EP2012771A2 - Nanoemulsionsimpfstoffe - Google Patents
NanoemulsionsimpfstoffeInfo
- Publication number
- EP2012771A2 EP2012771A2 EP07755465A EP07755465A EP2012771A2 EP 2012771 A2 EP2012771 A2 EP 2012771A2 EP 07755465 A EP07755465 A EP 07755465A EP 07755465 A EP07755465 A EP 07755465A EP 2012771 A2 EP2012771 A2 EP 2012771A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- composition
- vol
- subject
- immunity
- nanoemulsion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
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- 229960005486 vaccine Drugs 0.000 title abstract description 92
- 239000000203 mixture Substances 0.000 claims abstract description 550
- 230000036039 immunity Effects 0.000 claims abstract description 145
- 238000000034 method Methods 0.000 claims abstract description 130
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- 241000700605 Viruses Species 0.000 claims description 51
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 46
- 201000010099 disease Diseases 0.000 claims description 45
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- 239000003549 soybean oil Substances 0.000 description 188
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- 229960001927 cetylpyridinium chloride Drugs 0.000 description 163
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 163
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- -1 but not limited to Substances 0.000 description 134
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- 239000003921 oil Substances 0.000 description 112
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- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 104
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 86
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 58
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- MDYZKJNTKZIUSK-UHFFFAOYSA-N tyloxapol Chemical compound O=C.C1CO1.CC(C)(C)CC(C)(C)C1=CC=C(O)C=C1 MDYZKJNTKZIUSK-UHFFFAOYSA-N 0.000 description 52
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- 230000004224 protection Effects 0.000 description 29
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 28
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- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 26
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 25
- 244000068988 Glycine max Species 0.000 description 25
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 25
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- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 21
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- RUPBZQFQVRMKDG-UHFFFAOYSA-M Didecyldimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCC[N+](C)(C)CCCCCCCCCC RUPBZQFQVRMKDG-UHFFFAOYSA-M 0.000 description 19
- 230000010534 mechanism of action Effects 0.000 description 19
- 230000003472 neutralizing effect Effects 0.000 description 19
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 18
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 18
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- 241000282412 Homo Species 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 15
- 238000011161 development Methods 0.000 description 15
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- 150000003839 salts Chemical class 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 14
- 210000004443 dendritic cell Anatomy 0.000 description 14
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- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 12
- 241000700198 Cavia Species 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- IQDGSYLLQPDQDV-UHFFFAOYSA-N dimethylazanium;chloride Chemical compound Cl.CNC IQDGSYLLQPDQDV-UHFFFAOYSA-N 0.000 description 12
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- 125000000217 alkyl group Chemical group 0.000 description 10
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- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 10
- 235000003702 sterols Nutrition 0.000 description 10
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- BOBBYBHJYYJHHQ-UHFFFAOYSA-N 7-tert-butyl-6-(4-chlorophenyl)-2-sulfanylidene-1h-pyrido[2,3-d]pyrimidin-4-one Chemical compound CC(C)(C)C1=NC=2NC(=S)NC(=O)C=2C=C1C1=CC=C(Cl)C=C1 BOBBYBHJYYJHHQ-UHFFFAOYSA-N 0.000 description 9
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- 229960004670 didecyldimethylammonium chloride Drugs 0.000 description 9
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- 210000004988 splenocyte Anatomy 0.000 description 9
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 8
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- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 8
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24161—Methods of inactivation or attenuation
- C12N2710/24163—Methods of inactivation or attenuation by chemical treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention provides methods and compositions for the stimulation of immune responses. Specifically, the present invention provides methods of inducing an immune response to an agent (e.g., a bacteria or virus) in a subject (e.g., a human subject) and compositions useful in such methods (e.g., a nanoemulsion comprising a bacteria or virus or a component thereof). Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic and preventative medicine (e.g., vaccination)) and research applications.
- an agent e.g., a bacteria or virus
- compositions useful in such methods e.g., a nanoemulsion comprising a bacteria or virus or a component thereof.
- Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic and preventative medicine (e.g., vaccination)) and research applications.
- Immunization is a principal feature for improving the health of people. Despite the availability of a variety of successful vaccines against many common illnesses, infectious diseases remain a leading cause of health problems and death. Significant problems inherent in existing vaccines include the need for repeated immunizations, and the ineffectiveness of the current vaccine delivery systems for a broad spectrum of diseases. hi order to develop vaccines against pathogens that have been recalcitrant to vaccine development, and/or to overcome the failings of commercially available vaccines due to expense, complexity, and underutilization, new methods of antigen presentation and immunization must be developed that allow for fewer immunizations, more efficient usage, and/or fewer side effects to the vaccine. SUMMARY OF THE INVENTION
- the present invention provides methods and compositions for the stimulation of immune responses. Specifically, the present invention provides methods of inducing an immune response to an agent (e.g., a bacteria or virus) in a subject (e.g., a human subject) and compositions useful in such methods (e.g., a nanoemulsion comprising a bacteria or virus or a component thereof). Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic arid preventative medicine (e.g., vaccination)) and research applications.
- an agent e.g., a bacteria or virus
- compositions useful in such methods e.g., a nanoemulsion comprising a bacteria or virus or a component thereof.
- Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic arid preventative medicine (e.g., vaccination)) and research applications.
- the present invention provides a method of inducing an immune response to an orthopox virus in a subject comprising: providing a composition comprising a nanoemulsion and an immunogen, wherein the immunogen comprises orthopox virus inactivated by the nanoemulsion; and administering the composition to the subject under conditions such that the subject generates an immune response to the orthopox virus.
- the present invention is not limited by the nature of the immune response generated.
- immune responses may be generated and measured in a subject administered a composition comprising a nanoemulsion and an immunogen of the present invention including, but not limited to, activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, antigen presenting cells (APCs), macrophages, natural killer (NK) cells, etc.); up-regulated or down- regulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly (e.g., increased spleen cellularity); hyperplasia, mixed cellular infiltrates in various organs, and other responses (e.g., of cells) of the immune system that can be assessed with respect to immune stimulation known in the art.
- cells of the immune system e.g., B cells, T cells, dendritic cells, antigen presenting cells (APCs), macrophages, natural killer (NK) cells, etc.
- APCs antigen
- administering comprises contacting a mucosal surface of the subject with the composition.
- the present invention is not limited by the mucosal surface contacted.
- the mucosal surface comprises nasal mucosa.
- administrating comprises parenteral administration.
- the present invention is not limited by the route chosen for administration of a composition of the present invention.
- inducing an immune response induces immunity to the orthopox virus in the subject.
- the immunity comprises systemic immunity, hi some embodiments, the immunity comprises mucosal immunity.
- the immune response comprises increased expression of IFN- ⁇ in the subject.
- the immune response comprises a systemic IgG response to the inactivated orthopox virus.
- the immune response comprises a mucosal IgA response to the inactivated orthopox virus.
- the present invention is not limited by the type of orthopox virus used in a composition of the present invention. Indeed, a variety of orthopox viruses may be used including, but not limited to, variola virus, vaccinia virus, cowpox, monkeypox, gerbilpox, camelpox, among others.
- the orthopox virus inactivated by the nanoemulsion is administered to the subject under conditions such that between 10 and 10 3 pfu of the inactivated virus is present in a dose administered to the subject. However, the present invention is not limited to this amount of orthopox virus administered.
- more than 10 pfu of the inactivated virus e.g., 10 4 pfu, 10 s pfu, or more
- a 10% nanoemulsion solution is utilized to inactivate the orthopox virus.
- the present invention is not limited to this amount (e.g., percentage) of nanoemusion used to inactivate a orthopox virus.
- a composition comprising less than 10% nanoemulsion is used for inactivation.
- a composition comprising more than 10% nanoemulsion is used for inactivation.
- the nanoemulsion comprises W2o5EC.
- the present invention is not limited by the type of nanoemulsion utilized. Indeed, a variety of nanoemulsions are contemplated to be useful in the present invention.
- the nanoemulsion e.g., for generating an immune response (e.g., for use as a vaccine)
- the nanoemulsion comprises an oil-in-water emulsion, the oil-in-water emulsion comprising a discontinuous oil phase distributed in an aqueous phase, a first component comprising a solvent (e.g., an alcohol or glycerol), and a second component comprising a surfactant or a halogen-containing compound.
- a solvent e.g., an alcohol or glycerol
- the aqueous phase can comprise any type of aqueous phase including, but not limited to, water (e.g., diH2 ⁇ , distilled water, tap water) and solutions (e.g., phosphate buffered saline solution).
- the oil phase can comprise any type of oil including, but not limited to, plant oils (e.g., soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseed oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, and sunflower oil), animal oils (e.g., fish oil), flavor oil, water insoluble vitamins, mineral oil, and motor oil.
- plant oils e.g., soybean oil, avocado oil, flaxseed oil, coconut oil, cottonseed oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, and sunflower oil
- animal oils e.g., fish oil
- flavor oil water insoluble
- the oil phase comprises 30-90 vol% of the oil-in-water emulsion (i.e., constitutes 30-90% of the total volume of the final emulsion), more preferably 50-80%. While the present invention in not limited by the nature of the alcohol component, in some preferred embodiments, the alcohol is ethanol or methanol.
- the surfactant is a polysorbate surfactant (e.g., TWEEN 20, TWEEN 40, TWEEN 60, and TWEEN 80), a pheoxypolyethoxyethanol (e.g., TRITON X-100, X-301, X-165, X-102, and X-200, and TYLOXAPOL) or sodium dodecyl sulfate.
- a polysorbate surfactant e.g., TWEEN 20, TWEEN 40, TWEEN 60, and TWEEN 80
- a pheoxypolyethoxyethanol e.g., TRITON X-100, X-301, X-165, X-102, and X-200
- TYLOXAPOL sodium dodecyl sulfate
- the halogen-containing compound comprises a cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, tetradecyltrimethylammonium halides, cetylpyridinium chloride, cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide, cetyltrimethylammonium bromide, cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, or tetrad ecyltrimethyl
- Nanoemulsions of the present invention may further comprise third, fourth, fifth, etc. components.
- an additional component is a surfactant (e.g., a second surfactant), a germination enhancer, a phosphate based solvent (e.g., tributyl phosphate), a neutramingen, L-alanine, ammonium chloride, trypticase soy broth, yeast extract, L-ascorbic acid, lecithin, p-hyroxybenzoic acid methyl ester, sodium thiosulate, sodium citrate, inosine, sodium hyroxide, dextrose, and polyethylene glycol (e.g., PEG 200, PEG 2000, etc.).
- a surfactant e.g., a second surfactant
- a germination enhancer e.g., tributyl phosphate
- a neutramingen e.g., L-alanine, ammonium chloride
- the oil-in- water emulsion comprises a quaternary ammonium compound. In some preferred embodiments, the oil-in-water emulsion has no detectable toxicity to plants or animals (e.g., to humans). In other preferred embodiments, the oil-in-water emulsion causes no detectable irritation to plants or animals (e.g., to humans). In some embodiments, the oil-in-water emulsion further comprises any of the components described above.
- Quaternary ammonium compounds include, but are not limited to, N-alkyldimethyl benzyl ammonium saccharinate, l,3,5-Triazine-l,3,5(2H,4H,6H)-triethanol; 1-Decanaminium, N-decyl-N, N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride; 2-(2-(p- (Diisobuyl)cresosxy)ethoxy)ehyl dimethyl benzyl ammonium chloride; 2-(2-(p- (Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ammonium chloride; alkyl 1 or 3 benzyl- l-(2-hydroxethyl)-2-imidazolinium chloride; alkyl bis(2-hydroxyethyl) benzyl ammonium chloride; alkyl demethyl benzyl ammonium chloride
- the emulsion lacks any antimicrobial substances (i.e., the only antimicrobial composition is the emulsion itself).
- the nanoemulsion is X8P.
- the immunity protects the subject from displaying signs or symptoms of disease caused by an orthopox virus (e.g., vaccinia virus), hi some embodiments, the immunity protects the subject from challenge with a subsequent exposure to live orthopox virus.
- induction of an immune response protects a subject from morbidity and/or mortality associated with orthopox virus infection, hi some embodiments, the composition further comprises an adjuvant.
- the present invention is not limited by the type of adjuvant utilized. A number of adjuvants that find use in the present invention are described herein.
- the subject is a human.
- the present invention also provides a composition for stimulating an immune response comprising a nanoemulsion and an orthopox virus inactivated by the nanoemulsion, wherein the composition is configured to induce immunity to the orthopox virus in a subject.
- the nanoemulsion comprises W2o5EC.
- the composition provides a subject between 10 and 10 3 pfu of the inactivated virus when administered to the subject.
- the composition provides a subject between 10 3 and 10 pfu of the inactivated virus when administered to the subject.
- a dose of the composition that is administered to a subject comprises a 1% nanoemulsion solution.
- the inactivated orthopox virus is heat stable in the nanoemulsion.
- the orthopox virus is stable for greater than four weeks in the nanoemulsion.
- the orthopox virus is vaccinia virus.
- the present invention is not limited by the type of orthopox virus used. Indeed, a variety of orthopox viruses can be used in a composition for stimulating an immune response including, but not limited to, variola virus, cowpox, monkeypox, gerbilpox, and camelpox.
- the composition is diluted prior to administration to a subject.
- the subject is a human.
- the immunity is systemic immunity.
- the immunity is mucosal immunity.
- the composition further comprises an adjuvant.
- the present invention also provides a kit comprising a composition for stimulating an immune response comprising a nanoemulsion and an orthopox virus inactivated by the nanoemulsion, wherein the composition is configured to induce immunity to the orthopox virus in a subject, and instructions for administering the composition.
- the present invention provides a method of inducing an immune response to B. anthracis in a subject comprising providing a composition comprising a nanoemulsion and an immunogen, wherein the immunogen comprises a B. anthracis immunogen (e.g., recombinant protective antigen (rPA) of B. anthracis); and administering the composition to the subject under conditions such that the subject generates an immune response to B. anthracis.
- rPA recombinant protective antigen
- the present invention is not limited by the B. anthracis immunogen utilized.
- the immunogen is an isolated, purified or recombinant protein or peptide antigen, or derivative or variant thereof, selected from the group comprising, but not lmited to, protective antigen (PA), lethal factor (LF), edema factor (EF), and PA degradation products.
- PA protective antigen
- LF lethal factor
- EF edema factor
- PA degradation products PA degradation products
- immune responses may be generated and measured in a subject administered a composition comprising a nanoemulsion and an immunogen of the present invention including, but not limited to, activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, antigen presenting cells (APCs), macrophages, natural killer (NK) cells, etc.); up-regulated or down-regulated expression of markers and cytokines; stimulation of IgA, IgM 5 or IgG titer; splenomegaly (e.g., increased spleen cellularity); hyperplasia, mixed cellular infiltrates in various organs, and other responses (e.g., of cells) of the immune system that can be assessed with respect to immune stimulation known in the art.
- cells of the immune system e.g., B cells, T cells, dendritic cells, antigen presenting cells (APCs), macrophages, natural killer (NK) cells, etc.
- APCs antigen
- administering comprises contacting a mucosal surface of the subject with the composition.
- the present invention is not limited by the mucosal surface contacted.
- the mucosal surface comprises nasal mucosa.
- administrating comprises parenteral administration.
- the present invention is not limited by the route chosen for administration of a composition of the present invention.
- inducing an immune response induces immunity to B. anthracis in the subject.
- the immunity comprises systemic immunity.
- the immunity comprises mucosal immunity.
- the immune response comprises increased expression of IFN- ⁇ in the subject.
- the immune response comprises a systemic IgG response.
- the immune response comprises a mucosal IgA response.
- the composition comprises between 1 and 300 ⁇ g of rPA.
- the present invention is not limited to this amount of recombinant protective antigen administered.
- more than 300 ⁇ g of rPA is present in a dose administered to the subject.
- less than 1 ⁇ g of rPA is present in a dose administered to a subject.
- the composition comprises a 10% nanoemulsion solution.
- the present invention is not limited to this amount (e.g., percentage) of nanoemusion.
- a composition comprises less than 10% nanoemulsion.
- a composition comprises more than 10% nanoemulsion.
- a composition of the present invention comprises any of the nanoemulsions described herein.
- the nanoemulsion comprises W2o5EC.
- the present invention is not limited by the type of nanoemulsion utilized.
- the immunity protects the subject from displaying signs or symptoms of disease caused by B. anthrads..
- the immunity protects the subject from challenge with a subsequent exposure to live B. anthrads.
- induction of an immune response protects a subject from morbidity and/or mortality associated with B. anthrads infection.
- the composition further comprises an adjuvant.
- the present invention is not limited by the type of adjuvant utilized.
- the adjuvant is a CpG oligonucleotide. A number of other adjuvants that find use in the present invention are described herein.
- the subject is a human, hi some preferred embodiments, immunity protects said subject from displaying signs or symptoms of anthrax.
- the present invention also provides a composition for stimulating an immune response comprising a nanoemulsion and recombinant protective antigen of B. anthrads, wherein the composition is configured to induce immunity to B. anthrads in a subject, hi some embodiments, the nanoemulsion comprises W2o5EC.
- the composition provides the subject between 25 and 75 ⁇ g of the recombinant protective antigen when administered to the subject.
- a dose of the composition administered to the subject comprises a 1% nanoemulsion solution.
- the recombinant protective antigen is heat stable in the nanoemulsion. In some embodiments, the recombinant protective antigen is stable for greater than four weeks in the nanoemulsion.
- the composition is diluted prior to administration to a subject.
- the subject is a human.
- the immunity is systemic immunity.
- the immunity is mucosal immunity.
- the composition further comprises an adjuvant.
- the adjuvant comprises a CpG oligonucleotide.
- the present invention also provides a kit comprising a composition for stimulating an immune response comprising a nanoemulsion and recombinant protective antigen of B. anthracis, wherein the composition is configured to induce immunity to B. anthrads in a subject, and instructions for administering the composition.
- the kit further comprises a device for administering the composition.
- the present invention is not limited by the type of device utilized for administering the composition. Indeed, a variety of devices are contemplated to be useful in a kit including, but not limited to, a nasal applicator, a syringe, a nasal inhaler and a nasal mister.
- the kit comprises a composition comprising a nanoemulsion and a B.
- the present invention provides systems and methods for large scale administration (e.g., to a population of a town, village, city, state or country) of a composition of the present invention (e.g., in response to an attack using a Bacillus pathogen).
- the present invention provides a method of inducing an immune response to HW in a subject comprising providing a composition comprising a nanoemulsion and an immunogen, wherein the immunogen comprises recombinant gpl20; and administering the composition to the subject under conditions such that the subject generates an immune response to the HIV.
- the present invention is not limited by the type of immunogen utilized (e.g., recombinant gpl 20).
- the immunogen is an isolated, purified or recombinant Tat, Nef or other immunogenic HIV protein, or derivative thereof.
- the present invention is not limited by the nature of the immune response generated.
- immune responses may be generated and measured in a subject administered a composition comprising a nanoemulsion and an immunogen of the present invention including, but not limited to, activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, antigen presenting cells (APCs), macrophages, natural killer (NK) cells, etc.); up-regulated or down- regulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly (e.g., increased spleen cellularity); hyperplasia, mixed cellular infiltrates in various organs, and other responses (e.g., of cells) of the immune system that can be assessed with respect to immune stimulation known in the art.
- cells of the immune system e.g., B cells, T cells, dendritic cells, antigen presenting cells (APCs), macrophages, natural killer (NK) cells, etc.
- APCs antigen
- administering comprises contacting a mucosal surface of the subject with the composition.
- the present invention is not limited by the mucosal surface contacted, hi some preferred embodiments, the mucosal surface comprises nasal mucosa. In some embodiments, the mucosal surface comprises vaginal mucosa.
- administrating comprises parenteral administration. The present invention is not limited by the route chosen for administration of a composition of the present invention.
- inducing an immune response induces immunity to said HIV in said subject.
- the immunity comprises systemic immunity.
- the immunity comprises mucosal immunity.
- the immune response comprises increased expression of IFN- ⁇ in the subject.
- the immune response comprises a systemic IgG response. In some embodiments, the immune response comprises a mucosal IgA response. In some embodiments, the composition comprises between 15 and 75 ⁇ g of recombinant gpl20. However, the present invention is not limited to this amount of recombinant gpl20 administered. For example, in some embodiments, more than 75 ⁇ g of recombinant gpl20 is present in a dose administered to the subject, hi some embodiments, less than 15 ⁇ g of recombinant gpl20 is present in a dose administered to a subject. In some embodiments, the composition comprises a 10% nanoemulsion solution.
- a composition comprises less than 10% nanoemulsion. In some embodiments, a composition comprises more than 10% nanoemulsion. In some embodiments, the nanoemulsion comprises W 2 o5EC (See U.S. Pat. No. 6,015,832, hereby incorporated by reference in its entirety). In some embodiments, the nanoemulsion comprises X8P (See U.S. Pat. No. 6,015,832, hereby incorporated by reference in its entirety).
- the present invention also provides a kit comprising a composition for stimulating an immune response comprising a nanoemulsion and an HIV immunogen (e.g., recombinant gpl20), wherein the composition is configured to induce immunity to HIV in a subject, and instructions for administering the composition.
- the kit comprises a nanoemulsion in contact with an object (e.g., an applicator).
- the kit comprises a device for administering the composition.
- the present invention is not limited by the type of device included in the kit for administering the composition. Indeed, many different devices may be included in the kit including, but not limited to, a nasal applicator, a syringe, a nasal inhaler and a nasal mister.
- the kit comprises a vaginal applicator, vaginal mister or other type of device for vaginal administration (e.g., to the vaginal mucosa) of a composition of the present invention.
- a kit comprises a birth control device (e.g., a condom, an IUD, sponge, etc.) coated with a nanoemulsion composition of the present invention.
- a nanoemulsion composition of the present invention is mixed in a douche or a suppository or a lubricant (e.g., sexual lubricant).
- the present invention provides systems and methods (e.g., using a nanoemulsion composition of the present invention) for large scale administration (e.g., to a population of a city, village, town, state or country).
- large scale administrations are carried out in a manner that is easy to use (e.g., nasal administration) and that is culturally sensitive (e.g., so as not to offend those being administered a composition of the present invention).
- Figure 1 shows complete virus inactivation with nanoemulsion.
- PRA Plaque reduction assay
- B Luciferase assay of WWR-LUC Luciferase activity is presented in relative light units (RLU).
- C PCR analysis of lung DNA. Lane 1 : DNA size marker; lane 2: primers, no DNA; lane 3: no Taq; lane 4: 10 5 /Fk lung DNA; lanes 5-7: 10 5 /Fk/NE lung DNA; lanes 8-10: 10 5 /NE lung DNA; lane 11 : control - W DNA mixed with lung DNA. Arrows indicate amplified viral template and primers.
- Figure 2 shows immunogenicity of mucosal nanoemulsion vaccine in mice.
- A Development of serum anti-W IgG antibody response in mice vaccinated with various formulations of killed virus vaccine: 10 5 /NE (filled circle), 10 3 /NE (open circle), 10 5 /Fk/NE (filled triangle), 10 3 /Fk/NE (open triangle), 10 5 /Fk (filled diamond) and 10 3 /Fk (open diamond). Arrows indicate i.n administrations of the vaccine. Insert: Comparison of serum anti-W IgG after one or three vaccinations with 10 5 /NE vaccine. Data presented as mean of the individual anti-W IgG concentrations ⁇ sem.
- B Secretory anti-W IgA antibody in BAL. Results are presented as mean concentrations (+/-sem) of IgA obtained in assays performed with individual and pooled BAL fluids.
- Figure 3 shows virus neutralizing antibodies. Assays were performed with both individual and with pooled sera obtained after one, two and three vaccinations. Insert: Detection of virus neutralizing activity in BAL. Assays were performed with individual and pooled BAL fluids collected at the conclusion of the experiment at 16 weeks. Results were normalized and presented as NTso of the viral PRA.
- Figure 4 shows vaccinia-specific cellular immune responses.
- the data show a specific INF- ⁇ response to the virus in splenocytes from animals immunized with vaccinia virus inactivated by NE.
- Figure 5 shows intranasal challenge with live vaccine virus.
- A Survival curves for mice vaccinated with 10 pfu of killed WWR in various vaccine formulations: W/NE, W/Fk/NE and W/Fk, after i.n. challenge with IOXLDSO WWR-LUO.
- B Bioluminescence images of representative vaccinated (upper panel) and control mouse (lower panel). Images were recorded 2 to 5 days after challenge.
- Figure 6 shows the stability of vaccine preparation.
- A 0.5 ⁇ g of rPA protein was incubated 24 hours in saline and 1 % NE at room temperature and analyzed using non- reducing 10% PAGE. Silver staining demonstrated low molecular weight fragments after incubation of the antigen without nanoemulsion (saline).
- B Micrographs of the NE and rPA/NE mix show no alteration in the emulsion after mixing with antigen (400 * magnification).
- Figure 7 shows effect of nanoemulsion on the cellular uptake of rPA protein.
- Jaws II dendritic cells were incubated with either (A) medium, (B) 0.1 ⁇ g/ml of PA-FITC alone, (C) 0.1 ⁇ g/ml of PA-FITC mixed with 0.001% NE or (D) 1 ⁇ g/ml of PA-FITC mixed with 0.001 % NE. Green fluorescence indicates that the rPA was effectively internalized only when administered with nanoemulsion.
- Figure 8 shows time course of the serum anti-PA IgG in mice. Mice were intranasally immunized with two doses of vaccine (arrows).
- A Induction of the anti-PA IgG in CBAJJ mice vaccinated with 20 ⁇ g rPA and increasing concentration of NE. (A, insert). The anti-PA IgG subtypes in CBA/J mice immunized with rPA/NE. Data are presented as ratios of individual IgG2a, IgG2b and IgG3 titers versus IgGl titer.
- B Anti- PA IgG in Balb/c mice vaccinated with various formulations of rPA vaccine.
- Figure 9 shows anti-PA IgA and IgG antibodies in bronchial lavage.
- the anti-PA IgA (A) and anti-PA IgG (B) determined by ELISA of bronchial alveolar lavage (BAL) from Balb/c mice vaccinated with various formulations of vaccine.
- Anti-PA IgA and anti-PA IgG antibodies are expressed as the mean +/- sem of antibody concentrations.
- FIG 10 shows lethal toxin (LeTx) neutralization in vitro.
- RAW264.7 cells were treated with the anthrax LeTx that had been preincubated with a serial dilution of immune, pooled Balb/c sera. Bars represent the antibody dilution in which cells retain 50% viability (NC 50 ).
- Figure 11 shows PA-specific induction of splenocyte proliferation in vitro.
- Splenocytes isolated from immunized mice were stimulated with rPA (5 ⁇ g/ml) for 72 hours.
- Proliferation indexes were calculated as a ratio of the activity in rPA-stimulated cells to the activity in resting splenocytes.
- (*) Indicates statistical difference between groups (p ⁇ 0.05).
- Figure 12 shows immune response and survival of guinea pigs intranasally immunized with rPA/NE vaccine.
- Hartley guinea pigs were vaccinated with 2 doses of vaccine (day 1 and at 4 weeks as documented by arrows).
- A Anti-PA IgG in guinea pig serum. Antibody titers were determined at 3- to 4-week intervals with serum anti-PA IgG measured by ELISA (mean endpoint titers +/- sem).
- B Intradermal challenge. At 6 months, guinea pigs were i.d injected with 1000 x LD50 of Ames spores and mortality was monitored for 14 days.
- LeTx neutralization was performed at 22 weeks before the challenge.
- the antibody titer in which RAW264 cells retained 50% viability is determined from the cell viability obtained in at least two assays each performed in triplicate.
- (*) Indicates statistically significant difference as compared to unvaccinated animals (p ⁇ 0.001).
- Figure 13 shows immune response and intranasal challenge of guinea pigs intranasally vaccinated with rPA/NE vaccine. Hartley guinea pigs vaccinated on day 1 and at 4 weeks.
- A Anti-PA IgG and LeTx neutralizing antibody titers in serum. Antibody titers were determined at 3 and 6 weeks and are presented as the mean +/- sem of individual serum anti-PA IgG endpoint titers. The LeTx neutralization assay cell was performed before the challenge, with values representing mean titers in which RAW264 cells retained 50% viability (NCso).
- B and C Survival Curves after Intranasal Challenge.
- guinea pigs were infected with i.n. instillation of 10 LDso (B) and 100 x LD50 (C) of Ames spores, and animals were monitored up to 16 days.
- (*) Indicates p ⁇ 0.05 between all vaccinated groups as compared to unvaccinated animals.
- Figure 14 shows antibody response in mice intranasally vaccinated with two serotypes of recombinant gpl20 and nanoemulsion adjuvant.
- A Induction of serum anti- g ⁇ l20BaL IgG in mice immunized with gpl20 ⁇ aL mixed with 0.1%, 0.5% and 1% NE. Anti- gpl20 BaL IgG antibodies were measured at 6 weeks (after two doses) and 12 weeks (after three doses). Intranasal (i.n.) and intramuscular (i.m.) routes of immunization are indicated in the Figure.
- B Induction of anti-gpl20sFi62 IgG in mice i.n.
- Figure 15 shows nasal immunization with gpl20/NE induces mucosal IgA.
- A Secretory anti-gpl20 IgA in bronchial lavage (BAL), and (B) in serum and in the vaginal washes of mice vaccinated with gpl20BaL and NE adjuvant.
- Anti-gpl20 IgA concentration is presented as mean absorbance (OD 405 nm +/- s.d.) obtained in ELISA performed with 1 :2 diluted BAL fluids (A), undiluted vaginal washes, and 1:50 diluted serum (B).
- Statistically significant differences were observed between gpl20/saline and each gpl20/NE groups (p ⁇ 0.05).
- Figure 16 shows (A) Antigen-specific splenocyte proliferation. Splenocytes from immunized animals were stimulated in vitro with 2 ⁇ g/ml of autologous recombinant gpl 20BaL- Cell proliferation was normalized to controls and presented as mean +/- s.d. of individual proliferation indexes. The differences between the gpl20BaL/saline and the g ⁇ l20Ba-/NE groups were statistically significant (p ⁇ 0.05). (B) Antigen-specific activation of cytokine production in splenocytes in vitro.
- Splenocytes from immunized mice were activated with 2 ⁇ g/ml of autologous and heterologous serotypes of g ⁇ l20 (BaL and SFl 62, respectively) and with 20 ⁇ M of the V3 loop peptide.
- the released EFN- ⁇ was determined by ELISA and concentration is presented as a mean of individual samples +/- s.d.
- Figure 17 shows (A) Nasal immunization of guinea pig.
- Hartly guinea pigs (GP) were vaccinated in the prime-boost schedule with 50 ⁇ g gpl20sFi62 in 1 % NE.
- the serum IgG antibody response toward gpl20sFi62 and BaL serotypes was measured at six weeks.
- Anti-gpl20 IgG are presented as absorption values (OD 405 run, +/- s.d.) obtained in ELISA with 1:200 dilution of serum using plates coated with gpl20sFi62gp (autologous) and 120 ⁇ aL (heterologous) serotypes of antigen.
- B Neutralizing antibody produced by i.n.
- gpl20sFi62/NE The neutralization of laboratory strains and primary isolates of HIV were performed in the TZM-BL cell system. NT50 values represent the serum dilution at which relative luminescence units (RLU) were reduced 50% compared to virus control. Individual preimmune sera were used to evaluate nonspecific antiviral activity.
- RLU relative luminescence units
- the present invention provides methods and compositions for the stimulation of immune responses. Specifically, the present invention provides methods of inducing an immune response against a pathogen (e.g., vaccinia virus, Bacillus anthracis, HIV, etc.) in a subject (e.g., a human subject) and compositions useful in such methods (e.g., a nanoemulsion comprising a pathogen inactivated by the nanoemulsion, or an immunogenic portion thereof). Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic and preventative medicine (e.g., vaccination)) and research applications.
- the pathogen is mixed with the nanoemulsion prior to administration for a time period sufficient to inactivate the pathogen.
- protein components e.g., isolated or purified protein, or recombinant protein
- NE treatment e.g., neutralization of a pathogen with a NE of the present invention
- preserves important antigenic epitopes e.g., recognizable by a subject's immune system
- stabilizing their hydrophobic and hydrophilic components in the oil and water interface of the emulsion e.g., thereby providing one or more immunogens (e.g., stabilized antigens) against which a subject can mount an immune response).
- NE formulations penetrate the mucosa through pores, they carry immunogens to the submucosal location of dendritic cells (e.g., thereby initiating and/or stimulating an immune response).
- an immunogenic protein e.g., rPA from. B. anthrads, or g ⁇ l20 from HIV, etc.
- Dendritic cells avidly phagocytose NE oil droplets and this could provide a means to internalize immunogens (e.g., antigenic proteins or peptide fragments thereof) for antigen presentation. While other vaccines rely on inflammatory toxins or other immune stimuli for adjuvant activity (See, e.g., Holmgren and Czerkinsky, Nature Med. 2005, 11 ; 45-53), NEs have not been shown to be inflammatory when placed on the skin or mucous membranes in studies on animals and in humans.
- immunogens e.g., antigenic proteins or peptide fragments thereof
- NEs have not been shown to be inflammatory when placed on the skin or mucous membranes in studies on animals and in humans.
- a composition comprising a NE of the present invention may act as a "physical" adjuvant (e.g., that transports and/or presents immunogens (e.g., Vaccina proteins) to the immune system.
- an immunogen e.g., a NE inactivated pathogen (e.g., a virus (e.g., W)
- a "physical" adjuvant e.g., that transports and/or presents immunogens (e.g., Vaccina proteins) to the immune system.
- mucosal administration of a composition of the present invention generates mucosal (e.g., signs of mucosal immunity (e.g., generation of IgA antibody titers) as well as systemic immunity.
- Both cellular and humoral immunity play a role in protection against multiple pathogens and both can be induced with a NE composition (e.g., comprising a pathogen inactivated by the nanoemulsion, or an immunogenic portion of a pathogen) of the present invention.
- a NE composition e.g., comprising a pathogen inactivated by the nanoemulsion, or an immunogenic portion of a pathogen
- vaccinia-specific antibody titers are considered important for protective immunity in human subjects and in animal models of vaccination (See, e.g., Hammarlund et al, Nat. Med. 2003, 9; 1131 -1137).
- IFN- ⁇ Induction of IFN- ⁇ is suggestive of activation of specific MHC class I-restricted CD8+ T cells. These types of cells have been implicated in the recognition and clearance of Vaccinia infected cells, and for maintenance of immunity after vaccination (See, e.g., Earl et al, Nature, 2004; 482; 182-185; Hammarlund et al, Nat. Med. 2003, 9; 1131-1137; Edghill- Smith et all, Nature Med. 2005, 11; 740-747).
- administration e.g., mucosal administration
- a composition of the present invention e.g., NE-killed orthopox virus (e.g., W)
- NE-killed orthopox virus e.g., W
- administration results in the induction of both humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immune responses (e.g., against the orthopox virus).
- a composition of the present invention e.g., NE-killed orthopox virus (e.g., W) or a NE and one or more immunogens
- a vaccine e.g., a smallpox vaccine, an anthrax vaccine, an influenza vaccine, etc.
- a composition of the present invention induces (e.g., when administered to a subject) both systemic and mucosal immunity.
- administration of a composition of the present invention to a subject results in protection against an exposure (e.g., a lethal mucosal exposure) to a pathogen (e.g., a virus (e.g., an orthopox virus (e.g., W))).
- a pathogen e.g., a virus (e.g., an orthopox virus (e.g., W)
- mucosal administration e.g., vaccination
- pathogen infection e.g., that initiates at a mucosal surface
- pathogen infection e.g., that initiates at a mucosal surface
- the present invention provides compositions and methods for stimulating mucosal immunity (e.g., a protective IgA response) from a pathogen in a subject.
- the present invention provides a composition (e.g., comprising a NE and an immunogen) to serve as a mucosal vaccine.
- a composition e.g., comprising a NE and an immunogen
- This material can easily be produced from purified virus and/or bacteria and/or protein or recombinant protein and induces both mucosal and systemic immunity.
- the ability to produce this formulation rapidly and administer it via mucosal instillation provides a vaccine that can be used for
- microorganism refers to any species or type of microorganism, including but not limited to, bacteria, viruses, archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.
- microorganism encompasses both those organisms that are in and of themselves pathogenic to another organism (e.g., animals, including humans, and plants) and those organisms that produce agents that are pathogenic
- pathogen refers to an organism (e.g., biological agent), including microorganisms, that causes a disease state (e.g., infection, pathologic condition, disease, etc.) in another organism (e.g., animals and plants) ) by directly infecting the other organism, or by producing agents that causes disease in another organism (e.g., bacteria that produce pathogenic toxins and the like).
- a disease state e.g., infection, pathologic condition, disease, etc.
- animals and plants e.g., animals and plants
- agents that causes disease in another organism e.g., bacteria that produce pathogenic toxins and the like.
- Pathogens include, but are not limited to, viruses, bacteria, archaea, fungi, protozoans, mycoplasma, prions, and parasitic organisms.
- bacteria and "bacterium” refer to all prokaryotic organisms, including • those within all of the phyla in the Kingdom Procary ⁇ tae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.
- fungi is used in reference to eukaryotic organisms such as I molds and yeasts, including dimorphic fungi.
- disease and “pathologic condition” are used interchangeably, unless indicated otherwise herein, to describe a deviation from the condition regarded as normal or average for members of a species or group (e.g., humans), and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species or group.
- a deviation can manifest as a state, signs, and/or symptoms (e.g., diarrhea, nausea, fever, pain, blisters, boils, rash, immune suppression, inflammation, etc.) that are associated with any impairment of the normal state of a subject or of any of its organs or tissues that interrupts or modifies the performance of normal functions.
- a disease or pathological condition may be caused by or result from contact with a microorganism (e.g., a pathogen or other infective agent (e.g., a virus or bacteria)), may be responsive to environmental factors (e.g., malnutrition, industrial hazards, and/or climate), may be responsive to an inherent defect of the organism (e.g., genetic anomalies) or to combinations of these and other factors.
- a microorganism e.g., a pathogen or other infective agent (e.g., a virus or bacteria)
- environmental factors e.g., malnutrition, industrial hazards, and/or climate
- an inherent defect of the organism e.g., genetic anomalies
- compositions and methods of the present invention refer to an individual to be treated by (e.g., administered) the compositions and methods of the present invention.
- Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.
- the term “subject” generally refers to an individual who will be administered or who has been administered one or more compositions of the present invention (e.g., a composition for inducing an immune response).
- the terms “inactivating,” “inactivation” and grammatical equivalents when used in reference to a microorganism (e.g., a pathogen (e.g., a bacterium or a virus)), refer to the killing, elimination, neutralization and/or reducing of the capacity of the mircroorganism (e.g., a pathogen (e.g., a bacterium or a virus)) to infect and/or cause a pathological response and/or disease in a host.
- a microorganism e.g., a pathogen (e.g., a bacterium or a virus)
- NE nanoemulsion
- W nanoemulsion-inactivated vaccinia virus
- compositions comprising "NE- inactivated W,” “NE-killed V,” NE-neutralized V” or grammatical equivalents refer to compositions that, when administered to a subject, are characterized by the absence of, or significantly reduced presence of, VV replication (e.g., over a period of time (e.g., over a period of days, weeks, months, or longer)) within the host.
- fusigenic is intended to refer to an emulsion that is capable of fusing with the membrane of a microbial agent (e.g., a bacterium or bacterial spore). Specific examples of fusigenic emulsions are described herein.
- the term "lysogenic” refers to an emulsion (e.g., a nanoemulsion) that is capable of disrupting the membrane of a microbial agent (e.g., a virus (e.g., viral envelope) or a bacterium or bacterial spore).
- a microbial agent e.g., a virus (e.g., viral envelope) or a bacterium or bacterial spore.
- a microbial agent e.g., a virus (e.g., viral envelope) or a bacterium or bacterial spore.
- the presence of a Iysogenic and a fusigenic agent in the same composition produces an enhanced inactivating effect compared to either agent alone.
- Methods and compositions e.g., for inducing an immune response (e.g., used as a vaccine) using this improved antimicrobial composition are described in detail herein.
- emulsion includes classic oil-in-water or water in oil dispersions or droplets, as well as other lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., 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.
- lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.
- nanoemulsion refers to oil-in-water dispersions comprising small lipid structures.
- the nanoemulsions comprise an oil phase having droplets with a mean particle size of approximately 0.1 to 5 microns (e.g., 150 +/-25 nm in diameter), although smaller and larger particle sizes are contemplated.
- emulsion and nanoemulsion are often used herein, interchangeably, to refer to the nanoemulsions of the present invention.
- the terms "contact,” “contacted,” “expose,” and “exposed,” when used in reference to a nanoemulsion and a live microorganism refer to bringing one or more nanoemulsions into contact with a microorganism (e.g., a pathogen) such that the nanoemulsion inactivates the microorganism or pathogenic agent, if present.
- a microorganism e.g., a pathogen
- the present invention is not limited by the amount or type of nanoemulsion used for microorganism inactivation.
- a variety of nanoemulsion that find use in the present invention are described herein and elsewhere (e.g., nanoemulsions described in U.S. Pat. Apps. 20020045667 and 20040043041, and U.S. Pat. Nos.
- Ratios and amounts of nanoemulsion e.g., sufficient for inactivating the microorganism (e.g., virus inactivation)
- microorganisms e.g., sufficient to provide an antigenic composition (e.g., a composition capable of inducing an immune response)
- an antigenic composition e.g., a composition capable of inducing an immune response
- surfactant refers to any molecule having both a polar head group, which energetically prefers solvation by water, and a hydrophobic tail that is not well solvated by water.
- cationic surfactant refers to a surfactant with a cationic head group.
- anionic surfactant refers to a surfactant with an anionic head group.
- HLB Index Number refers to an index for correlating the chemical structure of surfactant molecules with their surface activity.
- the HLB Index Number may be calculated by a variety of empirical formulas as described, for example, by Meyers, (See, e.g., Meyers, Surfactant Science and Technology, VCH Publishers Inc., New York, pp. 231-245 (1992)), incorporated herein by reference.
- the HLB Index Number of a surfactant is the HLB Tndex Number assigned to that surfactant in McCutcheon's Volume 1 : Emulsifiers and Detergents North American Edition, 1996 (incorporated herein by reference).
- the HLB Index Number ranges from 0 to about 70 or more for commercial surfactants. Hydrophilic surfactants with high solubility in water and solubilizing properties are at the high end of the scale, while surfactants with low solubility in water that are good solubilizers of water in oils are at the low end of the scale.
- interaction enhancers refers to compounds that act to enhance the interaction of an emulsion with a microorganism (e.g., with a cell wall of a bacteria ⁇ e.g., a Gram negative bacteria) or with a viral envelope (e.g., Vaccinia virus envelope)).
- Contemplated interaction enhancers include, but are not limited to, chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and the like) and certain biological agents ⁇ e.g., bovine serum abulmin (BSA) and the like).
- buffer or “buffering agents” refer to materials, that when added to a solution, cause the solution to resist changes in pH.
- reducing agent and "electron donor” refer to a material that donates electrons to a second material to reduce the oxidation state of one or more of the second material's atoms.
- monovalent salt refers to any salt in which the metal ⁇ e.g., Na, K, or Li) has a net 1+ charge in solution (i.e., one more proton than electron).
- divalent salt refers to any salt in which a metal (e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.
- a metal e.g., Mg, Ca, or Sr
- 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.
- solution refers to an aqueous or non-aqueous mixture.
- a composition for inducing an immune response refers to a composition that, once administered to a subject (e.g., once, twice, three times or more (e.g., separated by weeks, months or years)), stimulates, generates and/or elicits an immune response in the subject (e.g., resulting in total or partial immunity to a microorganism (e.g., pathogen) capable of causing disease).
- the composition comprises a nanoemulsion and an imrnunogen.
- the composition comprising a nanoemulsion and an immunogen comprises one or more other compounds or agents including, but not limited to, therapeutic agents, physiologically tolerable liquids, gels, carriers, diluents, adjuvants, excipients, salicylates, steroids, immunosuppressants, immunostimulants, antibodies, cytokines, antibiotics, binders, fillers, preservatives, stabilizing agents, emulsifiers, and/or buffers.
- An immune response may be an innate (e.g., a non-specific) immune response or a learned (e.g., acquired) immune response (e.g.
- a composition comprising a nanoemulsion and an immunogen is administered to a subject as a vaccine (e.g., to prevent or attenuate a disease (e.g., by providing to the subject total or partial immunity against the disease or the total or partial attenuation (e.g., suppression) of a sign, symptom or condition of the disease.
- a vaccine e.g., to prevent or attenuate a disease (e.g., by providing to the subject total or partial immunity against the disease or the total or partial attenuation (e.g., suppression) of a sign, symptom or condition of the disease.
- adjuvant refers to any substance that can stimulate an immune response (e.g., a mucosal immune response). Some adjuvants can cause activation of a cell of the immune system (e.g., an adjuvant can cause an immune cell to produce and secrete a cytokine). Examples of adjuvants that can cause activation of a cell of the immune system include, but are not limited to, saponins purified from the bark of the Q.
- saponaria tree such as QS21 (a glycolipid that elutes in the 21.sup.st peak with HPLC fractionation; Aquila Biopharmaceuticals, hie, Worcester, Mass.); poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).
- QS21 a glycolipid that elutes in the 21.sup.st peak with HPLC fractionation; Aquila Bio
- compositions of the present invention e.g., comprising HF/ or an immunogenic epitope thereof (e.g., gpl20)
- one or more adjuvants e.g., to skew the immune response towards a ThI or Th2 type response.
- an amount effective to induce an immune response refers to the dosage level required (e.g., when administered to a subject) to stimulate, generate and/or elicit an immune response in the subject.
- An effective amount can be administered in one or more administrations (e.g., via the same or different route), applications or dosages and is not intended to be limited to a particular formulation or administration route.
- the term "under conditions such that said subject generates an immune response” refers to any qualitative or quantitative induction, generation, and/or stimulation of an immune response (e.g., innate or acquired).
- immune response refers to a response by the immune system of a subject.
- immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll receptor activation, lyrnphokine (e.g., cytokine (e.g., ThI or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion).
- lyrnphokine e.g., cytokine (e.g., ThI or Th2 type cytokines) or chemokine
- macrophage activation e.g., dendritic cell activation
- T cell activation e.g., CD4+ or CD8+ T cells
- NK cell activation e.g.
- immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells.
- an immunogen e.g., antigen (e.g., immunogenic polypeptide)
- CTL cytotoxic T lymphocyte
- B cell response e.g., antibody production
- T-helper lymphocyte response e.g., T-helper lymphocyte response
- DTH delayed type
- an immune response may be to immunogens that the subject's immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign).
- immunogens that the subject's immune system recognizes as foreign
- immune response refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade) cell- mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system) and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
- innate immune responses e.g., activation of Toll receptor signaling cascade
- T cells e.g., antigen-specific T cells
- B cells e.g., via generation and secretion
- immuno response is meant to encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response).
- an immunogen e.g., a pathogen
- acquired e.g., memory
- the term "immunity” refers to protection from disease (e.g., preventing or attenuating (e.g., suppression) of a sign, symptom or condition of the disease) upon exposure to a microorganism (e.g., pathogen) capable of causing the disease.
- Immunity can be innate (e.g., non-adaptive (e.g., non-acquired) immune responses that exist in the absence of a previous exposure to an antigen) and/or acquired (e.g., immune responses that are mediated by B and T cells following a previous exposure to antigen (e.g., that exhibit increased specificity and reactivity to the antigen)).
- immunogen refers to an agent (e.g., a microorganism (e.g., bacterium, virus or fungus) or portion thereof (e.g., a protein antigen (e.g., gpl20 or rPA))) that is capable of eliciting an immune response in a subject.
- immunogens elicit immunity against the immunogen (e.g., microorganism (e.g., pathogen or a pathogen product)) when administered in combination with a nanoemulsion of the present invention.
- pathogen product refers to any component or product derived from a pathogen including, but not limited to, polypeptides, peptides, proteins, nucleic acids, membrane fractions, and polysaccharides.
- the term "enhanced immunity” refers to an increase in the level of adaptive and/or acquired immunity in a subject to a given immunogen (e.g., microorganism (e.g., pathogen)) following administration of a composition (e.g., composition for inducing an immune response of the present invention) relative to the level of adaptive and/or acquired immunity in a subject that has not been administered the composition (e.g., composition for inducing an immune response of the present invention).
- a given immunogen e.g., microorganism (e.g., pathogen)
- the terms “purified” or “to purify” refer to the removal of contaminants or undesired compounds from a sample or composition.
- the term “substantially purified” refers to the removal of from about 70 to 90 %, ' up to 100%, of the contaminants or undesired compounds from a sample or composition.
- administering refers to the act of giving a composition of the present invention (e.g., a composition for inducing an immune response (e.g., a composition comprising a nanoemulsion and an immunogen)) to a subject.
- a composition of the present invention e.g., a composition for inducing an immune response (e.g., a composition comprising a nanoemulsion and an immunogen)
- routes of administration to the human body include, but are not limited to, through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intraperitoneally, etc.), topically, and the like.
- co-administration refers to the administration of at least two agent(s) (e.g., a composition comprising a nanoemulsion and an imrnunogen and one or more other agents - e.g., an adjuvant) or therapies to a subject.
- the co-administration of two or more agents or therapies is concurrent.
- a first agent/therapy is administered prior to a second agent/therapy.
- co-administration can be via the same or different route of administration.
- formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art.
- agents or therapies when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone.
- co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
- co-administration is preferable to elicit an immune response in a subject to two or more different immunogens (e.g., microorganisms (e.g., pathogens)) at or near the same time (e.g., when a subject is unlikely to be available for subsequent administration of a second, third, or more composition for inducing an immune response).
- immunogens e.g., microorganisms (e.g., pathogens)
- topically refers to application of a compositions of the present invention (e.g., a composition comprising a nanoemulsion and an immunogen) to the surface of the skin and/or mucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory, vaginal or nasal mucosa, and other tissues and cells which line hollow organs or body cavities).
- a compositions of the present invention e.g., a composition comprising a nanoemulsion and an immunogen
- compositions of the present invention are administered in the form of topical emulsions, injectable compositions, ingestible solutions, and the like.
- the form may be, for example, a spray (e.g., a nasal spray), a cream, or other viscous solution (e.g., a composition comprising a nanoernulsion and an immunogen in polyethylene glycol).
- a spray e.g., a nasal spray
- cream e.g., a cream, or other viscous solution
- pharmaceutically acceptable or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions (e.g., toxic, allergic or immunological reactions) when administered to a subject.
- the term "pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintrigrants (e.g., potato starch or sodium starch glycolate), polyethylethe glycol, and the like.
- wetting agents e.g., sodium lauryl sulfate
- dispersion media e.g., any and all solvents
- dispersion media e.g., coatings, sodium lauryl sulfate, isotonic and absorption delaying agents
- disintrigrants e.g., potato starch or sodium starch glycolate
- polyethylethe glycol polyethylethe glycol, and the like.
- the term "pharmaceutically acceptable salt” refers to any salt (e.g., obtained by reaction with an acid or a base) of a composition of the present invention that is physiologically tolerated in the target subject.
- Salts of the compositions of the present invention may be derived from inorganic or organic acids and bases.
- acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like.
- Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compositions of the invention and their pharmaceutically acceptable acid addition salts.
- bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NWW + , wherein W is CM alkyl, and the like.
- alkali metal e.g., sodium
- alkaline earth metal e.g., magnesium
- W is CM alkyl
- salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tos
- salts include anions of the compounds of the present invention compounded with a suitable cation such as Na , NH 4 + , and NW/ (wherein W is a Ci-4 alkyl group), and the like.
- a suitable cation such as Na , NH 4 + , and NW/ (wherein W is a Ci-4 alkyl group), and the like.
- salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable.
- salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
- salts of the compositions of the present invention are contemplated as being pharmaceutically acceptable.
- salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable composition.
- the term "at risk for disease” refers to a subject that is predisposed to experiencing a particular disease. This predisposition may be genetic (e.g., a particular genetic tendency to experience the disease, such as heritable disorders), or due to other factors (e.g., environmental conditions, exposures to detrimental compounds present in the environment, etc.). Thus, it is not intended that the present invention be limited to any particular risk (e.g., a subject may be "at risk for disease” simply by being exposed to and interacting with other people), nor is it intended that the present invention be limited to any particular disease.
- Nesal application means applied through the nose into the nasal or sinus passages or both.
- the application may, for example, be done by drops, sprays, mists, coatings or mixtures thereof applied to the nasal and sinus passages.
- vaginal application means applied into or through the vagina so as to contact vaginal mucosa.
- the application may contact the urethra, cervix, fornix, uterus or other area surrounding the vagina.
- the application may, for example, be done by drops, sprays, mists, coatings, lubricants or mixtures thereof applied to the vagina or surrounding tissue.
- kits refers to any delivery system for delivering materials.
- immunogenic agents e.g., compositions comprising a nanoemulsion and an immunogen
- such delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and/or supporting materials (e.g., written instructions for using the materials, etc.) from one location to another.
- kits include one or more enclosures (e.g., boxes) containing the relevant immunogenic agents (e.g., nanoemulsions) and/or supporting materials.
- fragment kit refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately.
- a first container may contain a composition comprising a nanoemulsion and an immunogen for a particular use, while a second container contains a second agent (e.g., an antibiotic or spray applicator).
- a second agent e.g., an antibiotic or spray applicator
- any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.”
- a “combined kit” refers to a delivery system containing all of the components of an immunogenic agent needed for a particular use in a single container (e.g., in a single box housing each of the desired components).
- kit includes both fragmented and combined kits.
- the present invention provides methods and compositions for the stimulation of immune responses. Specifically, the present invention provides methods of inducing an immune response to an agent (e.g., a bacteria or virus) in a subject (e.g., a human subject) and compositions useful in such methods (e.g., a nanoemulsion comprising a bacteria or virus or a component thereof). Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic and preventative medicine (e.g., vaccination)) and research applications.
- an agent e.g., a bacteria or virus
- compositions useful in such methods e.g., a nanoemulsion comprising a bacteria or virus or a component thereof.
- Compositions and methods of the present invention find use in, among other things, clinical (e.g. therapeutic and preventative medicine (e.g., vaccination)) and research applications.
- pathogenic microorganisms initiate infection by attaching to mucosal epithelial cells lining the gastro-intestinal, oropharyngeal, respiratory or genito-urinacy tracts.
- Some pathogens such as influenza virus, Bordetella pertussis, or Vibrio cholerae, remain at or within the mucosal tissue, while others, such as Salmonella typhi or hepatitis A virus, possess mechanisms permitting penetration into deeper tissues and spread systemically.
- Specific and non-specific defense mechanisms of the mucous membranes provide first line protection against both types of pathogen.
- Non-specific effectors include, for example, resident macrophages, antimicrobial peptides, lactofe ⁇ in and lysozyme, extremes of pH, bile acids, digestive enzymes, mucus, shedding of epithelial cells, flushing mechanisms (peristalsis, ciliary beating, micturation, etc) and competition from local flora.
- successful pathogens have generally evolved means to survive the non-specific defenses present at the site they infect and it is the secretory immune system that plays a major role in protecting against diseases caused by a number of bacterial and viral pathogens, and is a major effector against pathogens that are restricted to mucosal surfaces. For organisms that spread systemically, both local and systemic immune responses are likely needed for optimum immunity.
- orthopox e.g., smallpox, cowpox, monkeypox, gerbilpox, camelpox, and others
- W live Vaccinia virus
- these vaccines also produce infectious skin pustules (pox) and infrequent but severe side reactions limiting their use in individuals (and their close contacts) with immunodeficiency, eczema, atopic dermatitis, or heart disease (See, e.g., Eichner and Schwehm, Epidemiology. 2004, 15(3):258-60).
- orthopox vaccines e.g., smallpox, cowpox, monkeypox, gerbilpox, camelpox, and others
- a bioterrorist attack or outbreaks of other orthopox infections such as monkeypox or cowpox
- monkeypox or cowpox See, e.g., Edghill-Smith et al., Nature Med. 2005, 11; 740-747.
- the risk/benefit ratio of vaccination will require that new smallpox vaccines place a high priority on safety.
- a rapidly administered vaccine e.g., a mucosal vaccine.
- Such administration may, in addition to providing a vaccination and long-term immune protection, may provide a quick, on-site generator of immune responses (e.g., that reduce infection, symptoms and/or time course of disease).
- the present invention provides methods of inducing an immune response to orthopox viruses (e.g., vaccinia virus) in a subject (e.g., a human subject) and compositions useful in such methods (e.g., a nanoemulsion comprising an orthopox virus (e.g., vaccinia virus (W))).
- methods of inducing an immune response provided by the present invention are used for vaccination. Due to the rate of adverse events with existing orthopox (e.g., smallpox) vaccines, the present invention provides a significant improvement in orthopox (e.g., small pox) vaccination safety without compromising vaccine efficiency.
- the present invention describes the development of immunity (e.g., W immunity) in a subject after mucosal administration (e.g., mucosal vaccination) of a unique type of inactivated orthopox virus (e.g., W) preparation identified and characterized during development of the present invention.
- immunity e.g., W immunity
- mucosal administration e.g., mucosal vaccination
- W inactivated orthopox virus
- a formulation e.g., NE-killed W composition
- Mucosal administration of a composition comprising NE and W e.g., NE-killed W
- NE-killed W e.g., W
- NE-killed W e.g., W
- all animals were fully protected against an nasal instillation challenge with 10x LDso W (See, e.g., Example 7, Figure 5).
- 10x LDso W See, e.g., Example 7, Figure 5
- infection was completely prevented or was of a low level and self-limiting and infection resolved in four to five days.
- all naive animals died within this time period.
- mice administered even a single dose of a composition comprising NE-killed W developed significant serum concentrations of anti- W IgG 10 to 12 weeks after administration (See, e.g., Example 2). This level of response is comparable to the results obtained in Balb/c mice immunized by intramuscular injection with live W Wyeth at similar time point (See, e.g., Coulibaly et al., Virology, 2005; 341; 91-101).
- the present invention provides that a single administration (e.g., mucosal administration) of a composition comprising NE-killed W is sufficient to induce a protective immune response in a subject (e.g., protective immunity (e.g., mucosal and systemic immunity)).
- a subsequent administration e.g., one or more boost administrations subsequent to a primary administration
- administration of a composition comprising NE-killed W to a subject provides protective immunity against smallpox.
- NE treatment e.g., neutralization of an orthopox virus (e.g., VV) with a NE of the present invention
- preserves important viral neutralizing epitopes e.g., recognizable by a subject's immune system
- stabilizing their hydrophobic and hydrophilic components in the oil and water interface of the emulsion e.g., thereby providing one or more immunogens (e.g., stabilized antigens) against which a subject can mount an immune response.
- immunogens e.g., stabilized antigens
- NE formulations may carry viral proteins to the submucosal location of dendritic cells (e.g., thereby initiating and/or stimulating an immune response).
- Dendritic cells avidly phagocytose NE oil droplets and this could provide a means to internalize antigenic proteins for antigen presentation. While other vaccines rely on inflammatory toxins or other immune stimuli for adjuvant activity (See, e.g., Holmgren and Czerkinsky, Nature Med. 2005, 11; 45-53), NEs have not been shown to be inflammatory when placed on the skin or mucous membranes in studies on animals and in humans.
- a composition comprising a NE of the present invention may act as a "physical" adjuvant (e.g., that transports and/or presents orthopox antigens (e.g., Vaccina proteins) to the immune system.
- a composition comprising a NE of the present invention may act as a "physical" adjuvant (e.g., that transports and/or presents orthopox antigens (e.g., Vaccina proteins) to the immune system.
- mucosal administration of a composition of the present invention generates mucosal as well as systemic immunity (e.g., signs of mucosal immunity (e.g., generation of IgA antibody titers).
- Vaccinia-specific antibody titers are considered important for the estimate of protective immunity in human subjects and in animal models of vaccination (See, e.g., Hammarlund et al, Nat. Med. 2003, 9; 1131-1137).
- proteins important for the elicitation of neutralizing antibodies See, e.g., Galmiche et al, Virology, 1999, 254; 71-80; Hooper et al, Virology, 2003, 306; 181-195).
- administration e.g., mucosal administration
- a composition of the present invention e.g., NE-killed orthopox virus (e.g., W)
- humoral e.g., development of specific antibodies
- cellular e.g., cytotoxic T lymphocyte
- a composition of the present invention e.g., NE-killed orthopox virus (e.g., VV) is used as a smallpox vaccine.
- a composition of the present invention e.g., NE-killed orthopox virus (e.g., W) induces (e.g., when administered to a subject) both systemic and mucosal immunity.
- administration of a composition of the present invention to a subject results in protection against an exposure (e.g., a lethal mucosal exposure) to an orthopox virus (e.g., W).
- mucosal administration e.g., vaccination
- orthopox virus e.g., W
- W infection e.g., that initiates at a mucosal surface
- the present invention provides compositions and methods for stimulating mucosal immunity (e.g., a protective IgA response) from a pathogen in a subject.
- the present invention provides a composition (e.g., a NE- inactivated orthopox virus (e.g., W) formulation) to serve as a mucosal vaccine.
- a composition e.g., a NE- inactivated orthopox virus (e.g., W) formulation
- W NE- inactivated orthopox virus
- This material can easily be produced from purified virus (See, e.g., Example 1), and induces both mucosal and systemic immunity (See, e.g., Examples 2-7).
- the ability to produce this formulation rapidly and administer it via nasal instillation provides a vaccine that can be used in large-scale outbreaks or emergent situations.
- the present invention provides compositions for generating an immune response and methods of using the same (e.g., for use as a vaccine).
- a composition for generating an immune response comprises a NE and an immunogen (e.g., an orthopox virus (e.g., W) inactivated by the nanoemulsion).
- an immunogen e.g., an orthopox virus (e.g., W) inactivated by the nanoemulsion.
- an immunogen e.g., an orthopox virus (e.g., W) inactivated by the nanoemulsion.
- generation of an immune response e.g., resulting from administration of a composition comprising a nanoemulsion and an orthopox virus (e.g., W)
- an immune response provides total or partial immunity to the subject (e.g., from signs, symptoms or conditions of a disease (e.g., smallpox)).
- protection and/or immunity from disease e.g., the ability of a subject's immune system to prevent or attenuate (e.g., suppress) a sign, symptom or condition of disease) upon exposure to a nanoemulsion comprising an orthopox virus (e.g., W) is due to adaptive (e.g., acquired) immune responses (e.g., immune responses mediated by B and T cells following exposure to a NE comprising an orthopox virus (e.g., W) of the present invention (e.g., immune responses that exhibit increased specificity and reactivity to an orthopox virus (e.g., W)).
- adaptive immune responses e.g., immune responses mediated by B and T cells following exposure to a NE comprising an orthopox virus (e.g., W) of the present invention (e.g., immune responses that exhibit increased specificity and reactivity to an orthopox virus (e.g., W)).
- a NE comprising an immunogen e.g., an orthopox virus (e.g., W) inactivated by the NE
- an immunogen e.g., an orthopox virus (e.g., W) inactivated by the NE
- a composition comprising a NE and an immunogen comprises one or more other agents (e.g., a pharmaceutically acceptable carrier, adjuvant, excipient, and the like).
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a humoral immune response.
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a cellular (e.g., cytotoxic T lymphocyte) immune response, rather than a humoral response.
- a composition comprising a NE and an immunogen of the present invention induces both a cellular and humoral immune response.
- the present invention is not limited by the type of NE utilized (e.g., in an immunogenic composition comprising an immunogen. Indeed, a variety of NE compositions are contemplated to be useful in the present invention.
- a nanoemulsion (e.g., for inactivation of an orthopox virus (e.g., VV)) comprises (i) an aqueous phase; (ii) an oil phase; and at least one additional compound.
- these additional compounds are admixed into either the aqueous or oil phases of the composition.
- these additional compounds are admixed into a composition of previously em ⁇ lsified oil and aqueous phases.
- one or more additional compounds are admixed into an existing emulsion composition immediately prior to its use.
- one or more additional compounds are admixed into an existing emulsion composition prior to the compositions immediate use.
- Additional compounds suitable for use in a nanoemulsion of the present invention include, but are not limited to, one or more organic, and more particularly, organic phosphate based solvents, surfactants and detergents, cationic halogen containing compounds, germination enhancers, interaction enhancers, food additives (e.g., flavorings, sweetners, bulking agents, and the like) and pharmaceutically acceptable compounds.
- organic phosphate based solvents e.g., ethylene glycol, g., g., g., g., ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbio
- pathogenic microorganisms initiate infection by attaching to mucosal epithelial cells lining the gastro-intestinal, oropharyngeal, respiratory or genito-urinacy tracts.
- Some pathogens such as influenza virus, Bordetella pertussis, or Vibrio cholerae, remain at or within the mucosal tissue, while others, such as Salmonella typhi or hepatitis A virus, possess mechanisms permitting penetration into deeper tissues and spread systemically.
- Specific and non-specific defense mechanisms of the mucous membranes provide first line protection against both types of pathogen.
- Non-specific effectors include resident macrophages, antimicrobial peptides, lactoferrin and lysozyme, extremes of pH, bile acids, digestive enzymes, mucus, shedding of epithelial cells, flushing mechanisms (peristalsis, ciliary beating, micturation, etc) and competition from local flora.
- successful pathogens have generally evolved means to survive the non-specific defenses present at the site they infect and it is the secretory immune system which plays a major role in protecting against diseases caused by a number of bacterial and viral pathogens, and is probably a major effector against pathogens that are restricted to mucosal surfaces. For organisms that spread systemically, both local and systemic immune responses are desirable for optimum immunity.
- Anthrax is an infectious bacterial disease caused by Bacillis anthracis. It occurs most commonly in wild and domestic herbivores (sheep, goats, camels, antelope, cattle, etc.) but may also occur in humans. Infection can occur by cutaneous exposure, by ingestion (gastrointestinal anthrax), or by inhalation (pulmonary anthrax). 95% of anthrax infections in humans occur by cutaneous infection, either from contact with unvaccinated, infected animals in an agricultural setting, or by handling contaminated animal products (meat, leather, hides, hair, wool, etc.) in an industrial setting.
- Cutaneous anthrax is fatal in about 20% of cases if untreated, but it can usually be overcome with appropriate antimicrobial therapy. Inhalation or gastrointestinal anthrax infection is much more serious and much more difficult to treat. Inhalation anthrax results in repirat ⁇ ry shock and is fatal in 90%-100% of cases; gastrointestinal anthrax results in severe fever, nausea and vomiting, resulting in death in 25%-75% of cases.
- Anthrax vaccine Anthrax Vaccine Adsorbed (or AVA, commercial name BIOTHRAX)
- BIOTHRAX aluminum hydroxide
- the course of vaccination consists of six subcutaneous injections of 0.5 mL doses of vaccine over eighteen months, with annual boosters to maintain immunity. This vaccination is believed to provide immunity that is 90%-100% effective against aerosol anthrax challenge, based on animal studies and incidental human data. (See, e.g., Friedlander et al, supra).
- the vaccine strain employed a non- proteolytic, non-capsulated mutant strain of B. anthracis, V770-NP1-R, has several disadvantageous characteristics: Despite its mutations, the strain retains a sporogenic and fully toxogenic phenotype, and use of the whole strain in vaccine production results in lotto-lot variability in levels of protective antigen (PA), as well as inclusion of PA degradation products and other bacterial products (See, e.g., Farchaus, J., et al., Applied & Environmental Microbiol., 64(3):982-991 (1998)).
- PA protective antigen
- anthrax vaccine is contaminated with squalene (See, e.g., Garret, L., Big Battle Over Vaccine: Detractors Say Immunization for Antrhax Hazardous; Pentagon Says No, The Beacon Journal (Akron), Sunday JuI. 4, 1999, Section B, p. 1), and has resulted in hundreds of military personnel refusing to be vaccinated (See, e.g., Graham, B., Some in Military Fear Anthrax Inoculation Side Effects, The Plain Dealer (Cleveland), Nov. 26, 1998, Section: National, p. 6E; Air Force Reserve Pilots Quitting Due to Vaccine, The Plain Dealer (Cleveland), Feb. 27, 1999, Section: National, p. 6A).
- squalene See, e.g., Garret, L., Big Battle Over Vaccine: Detractors Say Immunization for Antrhax Hazardous; Pentagon Says No, The Beacon Journal (Akron), Sunday JuI. 4, 1999, Section B, p.
- compositions for immunization against anthrax, and other diseases caused by bacteria of the genus Bacillus that is effective to raise an immune response against B. anihracis.
- Such a composition would ideally be formulated without contaminants (e.g., capable of generating unwanted side effects) and would be effective without a need for a long course of vaccination.
- the present invention provides methods of inducing an immune response to bacteria of the genus Bacillus (e.g., B. anthracis) in a subject (e.g., a human subject) and compositions useful in such methods (e.g., a nanoemulsion comprising bacteria or bacterial components (e.g., isolated or recombinant proteins) of the genus Bacillus (e.g., B. anthracis)).
- methods of inducing an immune response provided by the present invention are used for vaccination. Due to the rate of adverse events with existing Bacillus (e.g., B. anthracis) vaccines, the present invention provides a significant improvement in Bacillus (e.g., B. anthracis) vaccination safety without compromising vaccine efficacy;
- the present invention describes the development of immunity (e.g., B. anthrads immunity) in a subject after mucosal administration (e.g., mucosal vaccination) with a composition comprising a nanoemulsion and an immunogenic protein from B. anthrads (e.g., rPA) generated and characterized during development of the present invention (See Examples 8-16).
- a composition comprising a nanoemulsion and an immunogenic protein from B. anthrads (e.g., rPA) generated and characterized during development of the present invention (See Examples 8-16).
- room temperature e.g., ih some embodiments, for more than 2 weeks, more preferably more than 3 weeks, even more preferably more than 4 weeks, and most preferably for more than 5 weeks
- Mucosal administration of a composition comprising NE and rPA to a subject resulted in high-titer mucosal and systemic antibody responses and specific ThI cellular immunity (See, e.g., Examples 11-12, 14-16).
- serum from mice immunized intranasally with a composition comprising NE and rPA was capable of neutralizing binding of PA to its receptor (ATR receptor) (See Example 13).
- Mice administered two doses and guinea pigs administered just a single dose of a composition comprising NE and rPA developed significant serum concentrations of anti-rPA IgG after administration (See, e.g., Example 11).
- mice administered this composition generated mucosal immune responses (e.g., IgA antibodies toward rP A) (See Example 12).
- the present invention provides that administration (e.g., mucosal administration) of a composition comprising NE and a B. anthrads immunogen (e.g., rPA) is sufficient to induce a protective immune response against B. anthrads in a subject (e.g., protective immunity (e.g., mucosal and systemic immunity)).
- a subsequent administration e.g., one or more boost administrations subsequent to a primary administration
- a subsequent administration e.g., one or more boost administrations subsequent to a primary administration
- anthrads immunogen e.g., rPA
- anthrads immunogen e.g., rPA
- rPA anthrads immunogen
- a subject provides protective immunity against anthrax (e.g., via a durable anti-PA IgG response).
- anthrads immunogen e.g., rPA
- intranasal instillations of NE alone or NE with CpG adjuvant was not able to induce an immune response against B. anthrads (See Examples 11-13).
- administration of rPA alone did not induce significant IgG or IgA antibody production in mice.
- anthracis stabilizes the rPA (See Example 9) and provides the proper environment for generation of an immune response.
- NE formulations are known to penetrate the mucosa through pores, they may carry immunogenic proteins to the submucosal location of dendritic cells (e.g., thereby initiating and/or stimulating an immune response).
- a NE when a NE is used to inactivate bacteria of the genus Bacillus (e.g., B.
- anthracis combining the bacteria and the NE preserves important immunogenic epitopes (e.g., recognizable by a subject's immune system), stabilizing their hydrophobic and hydrophilic components in the oil and water interface of the emulsion (e.g., thereby providing one or more immunogens (e.g., stabilized antigens) against which a subject can mount an immune response).
- important immunogenic epitopes e.g., recognizable by a subject's immune system
- stabilizing their hydrophobic and hydrophilic components in the oil and water interface of the emulsion e.g., thereby providing one or more immunogens (e.g., stabilized antigens) against which a subject can mount an immune response).
- Dendritic cells avidly phagocytose NE oil droplets and this could provide a means to internalize immunogenic proteins for antigen presentation. While other vaccines rely on inflammatory toxins or other immune stimuli for adjuvant activity (See, e.g., Holmgren and Czerkinsky, Nature Med. 2005, 11 ; 45-53), NEs have not been shown to be inflammatory when placed on the skin or mucous membranes in studies on animals and in humans.
- a composition comprising a NE of the present invention may act as a "physical" adjuvant (e.g., that transports and/or presents Bacillus proteins to the immune system (e.g., See Example 10)).
- a composition comprising a composition comprising NE and one or more Bacillus proteins may act as a "physical" adjuvant (e.g., that transports and/or presents Bacillus proteins to the immune system (e.g., See Example 10)).
- mucosal administration of a composition of the present invention generates mucosal as well as systemic immunity (e.g., signs of mucosal immunity (e.g., generation of IgA antibody titers).
- a composition of the present invention e.g., a composition comprising a NE and Bacillus proteins (e.g., rPA)
- anthrax vaccine e.g., a composition comprising a NE and Bacillus proteins (e.g., rPA)
- a composition of the present invention induces (e.g., when administered to a subject) both systemic and mucosal immunity.
- administration of a composition of the present invention to a subject results in protection against an exposure (e.g., a lethal mucosal exposure) to B. anthracis (See Examples 8 and 9) .
- an exposure e.g., a lethal mucosal exposure
- B. anthracis See Examples 8 and 9
- mucosal administration e.g., vaccination
- Bacillus infection e.g., that initiates at a mucosal surface.
- the present invention provides compositions and methods for stimulating mucosal immunity (e.g., a protective IgA response) from a pathogen in a subject.
- the present invention provides a composition (e.g., a composition comprising a NE and a B. anthracis immunogen (e.g., rP A)) to serve as a mucosal vaccine.
- a composition e.g., a composition comprising a NE and a B. anthracis immunogen (e.g., rP A)
- This material can easily be produced with NE and recombinant protein (See, e.g., Example 1), and induces both mucosal and systemic immunity (See, e.g., Examples 11-16).
- the ability to produce this formulation rapidly and administer it via nasal instillation provides a vaccine that can be used in large-scale outbreaks or emergent situations.
- the present invention provides a composition for generating an immune response comprising a NE and an immunogen (e.g., a purified, isolated or synthetic Bacillus protein or derivative or analogue thereof; or, bacteria of the genus Bacillus inactivated by the nanoemulsion).
- an immunogen e.g., a purified, isolated or synthetic Bacillus protein or derivative or analogue thereof; or, bacteria of the genus Bacillus inactivated by the nanoemulsion.
- an immunogen e.g., a purified, isolated or synthetic Bacillus protein or derivative or analogue thereof; or, bacteria of the genus Bacillus inactivated by the nanoemulsion.
- generation of an immune response e.g., resulting from administration of a composition comprising a nanoemulsion and a recombinant Bacillus protein
- generation of an immune response provides total or partial immunity to the subject (e.g., from signs, symptoms or conditions of a disease (e.g., anthrax)).
- protection and/or immunity from disease e.g., the ability of a subject's immune system to prevent or attenuate (e.g., suppress) a sign, symptom or condition of disease
- an immunogenic composition of the present invention is due to adaptive (e.g., acquired) immune responses (e.g., immune responses mediated by B and T cells following exposure to a NE comprising a recombinant Bacillus protein of the present invention (e.g., immune responses that exhibit increased specificity and reactivity to Bacillus).
- a NE comprising an immunogen is administered alone.
- a composition comprising a NE and an immunogen comprises one or more other agents (e.g., a pharmaceutically acceptable carrier, adjuvant, excipient, and the like).
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a humoral immune response.
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a cellular (e.g., cytotoxic T lymphocyte) immune response, rather than a humoral response.
- a composition comprising a NE and an immunogen of the present invention induces both a cellular and humoral immune response.
- the present invention is not limited by the type of NE utilized (e.g., in an immunogenic composition comprising an immunogen). Indeed, a variety of NE compositions are contemplated to be useful in the present invention.
- a nanoemulsion comprises (i) an aqueous phase; (ii) an oil phase; and at least one additional compound.
- these additional compounds are admixed into either the aqueous or oil phases of the composition.
- these additional compounds are admixed into a composition of previously emulsified oil and aqueous phases.
- one or more additional compounds are admixed into an existing emulsion composition immediately prior to its use.
- one or more additional compounds are admixed into an existing emulsion composition prior to the compositions immediate use.
- Additional compounds suitable for use in a nanoemulsion of the present invention include, but are not limited to, one or more organic, and more particularly, organic phosphate based solvents, surfactants and detergents, cationic halogen containing compounds, germination enhancers, interaction enhancers, food additives (e.g., flavorings, sweetners, bulking agents, and the like) and pharmaceutically acceptable compounds.
- organic phosphate based solvents e.g., ethylene glycol, g., g., g., g., ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, ethanol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbiol, sorbio
- the HIV envelope glycoprotein gpl20 is the viral protein that is used for attachment to the host cell. This attachment is mediated by the binding to two surface molecules of helper T cells and macrophages, known as CD4 and one of the two chemokine receptors CCR-4 or CXCR-5.
- the gpl20 protein is first expressed as a larger precursor molecule (gp 160), which is then cleaved post-translationally to yield gpl20 and g ⁇ 41.
- the gp 120 protein is retained on the surface of the virion by linkage to the gp41 molecule, which is inserted into the viral membrane.
- the g ⁇ l20 protein is the principal target of neutralizing antibodies.
- the most immunogenic regions of the gpl20 proteins (V3 loop) are also the most variable parts of the protein.
- the gpl20 protein also contains epitopes that are recognized by cytotoxic T lymphocytes (CTL). These effector cells are able to eliminate virus-infected cells, and therefore constitute a second major antiviral immune mechanism, m contrast to the target regions of neutralizing antibodies some CTL epitopes appear to be relatively conserved among different HIV strains. For this reason g ⁇ l20 and gp 160 are considered to be useful antigenic components in vaccines that aim at eliciting cell -mediated immune responses (particularly CTL).
- gpl20 immunogenic antigens have been developed: (1) purified gpl20 derived from HIV-infected tissue culture cells (referred to herein as “viral-derived gpl20”); (2) gpl20 made in cells infected with recombinant viruses, such as vaccinia or baculovirus (referred to herein as “live-virus-vector-derived gpl20 and gpl60"); (3) recombinant g ⁇ l20 made in mammalian cells (referred to herein as “recombinant mammalian gpl20”); (4) recombinant denatured polypeptides that represent all or various portions of gpl20 and gp41 (referred to herein as “recombinant denatured antigens”); and (5) peptides that represent small segments of gpl20 and gp41 (referred to herein as “peptides").
- viral-derived gpl20 purified gpl20 derived
- each of these immunogenic antigens are highly immunogenic as adjuvanted in a variety of species. They have generated antibodies capable of neutralizing the homologous isolate of HIV-I . Levels of neutralization have not (in general) reached the level of neutralizing titer found in infected humans and there has been much difficulty generating an immunogenic composition that generates immunity to more than one strain of HIV (e.g., other than the strain from which the immunogenic antigen was derived).
- HIV-I and HTV-2 are characterized by having a very high level of sequence diversity that is most pronounced in the gpl20 portion of the envelope. This sequence diversity is clustered in regions known as hypervariable regions.
- the present invention is well suited for delivery of a composition comprising a variety of HIV antigenic substances derived from a variety of HIV isolates.
- the present invention provides methods of inducing an immune response to HIV in a subject (e.g., a human subject) and compositions useful in such methods (e.g., a nanoemulsion comprising HIV or HIV components (e.g., isolated or recombinant HIV proteins).
- compositions useful in such methods e.g., a nanoemulsion comprising HIV or HIV components (e.g., isolated or recombinant HIV proteins).
- methods of inducing an immune response provided by the present invention are used for vaccination. Due to the rate of adverse events with existing HIV vaccines, the present invention provides a significant improvement in HIV vaccination safety without compromising vaccine efficacy.
- the present invention describes the development of immunity (e.g., HIV immunity) in a subject after mucosal administration (e.g., mucosal vaccination) with a composition comprising a nenoemulsion and an immunogenic protein from HIV (e.g., recombinant gpl20) generated and characterized during development of the present invention (See Examples 17-23).
- immunity e.g., HIV immunity
- mucosal administration e.g., mucosal vaccination
- an immunogenic protein from HIV e.g., recombinant gpl20
- room temperature e.g., in some embodiments, for more than 2 weeks, more preferably more than 3 weeks, even more preferably more than 4 weeks, and most preferably for more than 5 weeks
- an immune response against HIV in a subject e.g., that can be used either alone or as an adjuvant for inducing an anti-HIV immune response.
- Mucosal administration of a composition comprising NE and an HIV immunogen (e.g., recombinant gpl20) to a subject resulted in high-titer mucosal and systemic antibody responses and generated a ThI type cellular immune response (See, e.g., Examples 17, 18, and 21). Further, antibodies generated against one serotype of gpl20 cross-reacted with other gpl20 serotypes (See, e.g., Example 19).
- HIV immunogen e.g., recombinant gpl20
- mice immunized intranasally with a composition comprising NE and recombinant gpl20 generated mucosally secreted, anti-gpl20 specific IgA antibodies that were detectable in both bronchial as well as vaginal mucosal surfaces (See Example 20).
- mice administered a composition of the present invention generated a mucosal immune response to HIV.
- the immune response generated in mice administered a composition comprising a NE and recombinant gpl20 was also capable of neutralizing HIV (See Example 22).
- the present invention provides that administration (e.g., mucosal administration) of a composition comprising NE and an HIV immunogen (e.g., recombinant gpl20) is sufficient to induce a protective immune response against HIV in a subject (e.g., protective immunity (e.g., mucosal and systemic immunity)).
- a subsequent administration e.g., one or more boost administrations subsequent to a primary administration
- a subsequent administration e.g., one or more boost administrations subsequent to a primary administration
- administration of a composition comprising NE and an HIV immunogen (e.g., recombinant gpl20) to a subject provides protective immunity against AIDS.
- a NE and an HIV immunogen e.g., recombinant gpl20
- an HIV immunogen e.g., recombinant gpl20
- a NE formulations may carry immunogenic proteins to the submucosal location of dendritic cells (e.g., thereby initiating and/or stimulating an immune response).
- NE treatment e.g., neutralization of HIVwith a NE of the present ' invention
- preserves important viral neutralizing epitopes e.g., recognizable by a subject's immune system
- stabilizing their hydrophobic and hydrophilic components in the oil and water interface of the emulsion e.g., thereby providing one or more immunogens (e.g., stabilized antigens) against which a subject can mount an immune response).
- immunogens e.g., stabilized antigens
- NE formulations may carry viral proteins to the submucosal location of dendritic cells (e.g., thereby initiating and/or stimulating an immune response).
- Dendritic cells avidly phagocytose NE oil droplets and this could provide a means to internalize immunogenic proteins for antigen presentation. While other vaccines rely on inflammatory toxins or other immune stimuli for adjuvant activity (See, e.g., Holmgren and Czerkinsky, Nature Med. 2005, 11; 45-53), NEs have not been shown to be inflammatory when placed on the skin or mucous membranes in studies on animals and in humans.
- a composition comprising a NE of the present invention may act as a "physical" adjuvant (e.g., that transports and/or presents HIV proteins (e.g., gpl20) to the immune system.
- a "physical" adjuvant e.g., that transports and/or presents HIV proteins (e.g., gpl20) to the immune system.
- mucosal administration of a composition of the present invention generates mucosal as well as systemic immunity (e.g., signs of mucosal immunity (e.g., generation of IgA antibody titers).
- a composition of the present invention results in the induction of both humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immune responses (e.g., against HIV proteins (g ⁇ l20)).
- a composition of the present invention e.g., a composition comprising a NE and recombinant gpl20 from one or more serotypes of HIV is used as a AIDS vaccine.
- a composition of the present invention induces (e.g., when administered to a subject) both systemic and mucosal immunity.
- administration of a composition of the present invention to a subject results in protection against an exposure (e.g., a mucosal exposure) to HFV.
- mucosal administration e.g., vaccination
- HIV infection e.g., that initiates at a mucosal surface
- the present invention provides compositions and methods for stimulating mucosal immunity (e.g., a protective IgA response) from a pathogen in a subject.
- the present invention provides a composition (e.g., a composition comprising a NE and immunogenic protein antigens from HIV (e.g., g ⁇ l20) to serve as a mucosal vaccine.
- a composition e.g., a composition comprising a NE and immunogenic protein antigens from HIV (e.g., g ⁇ l20) to serve as a mucosal vaccine.
- This material can easily be produced with NE and HIV protein (e.g., viral-derived gpl20, live-virus-vector-derived gpl20 and gpl60, recombinant mammalian gpl20, recombinant denatured antigens, small peptide segments of gpl20 and gp41, V3 loop peptides (See, e.g., Example 17)), and induces both mucosal and systemic immunity (See, e.g., Examples 18-22).
- HIV protein e.g.,
- the present invention provides a composition for generating an immune response comprising a NE and an immunogen (e.g., a purified, isolated or synthetic HIV protein or derivative, variant, or analogue thereof; or, one or more serotypes of HIV inactivated by the nanoemulsion).
- an immunogen e.g., a purified, isolated or synthetic HIV protein or derivative, variant, or analogue thereof; or, one or more serotypes of HIV inactivated by the nanoemulsion.
- an immunogen e.g., a purified, isolated or synthetic HIV protein or derivative, variant, or analogue thereof; or, one or more serotypes of HIV inactivated by the nanoemulsion.
- generation of an immune response (e.g., resulting from administration of a composition comprising a nanoemulsion and an immunogen) provides total or partial immunity to the subject (e.g., from signs, symptoms or conditions of a disease (e.g., AIDS)).
- a disease e.g., AIDS
- protection and/or immunity from disease e.g., the ability of a subject's immune system to prevent or attenuate (e.g., suppress) a sign, symptom or condition of disease
- an immunogenic composition of the present invention is due to adaptive (e.g., acquired) immune responses (e.g., immune responses mediated by B and T cells following exposure to a NE comprising an immunogen of the present invention (e.g., immune responses that exhibit increased specificity and reactivity towards HIV).
- the compositions and methods of the present invention are used prophylactically or therapeutically to prevent or attenuate a sign, symptom or condition associated with AIDS.
- a NE comprising an immunogen is administered alone.
- a composition comprising a NE and an immunogen comprises one or more other agents (e.g., a pharmaceutically acceptable carrier, adjuvant, excipient, and the like), ⁇ n
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a humoral immune response.
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a cellular (e.g., cytotoxic T lymphocyte) immune response, rather than a humoral response.
- a composition comprising a NE and an immunogen of the present invention induces both a cellular and humoral immune response.
- compositions e.g., comprising a NE and an immunogen
- an immune response e.g., for use as a vaccine
- the present invention provides compositions for generating an immune response to bacterial pathogens (e.g., in vegetative or spore forms) including, but not limited to, Bacillus cereus, Bacillus circulans and Bacillus megaterium, Bacillus anthracis, bacteria of the genus Brucella, Vibrio cholera, Coxiella burnetii, Francisella tularensis, Chlamydia psittaci, Ricinus communis, Rickettsia prowazekii, bacterial of the genus Salmonella (e.g., S.
- the present invention provides compositions for generating an immune response to viral pathogens including, but not limited to, influenza A virus, avian influenza virus, H5N1 influenza virus, West Nile virus, SARS virus, Marburg virus, Arenaviruses.
- Nipah virus alphaviruses, f ⁇ loviruses, herpes simplex virus I 3 herpes simplex virus II, sendai, Sindbis, vaccinia, parvovirus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, hepatitis A virus, cytomegalovirus, human papilloma virus, picornavirus, hantavirus, junin virus, and ebola virus).
- the present invention provides compositions for generating an immune response to fungal pathogens, including, but not limited to, Candida albicnas and parapsilosis, Aspergillus fiimigatus and niger, Fusarium spp, Trychophyton spp.
- Bacteria for use in formulating a composition for generating an immune response of the present invention can be obtained from commercial sources, including, but not limited to, American Type Culture Collection (ATCC).
- ATCC American Type Culture Collection
- bacteria are passed in animals prior to being mixed with nanoemulsions in order to enhance their pathogenicity for each specific animal host for 5-10 passages (Sinai etal., J. Infect. Dis., 141:193 (1980)).
- the bacteria then are then isolated from the host animals, expanded in culture and stored at -80 0 C. Just before use, the bacteria are thawed and grown on an appropriate solid bacterial culture medium overnight.
- the bacteria are collected from the agar plate and suspended in a suitable liquid solution (e.g., Brain Heart Infusion (BHI) broth).
- a suitable liquid solution e.g., Brain Heart Infusion (BHI) broth.
- BHI Brain Heart Infusion
- concentration of bacteria is adjusted so that the bacteria count is approximately 1.5x10 colony forming units per ml (CFU/ml), based on the McFarland standard for bactericidal testing (Hendrichson and Krenz, 1991).
- Viruses for use in formulating a composition for generating an immune response of the present invention can be obtained from commercial sources, including, but not limited to, ATCC.
- viruses are passed in the prospective animal model for 5-10 times to enhance pathogenicity for each specific animal (Ginsberg and Johnson, Infect. Immun., 13:1221 (1976)).
- the virus is collected and propagated in tissue culture and then purified using density gradient concentration and ultracentrifugation (Garlinghouse et al, Lab Anim Sci., 37:437 (1987); and Mahy, Br. Med. Bull., 41 :50 (1985)).
- the Plaque Forming Units (PFU) are calculated in the appropriate tissue culture cells.
- Lethal dose and/or infectious dose for each pathogen can be calculated using any suitable method, including, but not limited to, by administering different doses of the pathogens to the animals by the infective route and identifying the doses which result in the expected result of either animal sickness or death based on previous publications (Fortier et al, Infect Immun., 59:2922 (1991); Jacoby, Exp Gerontol., 29:89 (1994); and Salit etal, Can J Microbiol., 30:1022 (1984)).
- nanoemulsion vaccine compositions of the present invention are not limited to any particular nanoemulsion. Any number of suitable nanoemulsion compositions may be utilized in the vaccine compositions of the present invention, including, but not limited to, those disclosed in Hamouda et al, J. Infect Dis., 180:1939 (1999); Hamouda and Baker, J. Appl. Microbiol., 89:397 (2000); and Donovan et al., Antivir. Chem. Chemother., 11:41 (2000), as well as those shown in Tables 1 and 2 and Figures 4 and 9.
- Preferred nanoemulsions of the present invention are those that are effective in killing or inactivating pathogens and that are non-toxic to animals.
- preferred emulsion formulations utilize non-toxic solvents, such as ethanol, and achieve more effective killing at lower concentrations of emulsion
- nanoemulsions utilized in the methods of the present invention are stable, and do not decompose even after long storage periods (e.g., one or more years).
- preferred emulsions maintain stability even after exposure to high temperature and freezing. This is especially useful if they are to be applied in extreme conditions (e.g., on a battlefield), hi some embodiments, one of the nanoemulsions described in Table 1 and or Figures 4 or 9 is utilized.
- the emulsions comprise (i) an aqueous phase; (ii) an oil phase; and at least one additional compound, hi some embodiments of the present invention, these additional compounds are admixed into either the aqueous or oil phases of the composition.
- these additional compounds are admixed into a composition of previously emulsified oil and aqueous phases. Li certain of these embodiments, one or more additional compounds are admixed into an existing emulsion composition immediately prior to its use. hi other embodiments, one or more additional compounds are admixed into an existing emulsion composition prior to the compositions immediate use.
- Additional compounds suitable for use in the compositions of the present invention include but are not limited to one or more, organic, and more particularly, organic phosphate based solvents, surfactants and detergents, quaternary ammonium containing compounds, cationic halogen containing compounds, germination enhancers, interaction enhancers, and pharmaceutically acceptable compounds.
- Certain exemplary embodiments of the various compounds contemplated for use in the compositions of the present invention are presented below. Y3EC 3% TYLOXAPOL; 1% Cetylpyridinium Chloride; 8% Ethanol; 64% Soybean oil; 24% Water
- the emulsions of the present invention contain (i) an aqueous phase and (ii) an oil phase containing ethanol as the organic solvent and optionally a germination enhancer, and (iii) TYLOXAPOL as the surfactant (preferably 2-5%, more preferably 3%).
- This formulation is highly efficacious against microbes and is also non-irritating and non-toxic to mammalian users (and can thus be contacted with mucosal membranes).
- the emulsions of the present invention comprise a first emulsion emulsified within a second emulsion, wherein (a) the first emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and an organic solvent; and (iii) a surfactant; and (b) the second emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and a cationic containing compound; and (iii) a surfactant.
- X8P comprises a water-in oil nanoemulsion, in which the oil phase was made from soybean oil, tri-n-butyl phosphate, and TRITON X-IOO in 80% water.
- XgW6()PC comprises a mixture of equal volumes of X8P with Wgo8P.
- Wgo8P is a liposome-like compound made of glycerol monostearate, refined soya sterols (e.g., GENEROL sterols), TWEEN 60, soybean oil, a cationic ion halogen-containing CPC and peppermint oil.
- the GENEROL family are a group of a polyethoxylated soya sterols (Henkel Corporation, Ambler, Pennsylvania). Emulsion formulations are given in Table 1 for certain embodiments of the present invention. These particular formulations maybe found in U.S. Pat. Nos. 5,700,679 (NN); 5,618,840; 5,549,901 (WgoSP); and 5,547,677,herein incorporated by reference in their entireties.
- the X8W(5oPC emulsion is manufactured by first making the Wg ⁇ 8P emulsion and X8P emulsions separately. A mixture of these two emulsions is then re-emulsified to produce a fresh emulsion composition termed X ⁇ WgoPC- Methods of producing such emulsions are described in U.S. Pat. Nos. 5,103,497 and 4,895,452 (herein incorporated by reference in their entireties). These compounds have broad-spectrum antimicrobial activity, and are able to inactivate vegetative bacteria through membrane disruption.
- compositions listed above are only exemplary and those of skill in the art will be able to alter the amounts of the components to arrive at a nanoemulsion composition suitable for the purposes of the present invention.
- Those skilled in the art will understand that the ratio of oil phase to water as well as the individual oil carrier, surfactant CPC and organic phosphate buffer, components of each composition may vary.
- compositions comprising X8P have a water to oil ratio of 4:1, it is understood that the X8P may be formulated to have more or less of a water phase. For example, in some embodiments, there is 3, 4, 5, 6, 7, 8, 9, 10, or more parts of the water phase to each part of the oil phase. The same holds true for the Wgo&P formulation.
- the ratio of Tri(N-butyl)phosphate:TRTTON X-100:soybean oil also maybe varied.
- Table 1 lists specific amounts of glycerol monooleate, polysorbate 60, GENEROL 122, cetylpyridinium chloride, and carrier oil for Wg ⁇ )8P, these are merely exemplary.
- An emulsion that has the properties of Wgo8P may be formulated that has different concentrations of each of these components or indeed different components that will fulfill the same function.
- the emulsion may have between about 80 to about lOOg of glycerol monooleate in the initial oil phase.
- the emulsion may have between about 15 to about 30 g polysorbate 60 in the initial oil phase.
- the composition may comprise between about 20 to about 30 g of a GENEROL sterol, in the initial oil phase.
- the nanoemulsions structure of the certain embodiments of the emulsions of the present invention may play a role in their biocidal activity as well as contributing to the non-toxicity of these emulsions.
- the active component in X8P, TRITON-X 100 shows less biocidal activity against virus at concentrations equivalent to 11 % X8P. Adding the oil phase to the detergent and solvent markedly reduces the toxicity of these agents in tissue culture at the same concentrations.
- the nanoemulsion enhances the interaction of its components with the pathogens thereby facilitating the inactivation of the pathogen and reducing the toxicity of the individual components. It should be noted that when all the components of X8P are combined in one composition but are not in a nanoemulsion structure, the mixture is not as effective as an antimicrobial as when the components are in a nanoemulsion structure.
- the inventive formulation comprise from about 3 to 8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 60 to 70 vol. % oil (e.g. , soybean oil), about 15 to 25 vol. % of aqueous phase (e.g., DiE ⁇ O or PBS), and in some formulations less than about 1 vol. % of IN NaOH.
- Some of these embodiments comprise PBS.
- one embodiment of the present invention comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 24 vol. % of D1H2O (designated herein as Y3EC).
- Another similar embodiment comprises about
- TYLOXAPOL 3.5 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, and about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 23.5 vol. % of DiH2 ⁇ (designated herein as Y3.5EC).
- Yet another embodiment comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 0.067 vol. % of IN NaOH, such that the pH of the formulation is about 7.1, about 64 vol. % of soybean oil, and about 23.93 vol. % of Dffl ⁇ O (designated herein as Y3EC pH 7.1). Still another embodiment comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC 3 about 0.67 vol. % of IN NaOH, such that the pH of the formulation is about 8.5, and about 64 vol. % of soybean oil, and about 23.33 vol.
- % of Dffl ⁇ O (designated herein as Y3EC pH 8.5).
- Another similar embodiment comprises about 4% TYLOXAPOL, about 8 vol. % ethanol, about 1% CPC, and about 64 vol. % of soybean oil, and about 23 vol. % of DiH2 ⁇ (designated herein as Y4EC).
- the formulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1 vol. % of CPC, and about 64 vol. % of soybean oil, and about 19 vol. % of DiH2 ⁇ (designated herein as Y8EC).
- a further embodiment comprises about 8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC 5 about 64 vol. % of soybean oil, and about 19 vol. % of Ix PBS (designated herein as Y8EC PBS).
- the inventive formulations comprise about 8 vol. % of ethanol, and about 1 vol. % of CPC, and about 64 vol. % of oil (e.g., soybean oil), and about 27 vol. % of aqueous phase (e.g., DiK ⁇ O or PBS) (designated herein as EC).
- some embodiments comprise from about 8 vol. % of sodium dodecyl sulfate (SDS), about 8 vol. % of tributyl phosphate (TBP), and about 64 vol. % of oil (e.g., soybean oil), and about 20 vol. % of aqueous phase (e.g., D1H2O or PBS)
- the inventive formulation comprise from about 1 to 2 vol. % of TRITON X-IOO, from about 1 to 2 vol. % of TYLOXAPOL, from about 7 to 8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 64 to 57.6 vol. % of oil (e.g., soybean oil), and about 23 vol. % of aqueous phase (e.g., D1H2O or PBS). Additionally, some of these formulations further comprise about 5 mM of L-alanine/Inosine, and about 10 mM ammonium chloride. Some of these formulations comprise PBS.
- one embodiment of the present invention comprises about 2 vol. % of TRITON X- 100, about 2 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % CPC, about 64 vol. % of soybean oil, and about 23 vol. % of aqueous phase D1H2O.
- the formulation comprises about 1.8 vol. % of TRITON X-IOO, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % of ethanol, about 0.9 vol.
- the formulations comprise from about 5 vol. % of TWEEN 80, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g., soybean oil), and about 22 vol. % of DiH2 ⁇ (designated herein as W 8O 5EC).
- the formulations comprise from about 5 vol. % of TWEEN 20, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g., soybean oil), and about 22 vol. % of DiH2 ⁇ (designated herein as W 2 o5EC).
- the formulations comprise from about 2 to 8 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g., soybean, or olive oil), and about 15 to 25 vol. % of aqueous phase (e.g., D1H2O or PBS).
- oil e.g., soybean, or olive oil
- aqueous phase e.g., D1H2O or PBS
- the present invention contemplates formulations comprising about 2 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 26 vol. % of D1H2O (designated herein as X2E).
- the formulations comprise about 3 vol.
- the formulations comprise about 4 vol. % TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 24 vol. % of D1H2O (designated herein as X4E).
- the formulations comprise about 5 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 23 vol. % of DiE ⁇ O (designated herein as X5E).
- Another embodiment of the present invention comprises about 6 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 22 vol. % of DiK ⁇ O
- the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 20 vol. % of DiB ⁇ O (designated herein as X8E). In still further embodiments of the present invention, the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of olive oil, and about 20 vol. % of DiH2 ⁇ (designated herein as X8E O). hi yet another embodiment comprises 8 vol. % of TRITON X-100, about 8 vol. % ethanol, about 1 vol.
- the formulations comprise from about 1 to 2 vol. % of TRITON X-100, from about 1 to 2 vol. % of TYLOXAPOL, from about 6 to 8 vol. % TBP, from about 0.5 to 1.0 vol. % of CPC, from about 60 to 70 vol. % of oil (e.g., soybean), and about 1 to 35 vol. % of aqueous phase (e.g., D1H2O or PBS). Additionally, certain of these formulations may comprise from about 1 to 5 vol.
- the formula comprises a casein hydrolysate ⁇ e.g., Neutramigen, or Progestimil, and the like).
- the inventive formulations further comprise from about 0.1 to 1.0 vol. % of sodium thiosulfate, and from about 0.1 to 1.0 vol. % of sodium citrate.
- PBS phosphate buffered saline
- one embodiment comprises about 2 vol. % of TRITON X-100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 23 vol. % of DiH2 ⁇ (designated herein as X2Y2EC).
- the inventive formulation comprises about 2 vol. % of TRITON X- 100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol. % of CPC, about 0.9 vol.
- the formulations comprise about 1.7 vol. % TRITON X-100, about 1.7 vol. % TYLOXAPOL, about 6.8 vol. % TBP, about 0.85% CPC, about 29.2% NEUTRAMIGEN, about 54.4 vol. % of soybean oil, and about 4.9 vol. % of DiH ⁇ O
- the formulations comprise about 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % of TBP, about 0.9 vol. % of CPC 5 about 5mM L- alanine/Inosine, about 1OmM ammonium chloride, about 57.6 vol. % of soybean oil, and the remainder vol. % of O.lx PBS (designated herein as 90% X2Y2 PC/GE).
- the formulations comprise about 1.8 vol. % of TRITON X-100, about 1.8 vol.
- the formulations comprise about 1.8 vol. % TRITON X-100, about 1.8 vol. % TYLOXAPOL, about 7.2 vol. % TBP, about 0.9 vol. % CPC, about 1 vol. % yeast extract, about 57.6 vol. % of soybean oil, and about 29.7 vol. % of DiH ⁇ O (designated herein as 90% X2Y2PC/YE).
- the inventive formulations comprise about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g., D1H2O or PBS).
- the inventive formulations comprise about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. % of CPC, about 64 vol. % of soybean, and about 24 vol. % of DiH ⁇ O
- the inventive formulations comprise from about 4 to 8 vol. % of TRITON X-IOO, from about 5 to 8 vol. % of TBP, about 30 to 70 vol. % of oil (e.g., soybean or olive oil), and about 0 to 30 vol. % of aqueous phase (e.g., DiH2 ⁇ or PBS). Additionally, certain of these embodiments further comprise about 1 vol.
- the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 20 vol. % of DiH2 ⁇ (designated herein as X8P).
- the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1% of CPC, about 64 vol. % of soybean oil, and about 19 vol. % of DiI ⁇ O
- the formulations comprise about 8 vol. % TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 50 vol. % of soybean oil, and about 33 vol. % of D1H2O (designated herein as ATB-XlOOl).
- the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % of CPC, about 50 vol. % of soybean oil, and about 32 vol. % of DiH2 ⁇ (designated herein as ATB-X002).
- Another embodiment of the present invention comprises about 4 vol. % TRITON X-100, about 4 vol. % of TBP, about 0.5 vol. % of CPC, about 32 vol. % of soybean oil, and about 59.5 vol. % of D1H2O (designated herein as 50%
- Still another related embodiment comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 0.5 vol. % CPC, about 64 vol. % of soybean oil, and about 19.5 vol. % of DiH2 ⁇ (designated herein as X8PC1/2).
- the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % of CPC, about 64 vol. % of soybean oil, and about 18 vol. % of D1H2O (designated herein as X8PC2).
- the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8% of TBP, about 1% of benzalkonium chloride, about 50 vol. % of soybean oil, and about 33 vol. % of DiH ⁇ O
- the formulation comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of cetylyridinium bromide, about 50 vol. % of soybean oil, and about 33 vol. % of DiH2 ⁇ (designated herein as X8P CPB).
- the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of cetyldimethyletylammonium bromide, about 50 vol. % of soybean oil, and about 33 vol.
- the present invention comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 500 ⁇ M EDTA, about 64 vol. % of soybean oil, and about 15.8 vol. % DiH ⁇ O (designated herein as X8PC EDTA).
- Additional similar embodiments comprise 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 10 mM ammonium chloride, about 5mM Inosine, about 5mM L-alanine, about 64 vol. % of soybean oil, and about 19 vol. % of DiH ⁇ O or PBS (designated herein as
- the inventive formulations further comprise about 5 vol. % of TRITON X-100, about 5% of TBP, about 1 vol. % of CPC, about 40 vol. % of soybean oil, and about 49 vol. % of Dffi ⁇ O (designated herein as
- the inventive formulations comprise about 2 vol. % TRITON X-100, about 6 vol. % TYLOXAPOL, about 8 vol. % ethanol, about 64 vol. % of soybean oil, and about 20 vol. % (designated herein as
- the formulations comprise about 8 vol. % of TRITON X-100, and about 8 vol. % of glycerol, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 25 vol. % of aqueous phase (e.g., D-H2O or
- Certain related embodiments further comprise about 1 vol. % L-ascorbic acid.
- one particular embodiment comprises about 8 vol. % of TRITON X-100, about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH ⁇ O (designated herein as X8G).
- the inventive formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of glycerol, about 1 vol. % of L- ascorbic acid, about 64 vol. % of soybean oil, and about 19 vol. % of D1H2O (designated herein as X8GV C ).
- the inventive formulations comprise about 8 vol. % of TRITON X-100, from about 0.5 to 0.8 vol. % of TWEEN 60, from about 0.5 to 2.0 vol. % of CPC, about 8 vol. % of TBP, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 25 vol. % of aqueous phase (e.g., DiE ⁇ O or PBS).
- the formulations comprise about 8 vol. % of TRITON X-100, about 0.70 vol. % of TWEEN 60, about 1 vol. % of CPC 5 about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 18.3 vol. % of DiH ⁇ O (designated herein as X8W60PCj).
- Another related embodiment comprises about 8 vol. % of TRITON X-100, about 0.71 vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 18.29 vol. % of D1H2O (designated herein as W6OO.7X8PC).
- the inventive formulations comprise from about 8 vol. % of TRITON X-100, about 0.7 vol. % of TWEEN 60, about 0.5 vol. % of CPC, about 8 vol. % of TBP, about 64 to 70 vol. % of soybean oil, and about 18.8 vol. % of DiH ⁇ O (designated herein as
- the present invention comprises about 8 vol. % of
- the formulations comprise about 0.71 vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 25.29 vol. % of DiE ⁇ O (designated herein as W6OO.7PC).
- the inventive formulations comprise about 2 vol. % of dioctyl sulfosuccinate, either about 8 vol. % of glycerol, or about 8 vol. % TBP, in addition to, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 20 to 30 vol. % of aqueous phase (e.g.; DiH ⁇ O or PBS).
- oil e.g., soybean or olive oil
- aqueous phase e.g.; DiH ⁇ O or PBS
- one embodiment of the present invention comprises about 2 vol. % of dioctyl sulfosuccinate, about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 26 vol. % of DiH2 ⁇ (designated herein as D2G).
- the inventive formulations comprise about 2 vol. % of dioctyl sulfosuccinate, and about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 26 vol. % of D1H2O (designated herein as D2P).
- the inventive formulations comprise about 8 to 10 vol. % of glycerol, and about 1 to 10 vol. % of CPC, about 50 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH2 ⁇ or PBS).
- the compositions further comprise about 1 vol.
- one particular embodiment comprises about 8 vol. % of glycerol, about 1 vol. % of CPC, about 64 voL-% of soybean oil, and about 27 vol. % of D1H2O (designated herein as GC).
- An additional related embodiment comprises about 10 vol. % of glycerol, about 10 vol. % of CPC, about 60 vol. % of soybean oil, and about 20 vol. % of DiH2 ⁇ (designated herein as GClO).
- the inventive formulations comprise about 10 vol. % of glycerol, about 1 vol. % of CPC, about 1 vol. % of L-ascorbic acid, about 64 vol. % of soybean or oil, and about 24 vol. % of DiH/jO (designated herein as GCV C ).
- the inventive formulations comprise about 8 to 10 vol. % of glycerol, about 8 to 10 vol. % of SDS, about 50 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH ⁇ O or
- compositions further comprise about 1 vol. % of lecithin, and about 1 vol. % of p-Hydroxybenzoic acid methyl ester.
- Exemplary embodiments of such formulations comprise about 8 vol. % SDS, 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH ⁇ O (designated herein as S8G).
- a related formulation comprises about 8 vol. % of glycerol, about 8 vol. % of SDS, about 1 vol. % of lecithin, about 1 vol. % of p-Hydroxybenzoic acid methyl ester, about 64 vol. % of soybean oil, and about 18 vol. % of D1H2O (designated herein as
- the inventive formulations comprise about 4 vol. % of TWEEN 80, about 4 vol. % of TYLOXAPOL, about 1 vol. % of CPC, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 19 vol. % of D1H2O (designated herein as Wg()4Y4EC).
- the inventive formulations comprise about 0.01 vol. % of CPC 5 about 0.08 vol. % of TYLOXAPOL, about 10 vol. % of ethanol, about 70 vol. % of soybean oil, and about 19.91 vol. % of DiH2 ⁇ (designated herein as
- the inventive formulations comprise about 8 vol. % of sodium lauryl sulfate, and about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol. % of D1H2O (designated herein as SLS8G).
- a candidate emulsion is suitable for use with the present invention.
- three criteria may be analyzed. Using the methods and standards described herein, candidate emulsions can be easily tested to determine if they are suitable.
- the desired ingredients are prepared using the methods described herein, to determine if an emulsion can be formed. If an emulsion cannot be formed, the candidate is rejected. For example, a candidate composition made of 4.5% sodium thiosulfate, 0.5% sodium citrate, 10% n-butanol, 64% soybean oil, and 21% D1H2O did not form an emulsion.
- the candidate emulsion should form a stable emulsion.
- An emulsion is stable if it remains in emulsion form for a sufficient period to allow its intended use. For example, for emulsions that are to be stored, shipped, etc., it may be desired that the composition remain in emulsion form for months to years. Typical emulsions that are relatively unstable, will lose their form within a day. For example, a candidate composition made of 8% 1-butanol, 5% TWEEN 10, 1% CPC, 64% soybean oil, and 22% D1H2O did not form a stable emulsion.
- the candidate emulsion should have efficacy for its intended use.
- an anti-bacterial emulsion should kill or disable pathogens to a detectable level.
- certain emulsions of the present invention have efficacy against specific microorganisms, but not against others.
- one is capable of determining the suitability of a particular candidate emulsion against the desired microorganism. Generally, this involves exposing the microorganism to the emulsion for one or more time periods in a side-by-side experiment with the appropriate control samples (e.g., a negative control such as water) and determining if, and to what degree, the emulsion kills or disables the microorganism.
- the appropriate control samples e.g., a negative control such as water
- a candidate composition made of 1% ammonium chloride, 5% TWEEN 20, 8% ethanol, 64% soybean oil, and 22% DiH 2 O was shown not to be an effective emulsion.
- the following candidate emulsions were shown to be effective using the methods described herein: 5% TWEEN 20, 5% Cetylpyridinium Chloride, 10% Glycerol, 60% Soybean Oil, and 20% diH 2 O (designated herein as
- W 2 o5GC5 1% Cetylpyridinium Chloride, 5% TWEEN 20, 10% Glycerol, 64% Soybean Oil, and 20% diH ⁇ O (designated herein as W2 ⁇ 5GC); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% Olive Oil, and 22% diH 2 O (designated herein as W2 ⁇ 5EC
- Cetylpyridinium Chloride 5% TWEEN 20, 8% Ethanol, 64% Cottonseed Oil, and 22% diH 2 O (designated herein as W2 ⁇ 5EC Cottonseed Oil); 8% Dextrose, 5% TWEEN 10, 1%
- Methocel K 2% Natrosol, 0.1% X8PC, O.lx PBS, 5 mM L-alanine, 5 mM Liosine, 10 mM Ammonium Chloride, and diH 2 O (designated herein as 0.1% X8PC/GE+2% Natrosol); 2%
- Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% Lard, and 22% diH 2 O (designated herein as W 2 Q5EC Lard); 1% Cetylpyridinium Chloride, 5% TWEEN 20, 8% Ethanol, 64% Mineral Oil, and 22% diH 2 O (designated herein as W 2 o5EC Mineral Oil);
- the emulsion comprises an aqueous phase.
- the emulsion comprises about 5 to 50, preferably 10 to 40, more preferably 15 to 30, vol. % aqueous phase, based on the total volume of the emulsion (although other concentrations are also contemplated).
- the aqueous phase comprises water at a pH of about 4 to 10, preferably about 6 to 8. The water is preferably deionized (hereinafter "DiH 2 O").
- the aqueous phase comprises phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the aqueous phase is sterile and pyrogen free.
- the emulsion comprises an oil phase.
- the oil phase (e.g., carrier oil) of the emulsion of the present invention comprises 30-90, preferably 60-80, and more preferably 60-70, vol. % of oil, based on the total volume of the emulsion (although other concentrations are also contemplated).
- Suitable oils include, but are not limited to, soybean oil, avocado oil, squalene oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, sunflower oil, fish oils, flavor oils, water insoluble vitamins and mixtures thereof. In particularly preferred embodiments, soybean oil is used.
- the oil phase is preferably distributed throughout the aqueous phase as droplets having a mean particle size in the range from about 1-2 microns, more preferably from 0.2 to 0.8, and most preferably about 0.8 microns.
- the aqueous phase can be distributed in the oil phase.
- the oil phase comprises 3-15, and preferably 5-10 vol. % of an organic solvent, based on the total volume of the emulsion. While the present invention is not limited to any particular mechanism, it is contemplated that the organic phosphate-based solvents employed in the emulsions serve to remove or disrupt the lipids in the membranes of the pathogens. Thus, any solvent that removes the sterols or phospholipids in the microbial membranes finds use in the methods of the present invention. Suitable organic solvents include, but are not limited to, organic phosphate based solvents or alcohols, hi some preferred embodiments, non-toxic alcohols (e.g., ethanol) are used as a solvent.
- the oil phase, and any additional compounds provided in the oil phase are preferably sterile and pyrogen free.
- the emulsions further comprises a surfactant or detergent.
- the emulsion comprises from about 3 to 15 %, and preferably about 10 % of one or more surfactants or detergents (although other concentrations are also contemplated). While the present invention is not limited to any particular mechanism, it is contemplated that surfactants, when present in the emulsions, help to stabilize the emulsions. Both non-ionic (non-anionic) and ionic surfactants are contemplated. Additionally, surfactants from the BRIJ family of surfactants find use in the compositions of the present invention. The surfactant can be provided in either the aqueous or the oil phase.
- Surfactants suitable for use with the emulsions include a variety of anionic and nonionic surfactants, as well as other emulsifying compounds that are capable of promoting the formation of oil-in-water emulsions.
- emulsifying compounds are relatively hydrophilic, and blends of emulsifying compounds can be used to achieve the necessary qualities.
- nonionic surfactants have advantages over ionic emulsifiers in that they are substantially more compatible with a broad pH range and often form more stable emulsions than do ionic ⁇ e.g., soap-type) emulsifiers.
- compositions of the present invention comprise one or more non-ionic surfactants such as polysorbate surfactants (e.g., polyoxyethylene ethers), polysorbate detergents, pheoxypolyethoxyethanols, and the like.
- polysorbate surfactants e.g., polyoxyethylene ethers
- polysorbate detergents include, but are not limited to, TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80, etc.
- TWEEN 60 polyoxyethylenesorbitan monostearate
- TWEEN 40 and TWEEN 80 comprise polysorbates that are used as emulsifiers in a number of pharmaceutical compositions. In some embodiments of the present invention, these compounds are also used as co-components with adjuvants. TWEEN surfactants also appear to have virucidal effects on lipid-enveloped viruses (See e.g., Eriksson et al, Blood Coagulation and Fibrinolysis 5 (Suppl. 3):S37-S44 (1994)).
- pheoxypolyethoxyethanols, and polymers thereof, useful in the present invention include, but are not limited to, TRITON (e.g., X-100, X-301, X-165, X-102, X-200), and TYLOXAPOL.
- TRITON X-100 is a strong non-ionic detergent and dispersing agent widely used to extract lipids and proteins from biological structures. It also has virucidal effect against broad spectrum of enveloped viruses (See e.g., Maha and Igarashi, Southeast Asian J. Trop. Med. Pub. Health 28:718 (1997); and Portocala et al., Virologie 27:261 (1976)). Due to this anti- viral activity, it is employed to inactivate viral pathogens in fresh frozen human plasma (See e.g., Horowitz et al, Blood 79:826 (1992)).
- the present invention is not limited to the surfactants disclosed herein. Additional surfactants and detergents useful in the compositions of the present invention may be ascertained from reference works (e.g., including, but not limited to, McCutheon's Volume 1 : Emulsions and Detergents - North American Edition, 2000) and commercial sources.
- the emulsions further comprise a cationic halogen containing compound.
- the emulsion comprises from about 0.5 to 1.0 wt. % or more of a cationic halogen containing compound, based on the total weight of the emulsion (although other concentrations are also contemplated).
- the cationic halogen-containing compound is preferably premixed with the oil phase; however, it should be understood that the cationic halogen-containing compound may be provided in combination with the emulsion composition in a distinct formulation.
- Suitable halogen containing compounds maybe selected from compounds comprising chloride, fluoride, bromide and iodide ions.
- suitable cationic halogen containing compounds include, but are not limited to, cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, or tetradecyltrimethylammonium halides.
- suitable cationic halogen containing compounds comprise, but are not limited to, cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide (CPB), and cetyltrimethylammonium bromide (CTAB), cetyidimethylethylammonium bromide, cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, and tetrad ecyltrimethylammonium bromide.
- CPC cetylpyridinium chloride
- CPC cetyltrimethylammonium chloride
- cetylbenzyldimethylammonium chloride cetylpyridinium bromide
- CTAB cetyltrimethylammonium bromide
- cetyidimethylethylammonium bromide cetyltributylphosphonium bromide
- the nanoemulsions further comprise a germination enhancer.
- the emulsions comprise from about 1 mM to 15 mM, and more preferably from about 5 mM to 1OmM of one or more germination enhancing compounds (although other concentrations are also contemplated).
- the germination enhancing compound is provided in the aqueous phase prior to formation of the emulsion. The present invention contemplates that when germination enhancers are added to the nanoemulsion compositions, the sporicidal properties of the nanoemulsions are enhanced.
- the present invention further contemplates that such germination enhancers initiate sporicidal activity near neutral pH (between pH 6 - 8, and preferably 7).
- neutral pH emulsions can be obtained, for example, by diluting with phosphate buffer saline (PBS) or by preparations of neutral emulsions.
- PBS phosphate buffer saline
- the sporicidal activity of the nanoemulsion preferentially occurs when the spores initiate germination.
- the emulsions utilized in the vaccines of the present invention have sporicidal activity.
- the fusigenic component of the emulsions acts to initiate germination and before reversion to the vegetative form is complete the lysogenic component of the emulsion acts to lyse the newly germinating spore.
- These components of the emulsion thus act in concert to leave the spore susceptible to disruption by the emulsions.
- the addition of germination enhancer further facilitates the • anti-sporicidal activity of the emulsions, for example, by speeding up the rate at which the sporicidal activity occurs.
- Germination of bacterial endospores and fungal spores is associated with increased metabolism and decreased resistance to heat and chemical reactants. For germination to occur, the spore must sense that the environment is adequate to support vegetation and reproduction.
- the amino acid L-alanine stimulates bacterial spore germination (See e.g., Hills, J. Gen. Micro. 4:38 (1950); and Halvorson and Church, Bacteriol Rev. 21:112 (1957)).
- L-alanine and L-proline have also been reported to initiate fungal spore germination (Yanagita, Arch Mikrobiol 26:329 (1957)).
- Simple ⁇ -amino acids, such as glycine and L-alanine occupy a central position in metabolism.
- Transamination or deamination of ⁇ -amino acids yields the glycogenic or ketogenic carbohydrates and the nitrogen needed for metabolism and growth.
- transamination or deamination of L-alanine yields pyruvate, which is the end product of glycolytic metabolism (Embden-Meyerhof-Parnas Pathway).
- Oxidation of pyruvate by pyruvate dehydrogenase complex yields acetyl-CoA, NADH, H + , and CO2.
- Acetyl-CoA is the initiator substrate for the tricarboxylic acid cycle (Kreb's Cycle), which in turns feeds the mitochondrial electron transport chain.
- Acetyl-CoA is also the ultimate carbon source for fatty acid synthesis as well as for sterol synthesis.
- Simple ⁇ -amino acids can provide the nitrogen, CO2, glycogenic and/or ketogenic equivalents required for germination and the metabolic activity that follows.
- suitable germination enhancing agents of the invention include, but are not limited to, ⁇ -amino acids comprising glycine and the L-enantiomers of alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof. Additional information on the effects of amino acids on germination may be found in U.S. Pat. No.
- the formulation comprises the germination enhancers L-alanine, CaC ⁇ , Inosine and NH4CI.
- the compositions further comprise one or more common forms of growth media (e.g., trypticase soy broth, and the like) that additionally may or may not itself comprise germination enhancers and buffers.
- a candidate germination enhancer should meet two criteria for inclusion in the compositions of the present invention: it should be capable of being associated with the emulsions disclosed herein and it should increase the rate of germination of a target spore when incorporated in the emulsions disclosed herein.
- One skilled in the art can determine whether a particular agent has the desired function of acting as an germination enhancer by applying such an agent in combination with the nanoemulsions disclosed herein to a target and comparing the inactivation of the target when contacted by the admixture with inactivation of like targets by the composition of the present invention without the agent. Any agent that increases germination, and thereby decreases or inhibits the growth of the organisms, is considered a suitable enhancer for use in the nanoemulsion compositions disclosed herein.
- addition of a germination enhancer (or growth medium) to a neutral emulsion composition produces a composition that is useful in inactivating bacterial spores in addition to enveloped viruses, Gram negative bacteria, and Gram positive bacteria for use in the vaccine compositions of the present invention.
- nanoemulsions comprise one or more compounds capable of increasing the interaction of the compositions (i.e., "interaction enhancer") with target pathogens (e.g., the cell wall of Gram negative bacteria such as Vibrio, Salmonella, Shigella and Pseudomonas).
- target pathogens e.g., the cell wall of Gram negative bacteria such as Vibrio, Salmonella, Shigella and Pseudomonas.
- the interaction enhancer is preferably premixed with the oil phase; however, in other embodiments the interaction enhancer is provided in combination with the compositions after emulsification.
- the interaction enhancer is a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA) or ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) in a buffer (e.g., tris buffer)).
- EDTA ethylenediaminetetraacetic acid
- EGTA ethylenebis(oxyethylenenitrilo)tetraacetic acid
- a buffer e.g., tris buffer
- chelating agents are merely exemplary interaction enhancing compounds. Indeed, other agents that increase the interaction of the nanoemulsions used in some embodiments of the present invention with microbial agents and/or pathogens are contemplated.
- the interaction enhancer is at a concentration of about 50 to about 250 ⁇ M.
- One skilled in the art will be able to determine whether a particular agent has the desired function of acting as an interaction enhancer by applying such an agent in combination with the compositions of the present invention to a target and comparing the inactivation of the target when contacted by the admixture with inactivation of like targets by the composition of the present invention without the agent.
- the addition of an interaction enhancer to nanoemulsion produces a composition that is useful in inactivating enveloped viruses, some Gram positive bacteria and some Gram negative bacteria for use in the vaccine compositions of the present invention.
- ⁇ anoemulsions of the present invention include a quaternary ammonium containing compound.
- Exemplary quaternary ammonium compounds include, but are not limited to, Alkyl dimethyl benzyl ammonium chloride, didecyl dimethyl ammonium chloride, Alkyl dimethyl benzyl and dialkyl dimethyl ammonium chloride, N 5 N- Dimethyl-2-hydroxypropylammonium chloride polymer, Didecyl dimethyl ammonium chloride, n- Alkyl dimethyl benzyl ammonium chloride, n-Alkyl dimethyl ethylbenzyl ammonium chloride,
- a nanoemulsion comprises one or more additional components that provide a desired property or functionality to the nanoemulsions. These components maybe incorporated into the aqueous phase or the oil phase of the nanoemulsions and/or may be added prior to or following emulsification.
- the nanoemulsions further comprise phenols (e.g., triclosan, phenyl phenol), acidifying agents (e.g., citric acid (e.g., 1.5-6%), acetic acid, lemon juice), alkylating agents (e.g., sodium hydroxide (e.g., 0.3%)), buffers (e.g., citrate buffer, acetate buffer, and other buffers useful to maintain a specific pH), and halogens (e.g., polyvinylpyrrolidone, sodium hypochlorite, hydrogen peroxide).
- phenols e.g., triclosan, phenyl phenol
- acidifying agents e.g., citric acid (e.g., 1.5-6%
- acetic acid e.g., lemon juice
- alkylating agents e.g., sodium hydroxide (e.g., 0.3%)
- buffers e.g., citrate buffer, acetate buffer, and other buffers
- Nanoemulsions of the present invention can be formed using classic emulsion forming techniques.
- the oil phase is mixed with the aqueous phase under relatively high shear forces (e.g., using high hydraulic and mechanical forces) to obtain an oil-in- water nanoemulsion.
- the emulsion is formed by blending the oil phase with an aqueous phase on a volume-to- volume basis ranging from about 1:9 to 5:1, preferably about 5:1 to 3:1, most preferably 4:1 , oil phase to aqueous phase.
- 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, NH). Methods of producing such emulsions are described in U.S. Pat. Nos. 5, 103,497 and 4,895,452, herein incorporated by reference in their entireties.
- compositions used in the methods of the present invention comprise droplets of an oily discontinuous phase dispersed in an aqueous continuous phase, such as water.
- nanoemulsions of the present invention are stable, and do not decompose even after long storage periods (e.g., greater than one or more years).
- nanoemulsions are stable (e.g., in some embodiments for greater than 3 months, in some embodiments for greater than 6 months, in some embodiments for greater than 12 months, in some embodiments for greater than 18 months) after combination with an immunogen (e.g., a pathogen), hi preferred embodiments, nanoemulsions of the present invention are non-toxic and safe when administered (e.g., via spraying or contacting mucosal surfaces, swallowed, inhaled, etc.) to a subject.
- an immunogen e.g., a pathogen
- a portion of the emulsion may be in the form of lipid structures including, but not limited to, unilamellar, multilamellar, and paucliamellar lipid vesicles, micelles, and lamellar phases.
- the emulsions of the present invention contain (i) an aqueous phase and (ii) an oil phase containing ethanol as the organic solvent and optionally a germination enhancer, and (iii) TYLOXAPOL as the surfactant (preferably 2-5%, more preferably 3%).
- This formulation is highly efficacious for inactivation of pathogens and is also non-irritating and non-toxic to mammalian subjects (e.g., and thus can be used for administration to a mucosal surface).
- the emulsions of the present invention comprise a first emulsion emulsified within a second emulsion, wherein (a) the first emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and an organic solvent; and (iii) a surfactant; and (b) the second emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and a cationic containing compound; and (iii) a surfactant.
- BCTP comprises a water-in oil nanoemulsion, in which the oil phase was made from soybean oil, tri-n-butyl phosphate, and TRITON X-IOO in 80% water.
- XsWeoPC comprises a mixture of equal volumes of BCTP with W ⁇ o8P.
- W 8 o8P is a liposome-like compound made of glycerol monostearate, refined oya sterols (e.g., GENEROL sterols), TWEEN 60, soybean oil, a cationic ion halogen-containing CPC and peppermint oil.
- the GENEROL family are a group of a polyethoxylated soya sterols (Henkel Corporation, Ambler, Pennsylvania).
- Exemplary emulsion formulations useful in the present invention are provided in Table IB. These particular formulations may be found in U.S. Pat. Nos. 5,700,679 (NN); 5,618,840; 5,549,901 (W ⁇ o8P); and 5,547,677, each of which is hereby incorporated by reference in their entireties.
- Certain other emulsion formulations are presented U.S. Pat. App. Serial No. 10/669,865, hereby incorporated by reference in its entirety.
- the X ⁇ W ⁇ oPC emulsion is manufactured by first making the Wso ⁇ P emulsion and BCTP emulsions separately. A mixture of these two emulsions is then re-emulsified to produce a fresh emulsion composition termed XsWeoPC. Methods of producing such emulsions are described in U.S. Pat. Nos. 5,103,497 and 4,895,452 (each of which is herein incorporated by reference in their entireties).
- compositions listed above are only exemplary and those of skill in the art will be able to alter the amounts of the components to arrive at a nanoemulsion composition suitable for the purposes of the present invention.
- Those skilled in the art will understand that the ratio of oil phase to water as well as the individual oil carrier, surfactant CPC and organic phosphate buffer, components of each composition may vary.
- compositions comprising BCTP have a water to oil ratio of 4:1, it is understood that the BCTP may be formulated to have more or less of a water phase. For example, in some embodiments, there is 3, 4, 5, 6, 7, 8, 9, 10, or more parts of the water phase to each part of the oil phase. The same holds true for the Wso8P formulation. Similarly, the ratio of Tri(N-butyl)phosphate:TRITON X-100:soybean oil also maybe varied.
- glycerol monooleate polysorbate 60
- GENEROL 122 cetylpyridinium chloride
- carrier oil for Wso8P
- the emulsion may have between about 80 to about lOOg of glycerol monooleate in the initial oil phase.
- the emulsion may have between about 15 to about 30 g polysorbate 60 in the initial oil phase.
- the composition may comprise between about 20 to about 30 g of a GENEROL sterol, in the initial oil phase.
- nanoemulsions can function both to inactivate a pathogen as well as to contribute to the non-toxicity of the emulsions.
- the active component in BCTP TRITON-Xl 00
- Adding the oil phase to the detergent and solvent markedly reduces the toxicity of these agents in tissue culture at the same concentrations.
- the nanoemulsion enhances the interaction of its components with the pathogens thereby facilitating the inactivation of the pathogen and reducing the toxicity of the individual components. Furthermore, when all the components of BCTP are combined in one composition but are not in a nanoemulsion structure, the mixture is not as effective at inactivating a pathogen as when the components are in a nanoemulsion structure.
- compositions recite various ratios and mixtures of active components.
- formulations are exemplary and that additional formulations comprising similar percent ranges of the recited components are within the scope of the present invention.
- a nanoemulsion comprises from about 3 to 8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 60 to 70 vol. % oil (e.g., soybean oil), about 15 to 25 vol. % of aqueous phase (e.g., DiH 2 O or PBS), and in some formulations less than about 1 vol. % of IN NaOH.
- CPC cetylpyridinium chloride
- oil e.g., soybean oil
- aqueous phase e.g., DiH 2 O or PBS
- PBS DiH 2 O
- one embodiment of the present invention comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 24 vol. % OfDiH 2 O (designated herein as Y3EC).
- Another similar embodiment comprises about 3.5 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, and about 1 vol. % of CPC, about 64 vol.
- Yet another embodiment comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 0.067 vol. % of IN NaOH, such that the pH of the formulation is about 7.1, about 64 vol. % of soybean oil, and about 23.93 vol. % OfDiH 2 O (designated herein as Y3EC pH 7.1). Still another embodiment comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 0.67 vol.
- the formulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1 vol. % of CPC, and about 64 vol. % of soybean oil, and about 19 vol. % OfDiH 2 O (designated herein as Y8EC).
- a further embodiment comprises about 8 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 19 vol. % of Ix PBS (designated herein as Y8EC PBS).
- a nanoemulsion comprises about 8 vol. % of ethanol, and about 1 vol. % of CPC, and about 64 vol. % of oil (e.g., soybean oil), and about 27 vol. % of aqueous phase (e.g., DiH 2 O or PBS) (designated herein as EC).
- oil e.g., soybean oil
- aqueous phase e.g., DiH 2 O or PBS
- a nanoemulsion comprises from about 8 vol. % of sodium dodecyl sulfate (SDS), about 8 vol. % of tributyl phosphate (TBP), and about 64 vol. % of oil (e.g., soybean oil), and about 20 vol. % of aqueous phase (e.g., DiHzO or PBS) (designated herein as S 8P).
- SDS sodium dodecyl sulfate
- TBP tributyl phosphate
- oil e.g., soybean oil
- aqueous phase e.g., DiHzO or PBS
- a nanoemulsion comprises from about 1 to 2 vol. % of TRITON X-100, from about 1 to 2 vol. % of TYLOXAPOL, from about 7 to 8 vol. % of ethanol, about 1 vol. % of cetylpyridinium chloride (CPC), about 64 to 57.6 vol. % of oil (e.g., soybean oil), and about 23 vol. % of aqueous phase (e.g., DiH 2 O or PBS). Additionally, some of these formulations further comprise about 5 mM of L-alanine/Lnosine, and about 10 mM ammonium chloride. Some of these formulations comprise PBS.
- one embodiment of the present invention comprises about 2 vol. % of TRITON X-100, about 2 vol. % of TYLOXAPOL, about 8 vol. % of ethanol, about 1 vol. % CPC, about 64 vol. % of soybean oil, and about 23 vol. % of aqueous phase DiH 2 O.
- the formulation comprises about 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % of ethanol, about 0.9 vol.
- a nanoemulsion comprises from about 5 vol. % of TWEEN 80, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g., soybean oil), and about 22 vol. % OfDiH 2 O (designated herein as Wso5EC).
- a nanoemulsion comprises from about 5 vol. % of TWEEN 20, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g., soybean oil), and about 22 vol. % of DffibO (designated herein as W 20 5EC).
- a nanoemulsion comprises from about 2 to 8 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g., soybean, or olive oil), and about 15 to 25 vol. % of aqueous phase (e.g., DiH 2 O or PBS).
- oil e.g., soybean, or olive oil
- aqueous phase e.g., DiH 2 O or PBS
- the present invention contemplates formulations comprising about 2 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 26 vol. % OfDiH 2 O (designated herein as X2E).
- a nanoemulsion comprises about 3 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 25 vol. % of DfflbO (designated herein as X3E).
- the formulations comprise about 4 vol. % Triton of X-100, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 24 vol. % OfDiH 2 O (designated herein as X4E).
- a nanoemulsion comprises about 5 vol. % of TRITON X-100, about 8 vol. % of ethanol, about 64 vol.
- a nanoemulsion comprises about 6 vol. % of TRITON X-IOO, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 22 vol. % of DiH 2 O (designated herein as X6E).
- a nanoemulsion comprises about 8 vol. % of TRITON X-IOO, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 O (designated herein as X8E).
- a nanoemulsion comprises about 8 vol. % of TRITON X-IOO, about 8 vol. % of ethanol, about 64 vol. % of olive oil, and about 20 vol. % OfDiH 2 O (designated herein as X8E O).
- a nanoemulsion comprises 8 vol. % of TRITON X-IOO, about 8 vol. % ethanol, about 1 vol. % CPC, about 64 vol. % of soybean oil, and about 19 vol. % of DiH 2 O (designated herein as X8EC).
- a nanoemulsion comprises from about 1 to 2 vol. % of TRITON X-IOO, from about 1 to 2 vol. % of TYLOXAPOL, from about 6 to 8 vol. % TBP, from about 0.5 to 1.0 vol. % of CPC, from about 60 to 70 vol. % of oil (e.g., soybean), and about 1 to 35 vol. % of aqueous phase (e.g., DiH 2 O or PBS). Additionally, certain of these nanoemulsions may comprise from about 1 to 5 vol. % of trypticase soy broth, from about 0.5 to 1.5 vol.
- a nanoemulsion further comprises from about 0.1 to 1.0 vol. % of sodium thiosulfate, and from about 0.1 to 1.0 vol. % of sodium citrate.
- PBS phosphate buffered saline
- one embodiment comprises about 2 vol.
- the inventive formulation comprises about 2 vol. % of TRITON X- 100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 23 vol. % OfDiH 2 O (designated herein as X2Y2EC).
- the inventive formulation comprises about 2 vol. % of TRITON X- 100, about 2 vol. % TYLOXAPOL, about 8 vol. % TBP, about 1 vol. % of CPC, about 0.9 vol. % of sodium thiosulfate, about 0.1 vol. % of sodium citrate, about 64 vol. % of soybean oil, and about 22 vol.
- a nanoemulsion comprises about 1.7 vol. % TRITON X-IOO, about 1.7 vol. % TYLOXAPOL, about 6.8 vol. % TBP, about 0.85% CPC, about 29.2% NEUTRAMIGEN, about 54.4 vol. % of soybean oil, and about 4.9 vol. % OfDiH 2 O (designated herein as 85% X2Y2PC/baby).
- a nanoemulsion comprises about 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol.
- a nanoemulsion comprises about 1.8 vol. % of TRITON X-100, about 1.8 vol. % of TYLOXAPOL, about 7.2 vol. % TBP, about 0.9 vol. % of CPC, and about 3 vol. % trypticase soy broth, about 57.6 vol. % of soybean oil, and about 27.7 vol.
- a nanoemulsion comprises about 1.8 vol. % TRITON X-100, about 1.8 vol. % TYLOXAPOL, about 7.2 vol. % TBP, about 0.9 vol. % CPC, about 1 vol. % yeast extract, about 57.6 vol. % of soybean oil, and about 29.7 vol. % OfDiH 2 O (designated herein as 90% X2Y2PC/YE).
- a nanoemulsion comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. % of CPC, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH2 ⁇ or PBS).
- a nanoemulsion comprises about 3 vol. % of TYLOXAPOL, about 8 vol. % of TBP, and about 1 vol. % of CPC, about 64 vol. % of soybean, and about 24 vol. % of DiHaO (designated herein as Y3PC).
- a nanoemulsion comprises from about 4 to 8 vol. % of TRITON X-100, from about 5 to 8 vol. % of TBP, about 30 to 70 vol. % of oil (e.g., soybean or olive oil), and about 0 to 30 vol. % of aqueous phase (e.g., DiH 2 O or PBS). Additionally, certain of these embodiments further comprise about 1 vol. % of CPC, about 1 vol. % of benzalkonium chloride, about 1 vol. % cetylyridinium bromide, about 1 vol.
- a nanoemulsion comprises about 8 vol. % of TRITON X- 100, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 O (designated herein as X8P).
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP 5 about 1% of CPC, about 64 vol.
- a nanoemulsion comprises about 8 vol. % TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 50 vol. % of soybean oil, and about 33 vol. % OfDiH 2 O (designated herein as ATB-XlOOl).
- the formulations comprise about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % of CPC, about 50 vol. % of soybean oil, and about 32 vol. % of DiH 2 O (designated herein as ATB-X002).
- a nanoemulsion comprises about 4 vol. % TRITON X-100, about 4 vol. % of TBP, about 0.5 vol. % of CPC, about 32 vol. % of soybean oil, and about 59.5 vol. % of DiH 2 O (designated herein as 50% X8PC).
- a nanoemulsion comprises about 8 vol. % of TRITON X- 100, about 8 vol. % of TBP, about 0.5 vol. % CPC, about 64 vol. % of soybean oil, and about 19.5 vol. % of DiH2 ⁇ (designated herein as X8PC1/2).
- a nanoemulsion comprises about 8 vol.
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 2 vol. % of CPC, about 64 vol. % of soybean oil, and about 18 vol. % of DiHaO (designated herein as X8PC2).
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8% of TBP, about 1% of benzalkonium chloride, about 50 vol. % of soybean oil, and about 33 vol. % Of DiH 2 O (designated herein as X8P BC).
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol.
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of cetyldimethyletylammonium bromide, about 50 vol. % of soybean oil, and about 33 vol. % OfDiH 2 O (designated herein as X8P CTAB).
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol.
- a nanoemulsion comprises 8 vol. % of TRITON X-100, about 8 vol. % of TBP, about 1 vol. % of CPC, about 10 mM ammonium chloride, about 5mM Inosine, about 5mM L-alanine, about 64 vol. % of soybean oil, and about 19 vol. % OfDiH 2 O or PBS (designated herein as X8PC GEi x ).
- a nanoemulsion comprises about 5 vol. % of TRITON X-100, about 5% of TBP, about 1 vol. % of CPC, about 40 vol. % of soybean oil, and about 49 vol. % OfDiH 2 O (designated herein as X5PsC).
- a nanoemulsion comprises about 2 vol. % TRITON X-100, about 6 vol. % TYLOXAPOL, about 8 vol. % ethanol, about 64 vol. % of soybean oil, and about 20 vol. % OfDiH 2 O (designated herein as X2Y6E).
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, and about 8 vol. % of glycerol, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 25 vol. % of aqueous phase (e.g., DiHaO or PBS).
- Certain nanoemulsion compositions e.g., used to generate an immune response (e.g., for use as a vaccine) comprise about 1 vol. % L-ascorbic acid.
- one particular embodiment comprises about 8 vol. % of TRITON X-100, about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol.
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 8 vol. % of glycerol, about 1 vol. % of L-ascorbic acid, about 64 vol. % of soybean oil, and about 19 vol. % OfDiH 2 O (designated herein as X8GV C ).
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, from about 0.5 to 0.8 vol. % of TWEEN 60, from about 0.5 to 2.0 vol. % of CPC, about 8 vol. % of TBP, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 25 vol. % of aqueous phase (e.g., DiH 2 O or PBS).
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 0.70 vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol.
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 0.71 vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 18.29 vol. % OfDiH 2 O (designated herein as W6OO.7X8PC).
- a nanoemulsion comprises from about 8 vol. % of TRITON X-100, about 0.7 vol. % of TWEEN 60, about 0.5 vol. % of CPC, about 8 vol.
- a nanoemulsion comprises about 8 vol. % of TRITON X-100, about 0.71 vol. % of TWEEN 60, about 2 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 17.3 vol. % OfDiH 2 O.
- a nanoemulsion comprises about 0.71 vol. % of TWEEN 60, about 1 vol. % of CPC, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 25.29 vol. % OfDiH 2 O (designated herein as W6OO.7PC).
- a nanoemulsion comprises about 2 vol. % of dioctyl sulfosuccinate, either about 8 vol. % of glycerol, or about 8 vol. % TBP, in addition to, about 60 to 70 vol. % of oil (e.g., soybean or olive oil), and about 20 to 30 vol. % of aqueous phase (e.g., DiH 2 O or PBS).
- a nanoemulsion comprises about 2 vol. % of dioctyl sulfosuccinate, about 8 vol. % of . glycerol, about 64 vol. % of soybean oil, and about 26 vol.
- a nanoemulsion comprises about 2 vol. % of dioctyl sulfosuccinate, and about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 26 vol. % OfDiH 2 O (designated herein as D2P).
- a nanoemulsion comprises about 8 to 10 vol. % of glycerol, and about 1 to 10 vol. % of CPC, about 50 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH 2 O or PBS). Additionally, in certain of these embodiments, a nanoemulsion further comprises about 1 vol. % of L-ascorbic acid. For example, in some embodiments, a nanoemulsion comprises about 8 vol. % of glycerol, about 1 vol. % of CPC, about 64 vol. % of soybean oil, and about 27 vol.
- a nanoemulsion comprises about 10 vol. % of glycerol, about 10 vol. % of CPC, about 60 vol. % of soybean oil, and about 20 vol. % OfDiH 2 O (designated herein as GClO).
- a nanoemulsion comprises about 10 vol. % of glycerol, about ⁇ vol. % of CPC, about 1 vol. % of L-ascorbic acid, about 64 vol. % of soybean or oil, and about 24 vol. % OfDiH 2 O (designated herein as GCV C ).
- a nanoemulsion comprises about 8 to 10 vol. % of glycerol, about 8 to 10 vol. % of SDS, about 50 to 70 vol. % of oil (e.g., soybean or olive oil), and about 15 to 30 vol. % of aqueous phase (e.g., DiH 2 O or PBS). Additionally, in certain of these embodiments, a nanoemulsion further comprise about 1 vol. % of lecithin, and about 1 vol. % of p-Hydroxybenzoic acid methyl ester. Exemplary embodiments of such formulations comprise about 8 vol. % SDS, 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol.
- a related formulation comprises about 8 vol. % of glycerol, about 8 vol. % of SDS, about 1 vol. % of lecithin, about 1 vol. % of p-Hydroxybenzoic acid methyl ester, about 64 vol. % of soybean oil, and about 18 vol. % OfDiH 2 O (designated herein as S8GL1B1).
- a nanoemulsion comprises about 4 vol. % of TWEEN 80, about 4 vol. % of TYLOXAPOL, about 1 vol. % of CPC, about 8 vol. % of ethanol, about 64 vol. % of soybean oil, and about 19 vol. % OfDiH 2 O (designated herein as Wso4Y4EC).
- a nanoemulsion comprises about 0.01 vol. % of CPC, about 0.08 vol. % of TYLOXAPOL, about 10 vol. % of ethanol, about 70 vol. % of soybean oil, and about 19.91 vol. % OfDiH 2 O (designated herein as Y.08EC.01).
- a nanoemulsion comprises about 8 vol. % of sodium lauryl sulfate, and about 8 vol. % of glycerol, about 64 vol. % of soybean oil, and about 20 vol. % OfDiH 2 O (designated herein as SLS8G).
- the specific formulations described above are simply examples to illustrate the variety of nanoemulsions that find use (e.g., to inactivate and/or neutralize a pathogen, and for generating an immune response in a subject (e.g., for use as a vaccine)) in the present invention.
- the present invention contemplates that many variations of the above formulations, as well as additional nanoemulsions, find use in the methods of the present invention.
- Candidate emulsions can be easily tested to determine if they are suitable.
- the desired ingredients are prepared using the methods described herein, to determine if an emulsion can be formed. If an emulsion cannot be formed, the candidate is rejected.
- a candidate composition made of 4.5% sodium thiosulfate, 0.5% sodium citrate, 10% n-butanol, 64% soybean oil, and 21% DiH 2 O does not form an emulsion.
- the candidate emulsion should form a stable emulsion.
- An emulsion is stable if it remains in emulsion form for a sufficient period to allow its intended use (e.g., to generate an immune response in a subject).
- Typical emulsions that are relatively unstable, will lose their form within a day.
- a candidate composition made of 8% 1-butanol, 5% Tween 10, 1% CPC, 64% soybean oil, and 22% DiHaO does not form a stable emulsion.
- Nanoemulsions that have been shown to be stable include, but are not limited to, 8 vol. % of TRITON X-IOO, about 8 vol. % of TBP, about 64 vol. % of soybean oil, and about 20 vol. % of DiH 2 O (designated herein as X8P); 5 vol. % of TWEEN 20, from about 8 vol. % of ethanol, from about 1 vol. % of CPC, about 64 vol. % of oil (e.g., soybean oil), and about 22 vol.
- % of DiH 2 O (designated herein as W2o5EC); 0.08% Triton X-IOO, 0.08% Glycerol, 0.01% Cetylpyridinium Chloride, 99% Butter, and 0.83% diH 2 O (designated herein as 1% X8GC Butter); 0.8% Triton X-IOO, 0.8% Glycerol, 0.1% Cetylpyridinium Chloride, 6.4% Soybean Oil, 1.9% diH 2 O, and 90% Butter (designated herein as 10% X8GC Butter); 2% W 20 5EC, 1% Natrosol 250L NF, and 97% diH 2 O (designated herein as 2% W 2 o5EC L GEL); 1% Cetylpyridinium Chloride, 5% Tween 20, 8% Ethanol, 64% 70 Viscosity Mineral Oil, and 22% diH 2 O (designated herein as W 2 o5EC 70 Mineral Oil); 1% Cetylpyridinium Chlor
- the candidate emulsion should have efficacy for its intended use.
- a nanoemuslion should inactivate (e.g., kill or inhibit growth of) a pathogen to a desired level (e.g., 1 log, 2 log, 3 log, 4 log, . . . reduction).
- a desired level e.g. 1 log, 2 log, 3 log, 4 log, . . . reduction.
- a candidate composition made of 1% ammonium chloride, 5% Tween 20, 8% ethanol, 64% soybean oil, and 22% DiH 2 O was shown not to be an effective emulsion.
- the nanoemulsions are non-toxic (e.g., to humans, plants, or animals), non-irritant (e.g., to humans, plants, or animals), and non-corrosive (e.g., to humans, plants, or animals or the environment), while possessing potency against a broad range of microorganisms including bacteria, fungi, viruses, and spores. While a number of the above described nanoemulsions meet these qualifications, the following description provides a number of preferred non-toxic, non-irritant, non- corrosive, anti-microbial nanoemulsions of the present invention (hereinafter in this section referred to as "non-toxic nanoemulsions").
- the non-toxic nanoemulsions comprise surfactant lipid preparations (SLPs) for use as broad-spectrum antimicrobial agents that are effective against bacteria and their spores, enveloped viruses, and fungi.
- SLPs surfactant lipid preparations
- these SLPs comprises a mixture of oils, detergents, solvents, and cationic halogen-containing compounds in addition to several ions that enhance their biocidal activities.
- SLPs are characterized as stable, non-irritant, and non-toxic compounds compared to commercially available bactericidal and sporicidal agents, which are highly irritant and/or toxic.
- ingredients for use in the non-toxic nanoemulsions include, but are not limited to: detergents (e.g., TRITON X-100 (5-15%) or other members of the TRITON family, TWEEN 60 (0.5-2%) or other members of the TWEEN family, or TYLOXAPOL (1-10%)); solvents (e.g., tributyl phosphate (5-15%)); alcohols (e.g., ethanol (5-15%) or glycerol (5- 15%)); oils (e.g., soybean oil (40-70%)); cationic halogen-containing compounds (e.g., cetylpyridinium chloride (0.5-2%), cetylpyridinium bromide (0.5-2%)), or cetyldimethylethyl ammonium bromide (0.5-2%)); quaternary ammonium compounds (e.g., benzalkonium chloride (0.5-2%), N-alkyldimethylbenzyl ammonium chloride (0.5-2%)); ions (calc
- Quaternary ammonium compounds for use in the present include, but are not limited to, N-alkyldimethyl benzyl ammonium saccharinate; 1,3,5-Triazine-1,3,5(2H,4H,6H)- triethanol; 1 -Decanaminium, N-decyl-N, N-dimethyl-, chloride (or) Didecyl dimethyl ammonium chloride; 2-(2-(p-(Diisobuyl)cresosxy)ethoxy)ehyl dimethyl benzyl ammonium chloride; 2-(2-(p-(Diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ar ⁇ monium chloride; alkyl 1 or 3 benzyl-1 -(2-hydroxethyl)-2-imidazolinium chloride; alkyl bis(2-hydroxyethyl) benzyl ammonium chloride; alkyl demethyl benzyl am
- the preferred non-toxic nanoemulsions are characterized by the following: they are approximately 200-800 nm in diameter, although both larger and smaller diameter nanoemulsions are contemplated; the charge depends on the ingredients; they are stable for relatively long periods of time (e.g., up to two years), with preservation of their biocidal activity; they are non-irritant and non-toxic compared to their individual components due, at least in part, to their oil contents that markedly reduce the toxicity of the detergents and the solvents; they are effective at concentrations as low as 0.1%; they have antimicrobial activity against most vegetative bacteria (including Gram-positive and Gram- negative organisms), fungi, and enveloped and nonenveloped viruses in 15 minutes (e.g., 99.99% killing); and they have sporicidal activity in 1-4 hours (e.g., 99.99% killing) when produced with germination enhancers.
- potential nanoemulsion compositions are tested in animal models of infectious diseases.
- animal models of infectious diseases The use of well-developed animal models provides a method of measuring the effectiveness and safety of a vaccine before administration to human subjects. Exemplary animal models of disease are shown in Table 3. These animals are commercially available (e.g., from Jackson Laboratories Charles River; Portage, MI).
- Bacillus cereus Animal models of Bacillus cereus (closely related to Bacillus anthracis) are utilized to test Anthrax vaccines of the present invention. Both bacteria are spore forming Gram positive rods and the disease syndrome produced by each bacteria is largely due to toxin production and the effects of these toxins on the infected host (Brown et al, J. Bact, 75:499 (1958); Burdon and Wende, J. Infect Dis., 107:224 (1960); Burdon et al, J. Infect. Dis., 117:307 (1967)). Bacillus cereus infection mimics the disease syndrome caused by Bacillus anthracis. Mice are reported to rapidly succumb to the effects of B. cereus toxin and are a useful model for acute infection. Guinea pigs develop a skin lesion subsequent to subcutaneous infection with B. cereus that resembles the cutaneous form of anthrax.
- Clostridium perfringens infection in both mice and guinea pigs has been used as a model system for the in vivo testing of antibiotic drugs (Stevens et al, Antimicrob. Agents Chemother., 31 :312 (1987); Stevens etal, J. Infect. Dis., 155:220 (1987); Alttemeier et al, Surgery, 28:621 (1950); Sandusky et al, Surgery, 28:632 (1950)). Clostridium tetani is well known to infect and cause disease in a variety of mammalian species.
- mice Mice, guinea pigs, and rabbits have all been used experimentally (Willis, Topley and Wilson's Principles of Bacteriology, Virology and Immunity. Wilson, G., A. Miles, and M.T. Parker, eds. pages 442-475 1983).
- Vibrio cholerae infection has been successfully initiated in mice, guinea pigs, and rabbits. According to published reports it is preferred to alter the normal intestinal bacterial flora for the infection to be established in these experimental hosts. This is accomplished by administration of antibiotics to suppress the normal intestinal flora and, in some cases, withholding food from the animals (Butterton et al, Infect. Immun., 64:4373 (1996); Levine et al, Microbiol. Rev., 47:510 (1983); Finkelstein et al, J. Infect. Dis., 114:203 (1964); Freter, J. Exp. Med., 104:411 (1956); and Freter, J. Infect. Dis., 97:57 (1955)).
- Shigella flexnerii infection has been successfully initiated in mice and guinea pigs.
- the normal intestinal bacterial flora be altered to aid in the establishment of infection in these experimental hosts. This is accomplished by administration of antibiotics to suppress the normal intestinal flora and, in some cases, withholding food from the animals (Levine et al, Microbiol. Rev., 47:510 (1983); Freter, J. Exp. Med., 104:411 (1956); Formal et al, J. Bact, 85:119 (1963); LaBrec et al, J. Bact 88:1503 (1964); Takeuchi etal, Am. J. Pathol., 47:1011 (1965)).
- mice and rats have been used extensively in experimental studies with Salmonella typhimurium and Salmonella enteriditis (Naughton et al, J. Appl. Bact., 81:651 (1996); Carter and Collins, J. Exp. Med., 139:1189 (1974); Collins, Infect. Immun., 5:191 (1972); Collins and Carter, Infect. Immun., 6:451 (1972)).
- Mice and rats are well established experimental models for infection with Sendai virus (Jacoby et al, Exp. Gerontol., 29:89 (1994); Massion et al, Am. J. Respir. Cell MoI. Biol. 9:361 (1993); Castleman et al, Am. J. Path., 129:277 (1987); Castleman, Am. J. Vet. Res., 44:1024 (1983); Mims and Murphy, Am. J. Path., 70:315 (1973)).
- Sindbis virus infection of mice is usually accomplished by intracerebral inoculation of newborn mice.
- weanling mice are inoculated subcutaneously in the footpad (Johnson et al, J. Infect. Dis., 125:257 (1972); Johnson, Am. J. Path., 46:929 (1965)).
- animals are housed for 3-5 days to rest from shipping and adapt to new housing environments before use in experiments.
- control animals are sacrificed and tissue is harvested to establish baseline parameters.
- Animals are anesthetized by any suitable method (e.g., including, but not limited to, inhalation of Isofluorane for short procedures or ketamine/xylazine injection for longer procedure).
- candidate nanoemulsion vaccines are evaluated using one of several suitable model systems.
- cell-mediated immune responses can be evaluated in vitro.
- an animal model may be used to evaluate in vivo immune response and immunity to pathogen challenge. Any suitable animal model may be utilized, including, but not limited to, those disclosed in Table 3.
- the amount of exposure of the pathogen to a nanoemulsion sufficient to inactivate the pathogen is investigated. It is contemplated that pathogens such as bacterial spores require longer periods of time for inactivation by the nanoemulsion in order to be sufficiently neutralized to allow for immunization.
- the time period required for inactivation may be investigated using any suitable method, including, but not limited to, those described in the illustrative examples below.
- the stability of emulsion-developed vaccines is evaluated, particularly over time and storage condition, to ensure that vaccines are effective long-term.
- the ability of other stabilizing materials (e.g., dendritic polymers) to enhance the stability and immunogenicity of vaccines is also evaluated.
- the ability of the vaccine to elicit an immune response and provide immunity is optimized.
- Non-limiting examples of methods for assaying vaccine effectiveness are described in Example 14 below.
- the timing and dosage of the vaccine can be varied and the most effective dosage arid administration schedule determined.
- the level of immune response is quantitated by measuring serum antibody levels.
- in vitro assays are used to monitor proliferation activity by measuring H 3 -thymidine uptake.
- ThI and Th2 cytokine responses are measured to qualitatively evaluate the immune response.
- levels of include IL-2, TNF-Y, IFN-Y, IL-4, IL-6, IL-Il, IL-12, etc.
- a composition of the present invention induces (e.g., when administered to a subject) both systemic and mucosal immunity.
- administration of a composition of the present invention to a subject results in protection against an exposure (e.g., a mucosal exposure) to HIV.
- mucosal administration e.g., vaccination
- HIV infection e.g., that initiates at a mucosal surface
- the present invention provides compositions and methods for stimulating mucosal immunity (e.g., a protective IgA response) from a pathogen in a subject.
- the present invention provides a composition (e.g., a composition comprising a NE and immunogenic protein antigens from HIV (e.g., g ⁇ l20) to serve as a mucosal vaccine.
- a composition e.g., a composition comprising a NE and immunogenic protein antigens from HIV (e.g., g ⁇ l20) to serve as a mucosal vaccine.
- This material can easily be produced with NE and HIV protein (e.g., viral-derived gpl20, live- virus- vector-derived gpl20 and gpl60, recombinant mammalian gpl20, recombinant denatured antigens, small peptide segments of gpl20 and gp41, V3 loop peptides, and induces both mucosal and systemic immunity.
- HIV protein e.g., viral-derived gpl20, live- virus- vector-derived gpl20 and gp
- the present invention provides a composition for generating an immune response comprising a NE and an immunogen (e.g., a purified, isolated or synthetic HIV protein or derivative, variant, or analogue thereof; or, one or more serotypes of HIV inactivated by the nanoemulsion).
- an immunogen e.g., a purified, isolated or synthetic HIV protein or derivative, variant, or analogue thereof; or, one or more serotypes of HIV inactivated by the nanoemulsion.
- an immunogen e.g., a purified, isolated or synthetic HIV protein or derivative, variant, or analogue thereof; or, one or more serotypes of HIV inactivated by the nanoemulsion.
- generation of an immune response (e.g., resulting from administration of a composition comprising a nanoemulsion and an immunogen) provides total or partial immunity to the subject (e.g., from signs, symptoms or conditions of a disease (e.g., AIDS)).
- a disease e.g., AIDS
- protection and/or immunity from disease e.g., the ability of a subject's immune system to prevent or attenuate (e.g., suppress) a sign, symptom or condition of disease
- an immunogenic composition of the present invention is due to adaptive (e.g., acquired) immune responses (e.g., immune responses mediated by B and T cells following exposure to a NE comprising an immunogen of the present invention (e.g., immune responses that exhibit increased specificity and reactivity towards HIV).
- the compositions and methods of the present invention are used prophylactically or therapeutically to prevent or attenuate a sign, symptom or condition associated with AIDS.
- a NE comprising an immunogen is administered alone.
- a composition comprising a NE and an immunogen comprises one or more other agents (e.g., a pharmaceutically acceptable carrier, adjuvant, excipient, and the like).
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a humoral immune response.
- a composition for stimulating an immune response of the present invention is administered in a manner to induce a cellular (e.g., cytotoxic T lymphocyte) immune response, rather than a humoral response.
- a composition comprising a NE and an immunogen of the present invention induces both a cellular and humoral immune response.
- the present invention is not limited by the type or strain of orthopox virus used (e.g., in a composition comprising a NE and immunogen (e.g., orthopox virus inactivated by the nanoemulsion).
- each ⁇ rthopox virus family member alone, or in combination with another family member may be used to generate a composition comprising a NE and an immunogen (e.g., used to generate an immune response) of the present invention.
- Orthopox virus family member include, but are not limited to, variola virus, vaccinia virus, cowpox, monkeypox, gergilpox, camelpox, and others.
- the present invention is not limited by the strain of vaccinia virus used.
- vaccinia virus strains are contemplated to be useful in the present invention including, but not limited to, classical strains of vaccinia virus (e.g., EM-63, Lister, New York City Board of Health, Elestree, and Temple of Heaven strains), attenuated strains (e.g., Ankara), non-replicating strains, modified strains (e.g., genetically or mechanically modified strains (e.g., to become more or less virulent)), Copenhagen strain, modified vaccinia Ankara, New York vaccinia virus, Vaccinia ViruswR and Vaccinia ViruswR-Luc, or other serially diluted strain of vaccinia virus.
- classical strains of vaccinia virus e.g., EM-63, Lister, New York City Board of Health, Elestree, and Temple of Heaven strains
- attenuated strains e.g., Ankara
- non-replicating strains e.g., modified
- a composition comprising a NE and immunogen may comprise one or more strains of vaccinia virus and/or other type of orthopox virus. Additionally, a composition comprising a NE and immunogen may comprise one or more strains of vaccinia virus, and, in addition, one or more strains of a non-vaccinia virus immunogen or immunogenic epitope thereof (e.g., a bacteria (e.g., B.
- immunogenic epitope thereof e.g., recombinant protective antigen
- a virus e.g., West Nile virus, Avian Influenza virus, Ebola virus, HSV, HPV, HCV, HIV, etc.
- an immunogenic epitope thereof e.g., gpl20
- the immunogen may comprise one or more antigens derived from a pathogen (e.g., orthopox virus).
- a pathogen e.g., orthopox virus
- the immunogen is a purified, recombinant, synthetic, or otherwise isolated protein (e.g., added to the NE to generate an immunogenic composition).
- the immunogenic protein may be a derivative, analogue or otherwise modified (e.g., PEGylated) form of a protein from a pathogen.
- the present invention is not limited by the type or strain of Bacillus used or immunogenic protein derived therefrom.
- Bacillus used or immunogenic protein derived therefrom 89 different strains of B. anthracis have been identified, ranging from virulent Ames and Vollum strains with biological warfare and bioterrorism applications to benign Sterne strain used for inoculations (See, e.g., Easterday et al., J Clin Microbiol. 200543(4): 1995-7).
- the strains differ in presence and activity of various genes, determining their virulence and production of antigens and toxins. Any one of these or yet to be identified or generated strains may be used in an immunogenic composition comprising a NE of the present invention.
- the immunogen may comprise one or more antigens derived from a pathogen (e.g., B. anthrads).
- the immunogen is a purified, recombinant, synthetic, or otherwise isolated protein (e.g., added to the NE to generate an immunogenic composition).
- the immunogenic protein may be a derivative, variant, analogue or otherwise modified form of a protein from a pathogen.
- the present invention is not limited by the type of protein (e.g., derived from bacteria of the genus Bacillus) used for generation of an immunogenic composition of the present invention.
- immunogenic proteins may be used including, but not limited to, protective antigen (PA), lethal factor (LF), edema factor (EF), PA degradation products (See, e.g., Farchaus, J., et al., Applied & Environmental Microbiol., 64(3):982-991 (1998)), as well as analogues, derivatives and modified forms thereof.
- PA protective antigen
- LF lethal factor
- EF edema factor
- PA degradation products See, e.g., Farchaus, J., et al., Applied & Environmental Microbiol., 64(3):982-991 (1998)
- analogues, derivatives and modified forms thereof See, e.g., Farchaus, J., et al., Applied & Environmental Microbiol., 64(3):982-991 (1998)
- Bacillus proteins of the present invention may be used in their native conformation, or more preferably, may be modified for vaccine use. These modifications may either be required for technical reasons relating to the method of purification, or they maybe used to biologically inactivate one or several functional properties of the Bacillus proteins (e.g., that would otherwise be toxic).
- the invention encompasses derivatives of Bacillus proteins that may be, for example, mutated proteins (e.g., that has undergone deletion, addition or substitution of one or more amino acids using well known techniques for site directed mutagenesis or any other conventional method).
- Bacillus proteins (e.g., rPA) of the present invention may be modified by chemical methods during a purification process to render the proteins stable and monomelic.
- One method to prevent oxidative aggregation of a protein is the use of chemical modifications of the protein's thiol groups.
- the disulphide bridges are reduced by treatment with a reducing agent such as DTT, ⁇ -mercaptoethanol, or gluthatione.
- a second step the resulting thiols are blocked by reaction with an alkylating agent (e.g., the protein can be carboxyamidated/carbamidomethylated using iodoacetamide).
- Each Bacillus family member alone, or in combination with another ' family member, may be used to generate a composition comprising a NE and an immunogen (e.g., used to generate an immune response) of the present invention.
- a composition comprising a NE and immunogen may comprise one or more strains of B. anthrads. Additionally, a composition comprising a NE and immunogen may comprise one or more strains of B. anthrads, and, in addition, one or more strains of a non-2?, anthrads immunogen (e.g., a virus such as West Nile virus, Avian Influenza virus, Ebola virus, HSV, HPV, HCV, HIV, etc. or an immunogenic epitope thereof (e.g., gpl20)).
- a virus such as West Nile virus, Avian Influenza virus, Ebola virus, HSV, HPV, HCV, HIV, etc.
- an immunogenic epitope thereof e.g., gpl20
- the present invention is not limited by the type (e.g., serotype, group, or clade) of HIV used or immunogenic protein derived therefrom.
- HIV-I HIV-I
- HIV-2 Both types are transmitted by sexual contact, through ! blood, and from mother to child, and they appear to cause clinically indistinguishable AIDS.
- HIV-2 is less easily transmitted, and the period between initial infection and illness is longer in the case of HIV-2.
- the predominant virus is HIV- 1, and generally when people refer to HIV without specifying the type of virus they will be referring to HIV-I .
- the relatively uncommon HIV-2 type is concentrated in West • Africa and is rarely found elsewhere.
- Each type is divided into groups, and each group is divided into subtypes and circulating recombinant forms (CRFs).
- the strains of HIV-I can be classified into three groups : the "major” group M, the "outlier” group O and the "new" group N.
- subtypes A, B, C, D, F, G, H, J and K are known to be at least nine genetically distinct subtypes (or clades) of HIV-I. These are subtypes A, B, C, D, F, G, H, J and K.
- any one of these or yet to be identified or generated serotypes, groups, or clades may be used in an immunogenic composition comprising a NE of the present invention.
- the immunogen may comprise one or more antigens derived from a pathogen (e.g., HIV).
- the immunogen is a purified, recombinant, synthetic, or otherwise isolated protein (e.g., added to the NE to generate an immunogenic composition).
- the immunogenic protein may be a derivative, analogue or otherwise modified form of a protein from a pathogen.
- the present invention is not limited by the type of protein (e.g., derived from HIV) used for generation of an immunogenic composition of the present invention. Indeed, a variety of immunogenic proteins may be used including, but not limited to, g ⁇ l60, gpl20, gp41 , Tat, and Nef; as well as analogues, derivatives and modified forms thereof.
- HIV proteins of the present invention may be used in their native conformation, or more preferably, may be modified for vaccine use. These modifications may either be required for technical reasons relating to the method of purification, or they may be used to biologically inactivate one or several functional properties of HIV protein.
- the invention encompasses derivatives of HIV proteins which may be, for example mutated proteins (e.g., that has undergone deletion, addition or substitution of one or more amino acids using well known techniques for site directed mutagenesis or any other conventional method.
- a HIV protein may be mutated so that it is biologically inactive while maintaining its immunogenic epitopes (See, e.g., Clements, Virology 235: 48-64, 1997).
- HIV proteins of the present invention may be modified by chemical methods during the purification process to render the proteins stable and monomelic.
- One method to prevent oxidative aggregation of a HIV protein is the use of chemical modifications of the protein's thiol groups.
- the disulphide bridges are reduced by treatment with a reducing agent such as DTT, ⁇ -mercaptoethanol, or gluthatione.
- the resulting thiols are blocked by reaction with an alkylating agent (e.g., the protein can be carboxyamidated/carbamidomethylated using iodoacetamide).
- Each HIV serotype, group or clade alone, or in combination with another family member, may be used to generate a composition comprising a NE and an immunogen (e.g., used to generate an immune response) of the present invention.
- a composition comprising a NE and immunogen may comprise one or more serotypes, groups or clades of HIV.
- a composition comprising a NE and immunogen may comprise one or more serotypes, groups or clades of HIV, and, in addition, one or more strains of a non-HIV immunogen (e.g., a virus such as West Nile virus, Avian Influenza virus, Ebola virus, HSV, HPV, HCV, , etc. or an immunogenic epitope thereof).
- a non-HIV immunogen e.g., a virus such as West Nile virus, Avian Influenza virus, Ebola virus, HSV, HPV, HCV, , etc. or an immunogenic epitope thereof.
- compositions comprising a NE and immunogen of the present invention may comprise one or more different agents in addition to the NE and immunogen.
- agents or cofactors include, but are not limited to, adjuvants, surfactants, additives, buffers, solubilizers, chelators, oils, salts, therapeutic agents, drugs, bioactive agents, antibacterials, and antimicrobial agents (e.g., antibiotics, antivirals, etc.).
- a composition comprising a NE and immunogen of the present invention comprises an agent and/or co-factor that enhance the ability of the immunogen to induce an immune response (e.g., an adjuvant).
- an agent and/or co-factor that enhance the ability of the immunogen to induce an immune response e.g., an adjuvant.
- the presence of one or more co-factors or agents reduces the amount of immunogen required for induction of an immune response (e.g., a protective immune respone (e.g., protective immunization)).
- the presence of one or more co-factors or agents can be used to skew the immune response towards a cellular (e.g., T cell mediated) or humoral (e.g., antibody mediated) immune response.
- the present invention is not limited by the type of co-factor or agent used in a therapeutic agent of the present invention.
- Adjuvants are described in general in Vaccine Design—the Subunit and Adjuvant Approach, edited by Powell and Newman; Plenum Press, New York, 1995.
- the present invention is not limited by the type of adjuvant utilized (e.g., for use in a composition (e.g., pharmaceutical composition) comprising a NE and immunogen).
- suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate.
- an adjuvant may be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.
- compositions comprising a NE and immunogen of the present invention comprises one or more adjuvants that induce a ThI- type response.
- a composition comprising a NE and immunogen of the present invention comprises one or more adjuvants that induce a Th2-type response.
- an immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system.
- Immune responses may be broadly categorized into two categories: humoral and cell mediated immune responses (e.g., traditionally characterized by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed ThI -type responses (cell- mediated response), and Th2-type immune responses (humoral response).
- Stimulation of an immune response can result from a direct or indirect response of a cell or component of the immune system to an intervention (e.g., exposure to an immunogen).
- Immune responses can be measured in many ways including activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells etc.); up-regulated or down-regulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly (including increased spleen celhilarity); hyperplasia and mixed cellular infiltrates in various organs.
- Other responses, cells, and components of the immune system that can be assessed with respect to immune stimulation are known in the art.
- compositions and methods of the present invention induce expression and secretion of cytokines (e.g., by macrophages, dendritid cells and CD4+ T cells). Modulation of expression of a particular cytokine can occur locally or systemically. It is known that cytokine profiles can determine T cell regulatory and effector functions in immune responses.
- ThI -type cytokines can be induced, and thus, the immunostimulatory compositions of the present invention can promote a ThI type antigen- specific immune response including cytotoxic T-cells.
- Th2-type cytokines can be induced thereby promoting a Th2 type antigen-specific immune response.
- Cytokines play a role in directing the T cell response.
- Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including B and other T cells. Most mature CD4+T helper cells express one of two cytokine profiles: ThI or Th2.
- ThI -type CD4+ T cells secrete IL-2, IL-3, IFN- ⁇ , GM-CSF and high levels of TNF- ⁇ .
- Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF- ⁇ .
- ThI type cytokines promote both cell-mediated immunity, and humoral immunity that is characterized by immunoglobulin class switching to IgG2a in mice and IgGl in humans. ThI responses may also be associated with delayed-type hypersensitivity and autoimmune disease. Th2 type cytokines induce primarily humoral immunity and induce class switching to IgGl and IgE.
- the antibody isotypes associated with ThI responses generally have neutralizing and opsonizing capabilities whereas those associated with Th2 responses are associated more with allergic responses.
- IL-12 and IFN- ⁇ are positive ThI and negative Th2 regulators.
- IL-12 promotes IFN- ⁇ production, and IFN- ⁇ provides positive feedback for IL-12.
- IL-4 and IL-10 appear important for the establishment of the Th2 cytokine profile and to down-regulate ThI cytokine production.
- the present invention provides a method of stimulating a ThI -type immune response in a subject comprising administering to a subject a composition comprising a NE and an immunogen.
- the present invention provides a method of stimulating a Th2-type immune response in a subject comprising administering to a subject a composition comprising a NE and an immunogen.
- adjuvants can be used (e.g., can be co-administered with a composition of the present invention) to skew an immune response toward either a ThI or Th2 type immune response.
- adjuvants that induce Th2 or weak ThI responses include, but are not limited to, alum, saponins, and SB-As4.
- adjuvants that induce ThI responses include but are not limited to MPL, MDP, ISCOMS, IL-12, IFN- ⁇ , and SB-AS2.
- ThI -type immunogens can be used (e.g., as an adjuvant) in compositions and methods of the present invention. These include, but are not limited to, the following.
- monophosphoryl lipid A e.g., in particular 3-de-O- acylated monophosphoryl lipid A (3D-MPL)
- 3D-MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains.
- diphosphoryl lipid A 5 and 3-O-deacylated variants thereof are used.
- Each of these immunogens can be purified and prepared by methods described in GB 2122204B, hereby incorporated by reference in its entirety.
- Other purified and synthetic lipopolysaccharides have been described (See, e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al., 1986, IntArch.Allergy.Immunol., 79(4):392-6; Hilgers et al., 1987, Immunology, 60(l):141-6; and EP 0 549 074, each of which is hereby incorporated by reference in its entirety).
- 3D-MPL is used in the form of a particulate formulation (e.g., having a small particle size less than 0.2 ⁇ m in diameter, described in EP 0 689454, hereby incorporated by reference in its entirety).
- saponins are used as an immunogen (e.g. ,ThI -type adjuvant) in a composition of the present invention.
- Saponins are well known adjuvants (See, e.g., Lacaille-Dubois and Wagner (1996) Phytomedicine vol 2 pp 363-386).
- Examples of saponins include Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof (See, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (l-2):l-55; and EP 0 362 279, each of which is hereby incorporated by reference in its entirety).
- haemolytic saponins QS7, QS 17, and QS21 HPLC purified fractions of Quil A; See, e.g., Kensil et al. (1991). J. Immunology 146,431-437, U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0 362 279, each of which is hereby incorporated by reference in its entirety).
- QS21 and polysorbate or cyclodextrin See, e.g., WO 99/10008, hereby incorporated by reference in its entirety.
- an immunogenic oligonucleotide containing unmethylated CpG dinucleotides (“CpG”) is used as an adjuvant in the present invention.
- CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA.
- CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis et al., J.Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.Immunol., 1998, 161(9):4463-6; and U.S. Pat. App. No.
- the immunostimulatory sequence is Purine-Purine-C-G-pyrimidine- pyrimidine; wherein the CG motif is not methylated.
- CpG oligonucleotides activate various immune subsets including natural killer cells (which produce EFN- ⁇ ) and macrophages.
- CpG oligonucleotides are formulated into a composition of the present invention for inducing an immune response.
- a free solution of CpG is co-administered together with an antigen (e.g., present within a NE solution (See, e.g., WO 96/02555; hereby incorporated by reference).
- a CpG oligonucleotide is covalently conjugated to an antigen (See, e.g., WO 98/16247, hereby incorporated by reference), or formulated with a carrier such as aluminium hydroxide (See, e.g., Brazolot-Millan et al., Proc.Natl.AcadSci., USA, 1998, 95(26), 15553-8).
- adjuvants such as Complete Freunds Adjuvant and Incomplete Freunds Adjuvant, cytokines (e.g., interleukins (e.g., IL-2, IFN- ⁇ , IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.), detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E.
- cytokines e.g., interleukins (e.g., IL-2, IFN- ⁇ , IL-4, etc.)
- macrophage colony stimulating factor e.g., tumor necrosis factor, etc.
- a bacterial ADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E.
- CT cholera toxin
- PT pertussis toxin
- CoIi heat-labile toxin particularly LT-K63 (where lysine is substituted for the wild- type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S 109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (See, e.g., WO93/13202 and WO92/19265, each of which is hereby incorporated by reference), and other immunogenic substances (e.g., that enhance the effectiveness of a composition of the present invention) are used with a composition comprising a NE and immunogen of the present invention.
- LT-K63 where lysine is substituted for the wild- type amino acid at position 63
- LT-R72 where arginine is substituted for the wild-type amino acid at position 72
- adjuvants that find use in the present invention include poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).
- PCPP polymer polymer
- Virus Research Institute, USA poly(di(carboxylatophenoxy)phosphazene
- MPL monophosphoryl lipid A
- MDP muramyl dipeptide
- t-MDP threonyl-muramyl
- Adjuvants may be added to a composition comprising a NE and an immunogen, or, the adjuvant may be formulated with carriers, for example liposomes, or metallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) prior to combining with or co-administration with a composition comprising a NE and an immunogen.
- carriers for example liposomes, or metallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) prior to combining with or co-administration with a composition comprising a NE and an immunogen.
- a composition comprising a NE and an immunogen comprises a single adjuvant. In other embodiments, a composition comprising a NE and an immunogen comprises two or more adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241; and WO . 94/00153, each of which is hereby incorporated by reference in its entirety).
- a composition comprising a NE and an immunogen of the present invention comprises one or more mucoadhesives (See, e.g., U.S. Pat. App. No. 20050281843, hereby incorporated by reference in its entirety).
- the present invention is not limited by the type of mucoadhesive utilized.
- mucoadhesives are contemplated to be useful in the present invention including, but not limited to, cross-linked derivatives of poly(acrylic acid) (e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (e.g., alginate and chitosan), hydroxypropyl methylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose.
- a mucoadhesive e.g., in a composition comprising a NE and immunogen
- a mucoadhesive enhances induction of an immune response in a subject (e.g., administered a composition of the present invention) due to an increase in duration and/or amount of exposure to an immunogen that a subject experiences when a mucoadhesive is used compared to the duration and/or amount of exposure to an immunogen in the absence of using the mucoadhesive.
- a composition of the present invention may comprise sterile aqueous preparations.
- Acceptable vehicles and solvents include, but are not limited to, water, Ringer's solution, phosphate buffered saline and isotonic sodium chloride solution.
- sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed mineral or non-mineral oil may be employed including synthetic mono-ordi-glycerides.
- fatty acids such as oleic acid find use in the preparation of injectables.
- Carrier formulations suitable for mucosal, subcutaneous, intramuscular, intraperitoneal, intravenous, or administration via other routes may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
- a composition comprising a NE and an immunogen of the present invention can be used therapeutically (e.g., to enhance an immune response) or as a prophylactic (e.g., for immunization (e.g., to prevent signs or symptoms of disease)).
- a composition comprising a NE and an immunogen of the present invention can be administered to a subject via a number of different delivery routes and methods.
- compositions of the present invention can be administered to a subject (e.g., mucosally (e.g., nasal mucosa, vaginal mucosa, etc.)) by multiple methods, including, but not limited to: being suspended in a solution and applied to a surface; being suspended in a solution and sprayed onto a surface using a spray applicator; being mixed with a mucoadhesive and applied (e.g., sprayed or wiped) onto a surface (e.g., mucosal surface); being placed on or impregnated onto a nasal and/or vaginal applicator and applied; being applied by a controlled-release mechanism; being applied as a liposome; or being applied on a polymer.
- a subject e.g., mucosally (e.g., nasal mucosa, vaginal mucosa, etc.)
- multiple methods including, but not limited to: being suspended in a solution and applied to a surface; being suspended in
- compositions of the present invention are administered mucosally (e.g., using standard techniques; See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995 (e.g., for mucosal delivery techniques, including intranasal, pulmonary, vaginal and rectal techniques), as well as European Publication No. 517,565 and Ilium et al., J. Controlled ReI., 1994, 29:133-141 (e.g.,for techniques of intranasal administration), each of which is hereby incorporated by reference in its entirety).
- mucosally e.g., using standard techniques; See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995 (e.g., for mucosal delivery techniques, including intranasal, pulmonary, vaginal and rectal techniques), as well as European Publication No. 517,565 and Ilium et
- compositions of the present invention may be administered dermally or transdermally, using standard techniques (See, e.g., Remington: The Science arid Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995).
- the present invention is not limited by the route of administration.
- mucosal vaccination is the preferred route of administration as it has been shown that mucosal administration of antigens has a greater efficacy of inducing protective immune responses at mucosal surfaces (e.g., mucosal immunity), the route of entry of many pathogens.
- mucosal vaccination such as intranasal vaccination, may induce mucosal immunity not only in the nasal mucosa, but also in distant mucosal sites such as the genital mucosa (See, e.g., Mestecky, Journal of Clinical Immunology, 7:265-276, 1987).
- mucosal vaccination in addition to inducing mucosal immune responses, mucosal vaccination also induces systemic immunity.
- non-parenteral administration e.g., muscosal administration of vaccines
- boost systemic immunity e.g., induced by parenteral or mucosal vaccination (e.g., in cases where multiple boosts are used to sustain a vigorous systemic immunity)
- a composition comprising a NE and an immunogen of the present invention may be used to protect or treat a subject susceptible to, or suffering from, disease by means of administering a composition of the present invention via a mucosal route (e.g., an oral/alimentary or nasal route).
- a mucosal route e.g., an oral/alimentary or nasal route.
- Alternative mucosal routes include intravaginal and intra-rectal routes.
- a nasal route of administration is used, termed "intranasal administration” or “intranasal vaccination” herein.
- Methods of intranasal vaccination are well known in the art, including the administration of a droplet or spray form of the vaccine into the nasopharynx of a sujbect to be immunized.
- a nebulized or aerosolized composition comprising a NE and immunogen.
- Enteric formulations such as gastro resistant capsules for oral administration, suppositories for rectal or vaginal administration also form part of this invention.
- Compositions of the present invention may also be administered via the oral route.
- a composition comprising a NE and an immunogen may comprise a pharmaceutically acceptable excipient and/or include alkaline buffers, or enteric capsules.
- Formulations for nasal delivery may include those with dextran or cyclodextran and saponin as an adjuvant.
- compositions of the present invention may also be administered via a vaginal route.
- a composition comprising a NE and an immunogen may comprise pharmaceutically acceptable excipients and/or emulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers of vaginal creams and suppositories.
- compositions of the present invention are administered via a rectal route.
- a composition comprising a NE and an immunogen may comprise excipients and/or waxes and polymers known in the art for forming rectal suppositories.
- the same route of administration (e.g., mucosal administration) is chosen for both a priming and boosting vaccination.
- multiple routes of administration are utilized (e.g., at the same time, or, alternatively, sequentially) in order to stimulate an immune response (e.g., using a composition comprising a NE and immunogen of the present invention).
- a composition comprising a NE and an immunogen is administered to a mucosal surface of a subject in either a priming or boosting vaccination regime.
- a composition comprising a NE and an immunogen is administered systemically in either a priming or boosting vaccination regime.
- a composition comprising a NE and an immunogen is administered to a subject in a priming vaccination regimen via mucosal administration and a boosting regimen via systemic administration.
- a composition comprising a NE and an immunogen is administered to a subject in a priming vaccination regimen via systemic administration and a boosting regimen via mucosal administration.
- systemic routes of administration include, but are not limited to, a parenteral, intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal or intravenous administration.
- a composition comprising a NE and an immunogen may be used for both prophylactic and therapeutic purposes.
- compositions of the present invention are administered by pulmonary delivery.
- a composition of the present invention can be delivered to the lungs of a subject (e.g., a human) via inhalation (e.g., thereby traversing across the lung epithelial lining to the blood stream (See, e.g., Adjei, et al. Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J. Pharmaceutics 1990; 63:135-144; Braquet, et al. J. Cardiovascular Pharmacology 1989 143-146; Hubbard, et al. (1989) Annals of Internal Medicine, Vol. Ill, pp.
- nebulizers metered dose inhalers
- powder inhalers all of which are familiar to those skilled in the art.
- Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.).
- each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants, surfactants, carriers and/or other agents useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
- a composition comprising a NE and an immunogen of the present invention may be used to protect and/or treat a subject susceptible to, or suffering from, a disease by means of administering a compositions comprising a NE and an immunogen by mucosal, intramuscular, intraperitoneal, intradermal, transdermal, pulmonary, intravenous, subcutaneous or other route of administration described herein.
- Methods of systemic administration of the vaccine preparations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (See, e.g., WO 99/27961, hereby incorporated by reference), or needleless pressure liquid jet device (See, e.g., U.S. Pat. No.
- the present invention may also be used to enhance the immunogenicity of antigens applied to the skin (transdermal or transcutaneous delivery, See, e.g., WO 98/20734 ; WO 98/28037, each of which are hereby incorporated by reference).
- the present invention provides a delivery device for systemic administration, pre-filled with the vaccine composition of the present invention.
- the present invention is not limited by the type of subject administered (e.g., in order to stimulate an immune response (e.g., in order to generate protective immunity (e.g., mucosal and/or systemic immunity))) a composition of the present invention. Indeed, a wide variety of subjects are contemplated to be benefited from administration of a composition of the present invention.
- the subject is a human.
- human subjects are of any age (e.g., adults, children, infants, etc.) that have been or are likely to become exposed to a microorganism.
- the human subjects are subjects that are more likely to receive a direct exposure to pathogenic microorganisms or that are more likely to display signs and symptoms of disease after exposure to a pathogen (e.g., immune suppressed subjects).
- the general public is administered (e.g., vaccinated with) a composition of the present invention (e.g., to prevent the occurrence or spread of disease).
- compositions and methods of the present invention are utilized to vaccinate a group of people (e.g., a population of a region, city, state and/or country) for their own health (e.g., to prevent or treat disease).
- the subjects are non-human mammals (e.g., pigs, cattle, goats, horses, sheep, or other livestock; or mice, rats, rabbits or other animal).
- compositions and methods of the present invention are utilized in research settings (e.g., with research animals).
- composition of the present invention may be formulated for administration by any route, such as mucosal, oral, topical, parenteral or other route described herein.
- the compositions may be in any one or more different forms including, but not limited to, tablets, capsules, powders, granules, lozenges, foams, creams or liquid preparations.
- Topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, foams, and aerosols, and may contain appropriate conventional additives such as preservatives, solvents (e.g., to assist penetration), and emollients in ointments and creams.
- Topical formulations may also include agents that enhance penetration of the active ingredients through the skin.
- agents include a binary combination of N- (hydroxyethyl) pyrrolidone and a cell-envelope disordering compound, a sugar ester in combination with a sulfoxide or phosphine oxide, and sucrose monooleate, decyl methyl sulfoxide, and alcohol.
- surfactants or wetting agents including, but not limited to, polyoxyethylene sorbitan mono-oleoate (Polysorbate 80); sorbitan mono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol polymer (Triton WR-1330); polyoxyethylene sorbitan tri-oleate (Tween 85); dioctyl sodium sulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and other pharmaceutically acceptable surfactants.
- surfactants or wetting agents including, but not limited to, polyoxyethylene sorbitan mono-oleoate (Polysorbate 80); sorbitan mono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol polymer (Triton WR-1330); polyoxyethylene sorbitan tri-oleate (Tween 85); dioctyl sodium sulfosuccinate; and sodium sarcosinate (Sar
- compositions may further comprise one or more alcohols, zinc-containing compounds, emollients, humectants, thickening and/or gelling agents, neutralizing agents, and surfactants.
- Water used in the formulations is preferably deionized water having a neutral pH.
- Additional additives in the topical formulations include, but are not limited to, silicone fluids, dyes, fragrances, pH adjusters, and vitamins.
- Topical formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation.
- the ointment base can comprise one or more of petrolatum, mineral oil, ceresin, lanolin alcohol, panthenol, glycerin, bisabolol, cocoa butter and the like.
- compositions of the present invention maybe formulated and used as foams.
- Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
- compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
- the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
- additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
- such materials when added, preferably do not unduly interfere with the biological activities of the components of the compositions of the present invention.
- the formulations can be sterilized and, if desired, mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like) that do not deleteriously interact with the NE and immunogen of the formulation.
- auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like
- immunostimulatory compositions of the present invention are administered in the form of a pharmaceutically acceptable salt.
- the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
- Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2- sulphonic, and benzene sulphonic.
- such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
- Suitable buffering agents include, but are not limited to, acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
- Suitable preservatives may include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
- a composition comprising a NE and an immunogen is coadministered with one or more antibiotics.
- one or more antibiotics may be administered with, before and/or after administration of a composition comprising a NE and an immunogen.
- the present invention is not limited by the type of antibiotic coadministered.
- antibiotics may be co-administered including, but not limited to, ⁇ -lactam antibiotics, penicillins (such as natural penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins), cephalosporins (first generation, second generation, and third generation cephalosporins), and other ⁇ - lactams (such as imipenem, monobactams,), ⁇ -lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinolines.
- penicillins such as natural penicillins, aminopenicillins, pen
- these agents include agents that inhibit cell wall synthesis ⁇ e.g., penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); and the imidazole antifungal agents ⁇ e.g., miconazole, ketoconazole and clotrimazole); agents that act directly to disrupt the cell membrane of the microorganism ⁇ e.g., detergents such as polmyxin and colistimethate and the antifungals nystatin and amphotericin B); agents that affect the ribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol, the tetracyclines, erthromycin and clindamycin); agents that alter protein synthesis and lead to cell death (e.g., aminoglycosides); agents that affect nucleic acid metabolism (e.g., the rifamycins and the quinolones); the antimetabolites (e.g., trimethoprim and
- the present invention also includes methods involving co-administration of a composition comprising a NE and an immunogen with one or more additional active and/or immunostimulatory agents (e.g., a composition comprising a NE and a different immnogen, an antibiotic, anti-oxidant, etc.).
- additional active and/or immunostimulatory agents e.g., a composition comprising a NE and a different immnogen, an antibiotic, anti-oxidant, etc.
- additional active and/or immunostimulatory agents e.g., a composition comprising a NE and a different immnogen, an antibiotic, anti-oxidant, etc.
- additional active and/or immunostimulatory agents e.g., a composition comprising a NE and a different immnogen, an antibiotic, anti-oxidant, etc.
- the agents may be administered concurrently or sequentially.
- the compositions described herein are administered prior to the other active agent(s).
- the pharmaceutical formulations and modes of administration
- the two or more co-administered agents may each be administered using different modes (e.g., routes) or different formulations.
- the additional agents to be co-administered e.g., antibiotics, adjuvants, etc.
- a composition comprising a NE and immunogen is administered to a subject via more than one route.
- a subject that would benefit from having a protective immune response (e.g., immunity) towards a pathogenic microorganism may benefit from receiving mucosal administration (e.g., nasal administration or other mucosal routes described herein) and, additionally, receiving one or more other routes of administration (e.g., parenteral or pulmonary administration (e.g., via a nebulizer, inhaler, or other methods described herein).
- mucosal administration e.g., nasal administration or other mucosal routes described herein
- one or more other routes of administration e.g., parenteral or pulmonary administration (e.g., via a nebulizer, inhaler, or other methods described herein).
- administration via mucosal route is sufficient to induce both mucosal as well as systemic immunity towards an immunogen or organism from which the immunogen is derived
- administration via multiple routes serves to provide both mucosal and systemic immunity.
- a subject administered a composition of the present invention via multiple routes of administration may have a stronger immune response to an immunogen than a subject administered a composition via just one route.
- Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compositions, increasing convenience to the subject and a physician.
- Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109, hereby incorporated by reference.
- Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
- lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides
- hydrogel release systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides
- sylastic systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides
- peptide based systems such as mono-di-and tri-glycerides
- wax coatings such as those described in U.S. Pat. Nos.
- a composition comprising a NE and an immunogen of the present invention comprises a suitable amount of the immunogen to induce an immune response in a subject when administered to the subject.
- the immune response is sufficient to provide the subject protection (e.g., immune protection) against a subsequent exposure to the immunogen or the microorganism (e.g., bacteria or virus) from which the immunogen was derived.
- the present invention is not limited by the amount of immunogen used.
- the amount of immunogen (e.g., virus or bacteria neutralized by the NE, or, recombinant protein) in a composition comprising a NE and immunogen (e.g., for use as an immunization dose) is selected as that amount which induces an immunoprotective response without significant, adverse side effects.
- the amount will vary depending upon' which specific immunogen or combination thereof is/are employed, and can vary from subject to subject, depending on a number of factors including, but not limited to, the species, age and general condition (e.g., health) of the subject, and the mode of administration. Procedures for determining the appropriate amount of immunogen administered to a subject to elicit an immune response (e.g., a protective immune response (e.g., protective immunity)) in a subject are well known to ⁇ those skilled in the art.
- an immune response e.g., a protective immune response (e.g., protective immunity)
- each dose (e.g., of a composition comprising a NE and an immunogen (e.g., administered to a subject to induce an immune response (e.g., a protective immune response (e.g., protective immunity))) comprises 0.05- 5000 ⁇ g of each immunogen (e.g., recombinant and/or purified protein), in some embodiments, each dose will comprise 1-500 ⁇ g, in some embodiments, each dose will comprise 350-750 ⁇ g, in some embodiments, each dose will comprise 50-200 ⁇ g, in some embodiments, each dose will comprise 25-75 ⁇ g of immunogen (e.g., recombinant and/or purifed protein).
- an immunogen e.g., administered to a subject to induce an immune response (e.g., a protective immune response (e.g., protective immunity)
- each dose will comprise 1-500 ⁇ g, in some embodiments, each dose will comprise 350-750 ⁇ g, in some embodiments, each dose will comprise 50-200 ⁇
- each dose comprises an amount of the immunogen sufficient to generate an immune response.
- An effective amount of the immunogen in a dose need not be quantified, as long as the amount of immunogen generates an immune response in a subject when administered to the subject.
- An optimal amount for a particular administration e.g., to induce an immune response (e.g., a protective immune response (e.g., protective immunity)) can be ascertained by one of skill in the art using standard studies involving observation of antibody titers and other responses in subjects.
- each dose e.g., of a composition comprising a NE and an immunogen (e.g., administered to a subject to induce and immune response)
- each dose is from 0.001 to 15% or more (e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more) by weight immunogen (e.g., neutralized bacteria or virus, or recombinant and/or purified protein).
- an initial or prime administration dose contains more immunogen than a subsequent boost dose
- each dose comprises between 10 and 10 9 pfu of the virus per dose; in some embodiments, each dose comprises between 10 and 10 pfu of the virus per dose; in some embodiments, each dose comprises between 10 and 10 pfu of the virus per dose; in some embodiments, each dose comprises between 10 2 and 10 4 pfu of the virus per dose; in some embodiments, each dose comprises 10 pfu of the virus per dose; in some embodiments, each dose comprises 10 pfu of the virus per dose; and in some embodiments, each dose comprises 10 4 pfu of the virus per dose. In some embodiments, each dose comprises more than 10 9 pfu of the virus per dose. In some preferred embodiments, each dose comprises 10 pfu of the virus per dose.
- each dose comprises 10 pfu of the virus per dose.
- each dose comprises between 10 and 10 10 bacteria per dose; in some embodiments, each dose comprises between 10 5 and 10 8 bacteria per dose; in some embodiments, each dose comprises between 10 3 and 10 5 bacteria per dose; in some embodiments, each dose comprises between 10 2 and 10 4 bacteria per dose; in some embodiments, each dose comprises 10 bacteria per dose; in some embodiments, each dose comprises 10 bacteria per dose; and in some embodiments, each dose comprises 10 4 bacteria per dose. In some embodiments, each dose comprises more than 10 10 bacteria per dose. In some embodiments, each dose comprises 10 3 bacteria per dose.
- the present invention is not limited by the amount of NE used to inactivate live microorganisms (e.g., a virus (e.g., one or more types of HFV)).
- a 0.1% - 5% NE solution is used, in some embodiments, a 5%-20% NE solution is used, in some embodiments, a 20% NE solution is used, and in some embodiments, a NE solution greater than 20% is used order to inactivate a pathogenic microorganism.
- a 10% NE solution is used.
- the present invention is not limited by the duration of time a live microorganism is incubated in a NE of the present invention in order to become inactivated.
- the microorganism is incubated for 1-3 hours in NE.
- the microorganism is incubated for 3-6 hours in NE.
- the microorganism is incubated for more than 6 hours in NE.
- the microorganism is incubated for 3 hours in NE (e.g., a 10% NE solution), hi some embodiments, the incubation is carried out at 37 0 C. In some embodiments, the incubation is carried out at a temperature greater than or less than 37 0 C.
- the present invention is also not limited by the amount of microorganism used for inactivation.
- the amount of microorganism may depend upon a number of factors including, but not limited to, the total amount of immunogenic composition (e.g., NE and immunogen) desired, the concentration of solution desired (e.g., prior to dilution for administration), the microorganism and the NE.
- the amount of microorganism used in an inactivation procedure is that amount that produces the desired amount of immunogen (e.g., as described herein) to be administered in a single dose (e.g., diluted from a concentrated stock) to a subject.
- a composition comprising a NE and an immunogen of the present invention is formulated in a concentrated dose that can be diluted prior to administration to a subject.
- dilutions of a concentrated composition may be administered to a subject such that the subject receives any one or more of the specific dosages provided herein.
- dilution of a concentrated composition may be made such that a subject is administered (e.g., in a single dose) a composition comprising 0.5-50% of the NE and immunogen present in the concentrated composition.
- a subject' is administered in a single dose a composition comprising 1% of the NE and immunogen present in the concentrated composition.
- Concentrated compositions are contemplated to be useful in a setting in which large numbers of subjects may be administered a composition of the present invention (e.g., an immunization clinic, hospital, school, etc.).
- a composition comprising a NE and an immunogen of the present invention e.g., a concentrated composition
- a composition comprising a NE and an immunogen of the present invention is stable at room temperature for more than 1 week, in some embodiments for more than 2 weeks, in some embodiments for more than 3 weeks, in some embodiments for more than 4 weeks, in some embodiments for more than 5 weeks, and in some embodiments for more than 6 weeks.
- the emulsion compositions of the invention will comprise at least 0.001% to 100%, preferably 0.01 to 90%, of emulsion per ml of liquid composition. It is envisioned that the formulations may comprise about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.025%, about 0.05%, about 0.075%, about 0.
- a subject may receive one or more boost administrations (e.g., around 2 weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around 4 months, around 6 months, around 9 months, around 1 year, around 2 years, around 3 years, around 5 years, around 10 years) subsequent to a first, second,third, fourth, fifth, sixth, seventh, eights, ninth, tenth, and/or more than tenth administration.
- boost administrations e.g., around 2 weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around 4 months, around 6 months, around 9 months, around 1 year, around 2 years, around 3 years, around 5 years, around 10 years
- reintroduction of an immunogen in a boost dose enables vigorous systemic immunity in a subject.
- the boost can be with the same formulation given for the primary immune response, or can be with a different formulation that contains the immunogen.
- the dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgment of a practitioner.
- Dosage units may be proportionately increased or decreased based on several factors including, but not limited to, the weight, age, and health status of the subject. In addition, dosage units may be increased or decreased for subsequent administrations (e.g., boost administrations).
- a composition comprising an immunogen of the present invention finds use where the nature of the infectious and/or disease causing agent (e.g., for which protective immunity is sought to be elicited) is known, as well as where the nature of the infectious and/or disease causing agent is unknown (e.g., in emerging disease (e.g., of pandemic proportion (e.g., influenza or other outbreaks of disease))).
- the nature of the infectious and/or disease causing agent e.g., for which protective immunity is sought to be elicited
- pandemic proportion e.g., influenza or other outbreaks of disease
- the present invention contemplates use of the compositions of the present invention in treatment of or prevention of (e.g., via immunization with an infectious and/or disease causing HIV or HIV- like agent neutralized via a NE of the present invention) infections associated with an emergent infectious and/or disease causing agent yet to be identified (e.g., isolated and/or cultured from a diseased person but without genetic, biochemical or other characterization of the infectious and/or disease causing agent).
- infections associated with an emergent infectious and/or disease causing agent yet to be identified (e.g., isolated and/or cultured from a diseased person but without genetic, biochemical or other characterization of the infectious and/or disease causing agent).
- compositions and methods of the present invention will find use in various settings, including research settings.
- compositions and methods of the present invention also find use in studies of the immune system (e.g., characterization of adaptive immune responses (e.g., protective immune responses (e.g., mucosal or systemic immunity))).
- Uses of the compositions and methods provided by the present invention encompass human and non-human subjects and samples from those subjects, and also encompass research applications using these subjects.
- Compositions and methods of the present invention are also useful in studying and optimizing nanoemulsions, immunogens, and other components and for screening for new components. Thus, it is not intended that the present invention be limited to any particular subject and/or application setting.
- compositions of the present invention are useful for preventing and/or treating a wide variety of diseases and infections caused by viruses, bacteria, parasites, and fungi, as well as for eliciting an immune response against a variety of antigens.
- the compositions can also be used in order to prepare antibodies, both polyclonal and monoclonal (e.g., for diagnostic purposes), as well as for immunopurification of an antigen of interest.
- polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) can be immunized with the compositions of the present invention. The animal is usually boosted 2-6 weeks later with one or more—administrations of the antigen. Polyclonal antisera can then be obtained from the immunized animal and used according to known procedures (See, e.g., Jurgens et al., J. Chrom. 1985, 348:363-370).
- the present invention provides a kit comprising a composition comprising a NE and an immunogen.
- the kit further provides a device for administering the composition.
- the present invention is not limited by the type of device included in the kit.
- the device is configured for nasal application of the composition of the present invention (e.g., a nasal applicator (e.g., a syringe) or nasal inhaler or nasal mister).
- a kit comprises a composition comprising a NE and an immunogen in a concentrated form (e.g., that can be diluted prior to administration to a subject).
- kits are present within a single container (e.g., vial or tube).
- each kit component is located in a single container (e.g., vial or tube).
- one or more kit component are located in a single container (e.g., vial or tube) with other components of the same kit being located in a separate container (e.g., vial or tube).
- a kit comprises a buffer.
- the kit further comprises instructions for use.
- Example 1 Materials and methods: Nanoemulsion inactivated Vaccinia virus
- mice Animals. Pathogen-free, 5 to 6-week-old, female Balb/c mice were purchased from Charles River Laboratories. Vaccination groups were housed separately, five animals to a cage, in accordance with the American Association for Accreditation of Laboratory Animal Care standards. All procedures involving mice were performed according to the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan.
- UUCA University Committee on Use and Care of Animals
- WWR exemplary vaccinia viruses
- WWR-LUC vaccinia viruses
- ATC American Type Culture Collection
- Recombinant WWR-LUC expresses firefly luciferase from the p7.5 early/late promoter and has been described (See, e.g., Luker et al., Virology. 2005, 341(2):284-300).
- WWR-LUC is not attenuated in vitro or in vivo because the virus was constructed with a method that does not require deletion of any viral genes (See, e.g., Blasco and Moss (1995). Gene 158(2), 157-162; Luker et al., Virology. 2005, 341(2):284-300).
- Virus was propogated on Vero cells infected at a multiplicity of infection of 0.5. Cells were harvested at 48 to 72 h and virus was isolated from culture supernatants and cells lysates. Cell lysates were obtained by rapidly freeze-thawing the cell pellet followed by homogenization in Dounce homogenizer in ImM Tris pH 9.0. Cell debris was removed by centrifugation at 2000 rpm.
- the purified virus stocks were obtained from clarified supernatants by layering on 4% to 40% sucrose gradients which were centrifuged for 1 hr at 25,000 x g. Turbid bands, containing viral particles, were collected, diluted in ImM Tris pH 9 and then concentrated by lhr centrifugation at 25,000 x g. Viral pellets were re-suspended in ImM Tris pH 9 and stored frozen at - ⁇ 80 0 C as virus stock.
- the WWR stocks were titered on Vero cells (See, e.g., Myc et al., Vaccine. 21:3801-3814). WWR has identical surface proteins as the native strain, but expresses luciferase protein during infection.
- NE (W 2 o5EC) was obtained from NANOBIO Corporation, Ann Arbor, MI (See U.S. Patent No. 6,015,832 issued to NANOBIO Corporation (Ann Arbor, MI), herein incorporated by reference in its entirety).
- the nanoemulsion was manufactured by emulsification of cetyl pyridum chloride (1%), Tween 20 (5%) and Ethanol (8%) in water with soybean oil (64%) using a high speed emulsifier. Resultant droplets have a mean particle size of 300 +/-25 ran in diameter.
- W2 ⁇ 5EC has been formulated with surfactants and food substances considered "Generally Recognized as Safe" (GRAS) by the FDA.
- W2 0 5EC can be economically manufactured under Good Manufacturing Practices (GMP) and is stable for at least 18 months at 4O 0 C.
- GMP Good Manufacturing Practices
- Vaccinia virus (W) neutralization data generated during the development of the present invention indicated that 1 hr incubation with 10% NE or 0.1% formalin was sufficient for inactivation of the virus (e.g., six log W titer reduction).
- formulations e.g., compositions
- an immune response e.g., vaccine formulations
- compositions e.g., for stimulating an immune response
- virus neutralization e.g., virus inactivation
- samples containing Ix 10 3 pfu to 5 xlO 5 pfu per dose of W were incubated for 3 hrs at 37°C in 10% W 2 o5EC NE 5 and were subsequently diluted to 1% NE for intranasal instillation (e.g., 1 x 10 3 to 1 x 10 5 pfu per dose).
- formalin (Sigma) inactivation of W was performed at RT for 3 hrs in 0.1% formalin.
- Formalin-killed virus was diluted in either saline or 1% NE to 10 3 or 10 pfu per dose to reduce the formalin to nontoxic concentrations for intranasal immunization. ' For every formulation in each experiment, virus inactivation by either NE or fomalin was confirmed in vitro by infecting Vero cells, followed with two subsequent passages of culture supernatants after 3-4 days of incubation. None of the control infections showed a presence of viral plaques. Additionally, PCR-detection assays of viral DNA in Vero cells and lungs of treated animals were performed as described below to confirm the absence of live, replicating virus.
- Immunization by scarification was performed in anesthetized mice by superficial scarification at the base of the tail. Before the procedure, hair was removed by a clipper to expose approximately 0.5-0.7 square centimeters and the naked skin was disinfected with 70% ethanol. A sterile bifurcate needle was used to superficially abrade the epidermis and 1 * 10 5 pfii dose of live WWR was applied in 10 ⁇ l PBS. Animals were immobilezed for up to 10 minutes to ensure virus absorption into the skin.
- Bioluminescence imaging was performed with a cryogenically-cooled CCD camera (IVIS) as described elsewhere (See, e.g., Luker et al., (2002). J Virol 76(23), 12149-12161; Cook and Griffin, (2003). J Virol 77(9), 5333-5338). Data for photon flux were quantified by region-of-interest (ROI) analysis of the head, chest and abdomen of infected mice. Background photon flux from an uninfected mouse injected with luciferin was subtracted from all measurements.
- ROI region-of-interest
- Blood samples were obtained from the saphenous vein at various time points during the course of trials conducted during the development of the present invention. Final samples were obtained by cardiac puncture from euthanized, premorbid mice. Serum was obtained from blood by centrifugation at 1500 x g for 5 minutes after the blood coagulated for 30-60 minutes at room temperature. Serum samples were stored at -20 0 C until used.
- BAL fluid was obtained from mice euthanized by isoflurane inhalation. After the trachea was dissected, a 22 gauge catheter (Angiocath, B-D) attached to a 1 ml syringe was inserted into the trachea. The lungs were infused twice with 0.5 ml of PBS containing 10 ⁇ M DTT and 0.5 mg/ml aprotinin. Approximately 1.0 ml of aspirate was recovered with a syringe. BAL samples were stored at -2O 0 C until analyzed. Murine splenocytes were mechanically isolated to obtain single-cell suspension in PBS.
- Red blood cells were removed by lysis with ACK buffer (150 mM NH 4 Cl, 10 mM KHCCb, 0.1 mM Na2EDTA), and the remaining cells washed twice in PBS.
- ACK buffer 150 mM NH 4 Cl, 10 mM KHCCb, 0.1 mM Na2EDTA
- splenocytes 2-4 x 10 /ml were resuspended in RPMI 1640 medium supplemented with 5 % FBS, 20OnM L- glutamine, and penicillin/streptomycin (100U/ml and lOO ⁇ g/ml).
- PCR amplification was performed with 10 ⁇ g of total cell or lung DNA using 0.5 ⁇ M of each primer, 0.2 mM of each dNTP, 2.5 mM of MgCl 2 , and 0.1 U/ ⁇ l of Tag DNA Polymerase (ROCHE Molecular Biochemicals, Indianapolis, IN). PCR reactions were carried out in a total volume of 20 ⁇ l, incubated at 94°C for 1 min, followed by 25 cycles with annealing at 55°C, extension at 72°C and denaturation at 94°C. PCR product analysis was performed using electrophoresis on 1% agarose gel in Tris— borate buffer for electrophoresis and ethidium bromide for DNA staining.
- Mouse anti- vaccinia antibodies were determined by ELISA. Microtiter 96-well flat bottom NUNC-PolySorp polystyrene plates were coated with a dilution of infected Vero cells lysate containing at least 5 x 10 4 pfu/well of vaccinia virus in PBS. Plates were incubated overnight at 4°C and fixed with 50% mixture of ethanol/acetone (EtOH/acetone) for 1 hour at -20 0 C.
- EtOH/acetone 50% mixture of ethanol/acetone
- the colorimetric reaction was performed with AP substrate SIGMAFAST (SIGMA, St. Louis, MO) according to the manufacturer's protocol. Spectrophotometric readouts were done using the SPECTRAMAX 340 ELISA reader (MOLECULAR DEVICES, Sunnyvale, CA) at 405 nm and reference wavelength of 690 nm. The endpoint titers and antibody concentrations were calculated as the serum dilution resulting in an absorbance greater than two standard deviations above the absorbance in control wells. The IgG antibody concentration was calculated according to the logarithmic transformation of the linear portion of the standard curve generated with the AP-conjugated anti-IgG antibody and multiplied by the serum dilution factor. The serum antibody concentrations are presented as a mean value +/- standard error (sem). Serum from the naive mice was used as a control for non-specific absorbance.
- Anti-W IgG antibody activity targeted toward alcohol denaturized versus formalin- alkylated viral epitopes was measured using ELISA, as described above with a few modifications.
- the 96 well plates were coated with 1 x 10 5 pfu/well of purified vaccinia virus and incubated overnight at 4°C. After virus was removed, wells were treated for 1 hour with either 50% EtOH/acetone at -20 0 C or with 1% formalin solution in PBS at 4 0 C. Plates were washed and blocked as described above.
- Neutralizing antibodies were determined with both a standard plaque reduction assay (PRA) (See, e.g., Newman et al., J. Chem. Microbiol. 2003, 3154-3157) and the inhibition of luciferase activity using recombinant WWR-LUC
- PRA plaque reduction assay
- the PRA was conducted by mixinglO ⁇ l of heat-inactivated mouse serum in serial, two-fold dilutions with lO ⁇ l of serum- free RPMI medium containing 200-300 pfu of W. Sera were incubated 6 hr at 37°C and subsequently placed in 0.5 ml of serum-free medium an overlaid on Vero cell monolayer.
- virus/serum inocula were removed and a fresh medium was placed on the cell monolayers. After 48 to 72 hrs, cells were fixed and stained with 0.1% crystal violet. Plaques were counted by two independent observers and the neutralization titer calculated using non-immune serum as a control.
- Vaccinia specific cytokine expression in splenocytes Spleens from vaccinated mice were harvested 12 weeks after initial vaccination. Splenocytes were obtained from mechanically disrupted spleens and suspended at 3 * 10 6 cells/ml in RPMI 1640 supplemented with 5% FBS, L-glutamine and penicillin/streptomycin. Cells were incubated with either 1 x 10 3 or 1 x 10 4 pfu per well of vaccinia virus for 72 hours at 37 0 C. Cell culture supernatants were harvested and analyzed for cytokine production. PHA-P (l ⁇ g per well) was incubated with the cells as a positive control. The EFN- ⁇ concentrations in splenocyte supernatants were determined using QU ANTIKINE M ELISA kits (R&D SYSTEMS Lac, Minneapolis, MN) according to the manufacturer's directions.
- Vaccinia virus challenge Immunized mice were challenged with live vaccinia virus to evaluate the effectiveness of the vaccine. Serum samples were collected two days before the vaccinia challenge and animals were weighed on the day of the challenge. Aliquots of purified recombinant WWR or WWR-LUC (sonicated and titered before use) were thawed and diluted in saline the day of the challenge.
- mice were anesthetized by inhalation of isoflurane and challenged intranasally with a 20 ⁇ l suspension of 2 ⁇ l ⁇ 6 pfu live WWR-LUC corresponding to 10 x LD50, or with live WWR doses ranging from 1 x 10 7 to 3.2 x 10 3 in 5 fold dilutions. Weight and body temperature were measured daily for 3 weeks following ' challenge. Mice that demonstrated a 30% loss in initial body weight were euthanized. Lethal dose (LDso) and the infectious dose (ID50) calculations were based on the animals death rates, and on the core body temperature and body weight loss, respectively (See, e.g., Reed and Muench, Am J Hyg 1938;27:493-7).
- LDso Lethal dose
- ID50 infectious dose
- IPLD logio Maximum dose - logioLDso controls.
- IPID index of protection against infection
- Nasal immunization with nanoemulsion-inactivated Vaccinia virus results in the induction specific systemic IgG response
- compositions of the present invention e.g., NE-killed W
- NE-killed W NE-killed W
- protective immunity similar to that seen in humans vaccinated by scarification with live, replicating W
- mice were intranasally (i.n.) immunized with six formulations containing either 10 5 or 10 3 pfu doses of W WR killed with NE (10 5 /NE and 10 3 /NE, respectively), formalin-killed virus mixed with 1% NE (10 5 /Fk/NE and 10 3 /Fk/NE, respectively), and formalin-killed virus in saline (10 5 ZFk and 10 3 /Fk). Control mice were treated with 1% NE alone. Antibody responses were characterized three weeks after initial vaccine administration (See Figure 2). Immunity was boosted with subsequent administrations, at 5 and 9 weeks ( Figure 2A).
- Immunizations with 10 3 /Fk/NE, and either 10 5 /Fk or 10 3 /Fk formulations of vaccine consistently produced low levels of anti-W antibodies, which did not increase significantly after booster administrations (See Figure 2A).
- a comparison of a single-dose with a three-dose schedule of immunization with 10 /NE showed that a single dose of vaccine produced significant ( ⁇ 4 ⁇ g/ml), albeit lower than three-dose, concentration of serum anti-W IgG at 12 weeks after immunization.
- the present invention provides that a single dose of W/NE vaccine maybe sufficient to initiate immune responses (e.g., mucosal or systemic immune responses), that can be enhanced by subsequent immunization (See Figure 2A insert). No specific anti-W antibodies were detected in any of the control mice.
- Mucosal immunity was assayed by W-specific secretory IgA antibody in bronchialalveolar fluids (BAL).
- Anti-W IgA was detected in BAL from animals immunized with either 10 3 /NE or 10 5 /NE. Animals vaccinated with formulations containing formalin-killed virus, whether diluted in saline or NE, did not produce measurable mucosal response despite the presence of serum anti-W IgG (See Figure 2B).
- compositions comprising NE-killed W generates mucosal immunity in a subject (e.g., as demonstrated by the presence of W-specific secretory IgA antibodies in the BAL of the subject) whereas compositions that do not contain NE-killed W (e.g., formalin-killed W) are not capable of generating mucosal immunity to W.
- the present invention provides that despite inactivation (e.g. complete neutralization) of W, nanoemulsions comprising inactivated W of the present invention retain important immunogenic eptitopes (e.g., recognized and responded to by the immune system (e.g., humoral immune system) of a subject).
- inactivation e.g. complete neutralization
- immune system e.g., humoral immune system
- VV- W R-L U C has identical surface proteins as the native strain, but expresses luciferase protein during infection. This allows for mortality assessment and monitoring of viral infection in challenged animals with imaging techniques. Comparison of antibodies in W- WR immunized animals versus both viral strains either in ELISA, Western blot or virus neutralization assays showed no difference between VV-WR and W-WR-LUC
- Example 6 Administration of NE-killed W generates W specific cellular immune responses
- Example 7 Subjects administered NE-killed W are protected against challenge with live, infectious W
- mice were nasally immunized with three doses of either 10 5 ME, 10 5 /Fk/NE or 10 5 /Fk vaccine. Control animals were treated with saline. At 12 weeks mice were challenged with 10 x LDso(2 x 10 6 pfo) of live W WR -LU O - Body weight and temperature were measured two times a day and animals were imaged for WWR-LUC luminescence once a day. All 10 /NE vaccinated mice survived viral challenge (See Figure 5A). Mice vaccinated with 10 5 /Fk/NE and 10 5 /Fk had 40% and 20% survival rates, respectively.
- mice immunized with NE vaccine did not have clinical evidence of illness and had average weight loss of 10 % or less, whereas surviving mice in control groups lost more than 25% of weigh at much lower doses of WWR.
- mice Pathogen-free, female Balb/c, CB A/3 mice (5-6 weeks old) and Hartley guinea pigs (females, 250 g) were purchased from Charles River Laboratories (Wilmington, MA). The mice and guinea pigs were housed in accordance with the American Association for Accreditation of Laboratory Animal Care standards. All procedures involving animals were performed according to the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan, the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch at Galveston, TX 5 and standard operating procedures at Battelle Memorial Institute, Columbus, OH.
- UUCA University Committee on Use and Care of Animals
- IACUC Institutional Animal Care and Use Committee
- Reagents Recombinant B. anthracis protective antigen (rPA) and lethal factor (rLF) were obtained from List Biological Laboratories, Inc. (Campbell, CA) and BEI Resource Repository (ATCC) as lyophilized preparations of purified proteins. After reconstitution in sterile MILLI-Q water (5 mg/ml), the aliquots were stored at -80 0 C.
- ODN oligonucleotide
- the bovine serum albumin (BSA), alkaline phosphatase (AP)-conjugated antibodies, goat anti-mouse IgG (#A-3562), and goat anti-mouse IgA ( ⁇ chain specific, #A-4937) were purchased from SIGMA, and goat anti-mouse IgE HPR-conjugate was bought from BETHYL (#A90-l 15P, Montgomery, TX).
- the Cell Proliferation Kit (XTT) was purchased from ROCHE DIAGNOSTICS (New Jersey, NY).
- rP A/ Adjuvant Formulations Nanoemulsion (formulation W2o5EC) was obtained from NANOBIO Corporation, Ann Arbor, MI.
- This nanoemulsion is manufactured by the emulsification of cetyl pyridum chloride (CPC, 1%), Tween 20 (5%), and ethanol (8%) in water with hot-pressed soybean oil (64%), using a high-speed emulsifier (e.g., prepared by a two-step procedure according to U.S. Patent No. 6,015,832 issued to NANOBIO Corporation (Ann Arbor, MI), herein incorporated by reference in its entirety).
- W2o5EC is formulated with surfactants and food substances considered 'Generally Recognized as Safe 1 (GRAS) by the FDA.
- W2o5EC can be manufactured under Good Manufacturing Practices (GMP) and is stable for at least 18 months at 40°C without any special storage conditions.
- Nanoemulsion diameter was determined by dynamic light scattering (DLS) using the NICOMP 380 ZLS (PSS NICOMP Particle Sizing Systems, Santa Barbara, CA). The mean droplet size was consistently below 400 nm.
- rPA/nanoemulsion formulations were prepared 30 to 60 minutes prior to immunization by mixing rPA protein solution with NE, using saline as diluent. Mice immunization studies were performed using a 20 ⁇ g dose of rPA mixed with nanoemulsion concentrations of 0.1% 0.5%, 1% and 2%.
- rPA aluminum hydroxide formulation
- SIGMA rPA aluminum hydroxide formulation
- Guinea pig immunization studies were performed with 10 ⁇ g, 50 ⁇ g and 100 ⁇ g doses of rPA mixed with 1% NE and saline as diluent.
- the immunization volume was 10 ⁇ l/nare for mice and 50 ⁇ l/nare for guinea pigs.
- Fluorescently labeled rPA protein was prepared using the FLUOROTAG FITC Conjugation Kit (#FITC1, SIGMA) according to manufacturer's protocol.
- Murine dendritic cells (Jaws II) were incubated for 30 min at 37 0 C with PA-FITC conjugate in PBS or with PA-FITC mixed in nanoemulsion. The 0.001 % NE concentration was chosen to ensure foil viability of cells growing as a monolayer culture. After incubation, cells were washed 3 times with PBS and fixed with 1.25% formalin in PBS. Cellular uptake was then analyzed by fluorescent microscopy. The microphotographs were taken with an OLYMPUS 1X70 microscope with an IXFLA inverted reflected fluorescence observation attachment. The images were processed using the SPOT basic and SPOT advanced programs.
- mice were immunized intranasally (i.n.) with either one or two administrations of experimental vaccine 3 weeks apart. Animals were monitored for adverse reactions, and antibody responses were measured at 3 to 4 week intervals over a period of up to 12 weeks.
- the immunizations were conducted by first anesthetizing the mice with Isoflurane, then holding them in an inverted position. rPA/NE mixes were applied to the nares with a pipette tip (10 ⁇ l per nare) and the animals were then allowed to inhale the material.
- Hartley guinea pigs were vaccinated intranasally (i.n.) with one or two administrations of vaccine (50 ⁇ l per nare) 4 weeks apart and antibody responses were measured at 3 to 4 week intervals over a period of up to 22 weeks.
- Blood Collection, Bronchial Alveolar Lavage (BAL) and Splenocytes Blood samples were obtained from the saphenous vein at various time points during the course of the trials. The terminal sample was obtained by cardiac puncture from euthanized, premorbid mice. Serum was obtained from blood by centrifogation at 1500 x g for 5 minutes after allowing it to coagulate for 30 to 60 minutes at ro ⁇ m temperature. Serum samples were stored at -20°C until analyzed.
- BAL fluid was obtained from mice euthanized by Isoflurane inhalation. After the trachea was dissected, a 22GA catheter (ANGIOCATH, B-D) attached to a syringe was inserted into the trachea. The lungs were infused twice with 0.5 ml of PBS containing 10 ⁇ M DTT and 0.5 mg/ml aprotinin (protease inhibitors) and approximately 1 ml of aspirate was recovered. BAL samples were stored at -2O 0 C for further study.
- Murine splenocytes were mechanically isolated to obtain single-cell suspension in PBS.
- Red blood cells (RBC) were removed by lysis with ACK buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA) 5 and the remaining cells were washed twice in PBS.
- ACK buffer 150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA
- splenocytes 2-4 x 10 /ml
- RPMI 1640 medium supplemented with 2% FBS, 200 nM L- glutamine, and penicillin/streptomycin (100 U/ml and 100 ⁇ g/ml).
- Mouse anti-PA-specific IgG and IgA levels were determined by ELISA.
- Microtiter plates (NUNC) were coated with 5 ⁇ g/ml (100 ⁇ l) of rPA in a coating buffer (50 mM sodium carbonate, 50 mM sodium bicarbonate, pH 9.6) and incubated overnight at 4 0 C. After the protein solution was removed, plates were blocked for 30 minutes with PBS containing 1% dry milk. The blocking solution was aspirated and plates were used immediately or stored sealed at 4°C until needed.
- Serum and BAL samples were serially diluted in 0.1% BSA in PBS, and 100 ⁇ l/well aliquots were incubated in rPA-coated plates for 1 hour at 37°C. Plates were washed three times with PBS-0.05% Tween 20, followed by 1 hour incubation with either anti-mouse IgG or anti- mouse IgA alkaline phosphatase (AP)-conjugated antibodies (ROCKLAND), then washed three times and incubated with AP substrate SIGMAFAST (SIGMA).
- SIGMA AP substrate SIGMAFAST
- the colorimetric reaction was stopped with 1 N NaOH according to the manufacturer's protocol, and readouts were performed using a SPECTRAMAX 340 ELISA reader (MOLECULAR DEVICES, Sunnyvale, CA) at 405 nm and the reference wavelength of 690 ran.
- the endpoint titers were recorded and, in case of BAL fluid, the final antibody concentrations were calculated (See Rhie et al.,2003 PNAS 100:10925-10930) from the standard curves obtained for each assay plate, using goat F(ab')2 anti-mouse IgG as a capturing agent and known concentrations of mouse IgG and IgA immunoglobulins, and detected with anti-IgG or anti-IgA-AP conjugates.
- Guinea pig anti-PA IgG was determined by the same method, except that rabbit anti-guinea pig IgG alkaline phosphatase (AP)-conjugate was used for detection (ROCKLAND). Antibody concentrations are presented as the mean +/- sem (standard error of the mean) of endpoint titers.
- Lethal Toxin (LeTx) Cytotoxicity and Neutralizing Antibodies Assay were performed using serial dilutions of the sera incubated for 1 hour with the LeTx (0.1 ⁇ g/ml rPA and 0.1 ⁇ g/ml rLF in PBS). The antibody-toxin mixtures were then added to RAW264.7 (20,000-30,000 cells/well) and incubated for 4-6 hours at 37 0 C. Cell viability was assessed with XTT assay. The serum titers resulting in 50% protection against LeTx cytotoxicity (neutralizing concentration NC ) were calculated from the cell viability curves and presented as the mean value of the individual sera.
- anthracis (Ames strain) spores were enumerated and diluted in PBS without calcium in magnesium for an i.n. spore challenge.
- Anesthetized guinea pigs were challenged by intranasal administration of either 1.2 x 10 6 or 1.2 x 10 7 spores, which corresponds, respectively, to an intranasal 10 * LDso and 100 x LD50 dose.
- Post-challenge observation of guinea pigs was performed as described above for intradermal challenge.
- the proliferation of mouse splenocytes was measured by an assay of the 5-bromo-2-deoxyuridine (BrdU) incorporation, using CELL PROLIFERATION ELISA, (ROCHE Molecular Biochemicals, Mannheim, Germany).
- the cells were incubated in the presence of rPA (5 ⁇ g/ml) or PHA-P mitogen (2 ⁇ g/ml) for 48 hours and then pulsed with BrdU for 24 hours.
- Cell proliferation was measured according to the manufacturer's instructions using a SPECTRA MAX 340 ELISA Reader at 370 nm and a reference wavelength of 492 nm.
- rPA with nanoemulsion did not appear to alter the antigen's protein structure, as it remained a single discrete band on a non-denaturing PAGE, corresponding to intact, full-length protein with a molecular weight of 83 kD (See Figure 6A).
- NE appeared to improve the stability of the rPA, which prevented the progressive degradation due to de- amidation that is observed of the antigens incubated in a buffer solution (See, e.g., Gupta et al., 2003. FEBS Letters 554:505-510; Zomber et aL, 2005 Journal of Biological Chemistry 280:39897-39906).
- the addition of the rPA protein did not alter either the size (359 +/-109 nm), appearance, or stability of the nanoemulsion as shown in photomicrographs of NE alone and mixed with antigen (See Figure 6B).
- Nanoemulsion Increases the Uptake of rPA by Dendritic Cells Without Inducing an
- NE adjuvant activity in vivo involves an increase in the antigen uptake by antigen presenting cells to nasal mucosa (e.g., without the indications of inflammation).
- CBA/J mice were immunized intranasally with 20 ⁇ g rPA mixed with either 0.1 %, 0.5%, 1 %, or 2% concentrations of NE.
- a rapid induction of anti-PA antibodies in serum was obtained in all vaccinated animals with some dependence on the concentration of the nanoemulsion.
- All CBA/J mice developed high titers of serum anti-PA IgG (endpoint titers ranging 10 4 to 10 5 ) at 5 weeks after only two administrations of the vaccine (at day 1 and at 3 weeks).
- the pattern of the IgG subtype antibodies indicated a prevalence of IgG2a and IgG2b over IgGl . Accordingly, in some embodiments, administration rPA mixed with NE to a subject induces ThI polarization of the immune response (See Figure 8A, insert).
- mice were immunized with 20 ⁇ g rPA mixed with 1% NE (rPA/NE) and compared to immunization with 20 ⁇ g rPA mixed with either MPL A (rPA/MPL A), unmethylated CpG ODN (rPA/CpG) or aluminum hydroxide (rPA/Alu) (See, e.g., Peterson et al., 2006. Infection and Immunity 74:1016-1024; Pittman et al., 2001. Vaccine 20:972-978; Pittman et al., 2002. Vaccine 20:2107-2115; Reuveny et al., 2001.
- MPL A rPA/MPL A
- rPA/CpG unmethylated CpG ODN
- rPA/Alu aluminum hydroxide
- mice immunized with rPA/NE were seropositive, with anti-PA IgG endpoint titers of at least 10 5 . This was compared to titers ranging from 10 2 -10 3 in the rPA/MPL A, rPA/CpG and rPA/Alu immunization groups (See Figure 8B). No anti-PA antibodies were detected in animals nasally immunized with rPA in saline.
- Serum was also analyzed for the presence of anti-PA IgE antibodies and revealed IgE anti-PA (detectable in at least 1 :80 dilution in dot blots) in mice intramuscularly immunized with rPA/Alu, but not in any other group (See Figure 8B 5 insert). This is consistent with reports of alum adjuvant-based vaccines inducing a Th2 response (See, e.g., Johansson et al., 2004. Vaccine 22:2873-2880; Lindblad, 2004. Vaccine 22:3658-3668).
- Example 12 Intranasal rPA/NE Vaccination Produces Mucosal Immunity.
- the present invention provides that significant mucosal immune responses are induced via nasal administration of a vaccine comprising rPA in NE, but not with intramuscular immunization. No inflammatory response was observed in histopathological examination of animals' nasal mucosa after administration of NE with or without antigen, indicating that the nanoemulsion is not pro-inflammatory.
- Example 13 rPA/NE Vaccines Produce Neutralizing Antibodies against Anthrax Toxin in Mice.
- PA antigen-specific cellular responses were measured in a proliferation assay (See Figure 5) and through the analysis of cytokine secretion from splenocytes stimulated in vitro with rPA (See Table 7 below). As shown in Figure 11 , rPA stimulated proliferation in splenocytes obtained from mice immunized with rPA/NE. No antigen-specific proliferation was detected in splenocytes from animals immunized with either rPA alone or rPA with CpG ODNs.
- PA-activated spleen cells showed extensive production of INF- ⁇ , TNF- ⁇ , and IL-2, but failed to produce IL-4 when compared to control (non-stimulated) cells.
- immunization e.g., nasal administration
- rPA/NE yields specific ThI -type polarized cellular responses (See Table 7).
- ThI ThI -type polarized cellular responses
- splenocyte cultures incubated with PHA induced significant proliferation and the secretion of both ThI and Th2 cytokines.
- Example 15 rPA/NE Vaccines Protect Guinea Pigs Against Intradermal Live Spore Challenge.
- guinea pigs were vaccinated intranasally with 10, 50, and 100 ⁇ g doses of rPA mixed with 1% NE. IgG responses were observed after a single vaccination and continued to increase after a second administration (at 4 weeks), producing endpoint antibody titers >1 x 10 . The animals were subsequently followed for 6 months to evaluate the duration of immunity. Nasal immunization in these animals produced durable immune responses with high antibody titers (>10 4 ) for at least 6 months (See Figure 12A). At 6 months, the animals were challenged intradermally (i.d.) with 1000 * LDso Ames strain spores.
- Example 16 rPA/NE Vaccines Protect Against Intranasal Spore Challenge.
- intranasal immunization was also tested in an inhalation challenge trial.
- Three groups of guinea pigs were immunized with formulations containing 10, 50, and 100 ⁇ g rPA mixed with 1% NE. Immunization produced 100% seroconversion and significant anti-PA IgG responses in immunized animals.
- a boost at 4 weeks resulted in the rapid increase of anti-PA IgG in the serum, producing endpoint antibody titers >1 x 10 in all groups.
- a LeTx neutralization assay before the challenge indicated a mean NC50 titers of 1-2 x 10 in all vaccinated groups (See Figure 13A).
- mice Pathogen-free, female Balb/c mice (5-6 weeks old) and Hartley guinea pigs (females, 250 g) were purchased from Charles River Laboratories (Wilmington, MA). The mice (five to a cage) and guinea pigs (one per cage) were housed in accordance with the American Association for Accreditation of Laboratory Animal Care standards. All procedures involving animals were performed according to the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan.
- UUCA University Committee on Use and Care of Animals
- Reagents Recombinant HIV gpl20 ⁇ aL and gpl20sFi62 serotype proteins produced in yeast were obtained from Dr. Joseph Sodorski via Dr. David Markovitz (Harvard Medical School and University of Michigan, respectively). The 5 mg/ml aliquots of the protein solutions in a sterile saline were stored at -80 0 C until used. The synthetic V3 loop peptide (BaL) was obtained from Dr. Steven King (University of Michigan).
- the 20-mer oligonucleotide (ODN) 5'-TCC ATG ACG TTC CGT ACG TT -3' (SEQ ID NO.: 4) (See, e.g., Moldoveanu et al., Vaccine 1998;16(11-12):1216-24), containing non-methylated CpG repeats, was synthesized by INTEGRATED DNA TECHNOLOGIES (IDT, Coralville, IA). ).
- the S. minnesota monophosphoryl lipid A (MPL A, #L-6638), PHA-P, BSA, DTT, and other chemicals used in buffers were purchased from SIGMA-ALD RICH Corporation (St. Louis, MO).
- the saline solution, phosphate buffered saline (PBS), cell culture media, and fetal bovine serum (FBS) was purchased from GIBCO (Grand Island, NY) and HYCLONE (Logan, UT), respectively.
- the alkaline phosphatase (AP)-conjugated antibodies, goat anti- mouse IgG (#A-3562), goat anti-mouse IgA ( ⁇ chain specific, #A-4937) were purchased from SIGMA, and rabbit anti-guinea pig IgG was bought from ROCKLAND (#606-408).
- NE oil-in- water nanoemulsion
- NE mean droplet size (about 300 + ⁇ /- 25 nm) was determined by dynamic light scattering (DLS) using the NICOMP 380 ZLS (PSS NICOMP Particle Sizing Systems, Santa Barbara, CA) gpl20/NE formulations were prepared by mixing gpl20 protein solution with NE, using saline as diluent.
- mice immunization studies were performed with a 20 ⁇ g dose of gpl20 mixed with 0.1%, 0.5% and 1% NE concentrations-
- either 5 ⁇ g of MPL A or 10 ⁇ g CpG ODN was added to the 20 ⁇ g gpl 20 in 1% NE or to the 20 ⁇ g gpl20 in saline.
- Guinea pig immunization study was performed using 50 ⁇ g dose gpl 20 mixed with 1% NE and saline as diluent.
- mice were immunized with two, and on one occasion with three, intranasal (i.n.) administrations of gpl 20/NE formulation at 3 weeks apart.
- the immunizations were performed by slowly applying gpl 20/NE mixes (10 ⁇ l per nare) to the nares of Isoflurane anesthetized mice. During delivery animals were held in the inverted position until droplets were completely inhaled.
- mice were immunized with gpl 20 in saline, and with either NE or saline alone.
- Intramuscular immunization was performed with two doses, 3 weeks apart, of 20 ⁇ g gpl20 injected in 50 ⁇ l of either saline or 1% NE.
- Hartley guinea pigs (3 animals per group) were anesthetized with Ketamine injection (40 mg/kg) and immunized intranasally with two i.n. administrations of gpl 20/NE mix (50 ⁇ l per nare) at 3 weeks apart.
- Blood samples were obtained either from the saphenous vein, at various time points during the course of the experiment, or by cardiac puncture from euthanized premorbid mice.
- Serum was obtained from coagulated blood (30-60 minutes at room temperature) by centrifugation at 1500 g for 5 minutes. Collected serum samples were heat inactivated at 56°C for 1 hour and stored at -20 0 C until analyzed.
- Mouse bronchial alveolar lavage fluid (BAL) was obtained from animals euthanized by inhalation of Isoflurane. The lung was infused twice with 0.5 ml of PBS with 10 ⁇ M DTT and 0.5 mg/ml aprotinin and approximately 1 ml of aspirate was recovered. BAL samples were stored at -2O 0 C until analyzed.
- Vaginal wash samples were collected from anesthetized mice by infusion of vaginal cavities with 100 ⁇ l of PBS with 10 ⁇ M DTT and 0.5 mg/ml aprotinin. The samples were centrifugated at 10,000 ⁇ g for 5 minutes at 4 0 C, and the supernatants were stored at -2O 0 C until analyzed.
- Murine splenocytes were mechanically isolated from the spleens to obtain single cell suspension in PBS.
- the red blood cells (RBC) were removed by lysis with ACK buffer (150 mM NH 4 Cl, 10 mM KHCO3, 0.1 mM NaaEDTA), and the remaining cells were washed twice in PBS.
- ACK buffer 150 mM NH 4 Cl, 10 mM KHCO3, 0.1 mM NaaEDTA
- splenocytes 2-4 x 10 cells/ml
- RPMI 1640 medium supplemented with 2% FBS, L-glutamine, and penicillin/streptomycin (100 U/ml and 100 ⁇ g/ml).
- mice determination of anti-gpl20 IgG and IgA antibodies.
- Mouse anti-gpl20-s ⁇ ecific IgG and IgA levels were determined by ELISA.
- Microtiter plates (MAXISORP; NALGE NUNC International, Rochester, NY) were coated with 5 ⁇ g/ml (100 ⁇ l) of either gpl20 ⁇ aL or gpl20sFi62 serotype envelope protein in the coating buffer (50 mM sodium carbonate, 50 mM sodium bicarbonate, pH 9.6) and incubated overnight at 4 0 C. After the protein solution was removed, plates were blocked for 30 minutes at 37°C with PBS- 1% dry milk solution.
- coating buffer 50 mM sodium carbonate, 50 mM sodium bicarbonate, pH 9.6
- the blocking solution was aspirated and plates were used immediately or stored sealed at 4°C until needed. Serum and BAL samples were serially diluted in 0.1% BSA in PBS, and 100 ⁇ l/well aliquots were incubated in gpl20 coated plates for 1 hour at 37 0 C. Plates were washed three times with PBS-0.05% Tween 20, followed by 1 hour incubation with either anti-mouse IgG or anti-mouse IgA alkaline phosphatase (AP)-conjugated antibodies, then washed three times and incubated with AP substrate SIGMAFAST (SIGMA, St. Louis, MO) according to the manufacturer's protocol.
- SIGMAFAST SIGMA, St. Louis, MO
- Spectrophotometric readouts were performed using the SPECTRA MAX 340 ELISA reader (MOLECULAR DEVICES, Sunnyvale, CA) at 405 ran and reference wavelength of 690 nm.
- Endpoint antibody titers were defined as the last reciprocal serial serum dilution at which the absorption at 405 nm was greater than two times absorbance above negative control.
- Guinea pig anti-gpl20 IgG was determined by the same method, except that rabbit anti-guinea pig IgG alkaline phosphatase (AP)-conjugate was used for detection (ROCKLAND).
- Antibody concentrations are presented as the mean +/- standard deviation (s.d.) of endpoint titers.
- HIV-I single-round neutralization assay An eight strain panel of clade B HIV-I used in this study contained the laboratory strains BaL, SF 162 and MN, and primary HIV-I isolates SSl 196.01, BGl 168.01, QH0692.42, 3988.25 and 5768.04 (Li 05).
- Virus neutralization was measured as a function of the reduction in luciferase reporter gene expression after a single round of virus infection in TZM-bl cells as described (See, e.g., Montefiori, editor. Evaluating neutralizing antibodies against HFV, SIV and SHIV in luciferase reporter gene assays. New York, NY: John Wiley & Sons, 2004).
- the TZM-bl cells are engineered to express CD4 and CCR5 and contain integrated reporter genes for firefly luciferase and E. coli ⁇ -galactosidase under control of an HIV-I LTR.
- Primary HIV- 1 isolates (TCIDso, 100 to 200) were incubated with serial dilutions of sera for 1 hour at 37°C. Subsequently virus/serum mixtures were added to the 96-well flat-bottom culture plate containing adherent TZM-bl cells. Control contained cells plus virus (virus control), and cells only (background control). Bioluminescence was measured after 48 hours using BRIGHT GLO substrate solution as described by the supplier (PROMEGA, Madison, WI).
- Neutralization titers (NT50) are the dilutions at which relative light units (RLU) were reduced by 50% compared to those of virus control wells after subtraction of background RLUs.
- Proliferation assay The proliferation of mouse splenocytes was measured by an assay of 5-bromo-2-deoxyuridine (BrdU) incorporation using a commercially available labeling and detection kit (Cell Proliferation ELISA, ROCHE Molecular Biochemicals, Mannheim, Germany). To assess antigen specific proliferation, cells (2 * 10 6 cell/ml) were incubated in medium alone and the presence of gpl20BaL (5 ⁇ g/ml), or as control with a PHA-P (2 ⁇ g/ml), for 48 hours and then pulsed with BrdU for 24 hours. Cell proliferation was measured according to the manufacturer's instructions using SPECTRA MAX 340 ELISA Reader (MOLECULAR DEVICES, Sunnyvale, CA) at 370 nm and reference wavelength of 492 nm.
- SPECTRA MAX 340 ELISA Reader MOLECULAR DEVICES, Sunnyvale, CA
- cytokine assays were performed using QUANTIKINE ELISA kits (R&D SYSTEMS, Inc., Minneapolis, MN) according to the manufacturer' s instructions:
- Nasal immunization with recombinant HIV gpl20 protein mixed with nanoemulsion induces potent IgG response in serum.
- NE has an adjuvant activity in the mucosal immunization with a recombinant HIVgpl20 protein
- Balb/c mice were intranasally (i.n.) immunized with either gpl20 ⁇ aL or gpl20sFi62 serotype of antigen. Effect of NE concentration was assessed using 20 ⁇ g of gpl20 ⁇ aL in saline or mixed with a 0.1%, 0.5% and 1% range of NE concentrations. Blood was collected at 6 weeks after two immunizations and at 12 weeks after three immunizations and analyzed for gpl20-specific antibodies by ELISA.
- mice immunized with either of gpl20Bai7NE preparations were seropositive after only two immunizations.
- the anti-gpl20 ⁇ aL IgG response showed concentration-dependent effect of NE, with lowest titers in gpl20 ⁇ aL/0.1% NE and highest in gpl20Bai/l% NE immunization groups (mean titers of 1.3 x 10 4 and 2.6 ⁇ 10 5 , respectively).
- Mice immunized with gpl20Bai/saline did not have detectable anti-gpl20Bai_ antibodies.
- the present invention provides that only two i.n. administrations of gpl20 ⁇ aL with NE adjuvant are required to mount a potent systemic IgG response in mice.
- NE is sufficient for robust mucosal adjuvanation.
- NE-produced immune responses were compared with the effects of known immunostimulants, unmethylated CpG ODN and MPL A.
- Mice were i.n. immunized with 20 ⁇ g gpl20sFi62 mixed with 1% NE (gpl20sFi62/NE) and compared to immunization with antigen mixed with either CpG ODN (gp!20 sFi62/CpG) or with MPL A (gpl20sFi62/MPL A).
- mice were immunized with a gpl20sFi62/NE and additionally with either CpG (gpl20 S Fi62/NE+CpG) or MPL A (gpl20s F i62/NE+MPL A). Similar to immunization with gpl20Bai, mice immunized with gpl20s F i62/NE responded with high anti-gpl20sFi62 IgG titers.
- CpG gpl20 S Fi62/NE+CpG
- MPL A gpl20s F i62/NE+MPL A
- Antibodies generated against one serotype of gpl20 cross-react with other gpl20 serotypes.
- NE adjuvant can produce a repertoire of IgG capable of recognizing both variable and conserved epitopes of the gpl20 immunogen (e.g., that participate in protective immunity against various types of HTV-I (See, e.g., Mascola, Curr MoI Med 2003;3(3):209-16).
- Nasal administration of pgl20/NE generates anti-gpl20 specific IgA antibodies detectable in bronchial and vaginal mucosal surfaces.
- the present invention provides that significant mucosal responses, both locally (e.g., in bronchial mucosa) and in distant sites (e.g., vaginal secretions), can be induced in response to i.n. immunization with antigen (e.g., gpl20) delivered with NE adjuvant.
- antigen e.g., gpl20
- Example 21 Cell mediated immune responses.
- Antigen-specific induction or IFN- ⁇ and the lack of detectable IL-4 expression evidences ThI polarization of the cellular immune response. No significant cytokine expression was detected in splenocytes from control mice or from mice immunized with gpl20 ⁇ aL in saline.
- guinea pigs were administered with two doses of gpl20sFi62 mixed with 1% NE. Immunization produced significant, albeit varied, levels of serum anti-gpl20 IgG antibodies in individual animals (See Figure 17A). As observed in mice, the guinea pig anti-gpl20 IgG cross-reacted with heterologous gpl20 immunogen. Immune sera from guinea pigs were tested for neutralizing activity against HIV-I . The breadth of the neutralizing response was evaluated in a panel of 8 viruses, including 3 laboratory strains and 5 primary HIV isolates.
- Neutralization of heterologous M-tropic strain HIVBQ L was comparable in all guinea pigs with NT50 greater than 50. No neutralization was observed with laboratory strain of T- tropic HIVMN virus. All five primary HIV isolates tested were effectively neutralized with sera from vaccinated guinea pigs. Neutralizing activity for the primary HIV isolates was comparable with both laboratory strains. The NT50 values for BG1168.1, SS1196.11 and 3988.25 ranged from 50 to 100 depending on the serum. The isolates QH0692.42 and 5768.4 were effectively neutralized with NT50 values grater than 100.
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BAKER J R JR ET AL: "Enhanced systemic and mucosal immune responses in mice immunized with recombinant Bacillus anthracis protective antigen (rPA) using a novel nanoemulsion adjuvant." JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 113, no. 2 Supplement, February 2004 (2004-02), page S292, XP002560178 & 60TH ANNUAL MEETING OF THE AMERICAN ACADEMY OF ALLERGY, ASTHMA AND IMMUNOLOGY (AAAAI); SAN FRANCISCO, CA, USA; MARCH 19-23, 2004 ISSN: 0091-6749 * |
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