgG and IgA) as well as the median and interquartile range of the ratio of H5- specific IgG or IgA (EU / pg) to total IgG or IgA at each timepoint per group. Inset shows nasal wash responses on an extended y axis.[0041 J FIGs. 8A-8F are graphs showing antibody-dependent cell-mediated cytotoxicity (ADCC), memory B cell, and memory T cell responses by group. FIG. 8A shows the induction of antibodies with ADCC capacity as fold-changes over Day 1. FIG. 8B shows the frequency of memory B cells producing anti-H5 IgG antibodies, reported as SFU per 1x10e6 cells. The median and 95% CI are shown. Each dot represents an individual. FIG. 8C shows the frequency of memory CD4 T cells producing IL-2 (net %) upon ex-vivo stimulation with an H5 peptide pool (A / Vietnam / 1203 / 2004 (clade 1)). FIG. 8D shows data from IFN-y producing cells. FIG. 8E shows the ability of rH5-NE (pooled Groups A-Cs) to induce multifunctional (MF) cells at each timepoint of the study. Data are shown in violin plots, and each dot represents one volunteer. MF cells (IL-2+ & IFN-Y+) are shown by the white circles, IFN-y-only producing cells are shown by the blue circles and IL-2-only producing cells are shown by the grey circles. IL-2-only and IFN- y-only cells are referred to as Single Functional (SF) cells. FIG. 8F displays the frequency of MF and SF cells by Groups A-C at days 57 (post-intranasal vaccination) and 255 (post-systemic-13-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 boost). Statistics from FIGs. 8B-8E are derived from Wilcoxon signed-rank tests *p<0.05, **p<0.01, ***p<0.005, **** p<0.0001.

[0042] FIGs. 9A-9D are graphs showing CD4 T cell responses to rH5 (clade 2.1) antigen. FIG. 9A shows the basic gating strategy used to identify memory CD4 T cells using CD54RA and CD62L. Memory CD4 T cells were defined as CD4+ CD62L low / high and CD45RA low / high, excluding double positive CD62+CD45RA+ cells. FIG. 9B shows a representative example of activated (CD69+) cells producing IL-2 upon stimulation with media (mock stimulation) or the rH5 (clade 2.1) antigen. In FIG. 9C shows the frequency of memory CD4 T cells producing IL-2 (net %) upon ex-vivo stimulation with rH5 (clade 2.1) antigen. The data is displayed in Violin plots. FIG. 9D shows similar data but from IFN-y producing cells. Data from volunteers vaccinated with the low-dose (Group A), middle-dose (Group B) and high-dose (Group C) of rH5-NE are shown in maroon, orange and yellow. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group D), are shown in cyan and gray colors. Statistics from FIG. 9C- 9D are derived from signed-rank tests. *p<0.05.DETAILED DESCRIPTIONI. Overview[00431 The present disclosure is directed to the surprising and unexpected discovery that a specified prime-boost influenza vaccine protocol results in remarkable immunoprotection and cross-protective immunity. The vaccine protocol comprises administration of an influenza vaccine primer followed by administration of an influenza vaccine booster, where either the primer or boosting vaccine comprises a nanoemulsion influenza vaccine, and where administration comprises a combination of intranasal and intramuscular administration.

[0044] In a first aspect, the priming vaccine is an intranasal (IN) nanoemulsion influenza vaccine, and the boosting vaccine an intramuscular (IM) vaccination of a nanoemulsion or conventional influenza vaccine. In a second aspect, the priming vaccine is intramuscular (IM) vaccination of a nanoemulsion or conventional influenza vaccine and the boosting vaccine is an intranasal (IN) nanoemulsion influenza vaccine. Thus, the key aspects of the disclosure are (1)-14-4924-5417-0743.1Atty. Dkt. No.: 038491-0359IN + IM influenza vaccine administration, or IM + IN influenza vaccine administration, and (2) where at least one of the IN or IM vaccines is a nanoemulsion influenza vaccine formulation. Given the long-standing challenges relating to lack of broad cross-protection for influenza vaccines, the present disclosure addresses an urgent and long standing need.[0045 Current influenza vaccines, including the seasonal influenza vaccine, do not exhibit broad cross protection. This is problematic as there are a very large number of influenza strains, and the lack of cross protection means that typical seasonal influenza vaccines do not protect against exposure to all influenza strains. Typically, seasons influenza vaccines contain antigens for three different influenza strains, and the three strains are selected each year by The World Health Organization (WHO) and the Food and Drug Administration's (FDA) Vaccines and Related Biological Products Advisory Committee (VRBPAC). The WHO's flu vaccine strain-selection committee chooses three or four strains that are predicted to be the most common in circulation during the upcoming flu season. For example, in 2024, the WHO recommended aB / Phuket / 3073 / 2013 (B / Yamagata lineage)-like virus for quadrivalent vaccines in the southern hemisphere. The VRBPAC makes the final decision on which viruses will be used in the United States' domestic flu vaccines. For example, in 2024, VRBPAC recommended the following viruses for the 2024-2025 flu season: (a) Egg-based vaccines: A / Victoria / 4897 / 2022 (HlNl)pdmO9-like vims, A / Thailand / 8 / 2022 (H3N2)-like vims, and B / Austria / 1359417 / 2021 (B / Victoria lineage)-like vims; and (b) Cell- or recombinant-based vaccines:A / Wisconsin / 67 / 2022 (HlNl)pdmO9-like vims and A / Massachusetts / 18 / 2022 (H3N2)-like vims. The seasonal flu vaccine usually changes each year because scientists monitor the evolution of flu vimses and determine how they have mutated and spread.

[0046] The lack of broad cross-protection for current influenza vaccines results in lower vaccination rates because there is a public perception that flu vaccines “do not work.” Challenger et al., “Identifying reasons for non-acceptance of influenza vaccine,” BMC Health Serv. Res., 23A 61 (2023) (“Doubts about vaccine efficacy was prevalent”). Thus, to increase effective influenza vaccination, notably in high risk populations, there is a need for influenza vaccines and vaccine dosing protocols thereof exhibiting cross-strain protection. The NE compositions and-15-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 dosing protocols of the present invention provide solutions to this need.

[0047] It has been surprisingly found that the influenza prime-boost vaccine protocol described herein, comprising a combination of IN and IM administration, wherein at least one of the IN and IM administered influenza vaccines is a NE influenza vaccine, can dramatically increase the immune response of a subject to the immunogen provided in the boosting vaccine, even when the prime and boost vaccines contain influenza immunogens from different influenza strains. Prior to the present disclosure, robust cross-administration route, cross-strain immune response potentiation to influenza had not been observed.

[0048] The methods comprise IN or IM administering to a subject at least one dose of a nanoemulsion (NE) vaccine, wherein the NE vaccine comprises droplets having an average diameter of less than about 1000 nm. The NE vaccine further comprises (a) an aqueous phase, (b) at least one oil, (c) at least one surfactant, (d) at least one organic solvent, (e) at least one influenza immunogen or recombinant influenza protein, or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, and (f) optionally comprising at least one chelating agent, or any combination thereof. The NE vaccine can be utilized as the priming vaccine, the boosting vaccine, or both the priming and boosting vaccine.

[0049] The human or animal subject can produce a protective immune response after at least one administration of the IN-administered NE vaccine and the IM-administered vaccine. In one embodiment, the subject undergoes seroconversion after a single administration of the IN- administered NE vaccine and the IM-administered vaccine. In a further embodiment, the subject is selected from adults, elderly subjects, juvenile subjects, infants, high risk subjects, pregnant women, and immunocompromised subjects.

[0050] The NE compositions of the disclosure function as a vaccine adjuvant. Adjuvants serve to: (1) bring the antigen — the substance that stimulates the specific protective immune response — into contact with the immune system and influence the type of immunity produced, as well as the quality of the immune response (magnitude or duration); (2) decrease the toxicity of certain antigens; (3) reduce the amount of antigen needed for a protective response; (4) reduce the number of doses required for protection; (5) provide greater cross-reactivity and protection to -16-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 heterologous influenza strains; (6) enhance immunity in poorly responding subsets of the population and / or (7) provide solubility to some vaccines components.

[0051] Experimental Data: As detailed in the experimental section below, surprisingly and unexpectedly, a robust cross-strain prime-boost effect was achieved using a multi-dose vaccination protocol with multiple different administration routes. Further and also surprisingly, as shown in FIG. 4B, intranasal delivery of an adjuvanted vaccine containing a heterologous strain can enhance the immune response to an IM vaccine containing a second antigen. As detailed in FIG. 4C-4E, significant neutralization titers were observed against multiple strains that did not appear in either the IN or the IM vaccine. These data represent the first demonstration of a cross-administration route, cross-strain immune response potentiation. Similar to that observed regarding the neutralization titers, antibody production unexpectedly followed a dose-dependent inverse correlation; the lower the dose of the IN prime vaccine, the greater the concomitant antibody production. As shown in FIG. 6A and 6B, a positive correlation between neutralization titer and HAl-binding antibody production was observed.[00521 Taken together, these data demonstrate that intranasal administration of a nanoemulsion adjuvanted vaccine promote immunological memory, manifesting in enhanced homologous H5N1 neutralization titers and increased production of H5 HA1 binding antibodies. Moreover, the effect surprisingly follows an inverse correlation between intranasal vaccine dose and immune response. Finally, the data demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC) and CD4 T cell responses against rH5 clade 2.1, indicating that immune responses are capable of neutralization in human subjects.II. Prime / Boost Vaccine Strategies & Immune Responses

[0053] It is hypothesized that IN administration stimulates a different immune response than IM administration, and that the combination of IN and IM influenza vaccine administration, wherein at least one of the prime and boost vaccines is a NE vaccine, produces the surprising synergistic and unexpected protective and cross-reactive response described herein. This synergistic protective and cross-reactive immune response resulting from the combination of IN and IM--17-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 administered influenza vaccines, where at least one of the vaccines is a NE vaccine, was not known or described prior to the present disclosure.

[0054] Prime-boost strategies have been utilized prior to the present invention, including a homologous prime-boost approach, where the same formulation used in both the prime and boost regimens, and a heterologous prime-boost approach, which involve different formulations used in the prime and boost regimens. However, the exact mechanism of the immune response following a prime and boost vaccination, including the mechanisms of differentiation of primary and secondary B and T cells after prime and boost, are not well understood. Even less is known about the evolution of innate responses after a primary and secondary vaccine encounter. Classically, innate immunity provides a first line of defense against invading pathogens and shapes adaptive immunity, which takes more time to develop. However, innate responses can differ between prime and boost, because (1) specific antibodies (Abs), and memory T cells influence innate cells upon re-exposure, and (2) innate cells themselves can functionally and intrinsically differ. Most textbooks still describe similar innate responses after one or more stimulations, independently of the immunological history, because of the short life span of responding innate cells, and the lack of known immune memory in the innate compartment.Palgen et al., Front. Immunol., 12 FA27 7 (Mar. 2021).

[0055] Thus, Applicant’s discovery that broad cross-protective immunity results from use of a heterologous prime-boost vaccine strategy, utilizing two different types of administration (IM and IN), where at least one of the vaccines is a NE influenza vaccine, was highly unexpected and surprising.III. Summary of the Experimental Results

[0056] The experimental results detailed in Example 1 illustrate the surprising discovery that a synergistic immunoprotective and cross-protective response results from administration of a combination of a priming and boosting influenza vaccine administration, wherein at least one of the prime or boosting vaccine is a NE influenza vaccine and wherein administration comprises a combination of IN and IM administration. Moreover, the protective response exhibits broad-18-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 cross-protection against numerous influenza strains, including for strains not having an antigen present in the IN or IM-administered vaccines.

[0057] FIG. l is a schematic illustrating the vaccine dosing timeline for the experiment. In particular, the figure details two IN influenza vaccine administrations at Days 0 and 29, followed by an IM influenza vaccine administration at Day 197. Serum antibody titers were assessed at days 1, 57, 197, and 225 following the first administration of the IN-administered NE vaccine.

[0058] There were five different study groups (Groups A-E), with each study group containing 8 human subjects. The IN-administered NE vaccine contained varying amounts (25 (Group A), 50 (Group B), or 100 pg (Group C)) of recombinant H5 (A / Indonesia, clade 2.1) combined with a NE adjuvant. Group D was a control group where a NE primer influenza adjuvant was not administered, and Group E was a control group where only placebo was administered.

[0059] Various data regarding the immune response generated by each dosing protocol tested for Groups A-E was evaluated, including the geometric mean antibody titers (GMT) (FIGs. 2A-2B), seroconversion rate (FIGs. 3A-3B), neutralization titers against numerous H5N1 strains, e.g., evaluating the ability of the IN-NE + IM dosing protocol to generate cross-protection (FIGs. 4A- E), production of H5-binding antibodies (FIGs. 5A-5B), and the correlation between antibody production and neutralization titers (FIGs. 6A-6B).

[0060] In particular, FIGS. 2A and 2B detail antibody generation, and FIGS. 3A and 3B detail seroconversion, against an influenza strain with an antigen present in one of the two vaccines.

[0061] As shown in FIG. 2A and FIG. 3A, protection against the Indonesia strain following administration of only the boost IM Vietnam vaccine was essentially absent without administration of the IN priming vaccine (Group D). However, significant protection was observed in subjects that received the prime-boost protocol (Groups A, B, and C). Interestingly, of Groups A-C, Groups A and C showed the highest level of antibody production against both the Indonesia strain, demonstrating that antibody generation is not directly correlated with the amount of antigen present in a vaccine. This same phenomena was observed with the seroconversion data, with Groups A and C showing a higher level of seroconversion as-19-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 compared to Group B. This data further highlights the unexpected nature of the discovery described herein.

[0062] Additionally, as shown in FIG. 2B and FIG. 3B, protection against the Vietnam strain present in the IM vaccine was also observed following administration of the IM vaccine containing a Vietnam strain antigen and the intranasal rH5 non-adjuvanted control (Group D).[0063 J However, surprisingly and unexpectedly, in subjects who also received the NE- adjuvanted IN priming vaccine containing the Indonesia strain antigen, protection against the Vietnam strain was robustly enhanced. For example, as shown in FIG. 2B, subjects who received the prime-boost administration protocol with a low dose of the IM vaccine (25 mcg of Vietnam strain antigen; Group A) exhibited a 100% seroconversion rate with respect to the Vietnam strain antigen, whereas subjects who received only a high dose of the IM vaccine (100 mcg of Vietnam strain antigen with IN control; Group D) exhibited a seroconversion rate of under 60% - a staggering seroconversion different of 40%.

[0064] These results are the first to demonstrate a robust cross-strain prime-boost effect achieved using a multi-dose vaccination protocol with multiple different administration routes.[0065| As shown in FIG. 4, the multidose vaccination protocol resulted in measurable neutralization titers against homologous and heterologous H5N1 influenza strains. Surprisingly and unexpectedly, a dose-dependent inverse correlation was observed with respect to the neutralization titers. That is, the highest neutralization titers were observed at the lowest tested doses. This was observed for titers against H5 A / Indonesia (Clade 2.1) (FIG. 4A), H5N1 A / Vietnam (Clade 1) (FIG. 4B), H5 A / Turkey (Clade 2.2) (FIG. 4C), H5 A / Egypt (Clade 2.2.1) (FIG. 4D), and H5 A / Anhui (Clade 2.3.4) (FIG. 4E).

[0066] Also surprisingly, as shown in FIG. 4B, subjects who received the prime-boost vaccination protocol with the IN adjuvanted vaccine containing 25 ug of the Indonesia strain antigen exhibited higher neutralization titers against the Vietnam strain than observed in subjects who received the non-adjuvanted IN adjuvanted vaccine containing 100 ug of the Indonesia strain antigen, both groups of subjects having received the IM dose of the Vietnam strain-20-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 vaccine.

[0067] These data suggest that the IN delivery of an influenza vaccine containing a heterologous strain can enhance the immune response to an IM influenza vaccine containing a second antigen, where at least one of the vaccines is a NE vaccine.

[0068] As detailed in FIG. 4C-4E, significant neutralization titers were observed against multiple strains that did not appear in either the IN or the IM vaccine. These data represent the first demonstration of a cross-administration route, cross-strain immune response potentiation.

[0069] Moreover, as shown in FIG. 5A and FIG. 5B, the prime-boost administration protocol resulted in the production of antibodies capable of binding to H5 Indonesia HA1 and H5 Vietnam HA1 strain antigens, as measured by surface plasmon resonance (SPR). Unexpectedly, intranasal administration of the adjuvanted Indonesia strain vaccine significantly enhanced the production of antibodies that bind to the H5 Vietnam HA1 antigen.

[0070] Similar to that observed regarding the neutralization titers, antibody production unexpectedly followed a dose-dependent inverse correlation: the lower the dose of the IN prime vaccine, the greater the concomitant antibody production.[0071 J As shown in FIG. 6A and 6B, a positive correlation between neutralization titer and HA1 -binding antibody production was observed with respect to both the Indonesia and Vietnam strains.

[0072] Taken together, these data demonstrate that administration of a NE adjuvanted influenza vaccine, as part of a prime-boost influenza vaccine protocol, promotes immunological memory, manifesting in enhanced homologous neutralization titers and increased production of antigen binding antibodies. Moreover, the effect surprisingly follows an inverse correlation between intranasal vaccine dose and immune response.IV. Methods of Inducing an Immune Response10073] Provided herein are methods of inducing an immune response to influenza in a subject by IN administering to the subject at least one dose of a priming influenza vaccine, followed by IM-21-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 administering to the subject at least one dose of a boosting influenza vaccine, where at least one of the priming and boosting vaccines comprises a nanoemulsion. Both the priming and boosting influenza vaccines comprise one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins. In some embodiments, administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.

[0074] In some embodiments, the immune response and / or protective immune response is measured by increased levels of cellular immunity, increased levels of humoral immunity, increased antibody titers, increased seroconversion rates, increased neutralization titers, or any combination thereof. Humoral immunity can be assessed by determining levels of IgG, IgA, IgM, IgE, or any combination thereof, in a sample obtained from a subject, such as a blood sample. Cellular immunity can be assessed by levels of antigen-specific cytotoxic T- lymphocytes, macrophages, and / or one or more cytokines released in response to the immunogen or protein. In some embodiments, the immune response and / or protective immune response comprises a Thl and / or Th2 response.10075J In some embodiments, following IN administration of a NE vaccine, the IN NE vaccine composition generates a mucosal immune response. The mucosal immune response may be demonstrated by an increase in antigen-specific IgA and / or IgG levels in nasal washes. Increases in IgG levels in nasal washes which are not proportional to an increase in serum antigen-specific IgG levels are indicative of local production of antigen-specific IgG by B-cells homed to mucosal surfaces. In some embodiments, IN administration of the NE vaccine composition results in long-lasting mucosal immunity, such as a protective immune response at least 30 weeks, at least 40 weeks, at least 45 weeks, at least 50 weeks, at least 55 weeks, or at least 60 weeks or more following administration.

[0076] The immune response of the subject can be measured by determining the titer and / or presence of antibodies against the immunogen after administration of the priming and / or-22-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 boosting influenza vaccine to evaluate the humoral response to the immunogen. Seroconversion refers to the development of specific antibodies to an immunogen and may be used to evaluate the presence of a protective immune response. Such antibody-based detection is often measured using Western blotting or enzyme-linked immunosorbent (ELISA) assays or hemagglutination inhibition assays (HAI). Persons of skill in the art would readily select and use appropriate detection methods.

[0077] Another method for determining the subject’s immune response is to determine the cellular immune response, such as through immunogen-specific cell responses, such as cytotoxic T lymphocytes, or immunogen-specific lymphocyte proliferation assay. Additionally, challenge by the pathogen may be used to determine the immune response, either in the subject, or, more likely, in an animal model. A person of skill in the art would be well versed in the methods of determining the immune response of a subject and the invention is not limited to any particular method.

[0078] In some embodiments, following IN and / or IM administration the NE vaccine composition generates a humoral immune response.[0079| In some embodiments, the IN and / or IM administered influenza vaccine composition, which can be a NE influenza vaccine or a non-NE influenza vaccine, is administered to the subject as a single dose. In some embodiments, the influenza vaccine composition is administered to the subject in at least two separate doses. In some embodiments, the at least two separate doses are separated in time by at least about 1 hour, at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 6 months, at least one year, or longer. In some embodiments, the at least two separate doses are separated in time by at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, or more.

[0080] In some embodiments, the IN NE vaccine is administered before the IM vaccine composition, and therefore the IN NE vaccine a priming vaccine and the IM vaccine is a -23-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 boosting vaccine. In some embodiments, the IM vaccine is administered before the IN NE vaccine composition, and therefore the IM vaccine is a priming vaccine and the IN NE vaccine is a boosting vaccine.

[0081] In some embodiments, administration comprises administering at least one (e.g., 1, 2, 3, 4, 5, or more) dose of a priming vaccine and at least one (e.g., 1, 2, 3, 4, 5, or more) dose of a boost vaccine. In some embodiments, a priming vaccine and a boost vaccine are formulated with the same adjuvant. In some embodiments, a priming vaccine and a boost vaccine are formulated with different adjuvants.

[0082] In some embodiments, a vaccine formulated for IM administration comprises one or more adjuvants selected from aluminum, aluminum phosphate, aluminum hydroxide, AS01, AS02, AS03, AS04, 3-O-desacyl-4’-monophosphoryl lipid A (MPL), MF59, aluminum hydroxyphosphate sulfate, and AS01B, cGAS / stimulator of interferon genes (STING) agonist, CpG, lipid nanoparticle (LNP), double stranded RNA (dsRNA), GLA, CDN, and virus-like particle (VLP).

[0083] In some embodiments, a first dose of a boosting vaccine is administered, either IM or IN, at least about 1 week, at least about two weeks, at least about three weeks, at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months or at least about 18 months or more following administration of at least one dose of the priming vaccine.

[0084] In some embodiments, a subject is (a) IN administered at least two doses of a priming vaccine comprising a NE adjuvant and one or more influenza immunogens or recombinant influenza proteins, or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, each dose separated in time by at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, -24-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 at least about 4 weeks, at least about 1 month, at least about 2 months, or at least about 6 months; and (b) following (a), is IM administered at least one dose of a boosting vaccine comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins.

[0085] In another aspect, a subject is (a) IM administered at least one dose of a priming vaccine comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, and (b) following (a), IN administered at least two doses of a boosting vaccine comprising a NE adjuvant and one or more influenza immunogens or recombinant influenza proteins, or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, each dose separated in time by at least about 1 day, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, or at least about 6 months.

[0086] The boosting vaccine can be administered at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, or at least about 1 year or longer following administration of the first dose of the priming vaccine. In some embodiments, the boosting vaccine is administered at least about 6 months following administration of the first dose of the priming vaccine. In some embodiments, the priming vaccine is administered in two separate doses. In some embodiments, the two separate doses are separated in time by at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, or at least about one month. In some embodiments, the boosting vaccine is administered as a single dose. In some embodiments, the boosting vaccine is IM administered as a single dose. In some embodiments, the boosting vaccine is IM administered as a single dose, wherein the single dose is lower than the dose required to achieve the same physiological effect when the priming vaccine is not administered.

[0087] In some embodiments, the IN and IM vaccine compositions comprise or encode the same influenza antigen. In some embodiments, each of the IN and IM vaccine compositions comprise-25-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 or encode an antigen from the same influenza strain. In some embodiments, each of the IN and IM vaccine compositions comprise or encode an antigen from different influenza strains.

[0088] In some embodiments, each of the IN and IM vaccine compositions contain from about 1 pg to about 300 pg of one or more influenza antigens, or any amount in-between these two values. In some embodiments, the NE vaccine composition comprises at least about 25 pg, at least about 50 pg, at least about 90 pg, or at least about 100 pg of one or more influenza antigens.

[0089] Provided herein is a method for inducing an immune response to influenza in a subject comprising: (a) IN administering to a subject at least one dose of a priming vaccine comprising (1) a NE adjuvant; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) IM administering to the subject at least one dose of a boosting vaccine comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, wherein administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.

[0090] In another aspect, provided herein is a method for inducing an immune response to influenza in a subject comprising: (a) IM administering to the subject at least one dose of a priming vaccine comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) IN administering to a subject at least one dose of a boosting vaccine comprising (1) a NE adjuvant; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins, wherein administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.-26-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[00911 In some embodiments, the NE adjuvant comprises: (i) droplets having an average diameter of less than about 1000 nm; (ii) an aqueous phase; (iii) at least one pharmaceutically acceptable oil; (iv) at least one non-ionic surfactant; (v) at least one cationic surfactant which is cetylpyridinium chloride (CPC); and (vi) at least one organic solvent which is an alcohol.[0092J In some embodiments, the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are the same. In some embodiments, the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are different. If both the IM and IN vaccines comprise a NE, then the NE can be the same or different in the two vaccines.[0093J In some embodiments, (a) sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a protective immune response; and (b) administration of at least one dose of the priming vaccine alone or at least one dose of the boosting vaccine alone, both administered at the same dose as when administered sequentially, does not result in a protective immune response. In some embodiments, sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a protective immune response that is greater than the magnitude of a protective immune response achieved following one or more administrations of the boosting vaccine alone at the same dose. In some embodiments, the protective immune response resulting from sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine is greater than the magnitude of a protective immune response achieved following at least two sequential administrations of the boosting vaccine alone at the same dose. In some embodiments, the protective immune response resulting from sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine is greater than the magnitude of a protective immune response achieved following at least three sequential administrations of the boosting vaccine alone at the same dose. In some embodiments, intranasal administration of the priming vaccine enhances the production of antibodies against the one or-27-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 more influenza immunogens or recombinant influenza proteins comprised in or produced from the boosting vaccine relative to that achieved following administration of the boosting vaccine in the absence of the priming vaccine.V. Overview regarding nanoemulsion vaccines

[0094] Nanoemulsions (NE) are oil-in-water emulsions composed of nanometer sized droplets with surfactant(s) at the oil-water interface. Because of their size, the NE droplets are pinocytosed by dendritic cells triggering cell maturation and efficient antigen presentation to the immune system.

[0095] In one aspect, the dosing protocols of the present disclosure allow for a robust “priming” effect by an IN NE vaccine, resulting in less antigen and / or fewer IM administrations being required to elicit a protective immune response in a subject, when compared to a dosing protocol that does not include priming with an IN NE vaccine. In another aspect, the NE vaccine is utilized as a boosting vaccine.10096] The NE vaccine adjuvant can be combined with an antigen or the NE vaccine adjuvant can be sequentially administered with an antigen. Alternatively, or in combination, the NE vaccine adjuvant can be administered to a subject having exposure to an antigen (i.e., prophylactic exposure, environmental exposure, etc.). Furthermore, additional adjuvants may be added to the NE vaccine. The NE may be combined with one or more commercial influenza vaccines, such as Fluvirin® and Fluzone®, or the NE may be sequentially administered with one or more commercial influenza vaccines.

[0997] The NE adjuvant comprises an aqueous phase, at least one oil, at least one surfactant or detergent, at least one organic solvent, at least one immunogen, and optionally at least one chelating agent. In one embodiment, the surfactant present in the NE vaccine is a cationic surfactant. More than one surfactant can be present in the NE vaccines of the invention. For example, the NE vaccines can comprise a cationic surfactant in combination with a non-ionic surfactant, or in combination with an anionic, and / or zwitterionic, and / or cationic surfactant and / or any combination thereof.-28-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[00981 The NE vaccine can be administered to a subject which has not previously received an influenza vaccine, and the NE vaccine can be administered to a subject who has previously received an influenza vaccine.

[0099] In one embodiment, the NE adjuvant comprises droplets having an average diameter of less than about 1000 nm and: (a) an aqueous phase; (b) about 1% oil to about 80% oil; (c) about 0.1% to about 50% organic solvent; (d) about 0.001% to about 10% of a surfactant; or (e) any combination thereof. In another embodiment, the nanoemulsion vaccine comprises: (a) an aqueous phase; (b) about 1% oil to about 80% oil; (c) about 0.1% to about 50% organic solvent; (d) about 0.001% to about 10% of a surfactant; (e) at least one influenza immunogen, recombinant influenza protein, or a combination thereof, or (f) any combination thereof.[0100| The quantities of each component present in the NE adjuvant and / or NE vaccine refer to a therapeutic nanoemulsion and / or nanoemulsion vaccine.

[0101] The NE can be formed using classic emulsion forming techniques. See e.g., U.S. 2004 / 0043041. In an exemplary method, an oil is mixed with the aqueous phase under relatively high shear forces (e.g., using high hydraulic and mechanical forces) to obtain a NE comprising oil droplets having an average diameter of less than about 1000 nm. Some embodiments employ a NE having an oil phase comprising an alcohol such as ethanol. The oil and aqueous phases can be blended using any apparatus capable of producing shear forces sufficient to form an emulsion, such as French Presses or high shear mixers (e.g., FDA approved high shear mixers are available, for example, from Admix, Inc., Manchester, N.H.). Methods of producing such emulsions are described in U.S. Pat. Nos. 5,103,497 and 4,895,452.

[0102] The nanoemulsions are stable, and do not deteriorate even after long storage periods. Certain nanoemulsions are non-toxic and safe when swallowed, inhaled, or contacted to the skin of a subject. The NE adjuvants and vaccines can be produced in large quantities and are stable for many months at a broad range of temperatures.A. Safety Profiles[0103| At present, there is no U.S. FDA approved seasonal influenza vaccine that contains an-29-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 adjuvant. A US FDA approved adjuvant used in various vaccines is Alum. There are three general types of aluminum-containing adjuvants: Aluminum hydroxide, Aluminum phosphate, and Potassium aluminum sulfate (often called “Alum”). The US licensed vaccines for children that contain aluminum adjuvants include diphtheria-tetanus-pertussis vaccine, and diphtheriatetanus-acellular pertussis vaccine.[0104| In addition to potential neurological effects, aluminum-containing vaccines have frequently resulted in local reactions such as redness, swelling and / or tenderness at the injection site. More severe local reactions such as large areas of swelling, sterile abscesses, subcutaneous (SC) nodules (small lumps under the skin some of which have inflammation in the tissue), and allergic responses are less common. Eickhoff et al., (2002). Workshop summary: Aluminum in vaccines. Vaccine, 20(Supplet 3): S 1 -S4. In contrast to aluminum-based vaccine adjuvants, the nanoemulsion vaccine adjuvants of the invention contain no components having any known adverse reactions or toxicity.

[0105] In contrast to currently known vaccine adjuvants such as Alum, the NE vaccine adjuvants can be administered intranasally as well as intramuscularly. Vaccine adjuvants such as Alum can only be administered intramuscularly (IM). Moreover, unlike many vaccine adjuvants currently in development, the NE vaccine adjuvants do not produce an inflammatory response. Inflammation caused by a vaccine adjuvant can be highly undesirable, particularly for a nasally administered vaccine.[0106| In another embodiment, upon administration the NE vaccine adjuvant does not any inflammation, minimal inflammation, or nominal inflammation correlated with the vaccine adjuvant, where “nominal” is defined as less than a 5% increase in inflammation.

[0107] The NE vaccine adjuvant (such as Wso5EC -adjuvant) are composed of water (USP), an oil such as highly refined soybean oil (USP), an alcohol such as anhydrous ethanol (USP), and surfactants, such as polysorbate 80 (NF) and the cationic surfactant cetylpyridinium chloride (USP). The nanoemulsion vaccine adjuvants are inherently antimicrobial and undergo endotoxin and microbial limit testing. All the ingredients are included on the FDA list of inactive ingredients for Approved Drug Products.-30-4924-5417-0743.1Atty. Dkt. No.: 038491-0359B. Stability

[0108] The NE adjuvant and vaccine can be stable at about 40°C and about 75% relative humidity for a time period of at least up to about 2 days, at least up to about 2 weeks, at least up to about 1 month, at least up to about 3 months, at least up to about 6 months, at least up to about 12 months, at least up to about 18 months, at least up to about 2 years, at least up to about 2.5 years, or at least up to about 3 years.[0109| In another aspect, the NE adjuvant and vaccine can be stable at about 25°C and about 60% relative humidity for a time period of at least up least up to about 2 days, at least up to about 2 weeks, to about 1 month, at least up to about 3 months, at least up to about 6 months, at least up to about 12 months, at least up to about 18 months, at least up to about 2 years, at least up to about 2.5 years, or at least up to about 3 years, at least up to about 3.5 years, at least up to about 4 years, at least up to about 4.5 years, or at least up to about 5 years.[011.0] Further, the NE adjuvant and vaccine can be stable at about 4°C for a time period of at least up to about 1 month, at least up to about 3 months, at least up to about 6 months, at least up to about 12 months, at least up to about 18 months, at least up to about 2 years, at least up to about 2.5 years, at least up to about 3 years, at least up to about 3.5 years, at least up to about 4 years, at least up to about 4.5 years, at least up to about 5 years, at least up to about 5.5 years, at least up to about 6 years, at least up to about 6.5 years, or at least up to about 7 years.[Gill] The NE adjuvant and vaccine can be stable at about -20°C for a time period of at least up to about 1 month, at least up to about 3 months, at least up to about 6 months, at least up to about 12 months, at least up to about 18 months, at least up to about 2 years, at least up to about 2.5 years, at least up to about 3 years, at least up to about 3.5 years, at least up to about 4 years, at least up to about 4.5 years, at least up to about 5 years, at least up to about 5.5 years, at least up to about 6 years, at least up to about 6.5 years, or at least up to about 7 years.C. Nanoemulsion Vaccine Components[0U2J The NE adjuvant and vaccine comprises droplets having an average diameter size, less than about 1,000 nm, less than about 950 nm, less than about 900 nm, less than about 850 nm,-31-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 less than about 800 nm, less than about 750 nm, less than about 700 nm, less than about 650 nm, less than about 600 nm, less than about 550 nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, less than about 350 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, or any combination thereof. In one embodiment, the droplets have an average diameter size greater than about 125 nm and less than or equal to about 600 nm. In a different embodiment, the droplets have an average diameter size greater than about 50 nm or greater than about 70 nm, and less than or equal to about 125 nm.

[0113] In a further embodiment, the NE adjuvant and vaccine comprises a non-ionic surfactant, such as a polysorbate surfactant, which may be polysorbate 80 or polysorbate 20, and may have a concentration of about 0.01% to about 5.0 %, or about 0.1% to about 3% of polysorbate 80. The NE adjuvant and vaccine may further comprise a cationic surfactant. The NE vaccine may further comprise at least one preservative. In another embodiment of the invention, the nanoemulsion vaccine comprises a chelating agent.1. Aqueous Phase

[0114] The aqueous phase can comprise any type of aqueous phase including, but not limited to, water (e.g., H2O, distilled water, purified water, water for injection, de-ionized water, tap water) and solutions (e g., phosphate buffered saline (PBS) solution). In certain embodiments, the aqueous phase comprises water at a pH of about 4 to 10, preferably about 6 to 8. The water can be deionized (hereinafter "DiH20"). In some embodiments the aqueous phase comprises phosphate buffered saline (PBS). The aqueous phase may further be sterile and pyrogen free.2. Organic Solvents[0115| Organic solvents in the NE vaccines include, but are not limited to, C1-C12 alcohol, diol, triol, dialkyl phosphate, tri-alkyl phosphate, such as tri-n-butyl phosphate, semi-synthetic derivatives thereof, and combinations thereof. In one aspect of the invention, the organic solvent is an alcohol chosen from a nonpolar solvent, a polar solvent, a protic solvent, or an aprotic solvent.[0116| Suitable organic solvents for the NE vaccine include, but are not limited to, ethanol,-32-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 methanol, isopropyl alcohol, glycerol, medium chain triglycerides, diethyl ether, ethyl acetate, acetone, dimethyl sulfoxide (DMSO), acetic acid, n-butanol, butylene glycol, perfumers alcohols, isopropanol, w-propanol, formic acid, propylene glycols, glycerol, sorbitol, industrial methylated spirit, triacetin, hexane, benzene, toluene, diethyl ether, chloroform, 1,4-dixoane, tetrahydrofuran, dichloromethane, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, formic acid, semi-synthetic derivatives thereof, and any combination thereof.3. Oil Phase|0117] The oil in the NE vaccine can be any cosmetically or pharmaceutically acceptable oil. The oil can be volatile or non-volatile, and may be chosen from animal oil, vegetable oil, natural oil, synthetic oil, hydrocarbon oils, silicone oils, semi-synthetic derivatives thereof, and combinations thereof.

[0118] Suitable oils include, but are not limited to, mineral oil, squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyl adipate, C12-15 alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate, Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil, Coconut oil, com oil, Cottonseed oil, Flaxseed oil, Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf-33-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine flower oil, lavender flower oil, manuka flower oil, Marhoram flower oil, orange flower oil, rose flower oil, ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil, sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl alcohol, semi-synthetic derivatives thereof, and any combinations thereof.4. Surfactants

[0119] The surfactant in the NE vaccine can be a pharmaceutically acceptable ionic surfactant, a pharmaceutically acceptable nonionic surfactant, a pharmaceutically acceptable cationic surfactant, a pharmaceutically acceptable anionic surfactant, or a pharmaceutically acceptable zwitterionic surfactant.

[0120] Exemplary useful surfactants are described in Applied Surfactants: Principles and Applications. Tharwat F. Tadros, Copyright 82005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30629-3), which is specifically incorporated by reference.[01211 Further, the surfactant can be a pharmaceutically acceptable ionic polymeric surfactant, a pharmaceutically acceptable nonionic polymeric surfactant, a pharmaceutically acceptable cationic polymeric surfactant, a pharmaceutically acceptable anionic polymeric surfactant, or a pharmaceutically acceptable zwitterionic polymeric surfactant. Examples of polymeric surfactants include, but are not limited to, a graft copolymer of a poly(methyl methacrylate) backbone with multiple (at least one) polyethylene oxide (PEG) side chain, polyhydroxystearic acid, an alkoxylated alkyl phenol formaldehyde condensate, a polyalkylene glycol modified polyester with fatty acid hydrophobes, a polyester, semi-synthetic derivatives thereof, or combinations thereof.[01221 Suitable surfactants include, but are not limited to, ethoxylated nonylphenol comprising 9 to 10 units of ethyleneglycol, ethoxylated undecanol comprising 8 units of ethyleneglycol,-34-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, ethoxylated hydrogenated ricin oils, sodium laurylsulfate, a diblock copolymer of ethyleneoxyde and propyleneoxy de, Ethylene Oxide-Propylene Oxide Block Copolymers, and tetra-functional block copolymers based on ethylene oxide and propylene oxide, Glyceryl monoesters, Glyceryl caprate, Glyceryl caprylate, Glyceryl cocate, Glyceryl erucate, Glyceryl hydroxysterate, Glyceryl isostearate, Glyceryl lanolate, Glyceryl laurate, Glyceryl linolate, Glyceryl myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate, Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thiglycolate, Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate, Glyceryl disterate, Glyceryl sesuioleate, Glyceryl stearate lactate, Polyoxyethylene cetyl / stearyl ether, Polyoxyethylene cholesterol ether, Polyoxyethylene laurate or dilaurate, Polyoxyethylene stearate or distearate, polyoxyethylene fatty ethers, Polyoxyethylene lauryl ether, Polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, a steroid, Cholesterol, Betasitosterol, Bisabolol, fatty acid esters of alcohols, isopropyl myristate, Aliphati-isopropyl n- butyrate, Isopropyl n-hexanoate, Isopropyl n-decanoate, Isoproppyl palmitate, Octyldodecyl myristate, alkoxylated alcohols, alkoxylated acids, alkoxylated amides, alkoxylated sugar derivatives, alkoxylated derivatives of natural oils and waxes, polyoxyethylene poly oxypropylene block copolymers, nonoxynol-14, PEG-8 laurate, PEG-6 Cocoamide, PEG-20 methylglucose sesqui stearate, PEG40 lanolin, PEG-40 castor oil, PEG-40 hydrogenated castor oil, polyoxyethylene fatty ethers, glyceryl diesters, polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, and polyoxyethylene lauryl ether, glyceryl dilaurate, glyceryl dimystate, glyceryl distearate, semi -synthetic derivatives thereof, or mixtures thereof.[01231 Additional suitable surfactants include, but are not limited to, non-ionic lipids, such as glyceryl laurate, glyceryl myristate, glyceryl dilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, and mixtures thereof.

[0124] Nonionic surfactants include, but are not limited to, an ethoxylated surfactant, an alcohol ethoxylated, an alkyl phenol ethoxylated, a fatty acid ethoxylated, a monoalkaolamide-35-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 ethoxylated, a sorbitan ester ethoxylated, a fatty amino ethoxylated, an ethylene oxi de-propylene oxide copolymer, Bis(polyethylene glycol bis[imidazoyl carbonyl]), nonoxynol-9, Bis(polyethylene glycol bis[imidazoyl carbonyl]), BrijE35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL, Decaethylene glycol monododecyl ether, N- Decanoyl-N-methylglucamine, n-Decyl alpha-D-glucopyranoside, Decyl beta-D- maltopyranoside, n-Dodecanoyl-N-methylglucamide, n-Dodecyl alpha-D-maltoside, n-Dodecyl beta-D-maltoside, n-Dodecyl beta-D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, n- Hexadecyl beta-D-maltoside, Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-O-(N-heptylcarbamoyl)-alpha- D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-beta-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-l, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillaja bark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85, Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol, Tergitol, Type TMN-10, Tergitol, Type TMN-6, Tetradecyl-beta-D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether, Triethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Triethylene glycol monohexadecyl-36-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 ether, Triethylene glycol monooctyl ether, Triethylene glycol monotetradecyl ether, Triton CF- 21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton® X- 100, Triton® X-l 14, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45, Triton® X- 705-70, TWEEN® 20, TWEEN® 21, TWEEN® 40, TWEEN® 60, TWEEN® 61, TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85, Tyloxapol, n-Undecyl beta-D-glucopyranoside, semisynthetic derivatives thereof, or combinations thereof.10125] In addition, the nonionic surfactant can be a poloxamer. Poloxamers are polymers made of a block of polyoxyethylene, followed by a block of polyoxypropylene, followed by a block of polyoxyethylene. The average number of units of polyoxyethylene and polyoxypropylene varies based on the number associated with the polymer. For example, the smallest polymer, Poloxamer 101, consists of a block with an average of 2 units of polyoxyethylene, a block with an average of 16 units of poly oxypropylene, followed by a block with an average of 2 units of polyoxyethylene. Poloxamers range from colorless liquids and pastes to white solids. In cosmetics and personal care products, Poloxamers are used in the formulation of skin cleansers, bath products, shampoos, hair conditioners, mouthwashes, eye makeup remover and other skin and hair products. Examples of Poloxamers include, but are not limited to, Poloxamer 101, Poloxamer 105, Poloxamer 108, Poloxamer 122, Poloxamer 123, Poloxamer 124, Poloxamer 181, Poloxamer 182, Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, Poloxamer 407, Poloxamer 105 Benzoate, and Poloxamer 182 Dibenzoate.

[0126] Suitable cationic surfactants include, but are not limited to, a quarternary ammonium compound, an alkyl trimethyl ammonium chloride compound, a dialkyl dimethyl ammonium chloride compound, a cationic halogen-containing compound, such as cetylpyridinium chloride, Benzalkonium chloride, Benzalkonium chloride, Benzyldimethylhexadecylammonium chloride,-37-4924-5417-0743.1Atty. Dkt. No.: 038491-0359Benzyldimethyltetradecylammonium chloride, Benzyldodecyldimethylammonium bromide, Benzyltrimethylammonium tetrachloroiodate, Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium bromide, Dodecyltrimethylammonium bromide, Ethylhexadecyldimethylammonium bromide, Girard's reagent T, Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium bromide, N,N’,N’-Polyoxyethylene(10)-N-tallow-l,3-diaminopropane, Thonzonium bromide, Trimethyl(tetradecyl)ammonium bromide, l,3,5-Triazine-l,3,5(2H,4H,6H)-triethanol, 1- Decanaminium, N-decyl-N, N-dimethyl-, chloride, Didecyl dimethyl ammonium chloride, 2-(2- (p-(Diisobutyl)cresosxy)ethoxy)ethyl dimethyl benzyl ammonium chloride, 2-(2-(p- (Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ammonium chloride, Alkyl 1 or 3 benzyl-1- (2-hydroxethyl)-2-imidazolinium chloride, Alkyl bi s(2-hydroxy ethyl) benzyl ammonium chloride, Alkyl demethyl benzyl ammonium chloride, Alkyl dimethyl 3, 4-di chlorobenzyl ammonium chloride (100% C12), Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (50% Cl 4, 40% Cl 2, 10% Cl 6), Alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (55% Cl 4, 23% C12, 20% Cl 6), Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride (100% C14), Alkyl dimethyl benzyl ammonium chloride (100% C16), Alkyl dimethyl benzyl ammonium chloride (41% C14, 28% C12), Alkyl dimethyl benzyl ammonium chloride (47% C12, 18% C14), Alkyl dimethyl benzyl ammonium chloride (55% C16, 20% C14), Alkyl dimethyl benzyl ammonium chloride (58% C14, 28% C16), Alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12), Alkyl dimethyl benzyl ammonium chloride (61% CH, 23% C14), Alkyl dimethyl benzyl ammonium chloride (61% C12, 23% Cl 4), Alkyl dimethyl benzyl ammonium chloride (65% Cl 2, 25% Cl 4), Alkyl dimethyl benzyl ammonium chloride (67% Cl 2, 24% Cl 4), Alkyl dimethyl benzyl ammonium chloride (67% C12, 25% C14), Alkyl dimethyl benzyl ammonium chloride (90% C14, 5% C12), Alkyl dimethyl benzyl ammonium chloride (93% C14, 4% C12), Alkyl dimethyl benzyl ammonium chloride (95% Cl 6, 5% Cl 8), Alkyl dimethyl benzyl ammonium chloride, Alkyl didecyl dimethyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride, Alkyl dimethyl benzyl ammonium chloride (Cl 2- 16), Alkyl dimethyl benzyl ammonium chloride (Cl 2- 18), Alkyl dimethyl benzyl ammonium chloride, dialkyl dimethyl benzyl ammonium chloride, Alkyl-38-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 dimethyl dimethybenzyl ammonium chloride, Alkyl dimethyl ethyl ammonium bromide (90% Cl 4, 5% Cl 6, 5% Cl 2), Alkyl dimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as in the fatty acids of soybean oil), Alkyl dimethyl ethylbenzyl ammonium chloride, Alkyl dimethyl ethylbenzyl ammonium chloride (60% C14), Alkyl dimethyl isopropylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3% C18), Alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1% C12), Alkyl trimethyl ammonium chloride (90% C18, 10% C16), Alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18), Di-(C8-10)-alkyl dimethyl ammonium chlorides, Dialkyl dimethyl ammonium chloride, Dialkyl methyl benzyl ammonium chloride, Didecyl dimethyl ammonium chloride, Diisodecyl dimethyl ammonium chloride, Dioctyl dimethyl ammonium chloride, Dodecyl bis (2-hydroxyethyl) octyl hydrogen ammonium chloride, Dodecyl dimethyl benzyl ammonium chloride, Dodecylcarbamoyl methyl dinethyl benzyl ammonium chloride, Heptadecyl hydroxyethylimidazolinium chloride, Hexahydro-1,3,5 - tris(2-hydroxyethyl)-s-triazine, Hexahydro-1, 3, 5-tris(2-hydroxyethyl)-s- triazine, Myristalkonium chloride (and) Quat RN1UM 14, N,N-Dimethyl-2- hydroxypropylammonium chloride polymer, n-Tetradecyl dimethyl benzyl ammonium chloride monohydrate, Octyl decyl dimethyl ammonium chloride, Octyl dodecyl dimethyl ammonium chloride, Octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride, Oxydiethylenebis(alkyl dimethyl ammonium chloride), Quaternary ammonium compounds, dicoco alkyldimethyl, chloride, Trimethoxysily propyl dimethyl octadecyl ammonium chloride, Trimethoxy silyl quats, Trimethyl dodecylbenzyl ammonium chloride, semi-synthetic derivatives thereof, and combinations thereof.

[0127] In one embodiment, the NE adjuvant or vaccine comprises a cationic surfactant which is cetylpyridinium chloride (CPC). CPC may have a concentration in the nanoemulsion and / or nanoemulsion vaccine of less than about 5.0% and greater than about 0.001%, or further, may have a concentration of less than about 5%, less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.90%, less than about 0.80%, less than about 0.70%, less than about 0.60%, less than about 0.50%, less than about 0.40%, less than about 0.30%, less than about 0.20%, less than about 0.10%, greater than about 0.001%, greater than about 0.002%, -39-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 greater than about 0.003%, greater than about 0.004%, greater than about 0.005%, greater than about 0.006%, greater than about 0.007%, greater than about 0.008%, greater than about 0.009%, and greater than about 0.010%.

[0128] In another embodiment of the invention, the NE adjuvant or vaccine comprises at least one cationic surfactant and at least one non-cationic surfactant. The non-cationic surfactant can be a nonionic surfactant, such as a polysorbate (Tween), such as polysorbate 80 or polysorbate 20. Tn one embodiment, the non-ionic surfactant is present in a concentration of about 0.01% to about 5.0%, or the non-ionic surfactant is present in a concentration of about 0.1% to about 3%. In yet another embodiment of the invention, the nanoemulsion vaccine comprises a cationic surfactant present in a concentration of about 0.01% to about 2%, in combination with a nonionic surfactant.5. Additional Ingredients

[0129] Additional compounds suitable for use in the NE adjuvant or vaccine include but are not limited to one or more solvents, such as an organic phosphate-based solvent, bulking agents, coloring agents, pharmaceutically acceptable excipients, a preservative, pH adjuster, buffer, chelating agent, etc. The additional compounds can be admixed into a previously emulsified nanoemulsion vaccine, or the additional compounds can be added to the original mixture to be emulsified. In certain of these embodiments, one or more additional compounds are admixed into an existing nanoemulsion composition immediately prior to its use.

[0130] The nanoemulsion vaccine may further comprise at least one pH adjuster. Suitable pH adjusters in the nanoemulsion vaccine of the invention include, but are not limited to, diethyanolamine, lactic acid, monoethanolamine, triethylanolamine, sodium hydroxide, sodium phosphate, semi-synthetic derivatives thereof, and combinations thereof.

[0131] In addition, the NE adjuvant or vaccine can comprise a chelating agent. In one embodiment of the invention, the chelating agent is present in an amount of about 0.0005% to about 1%. Examples of chelating agents include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), phytic acid, polyphosphoric acid, citric acid, gluconic-40-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 acid, acetic acid, lactic acid, and dimercaprol, and a preferred chelating agent is ethylenediaminetetraacetic acid.

[0132] The NE adjuvant or vaccine can comprise a buffering agent, such as a pharmaceutically acceptable buffering agent. In addition, the NE adjuvant or vaccine can comprise one or more emulsifying agents to aid in the formation of emulsions. Emulsifying agents include compounds that aggregate at the oil / water interface to form a kind of continuous membrane that prevents direct contact between two adjacent droplets. Certain embodiments of the present invention feature nanoemulsion vaccines that may readily be diluted with water or another aqueous phase to a desired concentration without impairing their desired properties.VI. Immunogens[01331 Any influenza immunogen may be used with the vaccines and methods of the present disclosure. The immunogen may be a part of the pathogen or the whole pathogen. For example, the immunogen may be a peptide, a glycoprotein, or an entire organism. The influenza immunogen may be native or recombinant, mutated and / or may comprise heterologous elements, such as a fusion polypeptide, to increase immunogenicity or aid in purification or formulation.[0134| In one embodiment, an immunogen from influenza virus may be used, including, but not limited to immunogens from influenza A virus, influenza B virus or influenza C virus. More specifically, the influenza pathogen may be, for example, one or more of (1) inactivated influenza virus, a recombinant immunogenic variant of an inactivated influenza virus, or an immunogenic fragment of an inactivated influenza virus; (2) H5N1, a recombinant immunogenic variant of H5N1, or an immunogenic fragment of H5N1; (3) H1N1, a recombinant immunogenic variant of H1N1, or an immunogenic fragment of H1N1; (4) H1N2, a recombinant immunogenic variant of H1N2, or an immunogenic fragment of H1N2; (5) H3N2, a recombinant immunogenic variant of H3N2, or an immunogenic fragment of H3N2; (6) H2N2, a recombinant immunogenic variant of H2N2, or an immunogenic fragment of H2N2; (7) H7N7, a recombinant immunogenic variant of H7N7, or an immunogenic fragment of H7N7; (8) H9N2, a recombinant immunogenic variant of H9N2, or an immunogenic fragment of H9N2; (9) H7N2, a recombinant immunogenic variant of H7N2, or an immunogenic fragment of H7N2; (10) H7N3, a recombinant-41-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 immunogenic variant of H7N3, or an immunogenic fragment of H7N3; (11) H10N7, a recombinant immunogenic variant of H10N7, or an immunogenic fragment of H10N7; (12) Hl, a recombinant immunogenic variant of Hl, or an immunogenic fragment of Hl; (13) H2, a recombinant immunogenic variant of H2, or an immunogenic fragment of H2; (14) H3, a recombinant immunogenic variant of H3, or an immunogenic fragment of H3; (15) H5, a recombinant immunogenic variant of H5, or an immunogenic fragment of H5; (16) H7, a recombinant immunogenic variant of H7, or an immunogenic fragment of H7; (17) H9, a recombinant immunogenic variant of H9, or an immunogenic fragment of H9; (18) Nl, a recombinant immunogenic variant of Nl, or an immunogenic fragment of Nl; (19) N2, a recombinant immunogenic variant of N2, or an immunogenic fragment of N2; (20) N3, a recombinant immunogenic variant of N3, or an immunogenic fragment of N3; (21) N7, a recombinant immunogenic variant of N7, or an immunogenic fragment of N7; (22) a seasonal influenza strain, a recombinant immunogenic variant of a seasonal influenza strain, or an immunogenic fragment of a seasonal influenza strain; (23) a pandemic influenza strain, a recombinant immunogenic variant of a pandemic influenza strain, or an immunogenic fragment of a pandemic influenza strain; (24) an influenza A virus strain, a recombinant immunogenic variant of an influenza A virus strain, or an immunogenic fragment of an influenza A virus strain; (25) an influenza B virus strain, a recombinant immunogenic variant of an influenza B virus strain, or an immunogenic fragment of an influenza B virus strain; (26) an influenza C virus strain, a recombinant immunogenic variant of an influenza C virus strain, or an immunogenic fragment of an influenza C virus strain; (27) A / New Caledonia / 20 / 99 lineage; (28) A / Fujian / 411 / 2002 lineage; (29) A / Kumamoto / 102 / 2002 lineage; (30) A / Wyoming / 3 / 2003 lineage; (31) A / Wellington / 1 / 2004 lineage; (32) A / Califomia / 7 / 2004 lineage; (33) A / New York / 55 / 2004 lineage; (34) A / Solomon Islands / 3 / 2006 lineage; (35) A / Wisconsin / 67 / 2005 lineage; (36) A / Hiroshima / 52 / 2005 lineage; (37) A / Brisbane / 10 / 2007 lineage; (38) A / Indonesia (39) A / Vietnam (40) B / Hong Kong / 330 / 2001 lineage; (41) B / Shandong / 7 / 97 lineage; (42) B / Hong Kong / 1434 / 2002 lineage; (43) B / Brisbane / 32 / 2002 lineage; (44) B / Shanghai / 361 / 2002 lineage; (45) B / Jiangsu / 10 / 2003 lineage; (46) B / Jilin / 20 / 2003 lineage; (47)B / Malaysia / 2506 / 2004 lineage; (48) B / Florida / 4 / 2006 lineage, (49) B / Victoria / 2 / 87 lineage, (50)-42-4924-5417-0743.1Atty. Dkt. No.: 038491-0359B / Yamagata / 16 / 88 lineage, (51) C / Aichi / 1 / 99 lineage, (52) C / Sao Paulo / 378 / 82 lineage, (53) C / Yamagata / 26 / 81 lineage, (54) C / Aichi / 1 / 81 lineage, (55) C / Aomori / 74 lineage, (56) C / Mississippi / 80 lineage, (57) recombinant protein or immunogenic fragment thereof derived from influenza virus HA, NA, Ml, NP, M2e, HA stalk, and / or M2; (58) any new strain or subtype that may arise due to antigenic drift and / or mutation; or (59) any combination thereof.VII. Pharmaceutical Compositions[0135| The nanoemulsion vaccines may be formulated into pharmaceutical compositions that comprise the NE vaccine in a therapeutically effective amount and suitable, pharmaceutically- acceptable excipients for pharmaceutically acceptable delivery. Such excipients are well known in the art.[0136 By the phrase “therapeutically effective amount” it is meant any amount of the NE vaccine that is effective in preventing, treating or ameliorating a disease caused by the pathogen associated with the immunogen administered in the composition comprising the nanoemulsion vaccine. By “protective immune response” it is meant that the immune response associated with prevention, treating, or amelioration of a disease. Complete prevention is not required, though is encompassed by the present invention. The immune response can be evaluated using the methods discussed herein or by any method known by a person of skill in the art.

[0137] Intranasal administration includes administration via the nose, either with or without concomitant inhalation during administration. Such administration is typically through contact by the composition comprising the NE vaccine with the nasal mucosa, nasal turbinates or sinus cavity. Administration by inhalation comprises intranasal administration, or may include oral inhalation. Such administration may also include contact with the oral mucosa, bronchial mucosa, and other epithelia.

[0138] An exemplary NE adjuvant composition according to the invention is designated “Wgo5EC” adjuvant. The composition of Wso5EC adjuvant is shown in the table below (Table 1). The mean droplet size for the Wso5EC adjuvant is ~400nm. All of the components of the NE are included on the FDA inactive ingredient list for Approved Drug Products.-43-4924-5417-0743.1Atty. Dkt. No.: 038491-0359

[0139] The nanoemulsion adjuvants are formed by emulsification of an oil, purified water, nonionic detergent, organic solvent and surfactant, such as a cationic surfactant. An exemplary specific nanoemulsion adjuvant is designated as “6O%W8o5EC”. The 6O%Wso5EC-adjuvant is composed of the ingredients shown in Table 2 below: purified water, USP; soybean oil USP; Dehydrated Alcohol, USP [anhydrous ethanol]; Polysorbate 80, NF and cetylpyridinium chloride, USP (CPCA11 components of this exemplary nanoemulsion are included on the FDA list of approved inactive ingredients for Approved Drug Products.

[0140] In some embodiments, a combination of vaccines comprises: (a) at least one dose of a priming vaccine comprising a NE vaccine formulated for IN administration, wherein the NE-44-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 vaccine comprises: (1) a NE adjuvant; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) at least one dose of a boosting vaccine formulated for IM administration, comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins.

[0141] In another aspect, a combination of vaccines comprises: (a) at least one dose of a priming vaccine formulated for IM administration, comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and (b) at least one dose of a boosting vaccine comprising a NE vaccine formulated for IN administration, wherein the NE vaccine comprises: (1) a NE adjuvant; and (2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins.[01421 In some embodiments, IN administration of at least one dose of the priming vaccine enhances the production of antibodies against the one or more influenza immunogens or recombinant influenza proteins comprised in or produced from the boosting vaccine relative to that achieved following administration of at least one dose of the boosting vaccine in the absence of the priming vaccine.VIII. Definitions[01431 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.-45-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[0144| It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0145] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0146] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0147] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.]0148| The terms "buffer" or "buffering agents" refer to materials which when added to a solution, cause the solution to resist changes in pH.

[0149] The terms "chelator" or "chelating agent" refer to any materials having more than one atom with a lone pair of electrons that are available to bond to a metal ion.

[0150] As used herein, the term "intranasal(ly)" refers to application of the compositions of the present invention to the surface of the skin and mucosal cells and tissues of the nasal passages,-46-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 e.g., nasal mucosa, sinus cavity, nasal turbinates, or other tissues and cells which line the nasal passages.|0151] As used herein, the term "intramuscular(ly)" refers to application of the compositions of the present invention into muscle tissue of a subject, preferably by injection.

[0152] The term "nanoemulsion," as used herein, includes dispersions or droplets, as well as other lipid structures that can form as a result of hydrophobic forces that drive apolar residues (i.e., long hydrocarbon chains) away from water and drive polar head groups toward water, when a water immiscible oily phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.[0153| The terms "pharmaceutically acceptable" or "pharmacologically acceptable," as used herein, refer to compositions that do not substantially produce adverse allergic or adverse immunological reactions when administered to a host (e.g., an animal or a human). Such formulations include any pharmaceutically acceptable dosage form. Examples of such pharmaceutically acceptable dosage forms include, but are not limited to, dips, sprays, seed dressings, stem injections, lyophilized dosage forms, sprays, and mists. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, wetting agents (e.g., sodium lauryl sulfate), isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like.

[0154] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.-47-4924-5417-0743.1Atty. Dkt. No.: 038491-0359]0155[ The term "subject" as used herein refers to organisms to be treated by the compositions of the present disclosure. Such organisms include animals (domesticated animal species, wild animals), and humans.

[0156] The term "surfactant" refers to any molecule having both a polar head group, which energetically prefers solvation by water, and a hydrophobic tail which is not well solvated by water. The term "cationic surfactant" refers to a surfactant with a cationic head group. The term "anionic surfactant" refers to a surfactant with an anionic head group. The term “ non-ionic surfactant: refers to a surfactant with uncharged head groups. The term “zwitterioninc surfactant refers to a surfactant with both cationic and anionic head groups.

[0157] As used herein, the term "systemically active drugs" is used broadly to indicate a substance or composition whose administration is not necessarily near the infection source and whose levels can be measured at sites quite distant from the site of administration (e.g., oral drug administration where levels of the drug are found in the bloodstream or in tissues or organs).

[0158] The invention is further described by reference to the following examples, which are provided for illustration only. The invention is not limited to the examples, but rather includes all variations that are evident from the teachings provided herein. All publicly available documents referenced herein, including but not limited to U.S. patents, are specifically incorporated by reference.EXAMPLEExample 1. Use of Intranasal adjuvanted H5 vaccine with intramuscular boost in generation of cross-protective immune responses[0159[ The present example illustrates the surprising discovery that an immune response to an intramuscularly (IM)-administered influenza vaccine composition can be significantly and unexpectedly boosted by first administering a primer vaccine, wherein the primer vaccine is intranasal (IN) administration of a nanoemulsion influenza vaccine composition. Further the experiment also details the generation of cross-protective immunity against influenza strains not present in the administered vaccines.-48-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[0160| FIG. l is a schematic illustrating the vaccine dosing timeline for experiment. In particular, the figure details two IN influenza vaccine administrations at Days 0 and 29, followed by an intramuscular influenza vaccine administration at Day 197. Serum antibody titers were assessed at days 1, 57, 197, and 225 following the first administration of the IN vaccine composition.[01611 Table 3 below details the five different study groups of the experiment, with each study group containing 8 human subjects (adults, aged 18-64 years). Groups A-C comprised subjects who were IN-administered a nanoemulsion influenza primer vaccine, with Groups A-C differing in the amount of influenza antigen present in the nanoemulsion (NE) adjuvant; Group A = 25 rH5 pg, Group B = 50 rH5 pg, and Group C = 100 rH5 pg. Prime: BW-1014, a nanoemulsion- adjuvanted recombinant H5 (A / Indonesia, clade 2.1); and Adjuvant: Oil-in-water nanoemulsion W805EC, a mucosal adjuvant. Group D was a control group where a nanoemulsion primer influenza adjuvant was not administered, and Group E was a control group where only placebo was administered (e.g., recombinant protein without a nanoemulsion).

[0162] Briefly, human subjects in Groups A-C were administered a vaccine composition comprising a nanoemul si on-adj uvanted recombinant H5 (A / Indonesia, clade 2.1) antigen (BW- 1014) via IN administration (Prime). The adjuvant was an oil-in-water nanoemulsion Wso5EC, as-49-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 shown in Table 1 and Table 2 (e.g., 20% W805EC). The subjects were administered the intranasal formulation on Day 0 and again on Day 29 following the first administration (See FIG. 1).

[0163] Following IN administration of the nanoemulsion vaccine, the subjects were further administered a boosting vaccine composition comprising H5N1 IIV (Sanofi Pasteur Inactivated Influenza Virus Vaccine, H5N1) via IM administration (IM) at 90 pg / 1 mL. The IM vaccine composition was administered on Day 197 following administration of the priming IN nanoemulsion vaccine composition. Group D was only administered the boosting vaccine composition, as detailed in Table 3.

[0164] Sanofi Pasteur Inactivated Influenza Virus Vaccine, H5N1, is available as a suspension in 5 mL multi-dose vials containing 5 doses. Each 1 mL dose is formulated to contain 90 micrograms (pg) hemagglutinin (HA) of the influenza virus strain A / Vietnam / 1203 / 2004 (H5N1, clade 1) and not more than 98.2 pg of thimerosal (approximately 50 pg of mercury / dose). Thimerosal, a mercury derivative, is added as a preservative.

[0165] Serum antibody titers were assessed for Groups A-E at days 1, 57, 197, and 225 following the first administration of the IN priming vaccine composition. Serum, nasal wash, and saliva samples were collected form the subjects over the course of the study.Materials and MethodsAssessment of cytokine production by T cells

[0166] Cryopreserved PBMC were thawed and rested for 4 hours (37°C, 5% CO2). PBMC were then washed and partitioned into four 2xl0e6 cell aliquots. Two aliquots were stimulated with (1) rH5 from A / Indonesia / 5 / 2005 (clade 2.1) (Fraunhofer USA Center for Molecular Biotechnology) at 3 pg / mL; and (2) a H5 HA peptide pool from A / Vietnam / 1203 / 2004 (clade 1) at 2 pg / mL of each peptide. The peptide pool consisted of a 93-peptide array (12- or 17-mers, with 1 laa overlap) from BEI Bioresources (NR- 18974). The other two aliquots served as negative (media) and positive (Staphylococcal enterotoxin B -SEB-; 10 mg / mL) controls.-50-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[0167| All samples received anti-CD107a-FITC (clone H4A3; Becton Dickinson Biosciences - BDB-) and anti-CD28 / CD49d co-stimulatory antibodies (BDB, USA). Two hours later, Brefeldin A and Monensin were added and incubated overnight (16 hours, at 37°C, 5% CO2). Next day the cells were stained for flow-cytometry. Briefly, cells were stained for viability (fixable yellow staining dye; 20 min; RT) and then with a surface antibody cocktail (30 min, RT) that included: CD62L-PE (clone DREG; eBiosciences), CD4-PerCP-Cy5.5 (clone: L200; BDB), CD19-BV570 (clone: HIB19; BioLegend), CD56-BV570 (clone: HCD56; BioLegend), CD3- BV650 (clone: SK7 BDB), CD8-A700 (RPA-T8; BDB), CD45RA-APC-H7 (clone: HI100; BDB). The cells were then fixed, permeabilized and stained with an intracellular cocktail (30 min, RT) including the next antibodies: CD69-ECD (clone: TP 1.55.3; BC), IFN-Y-PE-Cy7 (clone: B27; BDB), IL-17A-BV605 (clone: BL168; BioLegend), TNF-a-BV711 (clone: MAbl l; BioLegend), CD154-BV785 (clone: 24-31; BioLegend), IL-2-APC (clone: MQ1-17H12;BioLegend), and CD137-BV421 (clone: 4-1BB; BioLegend).Antibody-dependent cell-mediated cytotoxicity (ADCC) assay[G168| rH5 from A / Indonesia / 5 / 2005 (clade 2.1) (Fraunhofer USA Center for Molecular Biotechnology) was biotinylated (Abeam, USA) and then used to coat polystyrene-streptavidin beads (SA-beads; Spherotech, USA). For the assay, each reaction used 5 pL of SA-beads coated with -250 ng of rH5. Plasma from vaccinated volunteers (25 pL per reaction) was incubated with rH5-SA-beads (overnight, 4°C), the beads were subsequently washed 2x with assay media (cRPMI with 10% ultra-low IgG FBS), resuspended in 50 pL, and added into a 96-well plate (50 pL per well). 1.5xl0e6 Jurkat cells expressing the firefly luciferase gene under the control of NF AT response elements and constitutively expressing CD16a (V158) (BPS Bioscience) were added to the beads (50 pL) and incubated for 5 hours (37°C; 5% CO2). The luciferase signal was detected by a luminometer after cell lysis and the addition of luciferin substrate (BPS Bioscience). The assay was performed in triplicate wells.Memory B cell assay

[0169] Cryopreserved PBMC were thawed and polycl onally expanded (5-6 days) as described before. Expanded PBMC were harvested, counted, and seeded in quadruplicate wells (250,000-51-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 cells per well) of multi-screen plates coated with rH5 from A / Indonesia / 5 / 2005 (clade 2.1) (Fraunhofer USA Center for Molecular Biotechnology) at 3 pg / mL. Controls included wells coated with 1) goat anti-human IgA (Total IgA control; 5 pg / mL) (JIR, PA); 2) goat anti-human IgG (Total IgG control; 5 pg / mL) (JIR, PA); or 3) lx PBS. In Total IgG and IgA control wells, 2-fold dilutions of the cells, starting at 24,000, were seeded in duplicate. 5 hours later the plates were washed and incubated with goat-anti-human IgA-HRP (JIR, PA) or goat-anti-human IgG- HRP (JIR, PA). IgG and IgA Spot Forming Cells (SFC) were visualized with AEC substrate. Total and antigen-specific B memory SFC were calculated as SFC / 10e6 cells.Binding antibody measurements by Surface Plasmon Resonance (SPR)[0170J Steady-state equilibrium binding of post-H5Nl vaccinated human sera was monitored at 25 °C using a ProteOn SPR biosensor (Bio-Rad). The recombinant HA globular domain (rHAl- Hise) for the A / Indonesia / 05 / 2005 (clade 2.1) or from H5N1- A / Vietnam / 1203 / 2004 (clade 1) influenza virus strain was coupled to a GLC sensor chip with amine coupling with 1,000 RU in the test flow cells. Samples of 200 pl of sera at 10-fold dilutions were injected at a flow rate of 50 pl min1(120-s contact time) for association, and disassociation was performed over a 600-s interval. Responses from the protein surface were corrected for the response from a mock (no coating) surface and for responses from a separate, buffer-only injection. Binding antibodies were determined from two independent SPR runs.Results[01711 FIGs. 2A-2B are panels of graphs illustrating the geometric mean antibody titers (GMT) observed of samples obtained from subjects in the study: Study Groups A, B, C, D, and E. FIG. 2A is a panel of graphs showing the GMT observed against the Indonesia strain antigen for Groups A-E. FIG. 2B is a panel of graphs showing the GMT observed against the Vietnam strain antigen for Groups A-E.[0172 j FIG. 3A-3B are graphs depicting the seroconversion rate achieved in human subjects for Groups A-E using various tested dosing protocols. FIG. 3A shows the seroconversion rate achieved against the Indonesia strain antigen for Groups A-E. FIG. 3B shows the seroconversion-52-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 rate achieved against the Vietnam strain antigen for Groups A-E.

[0173] As shown in FIG. 2A and FIG. 3A, protection against the Indonesia strain following administration of only the boost IM Vietnam vaccine was essentially absent without administration of the IN priming vaccine (Group D). However, significant protection was observed in subjects that received the prime-boost protocol (Groups A, B, and C). Interestingly, of Groups A-C, Groups A and C showed the highest level of antibody production against both the Indonesia and Vietnam strains. This same phenomena was observed with the seroconversion data, with Groups A and C showing a higher level of seroconversion as compared to Group B.

[0174] Additionally, as shown in FIG. 2B and FIG. 3B, protection against Vietnam strain was also observed following administration of the IM vaccine containing a Vietnam strain antigen and the intranasal rH5 non-adjuvanted control (Group D).

[0175] However, surprisingly and unexpectedly, in subjects who also received the nanoemulsion-adjuvanted IN priming vaccine containing the Indonesia strain antigen, protection against the Vietnam strain was robustly enhanced. For example, as shown in FIG. 2B, subjects who received the prime-boost administration protocol with a low dose of the IM vaccine (25 mcg of Vietnam strain antigen; Group A) exhibited a 100% seroconversion rate with respect to the Vietnam strain antigen, whereas subjects who received only a high dose of the IM vaccine (100 mcg of Vietnam strain antigen with IN control; group D) exhibited a seroconversion rate of under 60% - a staggering seroconversion different of 40%.

[0176] These results are the first to demonstrate a robust cross-strain prime-boost effect achieved using a multi-dose vaccination protocol with multiple different administration routes.[0177! FIG. 4A-4E are graphs showing the effect of IN-nanoemulsion influenza vaccine primeboost dosing protocol on neutralization titers against numerous H5N1 strains, e.g., evaluating the ability of the IN-NE + IM dosing protocol to generate cross-protection. FIG. 4A is a graph illustrating the neutralization titers against H5 A / Indonesia (Clade 2.1) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with Group A showing the highest level of neutralization titers at Day 225. FIG. 4B is-53-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 a graph illustrating the neutralization titers against H5N1 A / Vietnam (Clade 1) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with Group A showing the highest level of neutralization titers at Day 225, although Group D showed modest levels. FIG. 4C is a graph illustrating the neutralization titers against H5 A / Turkey (Clade 2.2) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with all of Groups A-C showing levels of neutralization titers, with Group A being highest, next Group B, and finally Group C. FIG. 4D is a graph illustrating the neutralization titers against H5 A / Egypt (Clade 2.2.1) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with all of Groups A-C showing levels of neutralization titers, with Group A being highest, next Group B, and finally Group C. FIG. 4E is a graph illustrating the neutralization titers against H5 A / Anhui (Clade 2.3.4) as a function of dosing for Group A (25 pg), Group B (50 pg), Group C (100 pg), Group D (rH5 control), and Group D (control), with all of Groups A-C showing levels of neutralization titers, with Group A being highest, next Group B, and finally Group C.

[0178] As shown in FIG. 4, the multidose vaccination protocol resulted in measurable neutralization titers against homologous and heterologous H5N1 influenza strains. Surprisingly and unexpectedly, a dose-dependent inverse correlation was observed with respect to the neutralization titers. That is, the highest neutralization titers were observed at the lowest tested doses. This was observed for titers against H5 A / Indonesia (Clade 2.1) (FIG. 4A), H5N1 A / Vietnam (Clade 1) (FIG. 4B), H5 A / Turkey (Clade 2.2) (FIG. 4C), H5 A / Egypt (Clade 2.2.1) (FIG. 4D), and H5 A / Anhui (Clade 2.3.4) (FIG. 4E).

[0179] Also surprisingly, as shown in FIG. 4B, subjects who received the prime-boost vaccination protocol with the IN adjuvanted vaccine containing 25 ug of the Indonesia strain antigen exhibited higher neutralization titers against the Vietnam strain than observed in subjects who received the non-adjuvanted IN adjuvanted vaccine containing 100 ug of the Indonesia strain antigen, both groups of subjects having received the IM dose of the Vietnam strain vaccine.-54-4924-5417-0743.1Atty. Dkt. No.: 038491-0359]0180[ These data suggest that the intranasal delivery of an adjuvanted vaccine containing a heterologous strain can enhance the immune response to an IM vaccine containing a second antigen.

[0181] As detailed in FIG. 4C-4E, significant neutralization titers were observed against multiple strains that did not appear in either the IN or the IM vaccine. These data represent the first demonstration of a cross-administration route, cross-strain immune response potentiation.

[0182] FIGs. 5A-5B are graphs showing the production of H5-binding antibodies in serum samples as a function of dosing group (Group A-E), measured by surface plasmon resonance (SPR). FIG. 5A is a graph illustrating the amount of H5 Indonesia HA 1 -binding antibodies present in a serum sample as a function of dosing for Groups A-E, and FIG. 5B is a graph illustrating the amount of H5 Vietnam HA1 -binding antibodies present in a serum sample as a function of dosing for Groups A-E.

[0183] As shown in FIG. 5 A and FIG. 5B, the prime-boost administration protocol resulted in the production of antibodies capable of binding to H5 Indonesia HA1 andH5 Vietnam HA1 strain antigens, as measured by surface plasmon resonance (SPR). Unexpectedly, intranasal administration of the adjuvanted Indonesia strain vaccine significantly enhanced the production of antibodies that bind to the H5 Vietnam HA1 antigen.

[0184] Similar to that observed regarding the neutralization titers, antibody production unexpectedly followed a dose-dependent inverse correlation; the lower the dose of the IN prime vaccine, the greater the concomitant antibody production.

[0185] FIGs. 6A-6B are graphs showing the correlation between antibody production and neutralization titers achieved against the Indonesia and Vietnam strains. FIG. 6A is a graph showing the correlation between the neutralization titer against the Indonesia strain virus and production of H5 Indo HA 1-320 D-S RU-specific antibodies. FIG. 6B is a graph showing the correlation between the neutralization titer against the Vietnam strain virus and production of Vietnam HA 1-320 RU-specific antibodies.-55-4924-5417-0743.1Atty. Dkt. No.: 038491-0359]0186[ As shown in FIG. 6A and 6B, a positive correlation between neutralization titer and HA1 -binding antibody production was observed with respect to both the Indonesia and Vietnam strains.

[0187] Applicant measured rH5 (clade 2.1)-specific serum IgG and IgA and H5 stalk-specific serum IgG by ELISA (FIG. 7). FIG. 7 shows H5N1 clade 2.1 serum and nasal wash binding antibodies by group. FIG. 7A shows the H5 A / Indonesia (Clade 2.1)-specific serum IgG and IgA responses, as well as H5 stalk-specific IgG responses (EU / mL) as individual values and geometric mean concentration with 95% confidence intervals. FIG. 7B shows individual nasal wash responses (IgG and IgA) as well as the median and interquartile range of the ratio of H5- specific IgG or IgA (EU / pg) to total IgG or IgA at each timepoint per group. Inset shows nasal wash responses on an extended y axis. At baseline, IgA and IgG levels to rH5 were low, while anti-stalk IgG levels were elevated. Unlike with MN or HAI assays, significant GMT increases (p<0.05) from baseline were observed for most immunoglobulins measured at Days 57 and 197 in the rH5-NE groups. Significant GMT increases were measured from Day 197 to Day 225 in all groups (p<0.05) for IgG and IgA, though significant boost responses at this timepoint for H5 stalk-specific serum IgG were only measured in Group C and Group E (p<0.05).

[0188] Antibody-dependent cell-mediated cytotoxicity (ADCC) was assessed against rH5 clade 2.1. FIG. 8 shows ADCC, memory B cell, and memory T cell responses by group. Applicant defined ADCC seroconversion as >4-fold increase from baseline. FIG. 8A shows the induction of antibodies with ADCC capacity as fold-changes over Day 1. At the bottom of the figure, in blue text, shown is the percentage of volunteers that seroconverted (SC) at each timepoint in every group. SC was defined as a 4-fold increase in ADCC titers compared to Day 1 and the dotted line indicates this threshold. Applicant measured seroconversion in ADCC responses in all rH5-NE groups at Days 57 and 225 (p<0.05), while comparator groups developed ADCC responses only at Day 225 FIG. 8B shows the frequency of memory B cells producing anti-H5 IgG antibodies, reported as SFU per lxl0e6 cells. The median and 95% CI are shown. Each dot represents an individual. FIG. 8C shows the frequency of memory CD4 T cells producing IL-2 (net %) upon ex-vivo stimulation with an H5 peptide pool (A / Vietnam / 1203 / 2004 (clade 1)).-56-4924-5417-0743.1Atty. Dkt. No.: 038491-0359FIG. 8D shows data from IFN-y producing cells. In FIG. 8C and 8D, the data are shown in violin plots denoting the distribution of the data. Each dot represents one individual. In FIG. 8A-D, data from volunteers vaccinated with the low-dose (Group A), middle-dose (Group B) and high-dose (Group C) of rH5-NE is shown in maroon, orange, and yellow colors. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group E) are shown in cyan and gray colors. FIG. 8E shows the ability of rH5-NE (pooled Groups A-Cs) to induce multifunctional (MF) cells at each timepoint of the study. Data are shown in violin plots, and each dot represents one volunteer. MF cells (IL-2+ & IFN-y+) are shown by the white circles, IFN-y-only producing cells are shown by the blue circles and IL-2-only producing cells are shown by the grey circles. IL-2-only and IFN- y-only cells are referred to as Single Functional (SF) cells. FIG. 8F displays the frequency of MF and SF cells by Groups A-C at days 57 (post-intranasal vaccination) and 255 (post-systemic boost). The data in FIG. 8D is presented as percentage of the mean of MF and SF cells. White, blue, and gray areas of the pie show IFN-y-only, IL-2-only and MF cells, respectively. The yellow semicircle shows the added percentage of SF cells (LL-2-only plus IFN-y-only). Statistics from FIGs. 8B-8E are derived from Wilcoxon signed-rank tests *p<0.05, **p<0.01, ***p<0.005, **** p<0.0001. The percentage of participants with ADCC seroconversion at Day 57 was 50% (95% CI 11.8, 88.2) for Group A, 57% (95% CI 18.4, 90.1) for Group B, and 75% (95% CI 34.9, 96.8) for Group C. Seroconversion at Day 225 was 100% (95% CI 54.1, 100) for Group A, 86% (95% CI 42.1, 99.6) for Group B, 75% (95% CI 34.9, 96.8) for Group C, 33% (95% CI 4.3, 77.7) of Group D, and 50% (95% CI 11.8, 88.2) for Group E.

[0189] FIG. 9 shows CD4 T cell responses to rH5 (clade 2.1) antigen. In FIG. 9A, shown is the basic gating strategy used to identify memory CD4 T cells using CD54RA and CD62L. Memory CD4 T cells were defined as CD4+ CD62L low / high and CD45RA low / high, excluding double positive CD62+CD45RA+ cells. Activated (CD69+) memory CD4 T cells (CD4+, excluding CD45RA+ CD62L+ cells) were assessed for their ability to produce cytokines or upregulate additional activation markers (e.g., CD154, CD137) after ex-vivo stimulation with rH5 (clade 2.1). In FIG. 9B, shown is a representative example of activated (CD69+) cells producing IL-2 upon stimulation with media (mock stimulation) or the rH5 (clade 2.1) antigen. The data reported in the manuscript is the Net% (peptide-pool minus media). In the example the net percentage is-57-4924-5417-0743.1Atty. Dkt. No.: 038491-03590.116. In FIG. 9C, shown is the frequency of memory CD4 T cells producing IL-2 (net %) upon ex-vivo stimulation with rH5 (clade 2.1) antigen. The data is displayed in Violin plots. FIG. 9D shows similar data but from IFN-y producing cells. Data from volunteers vaccinated with the low-dose (Group A), middle-dose (Group B) and high-dose (Group C) of rH5-NE are shown in maroon, orange and yellow. Controls, including the unadjuvanted rH5 (Group D) and placebo (Group D), are shown in cyan and gray colors. Statistics from FIGs. 9C-9D are derived from signed-rank tests. *p<0.05. Memory CD4 T cells stimulated with the rH5 (clade 2.1) had IL-2 and IFN-y responses of lower magnitude than those identified in cells stimulated with the H5 peptide pool (clade 1), but the trends were similar.[0190| Taken together, these data demonstrate that intranasal administration of a nanoemulsion adjuvanted vaccine promote immunological memory, manifesting in enhanced homologous H5N1 neutralization titers and increased production of H5 HA1 binding antibodies. Moreover, the effect surprisingly follows an inverse correlation between intranasal vaccine dose and immune response. Finally, the data demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC) and CD4 T cell responses against rH5 clade 2.1, indicating that immune responses are capable of neutralization in human subjects.EQUIVALENTS[019 I J The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.-58-4924-5417-0743.1Atty. Dkt. No.: 038491-0359[01921 In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.INCORPORATION BY REFERENCE

[0193] 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, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.-59-4924-5417-0743.1

Claims

Atty. Dkt. No.: 038491-0359WHAT IS CLAIMED IS:

1. A method for inducing an immune response to influenza in a subject comprising:(a) intranasally administering to a subject at least one dose of a priming vaccine comprising:(1) a nanoemulsion comprising:(i) droplets having an average diameter of less than about 1000 nm;(ii) an aqueous phase;(iii) at least one pharmaceutically acceptable oil;(iv) at least one non-ionic surfactant;(v) at least one cationic surfactant; and(vi) at least one organic solvent which is an alcohol; and(2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and(b) intramuscularly administering to the subject at least one dose of a boosting vaccine comprising one or more influenza immunogens or recombinant influenza proteins, or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; wherein administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.

2. A method for inducing an immune response to influenza in a subject comprising:(a) intramuscularly administering to the subject at least one dose of a priming vaccine comprising one or more influenza immunogens or recombinant influenza proteins, or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins;(b) intranasally administering to a subject at least one dose of a boosting vaccine comprising:-60-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(1) a nanoemulsion comprising:(i) droplets having an average diameter of less than about 1000 nm;(ii) an aqueous phase;(iii) at least one pharmaceutically acceptable oil;(iv) at least one non-ionic surfactant;(v) at least one cationic surfactant; and(vi) at least one organic solvent which is an alcohol; and(2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and wherein administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.3 The method of claim 1 or 2, wherein the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are the same.

4. The method of claim 1 or 2, wherein the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are different.

5. The method of any one of claims 1-4, wherein:(a) at least two doses of the priming vaccine are administered to the subject;(b) at least two doses of the boosting vaccine are administered to the subject;(c) only one dose of the priming vaccine is administered to the subject;(d) only one dose of the boosting vaccine is administered to the subject; or(e) any combination thereof.

6. The method of any one of claims 1-5, wherein the vaccine administered-61-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 intramuscularly comprises a nanoemulsion influenza vaccine, which can be the same or different from the intranasally-administered nanoemulsion influenza vaccine.

7. The method of any one of claims 1-6, wherein a first dose of the boosting vaccine is administered at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months or at least 18 months or more following administration of at least one dose of the priming vaccine.

8. The method of any one of claims 1-7, wherein:(a) sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a protective immune response; and(b) administration of at least one dose of the priming vaccine alone or at least one dose of the boosting vaccine alone, both administered at the same dose as when administered sequentially, does not result in a protective immune response.

9. The method of any one of claims 1-8, wherein the protective immune response resulting from sequential administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine is greater than the magnitude of a protective immune response achieved following:(a) one or more administrations of the priming vaccine alone at the same dose; and / or one or more administrations of the boosting vaccine alone at the same dose; or(b) at least two sequential administrations of the priming vaccine alone at the same dose; or at least two sequential administrations of the boosting vaccine alone at the same dose; or(c) at least three sequential administrations of the priming vaccine alone at the same dose; or at least three sequential administrations of the boosting vaccine alone at the same dose.

10. The method of any one of claims 1-9, wherein administration of the priming-62-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 vaccine enhances the production of antibodies against the one or more influenza immunogens or recombinant influenza proteins comprised in or produced from the boosting vaccine relative to that achieved following administration of the boosting vaccine in the absence of the priming vaccine.

11. The method of any one of claims 1-10, wherein the immune response and / or protective immune response is measured by increased levels of cellular immunity, increased levels of humoral immunity, increased antibody titers, increased seroconversion rates, increased neutralization titers, or any combination thereof.

12. The method of claim 11, wherein the humoral immunity is assessed by levels of IgG, IgA, IgM, IgE, or any combination thereof.

13. The method of claim 11, wherein:(a) the cellular immunity is assessed by levels of antigen-specific cytotoxic T- lymphocytes, macrophages and / or one or more cytokines released in response to the influenza immunogen or protein; and / or(b) the immune response and / or protective immune response comprises a Thl and / or Th2 response.

14. The method of any one of claims 1-13, wherein a protective immune response is generated against an influenza strain, wherein an antigen of the influenza strain is not present in either the priming or boosting vaccine.

15. The method of any one of claims 1-16, wherein each of the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine is selected from the group consisting of:(1) inactivated influenza virus, a recombinant immunogenic variant of an inactivated influenza virus, or an immunogenic fragment of an inactivated influenza virus;-63-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(2) H5N1, a recombinant immunogenic variant of H5N1, or an immunogenic fragment of H5N 1 ;(3) H1N1, a recombinant immunogenic variant of H1N1, or an immunogenic fragment of H1N1;(4) H1N2, a recombinant immunogenic variant of H1N2, or an immunogenic fragment of H1N2;(5) H3N2, a recombinant immunogenic variant of H3N2, or an immunogenic fragment of H3N2;(6) H2N2, a recombinant immunogenic variant of H2N2, or an immunogenic fragment of H2N2;(7) H7N7, a recombinant immunogenic variant of H7N7, or an immunogenic fragment of H7N7;(8) H9N2, a recombinant immunogenic variant of H9N2, or an immunogenic fragment of H9N2;(9) H7N2, a recombinant immunogenic variant of H7N2, or an immunogenic fragment of H7N2;(10) H7N3, a recombinant immunogenic variant of H7N3, or an immunogenic fragment of H7N3;(11) H10N7, a recombinant immunogenic variant of H10N7, or an immunogenic fragment of H10N7;(12) Hl, a recombinant immunogenic variant of Hl, or an immunogenic fragment of Hl;(13) H2, a recombinant immunogenic variant of H2, or an immunogenic fragment of H2;(14) H3, a recombinant immunogenic variant of H3, or an immunogenic fragment of H3;(15) H5, a recombinant immunogenic variant of H5, or an immunogenic fragment of H5;-64-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(16) H7, a recombinant immunogenic variant of H7, or an immunogenic fragment of H7;(17) H9, a recombinant immunogenic variant of H9, or an immunogenic fragment of H9;(18) Nl, a recombinant immunogenic variant of Nl, or an immunogenic fragment of Nl;(19) N2, a recombinant immunogenic variant of N2, or an immunogenic fragment of N2;(20) N3, a recombinant immunogenic variant of N3, or an immunogenic fragment of N3;(21) N7, a recombinant immunogenic variant of N7, or an immunogenic fragment of N7;(22) a seasonal influenza strain, a recombinant immunogenic variant of a seasonal influenza strain, or an immunogenic fragment of a seasonal influenza strain;(23) a pandemic influenza strain, a recombinant immunogenic variant of a pandemic influenza strain, or an immunogenic fragment of a pandemic influenza strain;(24) an influenza A virus strain, a recombinant immunogenic variant of an influenza A virus strain, or an immunogenic fragment of an influenza A virus strain;(25) an influenza B virus strain, a recombinant immunogenic variant of an influenza B virus strain, or an immunogenic fragment of an influenza B virus strain;(26) an influenza C virus strain, a recombinant immunogenic variant of an influenza C virus strain, or an immunogenic fragment of an influenza C virus strain;(27) A / New Caledonia / 20 / 99 lineage;(28) A / Fujian / 411 / 2002 lineage;(29) A / Kumamoto / 102 / 2002 lineage;(30) A / Wyoming / 3 / 2003 lineage;(31) A / Wellington / 1 / 2004 lineage;(32) A / California / 7 / 2004 lineage;(33) A / New York / 55 / 2004 lineage;-65-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(34) A / Solomon Islands / 3 / 2006 lineage;(35) A / Wisconsin / 67 / 2005 lineage;(36) A / Hiroshima / 52 / 2005 lineage;(37) A / Brisbane / 10 / 2007 lineage;(38) A / Indonesia(39) A / Vietnam(40) B / Hong Kong / 330 / 2001 lineage;(41) B / Shandong / 7 / 97 lineage;(42) B / Hong Kong / 1434 / 2002 lineage;(43) B / Brisbane / 32 / 2002 lineage;(44) B / Shanghai / 361 / 2002 lineage;(45) B / Jiangsu / 10 / 2003 lineage;(46) B / Jilin / 20 / 2003 lineage;(47) B / Malaysia / 2506 / 2004 lineage;(48) B / Florida / 4 / 2006 lineage,(49) B / Victoria / 2 / 87 lineage,(50) B / Yamagata / 16 / 88 lineage,(51) C / Aichi / 1 / 99 lineage,(52) C / Sao Paulo / 378 / 82 lineage,(53) C / Yamagata / 26 / 81 lineage,(54) C / Aichi / 1 / 81 lineage,(55) C / Aomori / 74 lineage,(56) C / Mississippi / 80 lineage,(57) recombinant protein or fragment derived from influenza virus HA, NA, Ml, NP, M2e, HA stalk, and / or M2;(58) any new strain or subtype that may arise due to antigenic drift and / or mutation; or(59) any combination thereof.

16. The method of any one of claims 1-15, wherein the total influenza immunogen-66-4924-5417-0743.1Atty. Dkt. No.: 038491-0359 and / or influenza protein present:(a) in the priming vaccine from about 1 pg to about 300 pg, per dose;(b) in the priming vaccine from about 25 pg or about 100 pg,(c) in the boosting vaccine from about 20 pg to about 150 pg, per dose;(d) in the boosting vaccine at about 25 pg, about 50 pg, about 90 pg, or about 100 pg per dose; or(e) any combination thereof.

17. The method of any one of claims 1-16, wherein the pharmaceutically acceptable oil is selected from the group consisting of mineral oil, squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyl adipate, Cl 2- 15 alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate, Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil, Coconut oil, corn oil, Cottonseed oil, Flaxseed oil, Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine flower oil, lavender flower oil, manuka flower oil, Marhoram flower oil, orange flower oil, rose flower oil, ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil, sassafras-67-4924-5417-0743.1Atty. Dkt. No.: 038491-0359Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl alcohol, semi-synthetic derivatives thereof, and any combinations thereof.

18. The method of claim 17, wherein the pharmaceutically acceptable oil is soybean oil.

19. The method of any one of claims 1-18, wherein the organic solvent is a C1-C12 alcohol.

20. The method of claim 19, wherein the alcohol is ethanol.

21. The method of any one of claims 1-20, wherein the non-ionic surfactant is a polysorbate.

22. The method of claim 21, wherein the polysorbate is polysorbate 80, polysorbate 20, or any combination thereof.

23. The method of any one of claims 1-22, wherein the cationic surfactant is cetylpyridinium chloride (CPC).

24. The method of any one of claim 1-23, wherein the nanoemulsion comprises:(a) droplets having an average diameter of less than about 1000 nm;(b) an aqueous phase;(c) about 1% to about 80% (v / v) soybean oil;(d) about 0.001% to about 10% (v / v) of at least one non-ionic surfactant, which is Polysorbate 80, Polysorbate 20, or a combination thereof;(e) less than about 5% (v / v) cetylpyridinium chloride (CPC); and(f) about 0.01% to about 50% (v / v) ethanol.-68-4924-5417-0743.1Atty. Dkt. No.: 038491-035925. A combination of vaccines comprising:(a) at least one dose of a priming vaccine formulated for intranasal (IN) administration, wherein the vaccine comprises:(1) a nanoemulsion comprising:(i) droplets having an average diameter of less than about 1000 nm;(ii) an aqueous phase;(iii) at least one pharmaceutically acceptable oil;(iv) at least one non-ionic surfactant;(v) at least one cationic surfactant;(vi) at least one organic solvent which is an alcohol; and(2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and(b) at least one dose of a boosting vaccine formulated for intramuscular (IM) administration, comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins.

26. A combination of vaccines comprising:(a) at least one dose of a priming vaccine formulated for intramuscular (IM) administration, comprising one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins; and(b) at least one dose of a boosting vaccine formulated for intranasal (IN) administration, wherein the vaccine comprises:(1) a nanoemulsion comprising:(i) droplets having an average diameter of less than about 1000 nm;(ii) an aqueous phase;(iii) at least one pharmaceutically acceptable oil;-69-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(iv) at least one non-ionic surfactant;(v) at least one cationic surfactant;(vi) at least one organic solvent which is an alcohol; and(2) one or more influenza immunogens or recombinant influenza proteins or a polynucleotide encoding the one or more influenza immunogens or recombinant influenza proteins.

27. The combination of claim 25 or 26, wherein the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are the same.

28. The combination of claim 25 or 26, wherein the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine are different.

29. The combination of any one of claims 25-28, wherein each of the one or more influenza immunogens or recombinant influenza proteins of the priming vaccine and the one or more influenza immunogens or recombinant influenza proteins of the boosting vaccine is selected from the group consisting of(1) inactivated influenza virus, a recombinant immunogenic variant of an inactivated influenza virus, or an immunogenic fragment of an inactivated influenza virus;(2) H5N1, a recombinant immunogenic variant of H5N1, or an immunogenic fragment of H5N 1 ;(3) H1N1, a recombinant immunogenic variant of H1N1, or an immunogenic fragment of H INI;(4) H1N2, a recombinant immunogenic variant of H1N2, or an immunogenic fragment of H1N2;(5) H3N2, a recombinant immunogenic variant of H3N2, or an immunogenic fragment of H3N2;-70-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(6) H2N2, a recombinant immunogenic variant of H2N2, or an immunogenic fragment of H2N2;(7) H7N7, a recombinant immunogenic variant of H7N7, or an immunogenic fragment of H7N7;(8) H9N2, a recombinant immunogenic variant of H9N2, or an immunogenic fragment of H9N2;(9) H7N2, a recombinant immunogenic variant of H7N2, or an immunogenic fragment of H7N2;(10) H7N3, a recombinant immunogenic variant of H7N3, or an immunogenic fragment of H7N3;(11) H10N7, a recombinant immunogenic variant of H10N7, or an immunogenic fragment of H10N7;(12) Hl, a recombinant immunogenic variant of Hl, or an immunogenic fragment of Hl;(13) H2, a recombinant immunogenic variant of H2, or an immunogenic fragment of H2;(14) H3, a recombinant immunogenic variant of H3, or an immunogenic fragment of H3;(15) H5, a recombinant immunogenic variant of H5, or an immunogenic fragment of H5;(16) H7, a recombinant immunogenic variant of H7, or an immunogenic fragment of H7;(17) H9, a recombinant immunogenic variant of H9, or an immunogenic fragment of H9;(18) Nl, a recombinant immunogenic variant of Nl, or an immunogenic fragment of Nl;(19) N2, a recombinant immunogenic variant of N2, or an immunogenic fragment of N2;-71-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(20) N3, a recombinant immunogenic variant of N3, or an immunogenic fragment of N3;(21) N7, a recombinant immunogenic variant of N7, or an immunogenic fragment of N7;(22) a seasonal influenza strain, a recombinant immunogenic variant of a seasonal influenza strain, or an immunogenic fragment of a seasonal influenza strain;(23) a pandemic influenza strain, a recombinant immunogenic variant of a pandemic influenza strain, or an immunogenic fragment of a pandemic influenza strain;(24) an influenza A virus strain, a recombinant immunogenic variant of an influenza A virus strain, or an immunogenic fragment of an influenza A virus strain;(25) an influenza B virus strain, a recombinant immunogenic variant of an influenza B virus strain, or an immunogenic fragment of an influenza B virus strain;(26) an influenza C virus strain, a recombinant immunogenic variant of an influenza C virus strain, or an immunogenic fragment of an influenza C virus strain;(27) A / New Caledonia / 20 / 99 lineage;(28) A / Fujian / 411 / 2002 lineage;(29) A / Kumamoto / 102 / 2002 lineage;(30) A / Wyoming / 3 / 2003 lineage;(31) A / Wellington / 1 / 2004 lineage;(32) A / California / 7 / 2004 lineage;(33) A / New York / 55 / 2004 lineage;(34) A / Solomon Islands / 3 / 2006 lineage;(35) A / Wisconsin / 67 / 2005 lineage;(36) A / Hiroshima / 52 / 2005 lineage;(37) A / Brisbane / 10 / 2007 lineage;(38) A / Indonesia(39) A / Vietnam(40) B / Hong Kong / 330 / 2001 lineage;(41) B / Shandong / 7 / 97 lineage;-72-4924-5417-0743.1Atty. Dkt. No.: 038491-0359(42) B / Hong Kong / 1434 / 2002 lineage;(43) B / Brisbane / 32 / 2002 lineage;(44) B / Shanghai / 361 / 2002 lineage;(45) B / Jiangsu / 10 / 2003 lineage;(46) B / Jilin / 20 / 2003 lineage;(47) B / Malaysia / 2506 / 2004 lineage;(48) B / Florida / 4 / 2006 lineage,(49) B / Victoria / 2 / 87 lineage,(50) B / Yamagata / 16 / 88 lineage,(51) C / Aichi / 1 / 99 lineage,(52) C / Sao Paulo / 378 / 82 lineage,(53) C / Yamagata / 26 / 81 lineage,(54) C / Aichi / 1 / 81 lineage,(55) C / Aomori / 74 lineage,(56) C / Mississippi / 80 lineage,(57) recombinant protein or immunogenic fragment thereof derived from influenza virus HA, NA, Ml, NP, M2e, HA stalk, and / or M2;(58) any new strain or subtype that may arise due to antigenic drift and / or mutation; or(59) any combination thereof.

30. The combination of any one of claims 25-29, wherein the total influenza immunogen and / or influenza protein is present:(a) in the priming vaccine from about 1 pg to about 300 pg, per dose;(b) in the priming vaccine from about 25 pg or about 100 pg,(c) in the boosting vaccine from about 20 pg to about 150 pg, per dose;(d) in the boosting vaccine at about 25 pg, about 50 pg, about 90 pg, or about 100 pg per dose; or(e) any combination thereof.4924-5417-0743.1Atty. Dkt. No.: 038491-035931. The combination of any one of claims 25-30, wherein the pharmaceutically acceptable oil is selected from the group consisting of mineral oil, squalene oil, flavor oils, silicon oil, essential oils, water insoluble vitamins, Isopropyl stearate, Butyl stearate, Octyl palmitate, Cetyl palmitate, Tridecyl behenate, Diisopropyl adipate, Dioctyl sebacate, Menthyl anthranhilate, Cetyl octanoate, Octyl salicylate, Isopropyl myristate, neopentyl glycol dicarpate cetols, Ceraphyls®, Decyl oleate, diisopropyl adipate, Cl 2- 15 alkyl lactates, Cetyl lactate, Lauryl lactate, Isostearyl neopentanoate, Myristyl lactate, Isocetyl stearoyl stearate, Octyldodecyl stearoyl stearate, Hydrocarbon oils, Isoparaffin, Fluid paraffins, Isododecane, Petrolatum, Argan oil, Canola oil, Chile oil, Coconut oil, corn oil, Cottonseed oil, Flaxseed oil, Grape seed oil, Mustard oil, Olive oil, Palm oil, Palm kernel oil, Peanut oil, Pine seed oil, Poppy seed oil, Pumpkin seed oil, Rice bran oil, Safflower oil, Tea oil, Truffle oil, Vegetable oil, Apricot (kernel) oil, Jojoba oil (simmondsia chinensis seed oil), Grapeseed oil, Macadamia oil, Wheat germ oil, Almond oil, Rapeseed oil, Gourd oil, Soybean oil, Sesame oil, Hazelnut oil, Maize oil, Sunflower oil, Hemp oil, Bois oil, Kuki nut oil, Avocado oil, Walnut oil, Fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, common sage leaf oil, eucalyptus leaf oil, lemon grass leaf oil, melaleuca leaf oil, oregano leaf oil, patchouli leaf oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, Wintergreen leaf oil, flower oil, chamomile oil, clary sage oil, clove oil, geranium flower oil, hyssop flower oil, jasmine flower oil, lavender flower oil, manuka flower oil, Marhoram flower oil, orange flower oil, rose flower oil, ylang-ylang flower oil, Bark oil, cassia Bark oil, cinnamon bark oil, sassafras Bark oil, Wood oil, camphor wood oil, cedar wood oil, rosewood oil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, Oleic acid, Linoleic acid, Oleyl alcohol, Isostearyl alcohol, semi-synthetic derivatives thereof, and any combinations thereof.

32. The combination of claim 31, wherein the pharmaceutically acceptable oil is soybean oil.-74-4924-5417-0743.1Atty. Dkt. No.: 038491-035933. The combination of any one of claims 25-32, wherein the organic solvent is a Ci- C12 alcohol.

34. The combination of claim 33, wherein the alcohol is ethanol.

35. The combination of any one of claims 25-34, wherein the non-ionic surfactant is a polysorbate.

36. The combination of claim 35, wherein the polysorbate is polysorbate 80, polysorbate 20, or any combination thereof.

37. The combination of any one of claims 25-36, wherein the cationic surfactant is cetylpyridinium chloride (CPC).

38. The combination of any one of claim 25-37, wherein the nanoemulsion comprises:(a) droplets having an average diameter of less than about 1000 nm;(b) an aqueous phase;(c) about 1% to about 80% (v / v) soybean oil;(d) about 0.001% to about 10% (v / v) of at least one non-ionic surfactant, which is Polysorbate 80, Polysorbate 20, or a combination thereof;(e) less than about 5% (v / v) cetylpyridinium chloride (CPC); and(f) about 0.01% to about 50% (v / v) ethanol.

39. Use of a combination according to any one of claims 25-38 for the manufacture of a medicament, wherein administration of at least one dose of the priming vaccine and at least one dose of the boosting vaccine to a subject results in a greater immune response as compared to that generated by administration of the priming or boosting vaccine alone.-75-4924-5417-0743.1

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