CN101466370A

CN101466370A - Nanoemulsion vaccines

Info

Publication number: CN101466370A
Application number: CNA2007800219017A
Authority: CN
Inventors: 小J·R·贝克尔; A·比林斯卡; A·麦斯
Original assignee: University of Michigan
Current assignee: University of Michigan
Priority date: 2006-04-13
Filing date: 2007-04-13
Publication date: 2009-06-24

Abstract

The present invention provides methods and compositions for stimulation of an immune response. In particular, the present invention provides methods and compositions for using nanoemulsion compounds as mucosal adjuvants to induce immunity against environmental pathogens. Thus, in some embodiments, the present invention provides a nanoemulsion vaccine comprising a nanoemulsion and an inactivated pathogen or protein derived from said pathogen. The present invention thus provides improved vaccines against a variety of environmental and human-released pathogens.

Description

Nanoemulsion vaccines

In the U.S. Provisional Patent Application series number 60/791,800,60/791,759 of submission on April 13rd, 2006 and 60/791,758 priority, it all integrates with this paper with it to the present invention's requirement separately by reference to all.

Under the contract U54 AI57153-02 that contract MDA 972-97-1-0007 that ARPA authorizes and NIH authorize, under supporting, government finishes the present invention.Government has some right in the present invention.

Invention field

The invention provides the method and composition that is used for immune response stimulating.Especially, the invention provides the immunoreactive method and the compositions that is used for this method (nano-emulsion that for example, comprises antibacterial or virus or its component) of in experimenter (for example people experimenter), inducing to reagent (for example, antibacterial or virus).The compositions and methods of the invention are used for, and clinical (for example, therapeutic and preventative medical science (for example, inoculation)) and research are used, and other.

Background of invention

Immunity is a principal character of improving human health.Although the multiple successful vaccine of existing anti-many common diseases, the first cause that remains health problem and death of infectious disease.Inherent major issue comprises that needs repeat invalid for spectrum of diseases of immune and existing vaccine delivery system in the existing vaccine.

In order to develop the anti-vaccine that is difficult to carry out the pathogen of vaccine development, and/or overcome owing to expensive, the complicated and under-utilized failure that causes commercially available vaccine, must the new antigen presentation of exploitation and the method for immunity, for vaccine provides immune time still less, more effective use and/or side effect still less.

Summary of the invention

Therefore, in some embodiments, the invention provides the immunoreactive method of inducing in people experimenter vaccinia subgroup virus, this method comprises: provide to comprise nano-emulsion and combinations of immunogens thing, wherein said immunogen comprises by the vaccinia subgroup virus of nano-emulsion deactivation; With the experimenter is produced under the immunoreactive condition to vaccinia subgroup virus, to experimenter's applying said compositions.The immunoreactive character that the invention is not restricted to produce.In fact, can in use the experimenter who comprises nano-emulsion of the present invention and combinations of immunogens thing, produce and measure many kinds of immunoreation, comprising but be not limited to, activation, propagation or the differentiation of immune cell (for example, B cell, T cell, dendritic cell, antigen presenting cell (APC), macrophage, NK cell (NK) cell etc.); The rise of labelling and cytokine or the expression of downward modulation; The stimulation of IgA, IgM or IgG titre; Splenomegaly (for example, the splenocyte structure of increase); Hyperplasia in the various organs, blended cellular infiltration and according to immune other reactions of immunostimulation assessment known in the art (for example, cell).In some embodiments, using the mucomembranous surface that comprises the experimenter contacts with compositions.The present invention is not subject to the mucomembranous surface that is contacted.In some preferred embodiments, described mucomembranous surface comprises nasal mucosa.In some embodiments, use and comprise that parenteral uses.The present invention is not subject to the approach of selecting to be used to use compositions of the present invention.In some embodiments, induction of immunity is reflected at the immunity of having induced among the experimenter vaccinia subgroup virus.In some embodiments, described immunity comprises the general immunity.In some embodiments, described immunity comprises mucosal immunity.In some embodiments, described immunoreation comprises that the IFN-γ that increases among the experimenter expresses.In some embodiments, described immunoreation comprises the systemic IgG reaction to the vaccinia subgroup virus of deactivation.In some embodiments, described immunoreation comprises the mucosa IgA reaction to the vaccinia subgroup virus of deactivation.The present invention is not subject to the type of the vaccinia subgroup virus that uses in compositions of the present invention.In fact, can use multiple vaccinia subgroup virus, comprising but be not limited to alastrim virus, vaccinia virus, cowpox, monkeypox, gerbil jird pox (gerbilpox), camel pox or the like.In some embodiments, in the dosage that the experimenter is used, there is 10-10 ³Under the condition of the inactivation of viruses of pfu, the experimenter is used by the vaccinia subgroup virus of Emulsion deactivation.Yet the present invention is not subject to the vaccinia subgroup virus of using of this amount.For example, in some embodiments, exist in the dosage that the experimenter is used and surpass 10 ³The virus of the deactivation of pfu (for example, 10 ⁴Pfu, 10 ⁵Pfu or more).In some embodiments, the nano-emulsion solution deactivation vaccinia subgroup virus of use 10%.Yet the present invention is not subject to the nano-emulsion that is used for the deactivation vaccinia subgroup virus of this amount (for example, percent).For example, in some embodiments, comprise the compositions that is lower than 10% nano-emulsion and be used for deactivation.In some embodiments, comprise the compositions that surpasses 10% nano-emulsion and be used for deactivation.In some embodiments, described nano-emulsion comprises W ₂₀5EC.The present invention is not subject to the type of employed nano-emulsion.In fact, comprise that many kinds of nano-emulsioies are used for the present invention.For example, in some preferred embodiments, described nano-emulsion (for example, (for example be used to produce immunoreation, be used for as vaccine)) comprise oil in water emulsion, be included in the discontinuous oil phase that aqueous phase distributes oil in water emulsion, comprise first component of solvent (for example, ethanol or glycerol) and comprise second component of surfactant or halogen-containing chemical compound.Water comprises the water of any kind, comprising but be not limited to water (for example, diH ₂O, distilled water, tap water) and solution (for example, phosphate buffered saline(PBS)).Oil phase comprises the oil of any kind, comprising but not cloudy in, vegetable oil (for example, vegetable oil (canola oil), Semen Maydis oil, Oleum Brassicae campestris, safflower oil and Oleum Helianthi are drawn in Semen sojae atricolor Wang, American Avocado Tree oil, Semen Lini oil, Oleum Cocois, Oleum Gossypii semen, Squalene oil (squalene oil), olive oil, Kano), animal oil (for example, fish oil), flavored oils, water-fast vitamin, mineral oil and gasoline.In some preferred embodiments, oil phase comprises the oil in water emulsion (that is, forming the cumulative volume of the whole Emulsion of 30-90%) of 30-90 volume %, more preferably 50-80%.Though the present invention is not subject to the character of alkoxide component, in some preferred embodiments, described alcohol is ethanol or methanol.In addition, though the present invention is not subject to the character of surfactant, but in some preferred embodiments, described surfactant be the Polysorbate surfactant (for example, TWEEN 20, TWEEN 40, TWEEN 60 and TWEEN 80), the phenoxy group polyethoxy ethanol (for example, TRITON X-100, X-301, X-165, X-102 and X-200, and TYLOXAPOL) or sodium lauryl sulphate.Equally, though the present invention is not subject to the character of halogen contained compound, but in some preferred embodiments, described halogen contained compound comprises the halogenation cetyl pyridinium, the halogenation cetyltrimethyl ammonium, halogenation hexadecyldimethyl benzyl ammonium ethyl ammonium, halogenation hexadecyldimethyl benzyl ammonium benzyl ammonium, halogenation cetyl tributyl phosphorus, the halogenation dodecyl trimethyl ammonium, halogenation myristyl trimethyl ammonium, the halogenation cetyl pyridinium, hexadecyltrimethylammonium chloride, cetyl benzyl dimethyl ammonium chloride, brocide, cetrimonium bromide, bromination hexadecyldimethyl benzyl ammonium ethyl ammonium, bromination cetyl tributyl phosphorus, bromination dodecyl trimethyl ammonium or Cetrimide.Nano-emulsion of the present invention also comprises component such as the 3rd, the 4th, the 5th.In some preferred embodiments, other component be surfactant (for example, the second surface activating agent), sprout reinforcing agent (germination enhancer), (for example based on phosphatic solvent, Tributyl phosphate salt), neutramingen, L-alanine, ammonium chloride, tryptone meat peptone nutritional solution, yeast extract, L-ascorbic acid, lecithin, right-methyl hydroxybenzoate, sodium thiosulfate, sodium citrate, inosine, sodium hydroxide, glucose and Polyethylene Glycol (for example, PEG 200, PEG 2000 etc.).In some embodiments, described oil in water emulsion comprises quaternary ammonium compound.In some preferred embodiments, described oil in water emulsion does not have detectable toxicity to plant or animal (for example, to the people).In other preferred embodiments, described oil in water emulsion does not cause detectable stimulation to plant or animal (for example, to the people).In some embodiments, described oil in water emulsion also comprises any said components.Quaternary ammonium compound includes but not limited to saccharic acid N-alkyl dimethyl benzyl ammonium, 1,3,5-triazine-1,3,5 (2H, 4H, 6H)-three ethanol, 1-ten alkylammoniums, N-decyl-N, the N-dimethyl-, chlorine (or) DDAC, 2-(2-(right-(diisobutyl) toloxyl) ethyoxyl) ethyl dimethyl benzene ammonio methacrylate, 2-(2-(right-(diisobutyl) phenoxy group) ethyoxyl) ethyl dimethyl benzene ammonio methacrylate,

alkyl

1 or 3 benzyls-1-(2-ethoxy)-2-imidazolitm chloride quinoline; Alkyl two (2-ethoxy) benzyl ammonium chloride, alkyl demethyl benzyl ammonium chloride, alkyl dimethyl 3,4-dichloro-benzenes ammonio methacrylate (100% C12), alkyl dimethyl 3,4-dichloro-benzenes ammonio methacrylate (50% C14,40% C12,10% C16), alkyl dimethyl 3,4-dichloro-benzenes ammonio methacrylate (55% C14,23% C12,20% C16), alkyl dimethyl benzyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride (100% C14), alkyl dimethyl benzyl ammonium chloride (100% C16), alkyl dimethyl benzyl ammonium chloride (41%C14,28% C12), alkyl dimethyl benzyl ammonium chloride (47% C12,18% C14), alkyl dimethyl benzyl ammonium chloride (55% C16,20% C14), alkyl dimethyl benzyl ammonium chloride (58% C14,28% C16), alkyl dimethyl benzyl ammonium chloride (60% C14,25%C12), alkyl dimethyl benzyl ammonium chloride (61% C11,23% C14), alkyl dimethyl benzyl ammonium chloride (61% C12,23% C14), alkyl dimethyl benzyl ammonium chloride (65%C12,25% C14), alkyl dimethyl benzyl ammonium chloride (67% C12,24% C14), alkyl dimethyl benzyl ammonium chloride (67% C12,25% C14), alkyl dimethyl benzyl ammonium chloride (90% C14,5% C12), alkyl dimethyl benzyl ammonium chloride (93% C14,4% C12), alkyl dimethyl benzyl ammonium chloride (95% C16,5% C18), alkyl dimethyl benzyl ammonium chloride (with) DDAC, alkyl dimethyl benzyl ammonium chloride (as in fatty acid), alkyl dimethyl benzyl ammonium chloride (C12-C16), alkyl dimethyl benzyl ammonium chloride (C12-C18), alkyl dimethyl benzyl and dialkyl dimethyl ammonium chloride, alkyl dimethyl dimethyl benzene ammonio methacrylate, alkyl dimethyl ethyl ammonium bromide (90% C14,5% C16,5% C12), alkyl dimethyl ethyl ammonium bromide (blended alkyl and alkenyl in the fatty acid as soybean oil), alkyl dimethyl ethylamino benzonitrile ammonium chloride, alkyl dimethyl ethylamino benzonitrile ammonium chloride (60% C14), alkyl dimethyl cumene ammonio methacrylate (50% C12,30% C14,17% C16,3% C18), alkyl trimethyl ammonium chloride (58% C18,40% C16,1% C14,1% C12), alkyl trimethyl ammonium chloride (90% C18,10% C16), alkyl dimethyl (ethylbenzyl) ammonium chloride (C12-18), two-(C8-10)-alkyl-dimethyl ammonium chloride, dialkyl dimethyl ammonium chloride, dialkyl dimethyl ammonium chloride, dialkyl dimethyl ammonium chloride, dialkyl methyl benzyl ammonium chloride; DDAC, the diiso decyl alkyl dimethyl ammonium chloride, Quaternium 24, dodecyl two (2-ethoxy) octyl group hydrochlorinate ammonium, dodecyl dimethyl benzyl ammonium chloride, dodecyl carbamyl methyl dimethoxy base benzyl ammonium chloride, seven decyl ethoxy imidazolitm chloride quinolines, six hydrogen-1,3,5-three (2-ethoxy)-s-triazine, myristyl benzyl dimethyl ammonium chloride (myristalkonium chloride) (with) Quat RNIUM 14, N, N-dimethyl-2-hydroxypropyl ammonium chloride polymer, just-alkyl dimethyl benzyl ammonium chloride, just-alkyl dimethyl ethylamino benzonitrile ammonium chloride, just-the Zephiramine chloride monohydrate, the octyl-decyl alkyl dimethyl ammonium chloride, octyl group dodecyl dimethyl ammonium chloride, Octylphenoxy ethoxyethyl group dimethyl benzene ammonio methacrylate, oxygen diethylene base two (alkyl-dimethyl ammonium chloride), quaternary ammonium compound, two cocoa alkyl dimethyls, chloride, trimethoxy-silylpropyl dimethyl eight decyl ammonium chloride; Trimethoxysilyl quaternary ammonium compound, trimethyldodecane base benzyl ammonium chloride, just-dodecyl dimethyl ethylamino benzonitrile ammonium chloride, just-six decyl dimethyl benzene ammonio methacrylate, just-Zephiramine chloride, just-myristyl dimethyl ethyl benzyl ammonium chloride and decyl dimethyl benzene ammonio methacrylate just-eight.In some embodiments, described Emulsion does not contain any antimicrobial material (that is, antimicrobial compositions is an Emulsion itself).In some embodiments, described nano-emulsion is X8P.In some embodiments; described immunoprotection experimenter avoids showing the S or S of the disease that is caused by vaccinia subgroup virus (for example vaccinia virus); in some embodiments, described immunoprotection experimenter avoids being exposed to subsequently the attack of Bacillus anthracis alive.In some embodiments, induce the protection experimenter to avoid infecting relevant sickness rate and/or mortality rate with vaccinia subgroup virus to immunoreactive, in some embodiments, described compositions also comprises adjuvant.The present invention is not subject to the type of employed adjuvant.This paper has described and has been used for many adjuvants of the present invention.In some embodiments, described experimenter is the people.

The present invention also provides the compositions that is used for immune response stimulating, and it comprises nano-emulsion and by the vaccinia subgroup virus of this nano-emulsion deactivation, wherein prepares said composition to induce the immunity to vaccinia subgroup virus in the experimenter.In some embodiments, nano-emulsion comprises W ₂₀5EC.In some embodiments, when the experimenter was used, described compositions was the 10-10 of experimenter's confession ³The inactivation of viruses of pfu.In some embodiments, when the experimenter was used, described compositions was 10 of experimenter's confession ³-10 ⁵The inactivation of viruses of pfu.In some embodiments, the dosage of the compositions of using to the experimenter comprises 1% nano-emulsion solution.In some embodiments, the vaccinia subgroup virus of deactivation is heat-staple in described nano-emulsion.In some embodiments, vaccinia subgroup virus is stable above 4 weeks in described nano-emulsion.In some embodiments, described vaccinia subgroup virus is a vaccinia virus.The present invention is not subject to the type of employed vaccinia subgroup virus.In fact, many kinds of vaccinia subgroup viruses can be used for being used for immune response stimulating in the compositions, include but not limited to alastrim virus, cowpox, monkeypox and camel pox.In some embodiments, the described compositions of dilution before the experimenter is used.In some embodiments, described experimenter is the people.In some embodiments, immunity is the general immunity.In some embodiments, immunity is a mucosal immunity.In some embodiments, described compositions also comprises adjuvant.

The present invention also provides the test kit that comprises the compositions that is used for immune response stimulating and be used for the description of applying said compositions, described compositions comprises nano-emulsion and by the vaccinia subgroup virus of described nano-emulsion deactivation, wherein prepares described compositions to induce the immunity at vaccinia subgroup virus in the experimenter.

In some embodiments, the invention provides the immunoreactive method of in the experimenter, inducing at Bacillus anthracis (B.anthracis), this method comprises: provide to comprise nano-emulsion and combinations of immunogens thing, wherein immunogen comprises Bacillus anthracis immunogen (for example, the reorganization protective antigen (rPA) of Bacillus anthracis); With the experimenter is produced under the immunoreactive condition at Bacillus anthracis, to experimenter's applying said compositions.The present invention is not subject to employed Bacillus anthracis immunogen.For example, in some embodiments, immunogen is albumen or peptide antigen or derivatives thereof or a variant isolating, purification or reorganization, and it is selected from but is not limited to protective antigen (PA), lethal factor (LF), edema factor (EF) and PA catabolite.The present invention is not subject to the immunoreactive character that is produced.In fact, can in use the experimenter who comprises nano-emulsion of the present invention and combinations of immunogens thing, produce and measure many kinds of immunoreation, include but not limited to, activation, propagation or the differentiation of immune cell (for example, B cell, T cell, dendritic cell, antigen presenting cell (APC), macrophage, NK cell (NK) cell etc.); The rise of labelling and cytokine or the expression of downward modulation; The stimulation of IgA, IgM or IgG titre; Splenomegaly (for example, the splenocyte structure of increase); Hyperplasia in the various organs, blended cellular infiltration and according to other reactions in the immune system of immunostimulation assessment known in the art (for example, cell).In some embodiments, using the mucomembranous surface that comprises the experimenter contacts with compositions.The present invention is not subject to the mucomembranous surface that is contacted.In some preferred embodiments, described mucomembranous surface comprises nasal mucosa.In some embodiments, use and comprise that parenteral uses.The present invention is not subject to the approach of selecting to be used to use compositions of the present invention.In some embodiments, induction of immunity is reflected at the experimenter and has induced immunoreation to Bacillus anthracis.In some embodiments, described immunity comprises the general immunity.In some embodiments, described immunity comprises mucosal immunity.In some embodiments, described immunoreation comprises the expression of the IFN-γ that increases among the experimenter.In some embodiments, described immunoreation comprises the systemic IgG reaction.In some embodiments, described immunoreation comprises mucosa IgA reaction.In some embodiments, described compositions comprises 1-300 μ g rPA.Yet the present invention is not subject to the reorganization protective antigen of using of this amount.For example, in some embodiments, there is the rPA that surpasses 300 μ g in the dosage that the receptor is used.In some embodiments, there is the rPA that is lower than 1 μ g in the dosage that the receptor is used.In some embodiments, described compositions comprises 10% nano-emulsion solution.Yet the present invention is not subject to the nano-emulsion of this amount (for example, percent).For example, in some embodiments, compositions comprises and is lower than 10% nano-emulsion.In some embodiments, compositions comprises high 10% nano-emulsion.In some embodiments, compositions of the present invention comprises any nano-emulsion described herein.In some embodiments, described nano-emulsion comprises W ₂₀5EC.The present invention is not subject to the type of the nano-emulsion of using of this amount.

In some embodiments, described immunoprotection experimenter avoids showing the S or S of the disease that is caused by Bacillus anthracis.In some embodiments, described immunoprotection experimenter avoids being exposed to subsequently the attack of Bacillus anthracis alive.In some embodiments, immunoreactive inducing protects the experimenter to avoid sickness rate and/or the mortality rate relevant with anthrax bacillus infection.In some embodiments, described compositions also comprises adjuvant.The present invention is not subject to the type of employed adjuvant.In some embodiments, described adjuvant is the CpG oligonucleotide.This paper has described and has been used for many other adjuvants of the present invention.In some embodiments, described experimenter is the people, and in some preferred embodiments, the described experimenter of immunoprotection avoids showing the S or S of anthrax.

The present invention also provides the compositions that is used for immune response stimulating; it comprises the reorganization protective antigen of nano-emulsion and Bacillus anthracis; wherein prepare said composition to induce the immunity to Bacillus anthracis in the experimenter, in some embodiments, described nano-emulsion comprises W ₂₀5EC.In some embodiments, when the experimenter was used, described compositions provided the reorganization protective antigen of 25-75 μ g for the experimenter.In some embodiments, the dosage of the compositions that the experimenter is used comprises 1% nano-emulsion solution.In some embodiments, the reorganization protective antigen is heat-staple in nano-emulsion.In some embodiments, the reorganization protective antigen is stable above 4 weeks in nano-emulsion.In some embodiments, the described compositions of dilution before the experimenter is used.In some embodiments, described experimenter is the people.In some embodiments, described immunity is the general immunity.In some embodiments, described immunity is a mucosal immunity.In some embodiments, described compositions also comprises adjuvant.In some embodiments, described adjuvant comprises the CpG oligonucleotide.

The present invention also provides the test kit that comprises the compositions that is used for immune response stimulating and be used for the description of applying said compositions; described compositions comprises the reorganization protective antigen of nano-emulsion and Bacillus anthracis, wherein prepares described compositions to induce the immunity at Bacillus anthracis in the experimenter.In some embodiments, described test kit also comprises the device that is used for applying said compositions.The present invention is not subject to the type of the device that is used for applying said compositions.In fact, comprise that many kinds of devices can be used for test kit, comprising but be not limited to nose applicator (nasal applicator), syringe, nasal inhaler and nasal spray device.In some embodiments, described test kit comprises with device (applicator) and contacting, and comprises nano-emulsion and Bacillus anthracis combinations of immunogens thing.In some embodiments, the invention provides the system and method (for example the attack of bacillosis substance is used in response) that is used for using on a large scale (for example, to cities and towns, village, city, state or national crowd) compositions of the present invention.

In some embodiments, the invention provides the immunoreactive method of inducing at HIV in the experimenter, this method comprises: provide to comprise nano-emulsion and combinations of immunogens thing, wherein said immunogen comprises reorganization gp120; With make the experimenter produce at the immunoreactive condition of HIV under to experimenter's applying said compositions.The present invention is not subject to employed immunogenic type (for example, reorganization gp120).For example, in some embodiments, immunogen is Tat, Nef or other immunogenic HIV albumen or derivatives thereof isolating, purification or reorganization.The present invention is not subject to the immunoreactive character that is produced.In fact, can in use the experimenter who comprises nano-emulsion of the present invention and combinations of immunogens thing, produce and measure many kinds of immunoreation, include but not limited to, activation, propagation or the differentiation of immune cell (for example, B cell, T cell, dendritic cell, antigen presenting cell (APC), macrophage, NK cell (NK) cell etc.); The rise of labelling and cytokine or the expression of downward modulation; The stimulation of IgA, IgM or IgG titre; Splenomegaly (for example, the splenocyte structure of increase); Hyperplasia in the various organs, blended cellular infiltration and according to immune other reactions of immunostimulation assessment known in the art (for example, cell).In some embodiments, using the mucomembranous surface that comprises the experimenter contacts with described compositions.The present invention is not subject to the mucomembranous surface that is contacted.In some preferred embodiments, described mucomembranous surface comprises nasal mucosa.In some embodiments, described mucomembranous surface comprises vaginal mucosa.In some embodiments, use and comprise that parenteral uses.The present invention is not subject to the approach of selecting to be used to use compositions of the present invention.In some embodiments, induction of immunity is reflected at the immunity of inducing among the described experimenter at described HIV.In some embodiments, described immunity comprises the general immunity.In some embodiments, described immunity comprises mucosal immunity.In some embodiments, described immunoreation comprises the expression of the IFN-γ that increases among the experimenter.In some embodiments, described immunoreation comprises the systemic IgG reaction.In some embodiments, described immunoreation comprises mucosa IgA reaction.In some embodiments, described compositions comprises the reorganization gp120 of 15-75 μ g.Yet the present invention is not subject to the reorganization gp120 that uses of this amount.For example, in some embodiments, there is the reorganization gp120 that surpasses 75 μ g in the dosage that the experimenter is used.In some embodiments, there is the reorganization gp120 that is lower than 15 μ g in the dosage that the experimenter is used.In some embodiments, described compositions comprises 10% nano-emulsion solution.Yet the present invention is not subject to the nano-emulsion of this amount (for example, percent).For example, in some embodiments, compositions comprises and is lower than 10% nano-emulsion.In some embodiments, compositions comprises and surpasses 10% nano-emulsion.In some embodiments, described nano-emulsion comprises W ₂₀5EC (, all incorporating it into this paper by reference) referring to United States Patent (USP) 6,015,832.In some embodiments, described nano-emulsion comprises X8P (referring to United States Patent (USP) 6,015,832, all incorporating it into this paper by reference).

The invention provides the test kit that comprises the compositions that is used for immune response stimulating and be used to use the description of compositions, described compositions comprises nano-emulsion and HIV immunogen (gp120 for example recombinates), wherein prepares described compositions to induce the immunity at HIV in the experimenter.In some embodiments, described test kit comprises the nano-emulsion that contacts with object (for example applicator).In some embodiments, described test kit comprises the device that is used for applying said compositions.The present invention is not subject to the type of the device that the test kit that is used for applying said compositions comprises.In fact, comprise many different devices in the test kit, comprising but be not limited to nose applicator, syringe, nasal inhaler and nasal spray device.In some embodiments, described test kit comprises the device of the other types of colpostat, vagina aerosol apparatus or vaginal application (for example, to the vagina mucosa) compositions of the present invention.In some embodiments, test kit comprises the birth control device (for example, condom, IUD, continuous bar etc.) with nano-emulsion compositions bag quilt of the present invention.In some embodiments, nano-emulsion of the present invention is blended in washing liquid or suppository or the lubricant (for example, property with lubricator).In some embodiments, the invention provides the system and method (for example using nano-emulsion compositions of the present invention) that is used for using on a large scale (for example, to cities and towns, village, city, state or national crowd).In preferred embodiments, carrying out this type of in the mode of easy use (for example interanasal administration) and cultural sensitivity (for example, in order not offend the people who uses compositions of the present invention) uses on a large scale.

Summary of drawings

Following accompanying drawing constitutes the part of this description and is used to further specify some aspect of the present invention and embodiment.One or more by with reference among these figure can understand the present invention better in conjunction with the description of particular provided herein.

Fig. 1 shows the complete virus deactivation of using nano-emulsion.(A) VV _WRThe plaque number reduce algoscopy (Plaque reduction assay) (PRA).(B) VV _WR-LucLuciferase survey, fixed with relative light unit (RLU) expression uciferase activity.(C) pcr analysis of lung DNA.The big tick marks of swimming lane 1:DNA; Swimming lane 2: primer, no DNA; Swimming lane 3: no Taq; Swimming lane 4:10 ⁵/ Fk lung DNA; Swimming lane 5-7:10 ⁵/ Fk/NE lung DNA; Swimming lane 8-10:10 ⁵/ NE lung DNA; Swimming lane 11: the contrast-with the blended VV DNA of lung DNA.Arrow has been indicated the viral template and the primer of amplification.(D) using the VV that lives _WR-LucWith 10 ⁵The bioluminescence imaging of mice behind the viral intranasal infection that the NE of pfu kills.The purpose zone (ROI) that visible circle indication carrying out photon flux is analyzed in some images.

Fig. 2 shows the immunogenicity of mucosa nanoemulsion vaccines in mice.(A) use in the mice of several formulations inoculation of the viral vaccine kill, the generation of the anti-VV IgG of serum antibody response, described preparation is: 10 ⁵/ NE (filled circles), 10 ³/ NE (open circles), 10 ⁵/ Fk/NE (black triangle), 10 ³/ Fk/NE (hollow triangle), 10 ⁵/ Fk (solid diamond) and 10 ³/ Fk (empty solid diamond).The intranasal (i.n) of arrow indication vaccine is used.Illustration: 1 time or 3 inoculations 10 ⁵Behind the vaccine of/NE, the comparison of the anti-VV IgG of serum.Data are expressed as the meansigma methods ± sem of individual anti-VV IgG concentration.(B) the anti-VV IgA of the secreted among BAL antibody.The result be expressed as the mean concentration of using the IgA that obtains in individual and the mensuration that blended BAL liquid carries out (+/-sem).

Fig. 3 shows virucidin.Use individual serum and use the pooled serum that the back obtains in 1,2 and 3 time connects to measure.Illustration: the detection of the virucidin among the BAL.Individuality and blended BAL liquid that use was collected when the 16th week, experiment finished are measured.The result is carried out standardization and is expressed as the NT of viral PRA ₅₀

Fig. 4 shows the cowpox specific cell immunoreaction.With 10 ³With 10 ⁴The VV alive of pfu _WRThe vivoexpression of INF-γ in the splenocyte that stimulates.Data show is in the splenocyte of the animal of the vaccinia virus immunity of using the NE deactivation, at the specificity INF-gamma reaction of virus.

Fig. 5 shows the intranasal attack of using vaccine virus.(A) with 10 x LD ₂₀VV _WR-LucAfter intranasal is attacked, with 10 in the multiple bacterin preparation ⁵The VV that pfu kills _WRThe survival rate curve of the mice of inoculation, described several formulations is: VV/NE, VV/Fk/NE and VV/Fk.(B) mice (last figure) of representative inoculation and the bioluminescence image of control mice (figure below).2-5 days document images after attack.

Fig. 6 shows the stability of vaccine production thing.(A) the rPA protein 24 of incubation 0.5 μ g hour in the NE of saline solution and 1% at room temperature, and use the 10%PAGE of irreducibility that it is analyzed.Silver dyes the low molecular weight fraction behind the antigen incubation that is presented at no nano-emulsion (saline solution).(B) microphotograph of NE and rPA/NE mixture is presented at after antigen mixes, and does not have in the Emulsion to change (400 x amplification).

Fig. 7 shows the influence of nano-emulsion to the proteinic cellular uptake of rPA.With Jaws II dendritic cell with 0.1 independent μ g/ml PA-FITC of (A) culture medium, (B), (C) and the blended 0.1 μ g/ml PA-FITC of 0.001%NE or (D) with the blended 1 μ g/ml PA-FITC incubation of 0.001%NE.Green fluorescence is represented to have only when using with nano-emulsion, and rPA is just by effective internalization.

Fig. 8 shows the time graph of the anti-PA IgG of serum in the mice.Vaccine (arrow) intranasal immune mouse with two dosage.(A) inducing with antagonism PA IgG in the CBA/J mice of 20 μ g rPA and the ever-increasing NE inoculation of concentration.(A illustration).With the anti-PA IgG hypotype in the CBA/J mice of rPA/NE immunity.Data are expressed as the ratio of the titre of individual IgG2a, IgG2b and IgG3 to the titre of IgG1.(B) with the anti-PA IgG in the Balb/c mice of multiple rPA bacterin preparation inoculation.The result be expressed as the meansigma methods of the anti-PA IgG of individual serum terminal point titre+/-sem.(*) titre of rPA/NE inoculation acquisition and the significant difference (p＜0.05) between the antibody titer in other groups are used in expression.(B illustration) uses the record of the anti-PA IgE in the rPA/Alu mice immunized.The immune dot blotting of the dilution of the 1:10-1:80 of the pooled serum of rPA/NE (1), rPA/MPLA (2), rPA/CpG (3), contrast (4) and rPA/Alu (5) mice immunized of using by oneself.

Fig. 9 shows anti-PA IgA and the IgG antibody in the bronchial perfusate.Anti-PA IgA (A) and anti-PA IgG (B) that the ELISA of the bronchoalveolar lavage fluid (BAL) of the Balb/c mice by the multiple bacterin preparation inoculation of using by oneself measures.Anti-PA IgA and anti-PA IgG antibody be expressed as the meansigma methods of antibody concentration+/-sem.

Lethal toxin (LeTx) neutralization that Figure 10 display body is outer.With handling the RAW264.7 cell with the anthrax LeTx of the serial dilution thing precincubation of Balb/c serum immunity, blended.Mark bar (Bar) represents that wherein cell keeps 50% survival rate (NC ₅₀) antibody dilution.

The PA specificity of the outer splenocyte propagation of Figure 11 display body is induced.Stimulate from the isolating splenocyte of mice immunized 72 hours with rPA (5 μ g/ml).Proliferation index is calculated as the active ratio of the activity of rPA stimulated cells to the tranquillization splenocyte.Significant difference between (*) the expression group (p＜0.05).

Figure 12 shows immunoreation and the survival rate with the Cavia porcellus of rPA/NE vaccine intranasal immunity.With the vaccine of 2 dosage (as by indicated the 1st and the 4th day of arrow) inoculation Hartley Cavia porcellus.(A) the anti-PA IgG in the guinea pig serum.The antiserum PA IgG that measures by ELISA by the interval in 3 to 4 weeks measure antibody titer (average terminal point titre+/-sem).(B) Intradermal is attacked.At 6th month, with 1000 x LD ₅₀Ames spore subcutaneous injection Cavia porcellus, the monitoring mortality rate, carried out 14 days.For inoculation and control animal (B illustration), before attack, carry out LeTx neutralization the 22nd week.Determine that according to the cell survival rate that obtains at least twice mensuration (each time is to carry out in triplicate) wherein the RAW264 cell keeps 50% survival rate (NC ₅₀) antibody titer.(*) expression with do not inoculate animal and compare statistically-significant difference (p＜0.001).

Figure 13 shows with immunoreation of the intranasally inoculated Cavia porcellus of rPA/NE vaccine and intranasal attack.The 1st day and the 4th all Hartley Cavia porcelluss that inoculates.(A) anti-PA IgG and the LeTx NAT in the serum.Determine the titre of antibody in the 3rd and the 6th week, and be expressed as the meansigma methods of the anti-PA IgG of individual serum terminal point titre+/-sem.Carry out before attack among the LeTx and measure cell, numerical value represents wherein that the RAW264 cell keeps 50% survival rate (NC ₅₀) average titer.Survival rate curve after (B and C) intranasal is attacked.In the 7th week, pass through 10LD ₅₀(B) and 100 x LD ₅₀The intranasal of Ames spore (C) instils and infects Cavia porcellus, and the monitoring animal reaches 16 days.(*) expression is compared p between all inoculation group＜0.05 with nonvaccinated animal.

Figure 14 shows with the antibody response in the reorganization gp120 of two kinds of serotypes and the intranasally inoculated mice of nano-emulsion adjuvant.(A) use and 0.1%, 0.5% and the blended gp120 of 1%NE _BaLThe anti-gp120 of serum in the mice immunized _BaLIgG induces.Measure anti-gp120 in the 6th week (behind two dosage) and the 12nd week (behind three dosage) _BaLIgG antibody.The intranasal (i.n.) and intramuscular (i.m.) approach of indication immunity among the figure.(B) with the gp120 among the 1%NE among independent 1% NE of two dosage or that add CpG or MPL A _SF162Anti-gp120 in the intranasal immune mouse _SF162IgG induces.Measure anti-gp120 IgG antibody in the 6th week.Anti-gp120 antibody horizontal be expressed as terminal point titre in the serum of individual animals (+/-s.d.) meansigma methods.The cross reactivity of anti-gp120 antibody.Come personal (C) gp120 _BaLOr (D) gp120 _SF162The serum IgG of mice immunized and antigenic autoserum type and foreign serum type all react.Data are expressed as uses antigenic gp120 _SF162Or 120 _BaLThe titration curve of the blended anti-gp120 serum on the flat board of serotype bag quilt.

Figure 15 shows the nose immune induction mucosa IgA that uses gp120/NE.Use gp120 _BaLWith in (A) bronchial perfusate (BAL) neutralization (B) serum of the mice of NE adjuvant inoculation and the anti-gp120 IgA of secreted in the vaginal lotion.The concentration of anti-gp120 IgA be expressed as the average absorbance that obtains among the ELISA that the serum (B) of the BAL liquid (A) with 1:2 dilution, undiluted vaginal lotion and 1:50 dilution carries out (OD 405nm+/-s.d.).Between gp120/ saline solution and each gp120/NE group, observe statistically-significant difference (p＜0.05).

Figure 16 shows (A) antigenic specificity splenocyte propagation.With 2 μ g/ml from body weight group gp120 _BaLStimulated in vitro is from the splenocyte of the animal of immunity.Cell proliferation carried out standardization according to contrast and be expressed as the meansigma methods of individual proliferation index+/-s.d.Gp120 _BaL/ saline solution and gp120 _BaLDifference between the/NE group is statistics significant (p＜0.05).(B) antigenic specificity that cytokine is produced in the external splenocyte activates.Activate splenocyte with 2 μ g/ml from body and heterologous serotype gp120 (being respectively BaL and SF162) with the V3 cyclic peptide of 20 μ M from mice immunized.By the IFN-γ that ELISA measure to discharge, concentration represent the meansigma methods of individual sample+/-s.d.

Figure 17 shows the nose immunity of (A) Cavia porcellus.Initially-strengthened scheme in, with 50 μ g gp120 among the 1%NE _SF162Inoculation Hartly Cavia porcellus (GP).Measure gp120 in the 6th week _SF162With _BaLThe serum IgG antibody reaction of serotype.Anti-gp120 IgG is expressed as the serum of the dilution of using 1:200 and uses antigenic gp120 _SF162Gp (from body) and 120 _BaLThe absorption value that obtains among the ELISA that the flat board of (allogenic) serotype bag quilt carries out (OD405nm ,+/-s.d.).(B) by using gp120 _SF162The neutralizing antibody that the intranasal immunity of/NE produces.In the TZM-BL cell system, carry out HIV laboratory strains system and former generation separated strain neutralization.NT ₅₀Value representation is compared with virus control, and flat light emission (RLU) reduces by 50% o'clock serum dilution relatively.Serum is estimated non-specific antiviral activity before using individual immunity.

The general introduction of invention

The invention provides the method and composition for immune response stimulating. Especially, this Brightly provide inducing anti-disease substance (for example, cowpox disease in experimenter (for example people experimenter) Poison, anthrax spore bacillus, HIV etc.) immune response method and be used for the combination of the method Thing (for example, comprises nanometer breast by the pathogen of this nanoemulsion deactivation or its immune originality part Agent). The compositions and methods of the invention are used for clinical (for example, therapeutic and preventative medical science (example As, inoculation)) and the research application, and other. In some embodiments, will before using Pathogen is mixed the time that is enough to make the pathogen inactivation with nanoemulsion. In other embodiments, Will be from protein component (for example, protein separation or purifying or the restructuring egg of pathogen Mix with nanoemulsion in vain).

Although be not to implement essential to the invention and the present invention is not subject to the understanding of mechanism Any specific effect mechanism, but in some embodiments, NE processes (for example, with this Among the bright NE and pathogen) protection important antigen epi-position (for example, can be by experimenter's immunity System identification), in the oil of emulsion and hydrophilic interface, stablize their hydrophobicity and hydrophilic component (for example, thus provide one or more experimenters to produce the immune former of immune response for it (for example, stable antigen)). In other embodiments, because the NE preparation penetrates by the hole Mucous membrane, thus they with the former dendritic cells that are transported to mucous membrane lower floor location of immunity (for example, thereby Initial and/or immune response stimulating). Although the understanding to mechanism is not that enforcement the present invention institute is essential And the present invention be not subject to any specific effect mechanism, but in some embodiments, group Close NE and immune originality albumen (for example, from the rPA of anthrax spore bacillus or from HIV's Gp120 etc.) stable immunity is former, and is provided for producing the suitable immune originality material of immune response Material.

Dendritic cells are engulfed NE oil greedyly and are dripped, and this provides the internalization immunity former (for example, antigen The property albumen or its fragments of peptides) be used for the method for antigen submission. Although other vaccines depend on inflammation Toxin or other immunity stimulate the activity that is used for adjuvant (referring to, for example, Holmgren and Czerkinsky, Nature Med.2005,11; 45-53), but when to the animal and human Research in when placing on skin or the mucous membrane, NE had not demonstrated inflammation. Therefore, although to machine The understanding of system is not to implement essential to the invention and the present invention is not subject to any specific effect Mechanism, but in some embodiments, the composition that comprises NE of the present invention (for example comprises Former (for example, the NE pathogen that activates (for example, the virus (for example, VV)) of NE and immunity Composition) (for example, transhipment and/or submission immunity are former (for example, to can be used as " physics " adjuvant Cowpox albumen) to immune system). In some preferred embodiments, composition of the present invention Mucosal administration produce mucous membrane (for example, the signal of mucosal immunity (for example, IgA antibody titre Generation) and general immunity.

Cell and body fluid immunity all play an important role in the protection of anti-multiple pathogens, and can Use NE composition of the present invention (for example, to comprise by the pathogen of nanoemulsion deactivation or cause of disease The immune originality part of body) induces both. For example, it is believed that cowpox specificity antibody titre pair In the inoculation people experimenter in and animal model in protective immunity be important (referring to, for example, The people such as Hammarlund, Nat.Med.2003,9; 1131-1137). Several researchs are reflected Decided in and the important protein of the initiation of antibody (referring to, for example, the people such as Galmiche, Virology, 1999,254; 71-80; The people such as Hooper, Virology, 2003,306; 181-195). The smallpox vaccine (Dryvax) that gets the Green Light dilution thing in people volunteer recently Test confirms the formation of warts and generation and the cytotoxic T lymphocyte (CTL) of 2 kinds of specific antibodies The INF-γ t cell responses of inducing and improving strongly relevant (referring to, for example, Greenberg Deng the people, 2005,365; 398-409). The inducing of IFN-γ hinting specific MHC I class-The activation of restrictive CD8+T cell. The cell of these types involves the cell that cowpox infects Identification and removing, and the inoculation after the immunity keep (referring to, for example, the people such as Earl, Nature, 2004; 482; 182-185; The people such as Hammarlund, Nat.Med.2003,9; 1131-1137; The people such as Edghill-Smith, Nature Med.2005,11; 740-747).

Therefore, in some embodiments, the experimenter is used (for example, mucosal administration) originally (for example, the positive poxvirus (for example, VV)) that kills of NE causes body fluid (example the composition of invention As, the generation of specific antibodies) and cell (for example, cytotoxic T lymphocyte) immune response (for example, variola virus) both induce. In some preferred embodiments, the present invention Composition (for example, the positive poxvirus that kills of NE (for example, VV) or NE and one or more Immunity is former) as vaccine (for example, smallpox vaccine, live anthrax vaccine and influenza vaccine etc.).

In addition, in some embodiments, composition of the present invention (for example, comprise NE and The composition that immunity is former) induce (for example, when the experimenter is used) general and mucous membrane to exempt from Epidemic disease both both. Therefore, in some preferred embodiments, the experimenter is used of the present invention Composition causes uprising dew (for example, the lethal mucous membrane exposes) in pathogen (for example, virus (example Protection such as positive poxvirus (for example, VV)). Although the understanding to mechanism is not to implement this Bright necessary and the present invention is not subject to any specific effect mechanism, but mucosal administration (example Such as, inoculation) protection of antipathogen infection (for example, beginning at mucomembranous surface) is provided. Be difficult to stimulate the secretory IgA reaction and resist the cause of disease of invading at mucomembranous surface although proved so far The protection of body (referring to for example, the people such as Mestecky, Mucosal Immunology. the 3rd edition. (Academic Press, San Diego, 2005)), but the invention provides for tested Stimulate mucosal immunity (for example, protectiveness IgA reaction) composition and side from pathogen among the person Method.

In some embodiments, as the composition of mucosal vaccine (for example, the invention provides Comprise NE and the former composition of immunity). Can be easily from the virus of purifying and/or bacterium and/ Protein or the restructuring protein produce this material, described material is induced mucous membrane and general immunity. Produce fast said preparation ability and by mucous membrane instil use said preparation provide can be used for general Inoculation needs and is used for breaking out on a large scale or the vaccine of the emergency that happens suddenly

Definition

In order to help to understand the present invention, define below many terms and phrase:

As used herein, term " microorganism " refers to the microorganism of any species or type, Include but not limited to bacterium, virus, ancient bacterium, fungi, protozoan, mycoplasma, protein disease Poison and parasite. The term microorganism is included in another kind biology (for example, animal (comprising the people) And plant) in and they itself described biology is the microorganism of causing a disease and produces another kind life The reagent that thing causes a disease but itself be not the microorganism of directly causing a disease or infecting to other biological.

As used herein, term " pathogen " and grammer equivalent, be meant that biology (for example, biological reagent), comprise by the direct infection other biological or by being created in the reagent that causes disease in the another kind of biology (for example coming at another kind of biology, animal and plant) causes the microorganism (for example, producing the antibacterial of pathogenicity toxin etc.) of morbid state (for example, infection, pathological state, disease etc.) in." pathogen " includes, but not limited to virus, antibacterial, archeobacteria, fungus, protozoacide, mycoplasma, Protein virus and parasite.

Term " antibacterial " and " bacterium " are meant all prokaryotes, comprise-Prokaryota in all biology.This term is intended to comprise all microorganisms that are considered to antibacterial, comprises Mycoplasma, chlamydiaceae, actinomyces, streptomyces and Dermacentroxenus.This definition comprises the antibacterial of form of ownership, comprising coccus (cocci), bacillus (bacilli), spirillum (spirochete), spheroplast (spheroplast), protoplast etc.

As used herein, term " fungus " is used in reference to eukaryote for example mycete and yeast, comprises dimorphic fungi.

As used herein, unless point out in addition herein, otherwise term " disease " and " pathological state " are used interchangeably, description depart from it is believed that be species or colony (for example, the people) member is normal or average state, described depart under individual harmless condition most of these species or colony deleterious to affected individuality.Departing from like this can show as with any damage or the interference of experimenter's normal condition or change any damage of its organ or tissue of execution of normal function relevant state, sign and/or symptom (for example, diarrhoea, nauseating, fever, pain, vesicle, furuncle (boil), rash, immunosuppressant, inflammation etc.).Disease or pathological state can by or by with microorganism (for example, pathogen or other infectious agent are (for example, virus or antibacterial)) contact and cause, but its response environment factor (for example, malnutrition, industrial hazard and/or weather), the combination that it can respond biological birth defect (for example, genetic abnormality (geneticanomalies)) or respond these and other factors.

As used herein, term " host " or " experimenter " are meant the individuality with the compositions and methods of the invention treatments (for example using).The experimenter includes, but not limited to mammal (for example, Mus, ape, horse, cattle, pig, dog, cat etc.), and most preferably comprises the people.In description of the present invention, term " experimenter " is often referred to and will be applied or be applied one or more compositionss of the present invention individuality of (for example, being used for the induction of immunity reacted composition).

As used herein, term " makes deactivation ", " deactivation " and grammer equivalent, when (for example being used in reference to microorganism, pathogen (for example, antibacterial or virus)) time, be meant and kill, eliminate, neutralize and/or reduce pathological reaction and/or disease are infected and/or cause in microorganism (for example, pathogen (for example, antibacterial or virus)) in the host ability.For example, in some embodiments, the invention provides the compositions of the vaccinia virus (VV) that comprises nano-emulsion (NE) deactivation.Therefore, mentioned as this paper, comprising " VV of NE deactivation ", " V that NE kills ", " the neutral V of NE " or the compositions of grammer equivalent is meant, when the experimenter is used, it is characterized in that VV (for example duplicates among the host, in a period of time (for example, in a couple of days, several weeks, several months or longer time)) the compositions of the existence that does not exist or significantly reduce.

As used herein, term " confluent (fusigenic) " means the Emulsion that can merge with the film of microorganism reagent (microbial agent) (for example, antibacterial or bacterial spore).This paper has described the particular instance of confluent Emulsion.

The Emulsion (for example, nano-emulsion) of the film that as used herein, term " lysogenic " is meant can destroy microorganisms reagent (for example, virus (for example, peplos) or antibacterial or bacterial spore).In a preferred embodiment of the invention, the existence in same combination has produced with arbitrary independent reagent and has compared enhanced deactivation lysogeny with amalgamation reagent.This paper has described the method and composition (for example, being used for induction of immunity reaction (for example, as vaccine)) that uses this improved antimicrobial compositions in detail.

As used herein, term " Emulsion " comprises classical oil-in-water or Water-In-Oil dispersion or droplet, and as the result of hydrophobic force and other lipid conformations that form, when will be with the miscible oil phase of water mixed with water, described hydrophobic force driving non-polar residue (for example, long hydrocarbon chain) is away from water and drive polar head group towards water.These other lipid conformations include but not limited to monolayer, minority layer (paucilamellar) and multiwalled lipid carrier, micelle and layer (lamellar phase) mutually.Similarly, as used herein, term " nano-emulsion " is meant the oil in water dispersion that comprises little lipid conformation.For example, in some embodiments, described nano-emulsion comprise have mean particle size for about 0.1-5 micron (for example, diameter 150+/-oil phase of 25nm) droplet, yet comprise littler and bigger granular size.Term " Emulsion " and " nano-emulsion " are used interchangeably in this article usually, are meant nano-emulsion of the present invention.

As used herein, term " contact ", " contact ", " exposure " and " exposure ", when being used in reference to the microorganism of nano-emulsion and work, be meant one or more nano-emulsioies and microorganism (for example, pathogen) are contacted so that described nano-emulsion deactivation microorganism or pathogenicity reagent (if present).The present invention is not subject to the amount or the kind of the nano-emulsion that is used for bacteria inactivation rate.Be used for multiple nano-emulsion of the present invention and (for example be described in this paper and other places, U.S. Patent application 20020045667 and 20040043041 and United States Patent (USP) sequence number 6,015,832,6,506,803,6,635,676 and 6,559, the nano-emulsion of describing in 189, for all purposes, it all incorporates it into this paper separately by reference).(for example comprise nano-emulsion among the present invention, be enough to be used in deactivation microorganism (for example, inactivation of virus) and (for example, (for example be enough to provide antigenic composition with microorganism, can the induction of immunity reacted composition)) ratio and amount, include but not limited to ratio described herein and amount.

Term " surfactant " is meant any molecule that has very easily water-soluble polar head group and be difficult to water-soluble hydrophobic tail.Term " cationic surfactant " is meant the surfactant with cation head group.Term " anion surfactant " is meant the surfactant with anion head group.

Term " hydrophilic-lipophilic balance (HLB) index " and " HLB index " are the indexes of instigating the chemical constitution of surfactant molecule to be associated with their surface activity.Can pass through for example by Meyers, (referring to, for example, Meyers, Surfactant Science and Technology, VCH Publishers Inc., New York, pp.231-245 (1992)) (incorporating this paper by reference into) multiple empirical equation of describing is calculated the HLB index.As used herein, when suitable, the HLB index of surfactant is at McCutcheon ' s the 1st volume: Emulsifiers and Detergents North American Edition, assignment is given the HLB index of this surfactant in 1996 (the integrating with this paper by reference).For commercial surfactant, the HLB index 0 to about 70 or bigger scope in.The hydrophilic surfactant that has high dissolution and dissolution characteristics in water is high-end numerical range, and as the low side of the low deliquescent surfactant of in water, having of water in oil good solubilizing agent at numerical range.

As used herein, term " interaction enhancer (interactionenhancer) " is meant that work in order to agent and microorganism (for example, with the cell wall of antibacterial (for example, gram-negative bacteria) or with peplos (for example, vaccinia virus peplos)) interactional chemical compound.Included interaction enhancer includes but not limited to, chelating agen (for example ethylenediaminetetraacetic acid (EDTA), ethylenebis (oxygen ethylidene time amino) tetraacethyl (EGTA) etc.) and some biological reagent (for example, bovine serum albumin (BSA) etc.).

Term " buffer " or " buffer agent " cause the material of the variation of solution opposing pH when being meant in adding solution.

Term " Reducing agent " and " electron donor " are to point to the material that second material provides the state of oxidation of the one or more atoms of electronics to reduce by second material.

Term " monovalent salt " is meant that metal (for example, Na, K or Li) wherein has any salt of clean 1+ electric charge (that is, Duo than electronics a proton) in solution.

Term " divalent salts " is meant that metal (for example, Mg, Ca or Sr) wherein has any salt of clean 2+ electric charge in solution.

Term " chelating agen " or " chelating reagent " be meant have a plurality of contain can with any material of the atom of the lone electron pair of metal ion Cheng Jian.

Term " solution " is meant aqueous or non-aqueous mixtures.

As used herein, term " is used for the induction of immunity reacted composition " and is meant the experimenter (is for example being used, 1,2,3 times or more times are (for example, with several weeks, several months or several years interval)) after, in experimenter's moderate stimulation, generation and/or initiation immunoreation (for example, cause producing immunity wholly or in part at the microorganism that can cause disease (for example, pathogen)) compositions.In a preferred embodiment of the invention, described compositions comprises nano-emulsion and immunogen.In other preferred embodiments, comprise nano-emulsion and combinations of immunogens thing and comprise one or more other chemical compounds or reagent, include but not limited to that therapeutic agent, physiology can tolerate liquid, gel, carrier, diluent, adjuvant, excipient, Salicylate, steroid, immunosuppressant, immunostimulant, antibody, cytokine, antibiotic, binding agent, filler, antiseptic, stabilizing agent, emulsifying agent and/or buffer agent.Immunoreation can be congenital (for example, non-specific) immunoreation or posteriority are (for example, acquired) immunoreation is (for example, (for example in the experimenter, reduce infectiousness, sickness rate or mortality rate, pathogenic microbes causes by being exposed to) or the immunoreation of infection prevention, morbidity or dead generation in the experimenter (for example, by expose a pathogenic microbes causes)).Therefore, in some preferred embodiments, to comprise nano-emulsion and the combinations of immunogens thing (is for example used to the experimenter as vaccine, to prevent or (for example to weaken disease, immunity by anti-disease wholly or in part is provided to the experimenter or weakening wholly or in part (for example, suppressing)) to sign, symptom or the patient's condition of disease.

As used herein, term " adjuvant " is meant any material of immune response stimulating (for example, mucosal immunoreaction).Some adjuvants can cause the activation (for example, adjuvant can cause that immunocyte produces and secrete cytokines) of immune cell.The example that can cause the activated adjuvant of immune cell include, but not limited to from the saponin of the bark purification of soapbark (Q.saponaria) tree for example QS21 (use the glycolipid of HPLC fractionated eluting the 21.sup.st peak; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); Poly-(two (carboxylato phenoxy group) phosphonitrile ploy (di (carboxylatophenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA); The derivant of lipopolysaccharide is monophosphoryl lipid A (MPL for example; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyldipeptide (MDP; Ribi) and threonyl-muramyldipeptide (t-MDP; Ribi); OM-174 (the glycosamine disaccharide relevant with lipid A; OM PharmaSA, Meyrin, Switzerland); With leishmania elongation factor (Leishmaniaelongation factor) (the leishmania albumen of purification; Corixa Corporation, Seattle, Wash.).Conventional adjuvant is known in this area, and it comprises, for example, and aluminum phosphate or hydroxide salt (" Alumen ").In some embodiments, compositions of the present invention (for example, is comprised HIV or its immunogenicity epi-position (for example, gp120)) and uses (for example, so that the reaction of Th1 or Th2 type is tended in immunoreation) with one or more adjuvants.

As used herein, term " induction of immunity reaction effective dose " (for example, be used for induction of immunity reacted composition) is meant in experimenter's moderate stimulation, generation and/or causes the required dosage level (for example, when the experimenter is used) of immunoreation.Can use at one or many, use (for example, by identical or different approach) effective dose in application or the administration, effective dose is not intended to be subject to specific formulation or route of administration.

As used herein, term " in that described experimenter is produced under the immunoreactive condition " is meant that any qualitative or quantitative of immunoreation (for example, geneogenous or acquired) induce, produce and/or stimulate.

As used herein, term " immunoreation " is meant the reaction by experimenter's immune system generation.For example, immunoreation comprises, but (for example be not limited to To11 receptor activation, lymph element, cytokine (for example, the Th1 or the Th2 cell type factor) or chemotactic factor) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (for example, CD4+ or CD8+T cell), the detectable change (for example, increasing) in NK cell activation and/or the B cell activation (for example, production of antibodies and/or secretion).Immunoreactive other example comprises that immunogen (for example, antigen (for example, immunogenic polypeptide)) combination and the inducing cytotoxic T lymphocyte (" CTL ") to the MHC molecule reacts, (for example induce the B cell effect, production of antibodies) and/or the auxiliary lymphocyte reaction of T, and/or the antigenic delayed hypersensitivity (DTH) that anti-immunogenic polypeptide is originated reacts, immune cell (for example, the T cell, the B cell (for example, any stage of development (for example, plasma cell)) amplification) (for example, the growth of cell colony) and increase pass through antigenic processing and the submission that antigen presenting cell carries out.Immunoreation can be the immunogen (for example, from the non-autoantigen of microorganism (for example, pathogen) or be identified as the autoantigen of exotic) of exotic at experimenter's immune system recognition.Therefore, be to be understood that, as used herein, " immunoreation " is meant the immunoreation of any kind, (for example include but not limited to the innate immunity reaction, the activation of Toll receptor signal transductory cascade reaction), cell-mediated immunoreation (for example, by the T cell (for example, T cells with antigenic specificity) and the cell-mediated reaction of the immune non-thing opposite sex) and humoral immune reaction (for example, by the cell-mediated reaction of B (for example, by antibody being produced and is secreted into blood plasma, lymph and/or tissue fluid)).Term " immunoreation " to antigen and/or immunogenic reaction (for example means the immune system that comprises the experimenter, to immunogen (for example, pathogen) primary response and as acquired (for example, memory) reaction of the result of self adaptation immunoreation (adaptive immune response)) all aspects of ability.

As used herein, term " immunity " is meant after being exposed to the microorganism (for example, pathogen) that can cause disease, is protected from the infringement (for example, preventing or weaken sign, symptom or the patient's condition of (for example, suppressing) disease) of disease.Immunity can be geneogenous (for example, lack before be exposed to the non-habitual that exists under the antigenic situation (for example, non-acquired) immunoreation) and/or acquired (for example, formerly being exposed to behind the antigen by the cell-mediated immunoreation of B and T (for example show increase to antigenic specificity and reactivity)).

As used herein, term " immunogen " is meant and can causes immunoreactive reagent (for example, microorganism (for example, antibacterial, virus or fungus) or its part (for example, proteantigen (for example gp120 or rPA))) in the experimenter.In preferred embodiments, when with nano-emulsion combined administration of the present invention, immunogen causes the immunity of anti-this immunogen (for example, microorganism (for example, pathogen or pathogen product)).

As used herein, term " pathogen product " is meant any component or the product from pathogen, includes but not limited to polypeptide, peptide, protein, nucleic acid, film fraction and polysaccharide.

As used herein, term " enhanced immunity " is meant and (did not for example use said composition, be used for inducing immunoreactive compositions of the present invention) experimenter's adaptability and/or the level of acquired immunity compare, (for example using compositions, be used to induce immunoreactive compositions of the present invention) after, among the experimenter to the increase of the level of the adaptability of given immunogen (for example, microorganism (for example, pathogen)) and/or acquired immunity.

As used herein, term " purification " or " purification " are meant removes pollutant or undesired chemical compound from sample or compositions.As used herein, term " purification substantially " is meant from sample or compositions and removes about 70-90%, at the most 100% pollutant or undesired chemical compound.

As used herein, term administering " and " administration " be meant the behavior that compositions of the present invention (for example, being used for induction of immunity reacted composition (for example, comprising nano-emulsion and combinations of immunogens thing)) is given the experimenter.The exemplary approach that human body is used comprises, but be not limited to, by eyes (through eye), mouth (per os), skin (endermic), nose (per nasal), lung (suction), oral mucosa (through the oral cavity), ear, rectum, by injection (for example, intravenous ground, hypodermically, intraperitoneal ground etc.), part etc.

As used herein, term " is used " altogether and " altogether administration " is meant at least two kinds of reagent (for example, comprising nano-emulsion and immunogen and one or more other reagent-for example, adjuvant) or treatment are applied to the experimenter.In some embodiments, using altogether simultaneously of two or more reagent or treatment taken place.In other embodiments, before second reagent/treatment, use first reagent/treatment.In some embodiments, can use altogether by identical or different route of administration.Preparation of using and/or approach that those skilled in the art understand employed plurality of reagents or treatment can change.Can easily be identified for the proper dosage used altogether by those skilled in the art.In some embodiments, when using altogether reagent or when treatment, to use each reagent or treatment than being suitable for the lower dosage of dosage that they use separately.Therefore, the using altogether of reagent or treatment therein reduce potential deleterious (for example, deleterious) in the embodiment of the required dosage of reagent, and/or when using altogether of two or more reagent cause the experimenter by with other reagent use beneficial effect sensitization to a kind of reagent wherein altogether the time, need use altogether especially.In other embodiments, use altogether preferably and (for example in the experimenter, simultaneously or almost simultaneously cause two or more different immunogens, microorganism (for example, pathogen)) immunoreation (for example, when experimenter can not carry out subsequently the using when reacting of second, third or more compositionss) with induction of immunity.

As used herein, term " partly " to skin surface and/or mucomembranous cell and tissue (for example is meant, alveolar, oral cavity, tongue, chew its hetero-organization and the cell of usefulness (masticatory), vagina or nasal mucosa and covering hollow organ or body cavity internal layer) use compositions of the present invention (for example, comprising nano-emulsion and combinations of immunogens thing).

In some embodiments, but use compositions of the present invention with the form of local Emulsion, Injectable composition absorbent solution etc.When approach when being partial, described form can be for example spray (for example, nasal mist), emulsion or other viscous solutions (for example, comprise in the Polyethylene Glycol nano-emulsion and combinations of immunogens thing).

As used herein, term " pharmaceutically acceptable " or " pharmacology is last acceptable " are meant when the experimenter is used, and do not produce the compositions of adverse reaction (for example, toxicity, anaphylaxis or immunological response) basically.

As used herein, term " pharmaceutically acceptable carrier " is meant the arbitrary standards pharmaceutical carrier, comprise, but (for example be not limited to phosphate buffered saline(PBS), water and dissimilar wetting agent, sodium lauryl sulfate), any and all solvents, disperse medium, coating, sodium lauryl sulfate, etc. blend absorption delay agent, disintegrating agent (for example, potato starch or primojel), Polyethylene Glycol etc.Described compositions also can comprise stabilizing agent and antiseptic.The example of carrier, stabilizing agent and adjuvant be described and in this area be known (referring to for example, Martin, Remington ' s Pharmaceutical Sciences, the 15th edition, Mack Publ.Co., Easton, Pa. (1975) incorporate this paper by reference into).

As used herein, term " pharmaceutically acceptable salt " is meant the salt arbitrarily (for example, by obtaining with acid or alkali reaction) of the compositions of the present invention that can tolerate on the physiology in the target experimenter.Can obtain " salt " of compositions of the present invention from inorganic or organic bronsted lowry acids and bases bronsted lowry.The example of acid comprises, but be not limited to hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic, lactic acid, salicylic acid, succinic acid, p-methyl benzenesulfonic acid (toluene-p-sulfonic), tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethyl sulfonic acid, formic acid, benzoic acid, malonic acid, sulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid etc.Other acid, oxalic acid for example, although their this in pharmaceutically being unacceptable, can be used for preparing in obtaining compositions of the present invention as the salt of intermediate product and their pharmaceutically-acceptable acid addition.

The example of alkali includes but not limited to alkali metal (for example, sodium) hydroxide, alkaline-earth metal (for example, magnesium) hydroxide, ammonium and formula NW ₄ ⁺Chemical compound, wherein W is C _1-4Alkyl, or the like.

The example of salt includes but not limited to: acetate, adipic acid salt, alginate, aspartate, benzoate, benzene sulfonate, bisulphate, butyrate, citrate, camphorate, camsilate, cyclopentane propionate, diglucoside salt, lauryl sulfate, esilate, fumarate, flucoheptanoate, phosphoglycerol, Hemisulphate, enanthate, caproate, chloride, bromide, iodide, the 2-isethionate, lactate, maleate, mesylate, the 2-naphthalene sulfonate, nicotinate, oxalates, palmoate, pectate (pectinate), persulfate, phenpropionate, picrate, Pivalate, propionate, succinate, tartrate, rhodanate, toluene fulfonate, undecylate etc.Other examples of salt comprise and suitable cation Na for example ⁺, NH ₄ ⁺And NW ₄ ⁺(wherein W is C _1-4Alkyl) anion of bonded chemical compound of the present invention such as.For therapeutic use, the salt of chemical compound of the present invention is considered to pharmaceutically acceptable.Yet the salt of non-pharmaceutically acceptable bronsted lowry acids and bases bronsted lowry also can be used for for example preparation or the purification of pharmaceutically acceptable chemical compound.

For therapeutic use, the salt of compositions of the present invention is considered to pharmaceutically acceptable.Yet, also can be used for for example pharmaceutically acceptable preparation of compositions or purification as the salt of non-pharmaceutically acceptable bronsted lowry acids and bases bronsted lowry.

As used herein, term " is in the risk of disease " and is meant the experimenter who is easy to experience specified disease.This tendency can be genetic (for example, the specific genetic predisposition that the experience disease for example can hereditary disease), or owing to other factors (for example, environmental condition, be exposed to the hazardous compound that is present in the environment etc.).Therefore, do not wish that the present invention is subject to any specific risk (for example, experimenter can for no other reason than that be exposed to and interact and " being in the risk of disease " with other people), does not wish that the present invention is subject to any specific disease yet.

As used herein, " nose is used (Nasal application) " is meant by nose to enter nasal passage or Dou Tongdao or both application.Can for example carry out this application by drop, spray, mist agent (mist), coating or its mixture that is used for nose or Dou Tongdao.

As used herein, " vaginal application ", be meant use into or by vagina with the contact vaginal mucosa.This application can contact urethra, cervix uteri, fornix (fornix), uterus or other zones around vagina.Can for example carry out this application by drop, spray, mist agent, coating, lubricant or its mixture that is used for vagina or surrounding tissue.

As used herein, term " test kit " is meant any delivery system that is used for delivered substance.At immunogenicity reagent (for example, comprise nano-emulsion and combinations of immunogens thing) context in, this type of delivery system comprises and allows immunogenicity reagent and/or support material (technical instruction etc. that for example, is used for materials used) from the storage of position to another position, the system that transports or send.For example, test kit comprises one or more packing materials (for example, box) that comprise related immune originality reagent (for example, nano-emulsion) and/or support material.As used herein, term " part test kit (fragmented kit) " is meant the delivery system of the container that separates of the portions that comprises two or more each self-contained total reagent constituents.Can be together or dividually container is delivered to the purpose receiver.For example, first container can contain comprise be used for special-purpose comprise nano-emulsion and combinations of immunogens thing, and second container contains second reagent (for example, antibiotic or aerosol apparatus (spray applicator)).In fact, any delivery system of the container that separates that comprises the portions of two or more each self-contained total reagent component is included in the term " part test kit ".On the contrary, " composite reagent box " is meant in single container (for example, the single box of each required component being housed), contains the delivery system of all components of the required immunogenicity reagent of special-purpose.Term " test kit " comprises part and composite reagent box.

Detailed Description Of The Invention

The invention provides the method and composition that is used for immune response stimulating.Especially, the invention provides the immunoreactive method and the compositions that is used for this method (nano-emulsion that for example, comprises antibacterial or virus or its combination) of in experimenter (for example people experimenter), inducing to reagent (for example, antibacterial or virus).The compositions and methods of the invention are used for, and clinical (for example, therapeutic and preventative medical science (for example, inoculation)) and research are used, and other.

Several pathogenic microbes cause infection by the mucomembranous epithelial cell that invests gastrointestinal tract, oropharynx road, respiratory tract or genitourinary tract in being attached to.Some pathogen, for example influenza virus, Bordetella pertussis (Bordetella pertussis) or vibrio cholera (Vibriocholerae), be retained on the mucosal tissue or in it, yet other pathogen, for example, salmonella typhi (Salmonella typhi) or hepatitis A virus have and allow to penetrate into the more mechanism of deep tissues and general diffusion.The specificity of mucosa and nonspecific defense mechanism provide first line protection of anti-two types pathogen.The nonspecific effect device comprises, for example, resident macrophage, antimicrobial peptide, lactoferrin and lysozyme, extreme pH, bile acid, digestive enzyme, mucus, epithelially come off, wash machine-processed (flushing mechanism) (wriggling, ciliary beat, urinate etc.) and from the competition of local flora.Yet; successful pathogen has been evolved out the nonspecific defense that occurs on their positions of infecting of experience and the method for surviving usually; the secreted immune system plays main effect in being protected from the disease that is caused by many antibacterials and viral pathogen, antiviral main effects device is limited to mucomembranous surface.As if for the biology of general diffusion, best immunity needs local and general immunoreation.

The existing form of vaccinia subgroup virus (for example, variola, cowpox, monkeypox, gerbil jird pox (gerbilpox), camel pox and other) vaccine is out-of-date.。For example, the antismallpox vaccine that is used to realize all existing approvals that variola is eradicated is based on from skin of the calf that infects and the vaccinia virus alive (VV) that lymph node obtains.Although these vaccines have been given the long-acting immunity of anti-several different vaccinia subgroup viruses, make their material and do not satisfy the current standard that is used for people's vaccine.In addition, these vaccines also produce infectiousness skin pustule (variola) and rare but serious adverse, thereby limited their purposes in the individuality with immunodeficiency, eczema, atoipc dermatitis or heart disease (contacting) with the tight of them (referring to, for example, Eichner and Schwehm, Epidemiology.2004,15 (3): 258-60).

Expection vaccinia subgroup virus vaccine (for example, variola, cowpox, monkeypox, gerbil jird pox (gerbilpox), camel pox and other) the application in future can be that bio-terrorism is attacked or other vaccinia subgroup viruses infect, the for example reaction that breaks out of monkeypox or vaccinia virus (referring to, for example, people such as Edghill-Smith, Nature Med.2005,11; 740-747).Therefore, the risk/benefit assessment of the inoculation antismallpox vaccine of will looking for novelty is placed on safety the position of high-priority.In addition, in order to be used to the public health situation that happens suddenly, must substitute the vaccine of using by scarification with the vaccine of using fast (for example, mucosal vaccine).Except the protection of inoculation and permanent immunity can be provided, this type of is used can also can provide immunoreactive quick, on-the-spot generator (for example, reducing infectiousness, symptom and/or the course of disease of disease).

Therefore, the compositions that the invention provides the immunoreactive method of evoked response vaccinia subgroup virus (for example, vaccinia virus) in the experimenter (for example, people experimenter) and be used for this method (for example, the nano-emulsion that comprises vaccinia subgroup virus (for example, vaccinia virus (VV))).In preferred embodiments, the method with induction of immunity reaction provided by the invention is used for inoculation.Because the incidence rate of harmful incident of existing vaccinia subgroup virus (for example, variola) vaccine, the present invention provides significantly improving of vaccinia subgroup virus (for example, variola) inoculation safety under the situation that does not weaken efficacy of vaccines.

For example, the invention describes vaccinia subgroup virus in the deactivation of mucosal administration (for example, mucosal vaccination) unique types (for example, VV) behind the prepared product (in development process of the present invention, identify and characterize), the generation of immunity (for example, VV immunity) among the experimenter.VV with highly purified cell culture source mixes with nano-emulsion (NE) (surface activity antimicrobial material), thereby obtain at room temperature stable (for example, in some cases, stable above 2 weeks, more preferably surpassed for 3 weeks, even more preferably surpassed for 4 weeks and most preferably surpassed for 5 weeks) preparation (for example, the VV compositions that NE kills) and be used in and (for example induce anti-vaccinia subgroup virus among the experimenter, VV) immunoreactive preparation (for example, can use individually or be used to induce anti-VV immunoreation) as adjuvant.

Experimenter's mucosal administration is comprised NE and VV, and (for example, the VV that NE kills) compositions produces the mucosa of high titre and general antibody response and specific Th1 cellular immunization (referring to, for example, embodiment 2-4 and 6).In addition, all animals are protected fully and avoid using 10xLD ₅₀The nose instillation attack of VV (referring to, for example, embodiment 7, Fig. 5).In vaccinated animal, infect and to be prevented fully or have only low-level and self restriction, and infected at 4 to 5 days and to disappear.On the contrary, all non-immune animals are dead in this period.Use the VV-of 100 x LD _WRThe attack again that mice immunized is carried out subsequently proved conclusively protective immunity.Use in addition the mice of the compositions that comprises the VV that NE-kills of single dose use produce in 10 to 12 weeks of back significant anti-VV IgG serum-concentration (referring to, for example, embodiment 2).The result who obtains in the Balb/c mice of the reaction of this level and the VV Wyeth immunity of living by intramuscular injection at similar time point quite (referring to, for example, people such as Coulibaly, Virology, 2005; 341; 91-101).Therefore, in some embodiments, the invention provides: the compositions that single administration (for example, mucosal administration) comprises the VV that NE kills is enough to be subjected to examination to induce protective immunological reaction (for example, protective immunity (for example, mucosa and general immunity)) in advancing.In some embodiments, experimenter use subsequently (for example, the enhancing of the one or many after using is for the first time used) induced the enhanced immunoreation at VV in the experimenter.Therefore, the present invention proof is used the protective immunity that the compositions that comprises the VV that NE kills provides variola to the experimenter.

On the contrary, the intranasal that has or do not have the VV that the formalin of nano-emulsion kills instils and produces inconsistent and low antibody response, this antibody response in addition after immunity for the third time, still do not strengthen (referring to, for example, embodiment 2).Detect the active pattern of similar neutralization in serum and bronchoalveolar lavage fluid (BAL), neutralization is active not to be existed in the mice of the viral mucosal vaccination of killing with formalin yet.It is active not detect neutralization in the BAL of the animal of the inoculation by IP or SQ injection live virus.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action; but in some embodiments; NE (for example handles; with among the NE of the present invention and vaccinia subgroup virus (for example; VV)) (for example protected important viral neutralizing epitope; can be by experimenter's immune system recognition), the hydrophobic and hydrophilic component of in the oil of Emulsion and water interface, stablizing them (for example; thereby provide one or more experimenters to start immunoreactive immunogen (for example, stable antigen)) to it.In other embodiments, because known NE preparation passes mucosa by aperture, therefore, they can be transported to virus protein the localized dendritic cell of tela submucosa (for example, thereby cause and/or immune response stimulating).

Dendritic cell are wolfishly engulfed the NE oil droplet, and this can provide the internalization immunogenic protein to be used for the method for antigen presentation.Although other vaccines depend on the inflammatory toxin or be used for other immunostimulations of adjuvanticity (referring to, for example, Holmgren and Czerkinsky, Nature Med.2005,11; 45-53), but when placing on skin or the mucosa in the research to the animal and human, NE had not demonstrated inflammatory.Therefore, although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, (vaccinia subgroup virus that for example comprises the NE deactivation (for example to comprise the compositions of NE of the present invention, VV) compositions) (for example can be used as " physics " adjuvant, transhipment and/or the positive pox antigen of submission (for example, cowpox albumen) are to immune system).In some preferred embodiments, the mucosal administration of compositions of the present invention produces mucosa and general immunity (for example, the signal of mucosal immunity (for example, the generation of IgA antibody titer)).

Cell and humoral immunization all work in the protection of anti-vaccinia subgroup virus, use the NE preparation to induce these two kinds of immunity (referring to, for example, embodiment 2-4 and 6).It is believed that cowpox specific antibody titre among the people experimenter who estimates inoculation and the protective immunity in the zootype be important (referring to, for example, people such as Hammarlund, Nat.Med.2003,9; 1131-1137).Several research identified initiation important protein matter for neutralizing antibody (referring to, for example, people such as Galmiche, Virology, 1999,254; 71-80; People such as Hooper, Virology, 2003,306; 181-195).The INF-γ t cell responses of inducing and improving of the formation that the nearest test of the antismallpox vaccine that gets the Green Light (Dryvax) dilution in people volunteer confirms pustule and the generation of 2 kinds of specific antibodies and cytotoxic T cell (CTL) relevant strongly (referring to, for example, people such as Greenberg, 2005,365; 398-409).IFN-γ induces the activation that is hinting the specific restrictive CD8+T cell of MHCI class.The identification and the removing of the cell that the cowpox that involves the cell of these types infects, and immune the keeping in inoculation back (referring to, for example, people such as Earl, Nature, 2004; 482; 182-185; People such as Hammarlund, Nat.Med.2003,9; 1131-1137; People such as Edghill-Smith, NatureMed.2005,11; 740-747).

Therefore, in some embodiments, the experimenter (is for example used, mucosal administration) compositions of the present invention (for example, the vaccinia subgroup virus that NE kills (for example, VV)) causes body fluid (for example, the generation of specific antibodies) and cell (for example, cytotoxic T cell) both induce of immunoreation (for example, variola virus).In some preferred embodiments, (for example, the vaccinia subgroup virus (for example, VV)) that kills of NE is as antismallpox vaccine for compositions of the present invention.

In addition, in some embodiments, (for example, the vaccinia subgroup virus (for example, VV)) that kills of NE is induced (for example, when the experimenter is used) general and mucosal immunity to compositions of the present invention.Therefore, in some preferred embodiments, the experimenter is used compositions of the present invention cause uprising dew (for example, the lethal mucosa exposes) in the protection of vaccinia subgroup virus (for example, VV)).Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, mucosal administration (for example, inoculation) provides disease-resistant vaccinia subgroup virus (for example, VV) to infect the protection of (for example, beginning at mucomembranous surface).Although proved that so far the protection that is difficult to stimulate the secretory IgA, sIgA reaction and resists the pathogen of invading at mucomembranous surface is (referring to for example; people such as Mestecky; Mucosal Immunology. the 3rd edition. (Academic Press; San Diego; 2005)); be used in compositions and the method for experimenter's moderate stimulation but the invention provides from the mucosal immunity (for example, protectiveness IgA reaction) of pathogen.

In some embodiments, the invention provides compositions as mucosal vaccine (for example, the vaccinia subgroup virus of NE deactivation (for example, VV) preparation).Can be easily from the virus of purification produce this material (referring to, for example, embodiment 1), described material is induced mucosa and general immunity (referring to, for example, embodiment 2-7).Produce fast the ability of said preparation and instil and use said preparation and provide and can be used for breaking out on a large scale or the vaccine of the emergency that happens suddenly by mucosa

In some embodiments, the invention provides the method that is used to produce immunoreactive compositions and uses said composition (for example, to be used as vaccine).In some preferred embodiments, be used to produce immunoreactive compositions and comprise NE and immunogen (for example, by the vaccinia subgroup virus of nano-emulsion deactivation (for example, VV)).When the experimenter was used, compositions of the present invention was in (for example, the immunoreation of vaccinia subgroup virus (for example, VV)) of the anti-immunogen of experimenter's moderate stimulation.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, immunoreactive generation (for example, (for example comprise nano-emulsion and vaccinia subgroup virus by using, VV) compositions causes) generation provide to the experimenter wholly or in part the immunity (for example, sign, disease or the patient's condition according to disease (for example, variola)).Be not subjected to the constraint of any particular theory, (for example comprise vaccinia subgroup virus being exposed to, VV) behind the nano-emulsion at the protection of disease and/or immunity (for example, experimenter's immune system is prevented or (is for example weakened, inhibition) sign of disease, the ability of the symptom or the patient's condition) (for example owing to adaptability, acquired) immunoreation is (for example, of the present inventionly (for example comprise vaccinia subgroup virus being exposed to, VV) behind the NE, by the cell-mediated immunoreation of B and T (for example, show increase to vaccinia subgroup virus (for example, specificity VV) and reactive immunoreation)).

In some embodiments, use individually and comprise immunogen (for example, by the NE of the vaccinia subgroup virus of NE deactivation (for example, VV)).In some embodiments, comprise NE and immunogen and (for example, comprised one or more other reagent (for example, pharmaceutically acceptable carrier, adjuvant, excipient etc.) by the compositions of the vaccinia subgroup virus of NE deactivation (for example, VV)).In some embodiments, be used to stimulate immunoreactive compositions of the present invention to induce the immunoreactive mode of body fluid to use.In some embodiments, with inducing cell (for example, cytotoxic T cell) immunoreation but not the mode of humoral response use and be used to stimulate combinations of immunogens thing of the present invention.In some embodiments, comprise NE and combinations of immunogens thing inducing cell of the present invention and humoral immune reaction.

The type of the present invention is not subject to employed (for example, using in comprising immunogenic immunogenic composition) NE.In fact, multiple NE compositions can be used for the present invention.

For example, in some embodiments, nano-emulsion (for example, is used for vaccinia subgroup virus (for example, deactivation VV)) and comprises (i) water; (ii) oil phase; With at least a other chemical compound.In some embodiments of the present invention, these other compound is gone into the water or the oil phase of compositions.In other embodiments, these other compound is gone into to have before the compositions of emulsive oil and water.In certain embodiments, before be about to using, one or more other compound is gone into existing emulsion composition.In other embodiments, before being about to use compositions, one or more other compound is gone into existing emulsification composition.

Other chemical compounds that are suitable for nano-emulsion of the present invention include but not limited to one or more Organic substances, more particularly, based on solvent, surfactant and the detergent of organic phosphate, the chemical compound of cation halogen (cationic halogen containingcompound), sprouting reinforcing agent, interaction reinforcing agent, food additive (for example, flavoring agent, sweetener, extender etc.) and pharmaceutically acceptable chemical compound.Be provided for some exemplary of all cpds of compositions of the present invention below.

Several pathogenic microbes cause infection by the mucomembranous epithelial cell that is attached to built-in gastrointestinal tract, oropharynx road, respiratory tract or genitourinary tract.Some pathogen, for example influenza virus, Bordetella pertussis or vibrio cholera are retained on the mucosal tissue or in it, yet other pathogen, for example, salmonella typhi or hepatitis A virus have and allow to penetrate into the more mechanism of deep tissues and general diffusion.The specificity of mucosa and nonspecific defense mechanism provide first line protection of anti-two types pathogen.The nonspecific effect device comprises resident macrophage, antimicrobial peptide, lactoferrin and lysozyme, extreme pH, bile acid, digestive enzyme, mucus, epithelially comes off, wash machine-processed (wriggling, ciliary beat, urinate etc.) and from the competition of local flora.Yet; successful pathogen has been evolved out the nonspecific defense that occurs on their positions of infecting of experience and the method for surviving usually; the secreted immune system plays main effect in being protected from the disease that is caused by many antibacterials and viral pathogen, antiviral main effects device is limited to mucomembranous surface.For the biology of general diffusion, best immunity needs local and general immunoreation.

Anthrax is the infective bacterial diseases that is caused by Bacillus anthracis (Bacillis anthracis).It is main to occur in wild and herbivore that raise and train (sheep, goat, camel, Saigae Tataricae, cattle etc.), but also can occur in philtrum.Infection can expose by skin, by ingesting (gastrointestinal anthrax) or taking place by sucking (Pulmonary anthrax).The anthrax of philtrum 95% infects by skin infection and takes place, in agricultural environment, contacts with nonvaccinated, infected animal, or by in industrial environment, handling contaminated animal product (meat, leather, animal skin, hair, Pilus Caprae seu Ovis etc.).

If do not treat, the malignant pustule in about 20% case (Cutaneousanthrax) is fatal, but it can overcome by suitable antimicrobial therapy usually.Inhalational anthrax or gastrointestinal anthrax infect wants how seriously and more be difficult to treatment.Inhalational anthrax causes respiratory shock, is fatal in the case of 90%-100%; Gastrointestinal anthrax causes serious fever, feel sick and and vomiting, in the case of 25%-75%, cause death.

Developed the effective vaccine of anti-anthrax in generation nineteen fifty and nineteen sixty generation in the U.S., vaccine is in 1970 approvals that obtain FDA.

The airborne threat of anthrax still is in topnotch threat in history, because Bacillus anthracis has been accredited as the possible reagent that is used for biological warfare.Yet, only be in history high risk individuality for example needs such as veterinary, livestock management person, fleecer, meatworker consider to accept inoculation, the threat to the army personnel of disposing the probability of biological weapons made septic yanks adopt large-scale anthrax vaccination program in 1997, under this program, be intended to 2,400,000 army personnels in all service sectorss are used the anthrax vaccine.(referring to, for example, Secretaryof Defense, people such as Memorandum for Secretaries of the MilitaryDepartments, May 18,1998, Implementation of the AnthraxVaccination Program for the Total Force).

The anthrax vaccine of unique large-scale production, adsorptivity anthrax vaccine (AnthraxVaccine Adsorbed or AVA, trade name BIOTHRAX), be to have added≤0.02% formaldehyde and 0.0025% benzethonium chloride, be adsorbed to the non-infectious bacteria-free filtrate of attenuated strain of the Bacillus anthracis of aluminium hydroxide (Alumen) adjuvant.(referring to, for example, people such as Friedlander, JAMA, 282 (22): 2104-2106 (1999)).Seeded process is made up of the vaccine of 6 subcutaneous injection 0.5mL dosage in 18 months, strengthens annually to keep immunity.Based on zooscopy and accidental personal data, it is believed that this inoculation provides the immunity with the effective antiaero-sol anthrax attack of 90%-100%.(referring to, for example, people such as Friedlander, the same).

Though AVA has the effect of appropriateness, but employed vaccine strain is (non-proteolytic of Bacillus anthracis, not capsulated mutant, V770-NP1-R) have several disadvantageous features: though it is undergone mutation, but this strain system has kept product spore and complete toxogenic phenotype, in production of vaccine, use complete strain system cause protective antigen (PA) level batch and batch between difference, and comprise PA catabolite and other bacterial products (referring to, for example, Farchaus, J., Deng the people, Applied ﹠amp; Environmental Microbiol., 64 (3): 982-991 (1998)).In addition, report use the side effect that causes from the swelling of common injection site and touch a tender spot to general reaction (uncomfortable, tired, fever, wind and cold) and alopecia, myalgia, confirmed fatigue, toothache and gingiva pain, saliva thickness, the sample dermoreaction of burning, lose weight fast, blackout (blackout) and at least one death.(referring to, for example, Chicago Tribune, Mysterious illnesses strike some gulf vets, Mar.26,1992, p.2; The Washington Post, The Nation in Brief, Sep.29,2000, Section A, p.34).Some people claim the anthracolemus Seedling polluted by Squalene (referring to, for example, Garret, L., Big Battle Over Vaccine:DetractorsSay Immunization for Antrhax Hazardous; Pentagon Says No, TheBeacon Journal (Akron), Sunday Ju1.4,1999, Section B, p.1), its caused hundreds of army personnels refuse the inoculation (referring to, for example, Graham, B., Some inMilitary Fear Anthrax Inoculation Side Effects, The PlainDealer (Cleveland), the 26th volume, 1998, Section:Nationa1, p.6E; Air Force Reserve Pilots Quitting Due to Vaccine, The PlainDealer (Cleveland), Feb.27,1999, Section:Nationa1, p.6A).The grade height has been accepted from the court-martial of military service and is removed from office and do not accept the anthrax vaccine to major's army personnel.(referring to, for example, Eskenazi, M., How Anthrax Causes EarlyRetirement, TIME.com, Mar.31,2000.).

Therefore, exist being used for anti-anthrax and by the huge needs of the modified model compositions of the immunity of the bacterial disease of Bacillus, this modified model compositions produces the immunoreation of anti-Bacillus anthracis effectively.Can prepare this compositions of contamination-free (for example, can produce undesired side effect) ideally, it does not need for a long time inoculation and effectively.

Therefore, (for example the invention provides the experimenter, people experimenter) (for example induces in the antibacterial of Bacillus, Bacillus anthracis) immunoreactive method and the compositions that is used for this method are (for example, (for example comprise Bacillus, Bacillus anthracis) antibacterial or cell component (for example, isolating or the reorganization protein) nano-emulsion).In preferred embodiments, the method with induction of immunity reaction provided by the invention is used for inoculation.(ratio of) harmful incident for example, Bacillus anthracis, the present invention provides significantly improving of bacillus (for example, Bacillus anthracis) inoculation safety under the situation that does not weaken efficacy of vaccines because existing bacillus.

For example, the invention describes with comprising nano-emulsion and (for example from the immunogenic protein of Bacillus anthracis, rPA) mucosal administration (for example for compositions (immunogenic protein that produces and characterize in development process of the present invention), mucosal vaccination) after, the generation (referring to embodiment 8-16) of immunity (for example, Bacillus anthracis immunity) among the experimenter.Nano-emulsion (NE) (surface activity anti-biotic material) is mixed with reorganization protective antigen (rPA); obtain comprising the immunogenic composition of NE and rPA; it is at room temperature stable (for example; in some embodiments; stable above 2 weeks; more preferably surpassed for 3 weeks, even more preferably surpassed for 4 weeks and most preferably surpassed for 5 weeks) and be used in the immunoreation of inducing anti-Bacillus anthracis among the experimenter (for example, can separately or the immunoreation that is used to induce anti-anthrax bud bacillus as adjuvant).

The compositions that experimenter's mucosal administration is comprised NE and rPA cause high titre mucosa and general antibody response and specificity T h1 cellular immunization (referring to, for example, embodiment 11-12,14-16).In addition, come the personal compositions intranasal mice immunized that comprises NE and rPA serum can in and PA to the combination (referring to embodiment 13) of its receptor (ATR receptor).Used the mice of the compositions that comprises NE and rPA of two dosage and the Cavia porcellus of the compositions that comprises NE and rPA of only having used single dosage after using, produced significant anti-rPA IgG serum-concentration (referring to, for example, embodiment 11).In addition, the mice of having used said composition produces mucosal immunoreaction (for example, at rPA IgA antibody) (referring to embodiment 12).

Therefore; in some embodiments; the invention provides; (for example use; mucosal administration) (for example comprises NE and Bacillus anthracis immunogen; rPA) compositions is enough to induce the protective immunological reaction (for example, protective immunity (for example, mucosa and general immunity)) of anti-Bacillus anthracis in the experimenter.In some embodiments, use subsequently (for example, one or many is strengthened using after using for the first time) to the experimenter provides enhanced immunoreactive the inducing to Bacillus anthracis in the experimenter.Therefore, the present invention proof is used the experimenter and is comprised NE and Bacillus anthracis immunogen (for example, compositions rPA) provides the protective immunity (for example, by persistent anti-PA IgG reaction) of anti-anthrax.

On the contrary, independent NE or the NE immunoreation (referring to embodiment 11-13) of instiling and to induce anti-Bacillus anthracis with the intranasal of CpG adjuvant.

In addition, using rPA (for example, in saline solution) does not separately induce significant IgG or IgA antibody to produce in mice.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, combination NE and immunogenic protein (from the rPA of Bacillus anthracis) stablized rPA (referring to embodiment 9) and provided and be used to produce immunoreactive suitable environment.In other embodiments, because known NE preparation passes mucosa by aperture, so they can be transported to immunogenic protein the localized dendritic cell of tela submucosa (for example, thereby cause and/or immune response stimulating).In some embodiments; the antibacterial that is used for deactivation tooth born of the same parents Bacillus as NE (for example; Bacillus anthracis) time; combination antibacterial and NE (have for example protected important immunogenicity epi-position; can be by experimenter's immune system recognition), stablized the oil of Emulsion and in the water interface they hydrophobicity and hydrophilic component (for example; thereby provide one or more anti-experimenters to produce immunoreactive immunogen (for example, stable antigen)) to it.

Dendritic cell are wolfishly engulfed the NE oil droplet, and this can provide the internalization immunogenic protein to be used for the method for antigen presentation.Although other vaccines depend on the inflammatory toxin or be used for other immunostimulations of adjuvanticity (referring to, for example, Holmgren and Czerkinsky, Nature Med.2005,11; 45-53), but when placing on skin or the mucosa in to the research among the animal and human, NE had not demonstrated inflammatory.Therefore, although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, the compositions that comprises NE of the present invention (for example, (for example comprise NE and one or more bacillus albumen, rPA) compositions) can be used as " physics " adjuvant (for example, transhipment and/or submission bacillus albumen are to immune system (for example referring to embodiment 10)).In some preferred embodiments, the mucosal administration of compositions of the present invention produces mucosa and general immunity (for example, the signal of mucosal immunity (for example, the generation of IgA antibody titer)).

Cell and humoral immunization all (for example, Bacillus anthracis work in) the protection, are all induced (referring to, for example, embodiment 11-16) by the NE preparation in anti-bacillus.Therefore, in some embodiments, the experimenter is used (for example mucosal administration) compositions of the present invention cause body fluid (for example, the generation of specific antibodies) and cell (for example, cytotoxic T cell) immunoreation (for example, anti-bacillus is proteic).In some preferred embodiments, compositions of the present invention (for example, comprises NE and bacillus albumen (for example, compositions rPA)) as the anthrax vaccine.

In addition, in preferred embodiments, compositions of the present invention is induced (for example, when the experimenter is used) general and mucosal immunity.Therefore, in some preferred embodiments, the experimenter is used compositions of the present invention cause uprising dew (for example, deadly mucosa exposes) in the protection (referring to embodiment 8 and 9) of Bacillus anthracis.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, mucosal administration (for example, inoculation) provides anti-bacillus to infect (for example, causing at mucomembranous surface) protection.Though proved that so far the protection that is difficult to stimulate the secretory IgA, sIgA reaction and resists the pathogen of invading at mucomembranous surface is (referring to for example; people such as Mestecky; Mucosal Immunology. the 3rd edition. (Academic Press; San Diego; 2005)); be used in compositions and the method for experimenter's moderate stimulation but the invention provides from the mucosal immunity (for example, protectiveness IgA reaction) of pathogen.

In some embodiments, the compositions that the invention provides as mucosal vaccine (for example, comprises NE and Bacillus anthracis immunogen (for example, compositions rPA)).Available NE and recombiant protein easily produce this material (referring to, for example, embodiment 1), this material is induced mucosa and general immunity both (referring to, for example, embodiment 11-16).Produce fast said preparation and provide and can be used for breaking out on a large scale or the vaccine of urgent emergency case by the ability that the per nasal instillation is used said preparation.

In some preferred embodiments, the invention provides and be used to produce immunoreactive compositions, its comprise NE and immunogen (for example purification, isolating or synthetic bacillus albumen or derivatives thereof, variant or analog; Or by the antibacterial of the Bacillus of nano-emulsion deactivation).When the experimenter was used, compositions of the present invention stimulated anti-immunogenic immunoreation in vivo.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, immunoreactive generation (for example, owing to use and comprise that nano-emulsion and the proteic compositions of Recombinant spores bacillus cause) (for example provide immunity wholly or in part to the experimenter, sign, symptom and the patient's condition according to disease (for example, anthrax)).Be not subjected to the constraint of any particular theory; after being exposed to immunogenic composition of the present invention; to the protection of disease and/or immunity (for example; experimenter's immune system is prevented or (is for example weakened; inhibition) ability of sign, symptom or the patient's condition of disease) (for example comes from adaptability; acquired) immunoreation is (for example; be exposed to comprise the proteic NE of Recombinant spores bacillus of the present invention after; by B and the cell-mediated immunoreation (for example, showing specificity and the reactive immunoreation that increases) of T) to bacillus.

In some embodiments, use the NE that comprises immunogen (for example, Recombinant spores bacillus albumen) individually.In some embodiments, the compositions that comprises NE and immunogen (for example, Recombinant spores bacillus albumen) comprises one or more other reagent (for example, pharmaceutically acceptable carrier, adjuvant, excipient etc.).In some embodiments, be used to stimulate immunoreactive compositions of the present invention to induce the immunoreactive mode of body fluid to use.In some embodiments, with inducing cell (for example, cytotoxic T cell) immunoreation but not the mode of humoral response use and stimulate immunoreactive compositions of the present invention.In some embodiments, NE and combinations of immunogens thing inducing cell and the humoral immune reaction of comprising of the present invention.

The present invention is not subject to the employed NE type of (for example, comprising in the immunogenic immunogenic composition).In fact, multiple NE compositions can be used for the present invention.

For example, in some embodiments, nano-emulsion comprises (i) water; (ii) oil phase; With at least a other chemical compound.In some embodiments of the present invention, these other compound is gone into the water or the oil phase of compositions.In other embodiments, these other compound is gone into before in the compositions of emulsive oil phase and water.In some embodiment of these embodiments, one or more other chemical compounds before being about to use, it are mixed into existing emulsion composition.In other embodiments, one or more other chemical compound before being about to use, compositions is being mixed into existing emulsion composition.

Other chemical compounds that are suitable for nano-emulsion of the present invention comprise, but be not limited to, one or more are based on Organic substance, more particularly the chemical compound of the solvent of organic phosphate, surfactant and detergent, cation halogen, sprout reinforcing agent, interaction reinforcing agent, food additive (for example, flavoring agent, sweetener, extender etc.) and pharmaceutically acceptable chemical compound.Show some exemplary that relates to all cpds that is used for compositions of the present invention below.

HIV envelope glycoprotein gp120 is the virus protein that is used to be attached to host cell.Mediate this and adhere to by combining with two surface moleculars of helper T cell and phage (being called among CD4 and two chemokine receptors CCR-4 or the CXCR-5).Gp120 albumen at first is expressed as bigger precursor molecule (gp160), and the cutting of translation back obtains gp120 and gp41 then.Gp120 albumen is by being connected the surface that is retained in virion with the gp41 molecule that inserts viromembrane.

Gp120 albumen is the main target of neutralizing antibody.The proteic immunogenic zone of tool (V3 ring) of gp120 also is this most variable proteinic part.Gp120 albumen also comprises by the epi-position of cytotoxic T cell (CTL) identification.These effector lymphocytes can eliminate the cell of viral infection, thereby have constituted second main antiviral immunity mechanism.Opposite with the target area of neutralizing antibody, the performance between different HIV strain systems of number of C TL epi-position is conservative relatively.For this reason, gp120 and gp160 are considered to be intended to cause the useful antigenicity component in the vaccine of cell-mediated immunoreation (CTL especially).

Developed the gp120 immunity antigen of several types: (this paper is called " viral deutero-gp120 ") to the gp120 of the purification that the tissue culture cells that infect from HIV (1) obtains; (2) gp120 (this paper is called " gp120 and the gp160 in live vector source ") that for example produces in the cell of cowpox or baculovirus infection at recombinant virus; (3) the reorganization gp120 that produces in the mammalian cell (this paper is called " recombinant mammalian gp120 "); (4) represent all of gp120 and gp41 or the reorganization degeneration polypeptide of different piece (this paper is called " reorganization denatured antigen "); (5) represent the peptide (this paper is called " peptide ") of the small fragment of gp120 and gp41.

Generally speaking, each in these immunogenicity antigens has hyperimmunization originality as adjuvant at multiple species.They produced can in and the antibody of the homology separated strain of HIV-1.Neutral level not (substantially) reach that infected philtrum finds in and the level of titre, and in producing immunogenic composition, there are many difficulties, described immunogenic composition produces the immunity to a plurality of strain systems (for example, the strain system except the strain system that immunogenic antibodies is originated) of HIV.

When preparation HIV-1 vaccine, another factor that is difficult to especially overcome is a sequence polymorphism.HIV-1 and HIV-2 are characterised in that to have very high-caliber sequence polymorphism, and this is the most obvious in the gp120 of peplos part.This sequence polymorphism accumulates in the zone that is called hypervariable region.Many people propose to use the vaccine mixture that comprises the antigenicity substance that derives from multiple HIV separated strain, so that the protection of the anti-broad spectrum activity source of infection to be provided.The present invention is suitable for sending and comprises the multiple HIV antigenicity substance that derives from multiple HIV separated strain very much.

Therefore, still need to induce the immunogenic substance of the neutralizing antibody of anti-HIV, the preferred single raw material that uses the neutralizing antibody of inducing anti-broad-spectrum HIV separated strain.In addition, very need can induce to the general of HIV and the material of mucosal immunity because the people one of the surface of the most common HIV of being exposed to be vaginal mucosa.

Therefore, the invention provides and in experimenter (for example people experimenter), induce at the immunoreactive method of HIV and be used for the compositions (nano-emulsion that for example, comprises HIV or HIV component (for example, the HIV albumen of isolating or reorganization)) of this method.In some embodiments, the method by induction of immunity reaction provided by the invention is used for inoculation.Because the ratio of harmful incident of existing HIV vaccine under the situation of the effect that does not weaken vaccine, the invention provides significantly improving in the HIV inoculation safety.

For example, the invention describes with comprising nano-emulsion and (for example from the immunogenic protein of HIV, gp120 recombinates) (produce in development process of the present invention and characterize) mucosal administration is (for example, mucosal vaccination) after, the generation (referring to embodiment 17-23) of immunity (for example, HIV immunity) among the experimenter.Nano-emulsion (NE) (surface activity antimicrobial material) is mixed with reorganization gp120 from BaL or SF162 serotype, generation comprises the immunogenic composition of NE and reorganization gp120, it is at room temperature stable (for example, in some embodiments, stable above 2 weeks, more preferably surpassed for 3 weeks, even more preferably surpassed for 4 weeks and most preferably surpassed for 5 weeks) and be used in the immunoreation of inducing anti-HIV among the experimenter (for example, can use individually or be used to induce anti-HIV immunoreation) as adjuvant.

To experimenter's mucosal administration comprise NE and HIV immunogen (for example, reorganization gp120) compositions cause high titre mucosa and general antibody response and produce Th1 type cell immune response (referring to, for example,

embodiment

17,18 and 21).In addition, the antibody of a kind of serotype of the anti-gp120 of generation and other gp120 serotype cross reactions (referring to, for example, embodiment 19).In addition, produce the excretory anti-gp120 specificity IgA antibody of mucosa with the compositions intranasal mice immunized that comprises NE and reorganization gp120, it all can detected (referring to embodiment 20) on the sticking film surface of bronchus and vagina.Therefore, used the mucosal immunoreaction of the mice generation of compositions of the present invention at HIV.Used in the mice that comprises NE and reorganization gp120 the immunoreation that produces also can in and HIV (referring to embodiment 22).

Therefore; in some embodiments; the invention provides; (for example use; mucosal administration) (for example comprises NE and HIV immunogen; reorganization gp120) compositions is enough to induce anti-HIV in the experimenter protective immunological reaction (for example, protective immunity (for example, mucosa and general immunity)).In some embodiments, in the experimenter, provide enhanced immunoreactive inducing to the using subsequently of experimenter (for example, the one or many after initial using is strengthened using) to HIV.Therefore, the present invention's proof is used the protective immunity that the compositions that comprises NE and HIV immunogen (for example, reorganization gp120) provides anti-AIDS to the experimenter.

Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, combination NE and (for example from the HIV immunogen of the serotype of one or more HIV, reorganization gp120) stablize HIV immunogenic (for example, recombinate gp120) and provide and be used to produce immunoreactive suitable material.In other embodiments, because known NE preparation passes mucosa by aperture, so they can be transported to immunogenic protein the localized dendritic cell of tela submucosa (for example, thereby cause and/or immune response stimulating).

Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action; but in some embodiments; NE (for example handles; with among the NE of the present invention and HIV) (for example protected important viral neutralizing epitope; can be by the epi-position of experimenter's immune system recognition), the hydrophobic and hydrophilic component of in the oil of Emulsion and water interface, stablizing them (for example; thereby provide one or more experimenters to start immunoreactive immunogen (for example, stable anti-ization)) to it.In other embodiments, because known NE preparation passes mucosa by aperture, therefore, they can be transported to virus protein the localized dendritic cell of tela submucosa (for example, thereby cause and/or immune response stimulating).

Dendritic cell are wolfishly engulfed the NE oil droplet, and this can provide the internalization immunogenic protein to carry out the method for antigen presentation.Although other vaccines depend on the inflammatory toxin or be used for other immunostimulations of adjuvanticity (referring to, for example, Holmgren and Czerkinsky, Nature Med.2005,11; 45-53), but when placing on skin or the mucosa in the research to the animal and human, NE had not demonstrated inflammatory.Therefore, although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, the compositions that comprises NE of the present invention (for example comprises NE and from the Recombinant HIV albumen of one or more serotypes of HIV (for example, gp120) compositions) (for example can be used as " physics " adjuvant, transhipment and/or submission HIV albumen (for example, gp120) to immune system).In some preferred embodiments, the mucosal administration of compositions of the present invention produces mucosa and general immunity (for example, the signal of mucosal immunity (for example, the generation of IgA antibody titer)).

Cell and humoral immunization all work in the protection of anti-HIV, use the NE preparation to induce these two kinds of immunity (referring to, for example, embodiment 18-22).Therefore in some embodiments, the experimenter used (for example, mucosal administration) compositions of the present invention causes body fluid (for example, the generation of specific antibodies) and cell (for example, cytotoxic T cell) both induce of immunoreation (for example, anti-HIV albumen (gp120)).In some preferred embodiments, compositions of the present invention (for example, comprise NE and from the reorganization gp120 of one or more serotypes of HIV) is as the AIDS vaccine.

In addition, in some embodiments, compositions of the present invention is induced (for example, when the experimenter is used) general and mucosal immunity.Therefore, in some preferred embodiments, the experimenter is used compositions of the present invention cause uprising dew (for example, mucosa exposes) in the protection of HIV.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, mucosal administration (for example, inoculation) provides disease-resistant HIV to infect the protection of (for example, beginning at mucomembranous surface).Although proved that so far the protection that is difficult to stimulate the secretory IgA, sIgA reaction and resists the pathogen of invading at mucomembranous surface is (referring to for example; people such as Mestecky; Mucosal Immunology. the 3rd edition. (Academic Press; SanDiego; 2005)); be used in compositions and the method for experimenter's moderate stimulation but the invention provides from the mucosal immunity (for example, protectiveness IgA reaction) of pathogen.

In some embodiments, the invention provides compositions as mucosal vaccine (for example, comprise NE and from the compositions of the immunogenic protein antigen (gp120) of HIV).Available NE and HIV albumen are (for example, little fragments of peptides, the V3 cyclic peptide (referring to for example embodiment 17) of the gp120 in the gp120 of viral source, live vector source and gp160, recombinant mammalian gp120, reorganization denatured antigen, gp120 and gp41) easily produce this material, this material induce mucosa and general immunity (referring to, for example, embodiment 18-22).Produce fast said preparation and provide and can be used for using on a large scale the vaccine of the crowd of cities and towns, rural area, city, state or country (for example, to) by the ability that mucosa (for example, nose or vagina) instillation is used said preparation.

In some preferred embodiments, the invention provides and be used to produce immunoreactive compositions, its comprise NE and immunogen (for example purification, isolating or synthetic HIV albumen or derivatives thereof, variant or analog; Or by one or more serotypes of the HIV of nano-emulsion deactivation).When the experimenter was used, compositions of the present invention was in the anti-immunogenic immunoreation of experimenter's moderate stimulation.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, immunoreactive generation (for example, owing to use and comprise that nano-emulsion and combinations of immunogens thing cause) (for example provide immunity wholly or in part to the experimenter, according to disease (for example, sign AIDS), symptom or the patient's condition).Be not subjected to the constraint of any particular theory; after being exposed to immunity compositions of the present invention; to the protection of disease and/or immunity (for example; experimenter's immune system is prevented or (is for example weakened; suppress) ability of sign, symptom or the patient's condition of disease) come from adaptability (for example, acquired) immunoreation (for example, be exposed to comprise immunogenic NE of the present invention after; by B and the cell-mediated immunoreation (for example, showing specificity and the reactive immunoreation that increases) of T) to HIV.Therefore, in some embodiments, the preventative or therapeutic of the compositions and methods of the invention ground is used to prevent or weaken the sign relevant with AIDS, symptom or the patient's condition.

In some embodiments, use the NE that comprises immunogen (for example, Recombinant HIV albumen) individually.In some embodiments, the compositions that comprises NE and immunogen (for example, Recombinant HIV albumen) comprises one or more other reagent (for example, pharmaceutically acceptable carrier, adjuvant, excipient etc.).In some embodiments, be used to stimulate immunoreactive compositions of the present invention to induce the immunoreactive mode of body fluid to use.In some embodiments, with inducing cell (for example, cytotoxic T cell) immunoreation but not the mode of humoral response use and be used to stimulate combinations of immunogens thing of the present invention.In some embodiments, comprise NE and combinations of immunogens thing inducing cell of the present invention and humoral immune reaction.

B. pathogen

The invention is not restricted to the purposes of the pathogen of any particular type.In fact, the compositions (for example, comprising NE and immunogen) that is used to produce immunoreation at multiple pathogen (for example, as vaccine) within the scope of the invention.Therefore, in some embodiments, the invention provides and (for example be used to produce the directed toward bacteria pathogen, nutrition or spore form) immunoreactive compositions, described bacterial pathogens includes but not limited to wax shape bacillus (Bacilluscereus), bacillus circulans (Bacillus circulan) and Bacillus megatherium (Bacillus megaterium), Bacillus anthracis (Bacillus anthracis), the antibacterial of Brucella (Brucella), vibrio cholera (Vibrio cholera), Bai Shi rickettsia (Coxiella burnetii), soil draws hot Frances Salmonella (Francisella tularensis), chlamydia psittaci (Chlamydia psittaci), Semen Ricini (Ricinus communis), Rickettsia prowazekii (Rickettsiaprowazekii), the antibacterial of Salmonella (for example, salmonella typhi (S.typhi)), the antibacterial of Shigella (shigella), Cryptosporidium parvum (Cryptosporidiumparvum), melioidosis Burkholderia (Burkholderia pseudomallei), bacillus perfringens (Clostridium Perfringens), bacillus botulinus (Clostridiumbotulinum), vibrio cholera (Vibrio cholerae), streptococcus pyogenes (streptococcus pyogenes), streptococcus agalactiae (Streptococcusagalactiae), streptococcus pneumoniae (Streptococcus pneumonia), golden streptococcus (Staphylococcus aureus), Diplococcus gonorrhoeae (Neisseriagonorrhea), hemophilus influenza (Haemophilus influenzae), escherichia coli (Escherichia coli), Salmonella typhimurium (Salmonella typhimurium), shigella dysenteriae (Shigella dysenteriae), proteus mirabilis (Proteusmirabilis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), bacillus pestis (Yersinia pestis), yersinia enterocolitica (Yersiniaenterocolitica) and yersinia pseudotuberculosis (Yersiniapseudotuberculosis).In other embodiments, the invention provides the immunoreactive compositions that is used to produce at viral pathogen, described pathogen comprises, but be not limited to influenza A virus, avian influenza virus, the H5N1 influenza virus, west nile virus, SARS virus, Marburg virus (Marburg virus), arenavirus (Arenaviruses), Nipah virus, Alphavirus (alphaviruses), Filovirus (filoviruses), herpes simplex virus I, herpes simplex virus I I, Sendai virus, sindbis alphavirus, cowpox, parvovirus (parvovirus), the human immunodeficiency virus, hepatitis B virus, hepatitis C virus, hepatitis A virus, thin giant cell virus, the human papillomavirus, Pironavirus (picornavirus), Hantaan virus, Junin virus (junin virus) and Ebola virus).In other embodiments, the invention provides the immunoreactive compositions that is used to produce at fungal pathogens, described fungal pathogens includes but not limited to that Candida albicans (Candidaalbicnas) and level and smooth candida mycoderma (Candida parapsilosis), Aspergillus fumigatus (Aspergillus fumigatus) and aspergillus niger (Aspergillus niger), Fusarium (Fusarium spp.), trichophyta belong to (Trychophyton spp.).

Can (include but not limited to that American type culture collection (ATCC) obtains to be used for preparation and is used to produce immunoreactive antibacterial of the present invention from commercial source.In some embodiments, with before nano-emulsion mixes, antibacterial is gone down to posterity 5-10 generation to strengthen them for each specific animal reservoir's pathogenicity (people such as Sinai in animal, J.Infect.Dis., 141:193 (1980)). in some embodiments, separate described antibacterial from host animal then, increase, store down at-80 ℃ then by cultivation.Before being about to use, antibacterial is thawed, then overnight incubation on suitable solid bacteria culture medium.Second day, collect antibacterial from agar plate, it is suspended in the suitable liquid solution (for example, brain heart infusion (BHI) meat soup).Based on the McFarland standard that is used for bactericidal assay (Hendrichson and Krenz, 1991), the concentration of adjusting antibacterial is so that count of bacteria about 1.5 x 10 that are every ml ⁸Individual colony-forming units (CFU/ml).

Can obtain to be used to prepare the virus that is used to produce immunoreactive compositions of the present invention from commercial source (including, but are not limited to ATCC).In some embodiments, virus is gone down to posterity 5-10 time to strengthen the pathogenicity (Ginsberg and Johnson, Infect.Immun., 13:1221 (1976)) for specific separately animal place in the animal model of expection.In some embodiments, in tissue culture, collect and propagative viruses, use density gradient centrifugation and ultracentrifugation purified virus (people such as Garlinghouse, Lab Anim Sci., 37:437 (1987) then; And Mahy, Br.Med.Bull., 41:50 (1985)).In suitable tissue culture cells, calculate plaque-forming unit (PFU).

Can use any suitable method, include but not limited to, then based on before publication (people such as Fortier, Infect Immun., 59:2922 (1991) by animal being used the pathogen of (passing through route of infection) various dose; Jacoby, Exp Gerontol., 29:89 (1994); With Salit etal, Can J Microbiol., 30:1022 (1984)) identify that the dosage that causes Animal diseases or death calculates the fatal dose and/or the infective dose of each pathogen.

C. nano-emulsion

Nanoemulsion vaccines compositions of the present invention is not subject to any specific nano-emulsion.Can use many suitable nano-emulsion compositionss in the vaccine combination of the present invention, include, but are not limited to people such as Hamouda, J.Infect Dis., 180:1939 (1999); Hamouda and Baker, J.Appl.Microbiol., 89:397 (2000); With people such as Donovan, Antivir.Chem.Chemother., shown nano-emulsion in disclosed nano-emulsion and table 1 and table 2 and Fig. 4 and 9 among the 11:41 (2000).Preferred nano-emulsion of the present invention is effectively to kill or inactivating pathogens and be nontoxic nano-emulsion for animal.Therefore, preferred emulsion preparations uses innoxious solvent, for example ethanol, kill and wound with obtaining more to have effectively under lower Emulsion concentration, in preferred embodiments, the nano-emulsion that is used for method of the present invention is stable, and after long term store (for example, a year or for many years), do not decompose.In addition, preferred Emulsion in addition be exposed to high temperature and freezing after still keep stable.This they be used for extreme condition (for example, particularly useful in the time of afield), in some embodiments, use a kind of in the nano-emulsion of describing in table 1 and/or Fig. 4 or 9.

In some preferred embodiments, Emulsion comprises (i) water; (ii) oil phase; With at least a other chemical compound, in some embodiments of the present invention, these other compound is gone into the water or the oil phase of compositions.In other embodiments, these other compound is gone into to have before the compositions of emulsive oil and water.In certain embodiments, before be about to using, one or more other compound is gone into existing emulsion composition.In other embodiments, before being about to use compositions, one or more other compound is gone into existing emulsion composition.

Other the chemical compound that is suitable for compositions of the present invention is including but not limited to one or more Organic substances, more particularly, based on the chemical compound of solvent, surfactant and the detergent of organic phosphate, the chemical compound that comprises quaternary ammonium, cation halogen, sprout reinforcing agent, interaction reinforcing agent and pharmaceutically acceptable chemical compound.Show below and relate to some exemplary that is used for the different chemical compounds of compositions of the present invention.

Embodiments more of the present invention are used and are comprised alcoholic acid oil phase.For example, in some embodiments, Emulsion of the present invention comprises (i) water and (ii) and comprise ethanol as the oil phase of organic solvent and optional sprouting reinforcing agent with (iii) make the TYLOXAPOL (preferred 2-5%, more preferably 3%) of surfactant.The said preparation height is antimicrobial effectively, and is nonirritant and avirulent (thereby it can be contacted with mucosa) for the mammal user.

In some other embodiments, Emulsion of the present invention is included in emulsive first Emulsion in second Emulsion, and wherein (a) first Emulsion comprises (i) water; The oil phase that (ii) comprises oil and organic solvent; (iii) surfactant; (b) second Emulsion comprises (i) water; The oil phase that (ii) comprises the chemical compound of oil and cation; (iii) surfactant.

Following description provides many exemplary Emulsions, comprises being used for compositions X8P and X ₈W ₆₀The preparation of PC.X8P comprises water in oil emulsion, and wherein the TRITON X-100 from the water of Semen sojae atricolor, three-just-butyl phosphoric acid ester and 80% produces oil phase.X ₈W ₆₀PC comprises isopyknic X8P and W ₈₀The mixture of 8P.W ₈₀The liposome sample mixture that 8P is made by glyceryl monostearate, refining Generol 122 (for example, GENEROL sterin), TWEEN 60, soybean oil, cation halogen CPC and Oleum menthae.GENEROL family be one group of polyethoxylated Generol 122 (Henkel Corporation, Ambler, Pennsylvania).Provided the emulsion preparations that is used for certain embodiments of the present invention in the table 1.These specific compositions can see United States Patent (USP) 5,700,679 (NN); 5,618,840; 5,549,901 (W ₈₀8P) and 5,547,677, by reference with their whole this paper that incorporate into.

Make X8W ₆₀PC produces W at first respectively ₈₀8P Emulsion and X8P Emulsion.With the mixture reemulsification of these two kinds of Emulsions, be called X8W then thereby produce ₆₀The fresh emulsion composition of PC.The method that produces this type of Emulsion is described in United States Patent (USP) 5,103,497 and 4,895,452 (by integrating with this paper with quoting in full of they).These chemical compounds have broad-spectrum anti-microbial activity, can come deactivation vegetative bacteria (vegetative bacteria) by the destruction of film.

Above listed compositions be exemplary, those skilled in the art can change the amount of component, thereby realize to be fit to the nano-emulsion compositions of the object of the invention.It will be appreciated by those skilled in the art that oil phase can change the ratio of the component of water and indivedual oily carrier, surfactant CPC and organic phosphoric acid salt buffer agent, each compositions.

Although some comprises water that the compositions of X8P has 4:1 to the oil ratio example, be appreciated that X8P can be mixed with and has more or less water.For example, in some embodiments, 3,4,5,6,7,8,9,10 or more part of water to a part in the oil phase.For W ₈₀The 8P preparation also is like this.Similarly, three (N-butyl) phosphate ester: TRITON X-100: the ratio of soybean oil also can change.

Although having listed, table 1 is used for W ₈₀The specified quantitative of the glycerin mono-fatty acid ester of 8P, polysorbate60, GENEROL 122, hexadecylpyridinium chloride and substrate oil (carrier oil), but these only are exemplary.Preparation has W ₈₀The Emulsion of the character of 8P, described Emulsion have each component or the different component in fact in these components of variable concentrations, and it can realize identical functions.For example, Emulsion can have the about 100g glycerin mono-fatty acid ester of about 80-in initial oil phase.In other embodiments, Emulsion can have the about 30g polysorbate60 of about 15-in initial oil phase.In another embodiment, compositions can comprise the GENEROL sterin of the about 30g of about 20-in initial oil phase.

The nano-emulsion structure of some embodiment of Emulsion of the present invention works in their biocidal activity and helps the avirulence of these Emulsions.For example, the active component TRITON-X100 among the X8P shows lower biocidal activity when equaling the concentration of 11%X8P.Oil phase is added to detergent and solvent reduces when the same concentrations toxicity of these reagent in tissue culture significantly.Although (understanding to mechanism is not that enforcement is essential to the invention not to be bound by any theory, and the present invention is not subject to any specific mechanism), the hint nano-emulsion strengthens the interaction of its component and pathogen, thus the deactivation of promotion pathogen and reduce the toxicity of individual components.Should be pointed out that when all components and incorporate into a kind of compositions but not in the nano-emulsion structure time, then mixture is effective not as the antimicrobial acivity when component is in the nano-emulsion structure X8P.

Many other embodiments are provided with type of preparation below with similar compositions.The effect of many these based compositions as anti-biotic material is provided among Fig. 9.Following compositions has been quoted from the multiple ratio and the mixture of active component.Those skilled in the art are to be understood that the preparation of following citation is exemplary, comprise citation component similar percentage ranges other preparations within the scope of the invention.

In certain embodiments of the invention, preparation of the present invention comprise the hexadecylpyridinium chloride (CPC) of the ethanol of the TYLOXAPOL of about 3-8 volume %, about 8 volume %, about 1 volume %, approximately 60-70 volume % oil (for example, soybean oil), water (for example, the DiH of about 15-25 volume % ₂O or PBS) and in some preparations, be lower than the 1NNaOH of about 1 volume %.Some embodiments in these embodiments comprise PBS.Add 1N NaOH and/or PBS in some embodiments of these embodiments and make the user advantageously control the pH of preparation, the scope of pH is about 10.0 at about 4.0-like this, more preferably about 7.1-8.5.For example, one embodiment of the invention comprise the soybean oil of the CPC of the ethanol of the TYLOXAPOL of about 3 volume %, about 8 volume %, about 1 volume %, about 64 volume % and the DiH of about 24 volume % ₂O (this paper is called Y3EC).Another similar embodiment comprises ethanol and the soybean oil of the CPC of about 1 volume %, about 64 volume % and the DiH of about 23.5 volume % of the TYLOXAPOL of about 3.5 volume %, about 8 volume % ₂O (this paper is called Y3.5EC).Another embodiment comprises the CPC of the ethanol of the TYLOXAPOL of about 3 volume %, about 8 volume %, about 1 volume %, the 1N NaOH of about 0.067 volume % (pH of preparation is about 7.1 like this), the soybean oil of about 64 volume % and the DiH of about 23.93 volume % ₂O (this paper is called Y3EC pH 7.1).Another embodiment comprises the CPC of the ethanol of the TYLOXAPOL of about 3 volume %, about 8 volume %, about 1 volume %, the 1N NaOH of about 0.67 volume % (pH of preparation is about 8.5 like this) and the soybean oil of about 64 volume % and the DiH of about 23.33 volume % ₂O (this paper is called Y3EC pH 8.5).Another similar embodiment comprises the ethanol of about 4%TYLOXAPOL, about 8 volume %, about 1%CPC and the soybean oil of about 64 volume % and the DiH of about 23 volume % ₂O (this paper is called Y4EC).Preparation comprises CPC and the soybean oil of about 64 volume % and the DiH of about 19 volume % of about 8%TYLOXAPOL, about 8% ethanol, about 1 volume % in another embodiment ₂O (this paper is called Y8EC).Another embodiment comprises the soybean oil of the CPC of the ethanol of the TYLOXAPOL of about 8 volume %, about 8 volume %, about 1 volume %, about 64 volume % and the 1x PBS (this paper is called Y8EC PBS) of about 19 volume %.

In some embodiments of the present invention, preparation of the present invention comprises the ethanol of about 8 volume % and CPC and the oil (for example, soybean oil) of about 64 volume % and water (for example, the DiH of about 27 volume % of about 1 volume % ₂O or PBS) (this paper is called EC).

In the present invention, some embodiments comprise water (for example, the DiH of the Tributyl phosphate ester (TBP) of the sodium lauryl sulphate (SDS) of about 8 volume %, about 8 volume % and the oil of about 64 volume % (for example, soybean oil) and about 20 volume % ₂O or PBS) (this paper is called S8P).

In certain embodiments of the invention, the oil that preparation of the present invention comprises the hexadecylpyridinium chloride (CPC) of the ethanol of the TYLOXAPOL of the TRITON X-100 of about 1 to 2 volume %, about 1 to 2 volume %, about 7 to 8 volume %, about 1 volume %, about 64 to 57.6 volume % (for example, soybean oil), water (for example, DiH with about 23 volume % ₂O or PBS).In addition, some in these preparations also comprise about 5mM L-alanine/inosine and about 10mM ammonium chloride.In these preparations some comprise PBS.Adding PBS in some embodiments in these embodiments makes the user advantageously control the pH of preparation.For example, one embodiment of the invention comprise the ethanol of the TYLOXAPOL of the TRITON X-100 of about 2 volume %, about 2 volume %, about 8 volume %, the soybean oil of about 1 volume %CPC, about 64 volume % and the water DiH of about 23 volume % ₂O.Preparation comprises soybean oil and the residue 1x PBS (this paper is called 90% X2Y2EC/GE) of the CPC of the ethanol of the TYLOXAPOL of the TRITON X-100 of about 1.8 volume %, about 1.8 volume %, about 7.2 volume %, about 0.9 volume %, about 5mM L-alanine/inosine and about 10mM ammonium chloride, about 57.6 volume % in another embodiment.

In alternative embodiment of the present invention, preparation comprises the DiH of the CPC of the ethanol of the TWEEN 80 of about 5 volume %, about 8 volume %, about 1 volume %, the oil of about 64 volume % (for example, soybean oil) and about 22 volume % ₂(this paper is called W to O ₈₀5EC).

In other embodiments of the present invention, preparation comprises the DiH of the CPC of the ethanol of the TWEEN 20 of about 5 volume %, about 8 volume %, about 1 volume %, the oil of about 64 volume % (for example, soybean oil) and about 22 volume % ₂(this paper is called W to O ₂₀5EC).

In other embodiments of the present invention, the oil that preparation comprises the CPC of the ethanol of the TRITON X-100 of about 2 to 8 volume %, about 8 volume %, about 1 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 15 to 25 volume % ₂O or PBS).For example, the present invention relates to comprise the soybean oil of the ethanol of the TRITONX-100 of about 2 volume %, about 8 volume %, about 64 volume % and the DiH of about 26 volume % ₂The preparation of O (this paper is called X2E).In other similar embodiments, preparation comprises the soybean oil of the ethanol of the TRITON X-100 of about 3 volume %, about 8 volume %, about 64 volume % and the DiH of about 25 volume % ₂O (this paper is called X3E).In other embodiments, preparation comprises about 4 volume %TRITON X-100, the ethanol of about 8 volume %, the soybean oil of about 64 volume % and the DiH of about 24 volume % ₂O (this paper is called X4E).In other embodiments, preparation comprises the soybean oil of the ethanol of the TRITON X-100 of about 5 volume %, about 8 volume %, about 64 volume % and the DiH of about 23 volume % ₂O (this paper is called X5E).Another embodiment of the invention comprises the soybean oil of the ethanol of the TRITON X-100 of about 6 volume %, about 8 volume %, about 64 volume % and the DiH of about 22 volume % ₂O (this paper is called X6E).In other embodiments of the present invention, preparation comprises the soybean oil of the ethanol of the TRITON X-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8E).In other embodiments of the present invention, preparation comprises the olive oil of the ethanol of the TRITON X-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8E O).Another embodiment comprises the TRITON X-100 of 8 volume %, about 8 volume % ethanol, the soybean oil of about 1 volume %CPC, about 64 volume % and the DiH of about 19 volume % ₂O (this paper is called X8EC).

In alternate embodiment of the present invention, the oil that preparation comprises the TYLOXAPOL of the TRITON X-100 of about 1 to 2 volume %, about 1 to 2 volume %, about 6 to 8 volume %TBP, the CPC of about 0.5 to 1.0 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor) and the water of about 1 to 35 volume % (for example, DiH ₂O or PBS).In addition, some preparation in these preparations can comprise the liquid infant formula of the yeast extract of the tryptone meat peptone nutritional solution of about 1 to 5 volume %, about 0.5 to 1.5 volume %, about 5mM L-alanine/inosine, about 10mM ammonium chloride and about 20-40 volume %.Comprise in the embodiment of liquid infant formula at some, this prescription comprises casein hydrolysate (for example, Neutramigen or Progestimil etc.).Some embodiments in these embodiments, preparation of the present invention also comprise the sodium thiosulfate of about 0.1 to 1.0 volume % and the sodium citrate of about 0.1 to 1.0 volume %.Other similar embodiments that comprise these solvents use phosphate buffered saline(PBS) (PBS) as water.For example, an embodiment comprises the TRITONX-100 of about 2 volume %, about 2 volume %TYLOXAPOL, about 8 volume %TBP, the soybean oil of the CPC of about 1 volume %, about 64 volume % and the DiH of about 23 volume % ₂O (this paper is called X2Y2EC).In other embodiments, preparation of the present invention comprises the TRITON X-100 of about 2 volume %, about 2 volume %TYLOXAPOL, about 8 volume %TBP, the sodium thiosulfate of the CPC of about 1 volume %, about 0.9 volume %, the sodium citrate of about 0.1 volume %, the soybean oil of about 64 volume % and the DiH of about 22 volume % ₂O (this paper is called X2Y2PCSTS1).In another similar embodiment, preparation comprises about 1.7 volume %TRITONX-100, about 1.7 volume %TYLOXAPOL, about 6.8 volume %TBP, about 0.85%CPC, the soybean oil of about 29.2%NEUTRAMIGEN, about 54.4 volume % and the DiH of about 4.9 volume % ₂O (this paper is called 85% X2Y2PC/baby).In another embodiment of the invention, preparation comprises the CPC of the TBP of the TYLOXAPOL of the TRITON X-100 of about 1.8 volume %, about 1.8 volume %, about 7.2 volume %, about 0.9 volume %, about 5mM L-alanine/inosine, about 10mM ammonium chloride, the soybean oil of about 57.6 volume % and the 0.1x PBS (this paper is called 90% X2Y2 PC/GE) of residual volume %.In another embodiment, preparation comprises the TYLOXAPOL of the TRITON X-100 of about 1.8 volume %, about 1.8 volume %, the CPC of about 7.2 volume %TBP, about 0.9 volume %, with the soybean oil of the tryptone meat peptone nutritional solution of about 3 volume %, about 57.6 volume % and the DiH of about 27.7 volume % ₂O (this paper is called 90% X2Y2PC/TSB).In another embodiment of the present invention, preparation comprises about 1.8 volume %TRITON X-100, about 1.8 volume %TYLOXAPOL, about 7.2 volume %TBP, about 0.9 volume %CPC, about 1 volume % yeast extract, the soybean oil of about 57.6 volume % and the DiH of about 29.7 volume % ₂O (this paper is called 90% X2Y2PC/YE).

In some embodiments of the present invention, the oil that preparation of the present invention comprises the TBP of the TYLOXAPOL of about 3 volume %, about 8 volume % and the CPC of about 1 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 15 to 30 volume % ₂O or PBS).In specific embodiment of the present invention, preparation of the present invention comprises TBP and the Semen sojae atricolor of the CPC of about 1 volume %, about 64 volume % and the DiH of about 24 volume % of the TYLOXAPOL of about 3 volume %, about 8 volume % ₂O (this paper is called Y3PC).

In some embodiments of the present invention, the oil that preparation of the present invention comprises the TBP of the TRITON X-100 of about 4 to 8 volume %, about 5 to 8 volume %, about 30 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 0 to 30 volume % ₂O or PBS).In addition, some embodiment in these embodiments, the bromination hexadecyldimethyl benzyl ammonium ethyl ammonium, 500 μ M EDTA, about 10mM ammonium chloride, about 5mM inosine and the about 5mM L-alanine that also comprise the chloro-hexadecane yl pyridines of the benzalkonium chloride of the CPC of about 1 volume %, about 1 volume %, about 1 volume %, about 1 volume %.For example, in some embodiment of these embodiments, preparation of the present invention comprises the soybean oil of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8P).In another embodiment of the invention, preparation of the present invention comprises the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, the soybean oil of about 1%ofCPC, about 64 volume % and the DiH of about 19 volume % ₂O (this paper is called X8PC).In another embodiment, preparation comprises the soybean oil of about 8 volume %TRITONX-100, the CPC of the TBP of about 8 volume %, about 1 volume %, about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called ATB-X1001).In another embodiment, preparation comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 2 volume %, about 50 volume % and the DiH of about 32 volume % ₂O (this paper is called ATB-X002).Another embodiment of the invention comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 4 volume %, about 4 volume %, about 0.5 volume %, about 32 volume % and the DiH of about 59.5 volume % ₂O (this paper is called 50% X8PC).Another relevant embodiment comprises the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, the soybean oil of about 0.5 volume %CPC, about 64 volume % and the DiH of about 19.5 volume % ₂(this paper is called X8PC to O _1/2).In some embodiments of the present invention, preparation of the present invention comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 2 volume %, about 64 volume % and the DiH of about 18 volume % ₂O (this paper is called X8PC2).In other embodiments, preparation of the present invention comprises the TRITON X-100 of about 8 volume %, about 8% TBP, about 1% benzalkonium chloride, the soybean oil of about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called X8P BC).In alternate embodiment of the present invention, preparation comprises the soybean oil of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, the bromohexadecane yl pyridines of about 1 volume %, about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called X8P CPB).In another exemplary of the present invention, preparation comprises the soybean oil of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, the bromination hexadecyldimethyl benzyl ammonium ethyl ammonium of about 1 volume %, about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called X8P CTAB).In other embodiments, the present invention comprises the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 1 volume %, soybean oil and the about 15.8 volume %DiH of about 500 μ M EDTA, about 64 volume % ₂O (this paper is called X8PC EDTA).Other similar embodiment comprises the CPC of the TBP of the TRITON X-100 of 8 volume %, about 8 volume %, about 1 volume %, about 10mM ammonium chloride, about 5mM inosine, about 5mM L-alanine, the soybean oil of about 64 volume % and the DiH of about 19 volume % ₂(this paper is called X8PC GE for O or PBS _1x).In another embodiment of the invention, preparation of the present invention also comprises the TRITON X-100 of about 5 volume %, the soybean oil of the CPC of about 5% TBP, about 1 volume %, about 40 volume % and the DiH of about 49 volume % ₂(this paper is called X5P to O ₅C).

In some embodiments of the present invention, preparation of the present invention comprises the soybean oil of the ethanol of the TYLOXAPOL of the TRITON X-100 of about 2 volume %, about 6 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X2Y6E).

In other embodiment of the present invention, preparation comprises the TRITONX-100 of about 8 volume % and the glycerol of about 8 volume %, the oil (for example, Semen sojae atricolor or olive oil) of about 60 to 70 volume % and water (for example, the DiH of about 15 to 25 volume % ₂O or PBS).The embodiment that some is relevant also comprises about 1 volume % L-ascorbic acid really.For example, a specific embodiment comprises the soybean oil of the glycerol of the TRITONX-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8G).In another embodiment, preparation of the present invention comprises the soybean oil of the glycerol of the TRITON X-100 of about 8 volume %, about 8 volume %, the L-ascorbic acid of about 1 volume %, about 64 volume % and the DiH of about 19 volume % ₂(this paper is called X8GV to O _C).

The oil that preparation of the present invention in other embodiments comprises the TBP of the CPC of the TWEEN 60 of the TRITONX-100 of about 8 volume %, about 0.5 to 0.8 volume %, about 0.5 to 2.0 volume %, about 8 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil), water (for example, DiH with about 15 to 25 volume % ₂O or PBS).For example, in a specific embodiment, preparation comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.70 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 18.3 volume % ₂(this paper is called X8W60PC to O ₁).Another relevant embodiment comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.71 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 18.29 volume % ₂(this paper is called W60 to O _0.7X8PC).In other embodiments, preparation of the present invention comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.7 volume %, about 0.5 volume %, about 8 volume %, about 64 to 70 volume % and the DiH of about 18.8 volume % ₂(this paper is called X8W60PC to O ₂).In other embodiments, the present invention comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.71 volume %, about 2 volume %, about 8 volume %, about 64 volume % and the DiH of about 17.3 volume % ₂O.In another embodiment of the invention, preparation comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of about 0.71 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 25.29 volume % ₂(this paper is called W60 to O _0.7PC).

In another embodiment of the invention, preparation of the present invention comprises glycerol or the about 8 volume %TBP of the dioctyl sulfosuccinate of about 2 volume %, about 8 volume %, in addition, the oil of about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 20 to 30 volume % ₂O or PBS).For example, one embodiment of the invention comprise the soybean oil of the glycerol of the dioctyl sulfosuccinate of about 2 volume %, about 8 volume %, about 64 volume % and the DiH of about 26 volume % ₂O (this paper is called D2G).In another related embodiment, preparation of the present invention comprises dioctyl sulfosuccinate and the soybean oil of the TBP of about 8 volume %, about 64 volume % and the DiH of about 26 volume % of about 2 volume % ₂O (this paper is called D2P).

In other embodiments of the present invention, the oil that preparation of the present invention comprises the glycerol of about 8 to 10 volume % and the CPC of about 1 to 10 volume %, about 50 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 15 to 30 volume % ₂O or PBS).In addition, in some embodiment of these embodiments, compositions also comprises the L-ascorbic acid of about 1 volume %.For example, a specific embodiment comprises the soybean oil of the CPC of the glycerol of about 8 volume %, about 1 volume %, about 64 volume % and the DiH of about 27 volume % ₂O (this paper is called GC).Another relevant embodiment comprises the soybean oil of the CPC of the glycerol of about 10 volume %, about 10 volume %, about 60 volume % and the DiH of about 20 volume % ₂O (this paper is called GC10).In another embodiment of the invention, preparation of the present invention comprises the Semen sojae atricolor of the CPC of the glycerol of about 10 volume %, about 1 volume %, the L-ascorbic acid of about 1 volume %, about 64 volume % or the DiH of oil and about 24 volume % ₂(this paper is called GCV to O _C).

In some embodiments of the present invention, the oil that preparation of the present invention comprises the SDS of the glycerol of about 8 to 10 volume %, about 8 to 10 volume %, about 50 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 15 to 30 volume % ₂O or PBS).In addition, in some embodiment of these embodiments, compositions also comprises the lecithin of about 1 volume % and the methyl parahydroxybenzoate of about 1 volume %.The exemplary of this type of reagent comprises the soybean oil of the glycerol of SDS, the 8 volume % of about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called S8G).Related preparations comprises the soybean oil of the methyl parahydroxybenzoate of the lecithin of the SDS of the glycerol of about 8 volume %, about 8 volume %, about 1 volume %, about 1 volume %, about 64 volume % and the DiH of about 18 volume % ₂O (this paper is called S8GL1B1).

In another embodiment of the invention, preparation of the present invention comprises the soybean oil of the ethanol of the CPC of the TYLOXAPOL of the TWEEN 80 of about 4 volume %, about 4 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 19 volume % ₂(this paper is called W to O ₈₀4Y4EC).

In some embodiments of the present invention, preparation of the present invention comprises the soybean oil of the ethanol of the TYLOXAPOL of the CPC of about 0.01 volume %, about 0.08 volume %, about 10 volume %, about 70 volume % and the DiH of about 19.91 volume % ₂O (Y.08EC.01 this paper be called).

In another embodiment of the invention, preparation of the present invention comprises the sodium lauryl sulfate of about 8 volume % and the glycerol of about 8 volume %, the soybean oil of about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called SLS8G).

Above-mentioned specific formulation just illustrates the simple examples of the multiple compositions of using among the present invention.Other nano-emulsioies that the present invention includes many variants of above-mentioned preparation and be used for method of the present invention.In order to determine whether candidate's Emulsion is suitable for the present invention, analyzes 3 standards.Whether by using method described herein and standard, it is suitable to determine them easily to detect candidate's Emulsion.At first, the component that uses method preparation described herein to want is to determine whether forming Emulsion.If can not form Emulsion, discard this material standed for.For example, by 4.5% sodium thiosulfate, 0.5% sodium citrate, 10% n-butyl alcohol, 64% soybean oil and 21% DiH ₂The modifying composition of O preparation does not form Emulsion.

The second, in preferred embodiments, candidate's Emulsion should form stable Emulsion.If it keeps being enough to allow time of its purposes of wanting with the form of Emulsion, then Emulsion is stable.For example, for wait to store, the Emulsion of transportation etc., wish that compositions keeps the several months to the several years with the form of Emulsion.Unsettled relatively typical Emulsion will lose their form in one day.For example, by 8%1-butanols, 5% TWEEN 10,1%CPC, 64% soybean oil and 22%DiH ₂The candidate set compound of O preparation does not form stable Emulsion.By using method described herein to show that following candidate's Emulsion is stable: 0.08% TRITON X-100,0.08% glycerol, 0.01% hexadecylpyridinium chloride, 99% butter and 0.83% diH ₂O (this paper is called 1% X8GC butter); 0.8%TRITON X-100,0.8% glycerol, 0.1% hexadecylpyridinium chloride, 6.4% soybean oil, 1.9%diH ₂O and 90% butter (this paper is called the 10%X8GC butter); 2%W ₂₀5EC, 1%Natrosol250L NF and 97% diH ₂(this paper is called 2% W to O ₂₀5EC L GEL); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% 70 thickness mineral oil and 22% diH ₂(this paper is called W to O ₂₀5EC 70 mineral oil); 1% hexadecylpyridinium chloride, 5%TWEEN 20,8% ethanol, 64% 350 thickness mineral oil and 22% diH ₂(this paper is called W to O ₂₀5EC 350 mineral oil).

The 3rd, candidate's Emulsion should have effect for its purposes of wanting.For example, antibiotic Emulsion should kill or make the pathogen incapacitation to detectable level.As shown in this paper, some Emulsion of the present invention has the effect of anti-specified microorganisms, but does not have the effect of anti-other microorganisms.By using method described herein, people can determine the fitness of the anti-purpose microorganism of particular candidate Emulsion.Usually, this is included in and (for example uses suitable control sample, negative control is water for example) parallel experiment in, microbial exposure is carried out one or more periods in Emulsion, determine then whether Emulsion kills or make the microorganism incapacitation and determine to carry out the degree of this ability.For example, show by 1% ammonium chloride, 5% TWEEN 20,8% ethanol, 64% soybean oil and 22% DiH ₂The candidate set compound of O preparation is not effective Emulsion.Show that with method described herein following candidate's Emulsion is effective Emulsion: 5% TWEEN 20,5% hexadecylpyridinium chloride, 10% glycerol, 60% soybean oil and 20% diH ₂(this paper is called W to O ₂₀5GC5); 1% hexadecylpyridinium chloride, 5% TWEEN 20,10% glycerol, 64% soybean oil and 20% diH ₂(this paper is called W to O ₂₀5GC); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% olive oil and 22%diH ₂(this paper is called W to O ₂₀The 5EC olive oil); 1% hexadecylpyridinium chloride, 5%TWEEN20,8% ethanol, 64% Semen Lini oil and 22% diH ₂(this paper is called W to O ₂₀The 5EC Semen Lini oil); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Semen Maydis oil and 22% diH ₂(this paper is called W to O ₂₀The 5EC Semen Maydis oil); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Oleum Cocois and 22% diH ₂(this paper is called W to O ₂₀The 5EC Oleum Cocois); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Oleum Gossypii semen and 22% diH ₂(this paper is called W to O ₂₀5EC Oleum Gossypii semen); 8% glucose, 5% TWEEN 10,1% hexadecylpyridinium chloride, 64% soybean oil and 22%diH ₂(this paper is called W to O ₂₀The 5C glucose); 8% PEG 200,5% TWEEN10,1% hexadecylpyridinium chloride, 64% soybean oil and 22% diH ₂(this paper is called W to O ₂₀5C PEG200); 8% methanol, 5% TWEEN 10,1% hexadecylpyridinium chloride, 64% soybean oil and 22%diH ₂(this paper is called W to O ₂₀5C methanol); 8% PEG 1000,5% TWEEN 10,1% hexadecylpyridinium chloride, 64% soybean oil and 22% diH ₂(this paper is called W to O ₂₀5C PEG 1000); 2%W ₂₀5EC, 2%Natrosol 250H NF and 96%diH ₂(this paper is called 2%W to O ₂₀5EC Natrosol2 is also referred to as 2%W ₂₀5EC GEL); 2%W ₂₀5EC, 1% Natrosol 250H NF and 97% diH ₂(this paper is called 2%W to O ₂₀5EC Natrosol 1); 2% W ₂₀5EC, 3% Natrosol 250H NF and 95% diH ₂(this paper is called 2% W to O ₂₀5EC Natrosol 3); 2% W ₂₀5EC, 0.5%Natrosol 250H NF and 97.5%diH ₂(this paper is called 2%W to O ₂₀5EC Natrosol 0.5); 2%W ₂₀5EC, 2%Methocel A and 96% diH ₂(this paper is called 2% W to O ₂₀5EC MethocelA); 2%W ₂₀5EC, 2%Methocel K and 96% diH ₂(this paper is called 2%W to O ₂₀5EC MethocelK); 2% Natrosol, 0.1% X8PC, 0.1x PBS, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride and diH ₂O (this paper is called 0.1%X8PC/GE+2%Natrosol); 2%Natrosol, 0.8% TRITON X-100,0.8% Tributyl phosphate ester, 6.4% soybean oil, 0.1% hexadecylpyridinium chloride, 0.1 x PBS, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride and diH ₂O (this paper is called 10% X8PC/GE+2% Natrosol); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Adeps Sus domestica (Lard) and 22% diH ₂(this paper is called W to O ₂₀5EC Adeps Sus domestica); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% mineral oil and 22% diH ₂(this paper is called W to O ₂₀5EC mineral oil); 0.1% hexadecylpyridinium chloride, 2% nerolidol (Nerolidol), 5% TWEEN 20,10% ethanol, 64% soybean oil and 18.9% diH ₂(this paper is called W to O ₂₀5EC _0.1N); 0.1% hexadecylpyridinium chloride, 2% farnesol (Farnesol), 5% TWEEN 20,10% ethanol, 64% soybean oil and 18.9%diH ₂(this paper is called W to O ₂₀5EC _0.1F); 0.1% hexadecylpyridinium chloride, 5% TWEEN 20,10% ethanol, 64% soybean oil and 20.9% diH ₂(this paper is called W to O ₂₀5EC _0.1); 10% hexadecylpyridinium chloride, 8% Tributyl phosphate ester, 8% TRITON X-100,54% soybean oil and 20%diH ₂(this paper is called X8PC to O ₁₀); 5% hexadecylpyridinium chloride, 8%TRITON X-100,8% Tributyl phosphate ester, 59% soybean oil and 20% diH ₂(this paper is called X8PC to O ₅); 0.02% hexadecylpyridinium chloride, 0.1% TWEEN 20,10% ethanol, 70% soybean oil and 19.88%diH ₂(this paper is called W to O ₂₀O.1EC _0.02); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% glycerol, 64% Mobil 1 and 22% diH ₂(this paper is called W to O ₂₀5GC Mobil 1); 7.2%TRITON X-100,7.2% Tributyl phosphate ester, 0.9% hexadecylpyridinium chloride, 57.6% soybean oil, 0.1x PBS, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride and 25.87%diH ₂O (this paper is called 90% X8PC/GE); 7.2% TRITON X-100,7.2% Tributyl phosphate ester, 0.9% hexadecylpyridinium chloride, 57.6% soybean oil, 1% EDTA, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride, 0.1x PBS and diH ₂O (this paper is called 90%X8PC/GEEDTA); With 7.2%TRI TON X-100,7.2% Tributyl phosphate ester, 0.9% hexadecylpyridinium chloride, 57.6% soybean oil, 1% sodium thiosulfate, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride, 0.1x PBS; And diH ₂O (this paper is called 90% X8PC/GE STS).

1. water

In some embodiments, Emulsion comprises water.In some preferred embodiment, it is about 5 to 50 that the cumulative volume of Emulsion emulsion-based (although also relating to other concentration) comprises, preferred 10 to 40, more preferably the water of 15 to 30 volume %.In preferred embodiments, it is about 4 to 10 that water comprises pH, preferably approximately 6 to 8 water.The preferably deionized (" DiH hereinafter of water ₂O ").In some embodiments, water comprises phosphate buffered saline(PBS) (PBS).In some preferred embodiments, water is aseptic and pyrogen-free.

2. oil phase

In some embodiments, Emulsion comprises oil phase.In some preferred embodiment, the cumulative volume (although also relating to other concentration) of the oil phase of Emulsion of the present invention (for example, substrate oil) emulsion-based comprises 30-90, preferably 60-80 and the more preferably oil of 60-70 volume %.Suitable oil includes but not limited to that soybean oil, American Avocado Tree oil, Squalene oil, Squalene oil, olive oil, Kano draw vegetable oil, Semen Maydis oil, Oleum Brassicae campestris, safflower oil, Oleum Helianthi, fish oil, flavored oils, water solublity only to give birth to plain and its mixture.In particularly preferred embodiment, use soybean oil.In a preferred embodiment of the invention, oil phase is preferably to have about 1-2 micron, more preferably in 0.2 to 0.8 scope and the droplet of most preferably about 0.8 micron mean particle size be distributed in aqueous phase.In other embodiments, water can be distributed in the oil phase.

In some embodiments, the cumulative volume of oil phase emulsion-based comprises the organic solvent of 3-15 and preferred 5-10 volume %.Although the present invention is not subject to any specific mechanism, comprise that the solvent based on organophosphorus ester that will use in the Emulsion is used for removing or destroy the lipid of the film of pathogen.Therefore, remove sterin in the microbial film or any solvent of phospholipid and can be used for method of the present invention.Appropriate organic solvent includes but not limited to that based on the solvent of organophosphorus ester or alcohol in some preferred embodiments, avirulence alcohol (for example, ethanol) is as adjuvant.The chemical compound of any other that provides in oil phase and the oil phase preferably aseptic with pyrogen-free.

3. surfactant and detergent

In some embodiments, Emulsion also comprises surfactant or detergent.In some preferred embodiments, Emulsion comprises about 3 to 15% and preferably approximately 10% one or more surfactants or detergent (although also relating to other concentration).Although the present invention is not subject to any specific mechanism, when comprising in being present in Emulsion, surfactant helps stable emulsion.Comprise nonionic (non-anion) and ionic surface active agent.In addition, the surfactant from the BRIJ family of surfactant is used for compositions of the present invention.Form with liquid phase or oil phase provides surfactant.The surfactant that is suitable for Emulsion comprises multiple anion and non-ionic surface active agent, and other emulsifying agents that can promote the formation of oil in water emulsion.Usually, emulsifying agent is hydrophilic relatively, and the mixture of emulsifying agent can be used for obtaining essential character.In some preparations, non-ionic surface active agent has the ionic surfactant of being better than, because compare with ion (for example, the soap type) emulsifying agent, and the more stable Emulsion of they more compatible with the pH scope of broadness basically and common formation.Therefore, in some preferred embodiment, compositions of the present invention comprises one or more non-ionic surface active agents, for example Polysorbate surfactant (for example, polyoxyethylene groups ether), Polysorbate detergent, polyoxy polyethoxy ethanol etc.The example that is used for Polysorbate detergent of the present invention includes but not limited to TWEEN 20, TWEEN 40, TWEEN 60, TWEEN 80 etc.

TWEEN 60 (polyoxyethylene sorbitan monostearate) and TWEEN 20, TWEEN40 and TWEEN 80 are included in the Polysorbate that is used as emulsifying agent in many pharmaceutical compositions together.In some embodiments of the present invention, these chemical compounds also are used as component altogether with adjuvant.As if the TWEEN surfactant also have the effect of killing the virus to lipid-coated virus (referring to for example, people such as Eriksson, Blood Coagulation and Fibtinolysis 5 (Suppl.3): S37-S44 (1994)).

The example that is used for phenoxy group polyethoxy ethanol of the present invention and its polymer includes but not limited to TRITON (for example, X-100, X-301, X-165, X-102, X-200) and TYLOXAPOL.TRITON X-100 is widely used in to extract lipid and proteinic strong nonionic detergent and dispersant from biological structure.Its also have anti-wide spectrum enveloped virus the effect of killing the virus (referring to for example, Maha and Igarashi, Southeast Asian J.Trop.Med.Pub.Health28:718 (1997); With people such as Portocala, Virologie 27:261 (1976)).Because this antiviral activity, it is used for the inactivation viral pathogen of FF human plasma of living (referring to for example, people such as Horowitz, Blood 79:826 (1992)).

The present invention is not subject to surfactant disclosed herein.Can from reference work (for example, including but not limited to McCutheon ' s the 1st volume: Emulsions and Detergents-North American Edition, 2000) and commercial source, be identified for other surfactants of the present invention and detergent.

4. the chemical compound of cation halogen

In some embodiments, emulsifying agent also comprises the chemical compound of cation halogen.In some preferred embodiments, the gross weight of Emulsion emulsion-based (although also relating to other concentration) comprises about 0.5 to 1.0wt.% or the chemical compound of more cation halogen.In preferred embodiments, the chemical compound with the cation halogen preferably mixes with oil phase; Yet, should be appreciated that and the chemical compound of cation halogen can be provided in the different preparations with emulsion composition.That suitable halogen contained compound can be selected from is chloride, the chemical compound of fluorine, bromine and iodine example.In preferred embodiments, the chemical compound of suitable cation halogen includes but not limited to cetyl halogenation pyridine, cetyl trimethyl ammonium halide, hexadecyldimethyl benzyl ammonium ethyl ammonium halide, hexadecyldimethyl benzyl ammonium benzyl ammonium halide, cetyl tributyl phosphorus Halides, dodecyl trimethyl-ammonium halide or myristyl trimethyl-ammonium halide.In some specific embodiments, the chemical compound of suitable cation halogen comprises Jie and is not limited to hexadecylpyridinium chloride (CPC), hexadecyltrimethylammonium chloride, cetyl benzyl dimethyl ammonium chloride, cetyl pyridinium bromide (CPB) and cetyl trimethyl ammonium bromide (CTAB), the cetyldimethylethylambromide bromide ammonium, hexadecyltri-n-butylphosp, Dodecyl trimethyl ammonium chloride and Tetradecyl Trimethyl Ammonium Bromide. in particularly preferred embodiment, the cation halogen compounds is CPC, although compositions of the present invention is not subject to the preparation of the chemical compound with any specific cation.

5. sprouting reinforcing agent

In other embodiments of the present invention, nano-emulsion also comprises the sprouting reinforcing agent.In some preferred embodiments, Emulsion comprise about 1mM to 15mM and more preferably approximately one or more of 5mM to 10mM strengthen the chemical compound of sprouting (although also relating to other concentration).In preferred embodiments, before the preparation of Emulsion, provide the chemical compound that strengthens sprouting at aqueous phase.The present invention includes, when in the nano-emulsion compositions, adding the sprouting reinforcing agent, strengthen the spore characteristic extremely of nano-emulsion.The present invention comprises that also this type of is sprouted reinforcing agent and is locating to cause spore activity extremely near neutral pH (pH6-8 and preferred 7).Can be for example by obtaining this type of neutral pH Emulsion with phosphate buffered saline(PBS) (PBS) dilution or by preparing neutral Emulsion.The spore activity of killing of nano-emulsion preferably takes place when spore begins to sprout.

In specific embodiments, proved that the Emulsion that is used for vaccine of the present invention has spore activity extremely.Although the present invention be not subject to any specific mechanism and to the understanding of mechanism be not implement essential to the invention, but acting on to cause, the amalgamation component (fusigenic component) that it is believed that Emulsion sprouts, reverse finish to the trophosome form before, the lysogeny component of Emulsion acts on cracking and sprouts spore again.Therefore, these component synergism of Emulsion are so that spore is easy to be subjected to the destruction of Emulsion.The adding of sprouting reinforcing agent is also by for example quickening to kill the anti-spore activity of killing that the active speed that takes place of spore promotes Emulsion.

The sprouting of bacterial endospore and fungal spore and increase metabolism and relevant to the resistance of heat and chemical reagent minimizing.For the sprouting that takes place, spore must feel that environment is enough to support growth and reproduction.Aminoacid L-alanine stimulate bacterial spore sprout (referring to for example, Hills, J.Gen.Micro.4:38 (1950); And Halvorson and Church, Bacteriol Rev.21:112 (1957)).L-alanine and L proline also cause fungus spore germination (Yanagita, Arch Mikrobiol 26:329 (1957)) according to reports.Simple alpha amino acid is the glycosides propylhomoserin L-alanine position of plying in the centre in metabolism for example.The transamination of alpha amino acid or deaminizating have produced metabolism and have grown the carbohydrate and the nitrogen of required glycogenic or living ketone.For example, the transamination of L-alanine or deaminizating produce the pyruvate as the end-product of glycolysis metabolism (embden-Meyerhof-Parnas pathway).The acid of pyruvate dehydrogenase complex oxide acetylacetonate produces acetyl-CoA, NADH, H ⁺And CO ₂Acetyl-CoA is the initial substrate of tricarboxylic acid cycle (Kreb ' s circulation), and it is supply line plastochondria electron transport chain conversely.Acetyl-CoA still is the synthetic and synthetic final carbon source of sterin of fatty acid.Simple alpha amino acid can provide to be sprouted and required nitrogen, the CO of metabolic activity subsequently ₂, give birth to glycogen and/or give birth to the equivalent of ketone.

In certain embodiments, suitable sprouting reinforcing agent of the present invention includes but not limited to alpha amino acid, comprises L-enantiomer, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine and its Arrcostab of glycine and alanine.About aminoacid other information of the effect of sprouting are found in United States Patent (USP) 5,510,104; Integrate with this paper by quoting in full.In some embodiments, also can use glucose, fructose, agedoite, sodium chloride (NaCl), ammonium chloride (NH ₄Cl), calcium chloride (CaCl ₂) and the mixture of potassium chloride (KCl).In particularly preferred embodiment of the present invention, preparation comprises sprouts reinforcing agent L-alanine, CaCl ₂, inosine and NH ₄Cl.In some embodiments, compositions also comprises the growth medium (for example, tryptone meat peptone nutritional solution etc.) of one or more forms, and it additionally comprises or itself can not comprise sprouting reinforcing agent and buffer agent.

Above-claimed cpd is exemplary sprouting reinforcing agent, is to be understood that other known sprouting reinforcing agents can be used for the nano-emulsion that uses in embodiments more of the present invention.The candidate sprouts reinforcing agent should satisfy two standards to be included in the compositions of the present invention: it should combine with Emulsion disclosed herein and when incorporating Emulsion disclosed herein into, it should increase the germination rate of target spore.Those skilled in the art are by using particular agent and nano-emulsion disclosed herein to target, to work as when contacting deactivation that the deactivation of target and the compositions of the present invention that does not contain described reagent are produced same target then relatively, can determine whether this reagent has as the function of wanting of sprouting reinforcing agent with mixture.Increase and sprout, thereby any reagent of the growth of minimizing or inhibition biology is considered to be used for the suitable reinforcing agent of nano-emulsion compositions disclosed herein.

In other embodiments, in neutral emulsion composition, add and sprout reinforcing agent (or growth medium) generation compositions, described compositions is used for the deactivation bacterial spore, and enveloped virus, gram-negative bacteria and gram-positive bacteria, is used for vaccine combination of the present invention.

6. interaction reinforcing agent

In other embodiments, nano-emulsion comprises one or more (for example can increase compositions and target pathogen, gram-negative bacteria is vibrio (Vibrio), Salmonella (Salmonella), shigella (Shigella) and pseudomonas (cell wall of Pseudomonas) for example) interactional chemical compound (that is, " interaction reinforcing agent ").In preferred embodiments, preferably with interaction reinforcing agent and oil phase premixing; Yet, in other embodiments, after emulsifying, the interaction reinforcing agent is provided with compositions.In some preferred embodiment, the interaction reinforcing agent is chelating agen (for example ethylenediaminetetraacetic acid (EDTA) in buffer (for example, tris buffer) or ethylenebis (oxygen ethylidene time amino) tetraacethyl (EGTA)).Be appreciated that chelating agen is the exemplary interactional chemical compound of enhancing.In fact, comprise that increase is used for the nano-emulsion of embodiments more of the present invention and interactional other reagent of microorganism reagent and/or pathogen.In particularly preferred embodiment, the interaction reinforcing agent exists with about 50 concentration to about 250 μ M.Those skilled in the art are by using particular agent and nano-emulsion disclosed herein to target, to work as when contacting deactivation that the deactivation of target and the compositions of the present invention that does not contain described reagent are produced same target then relatively, can determine whether this reagent has the function of wanting as the interaction reinforcing agent with mixture.Increase the interaction of Emulsion and antibacterial, thereby reduce or suppress any reagent of the growth (comparing) of antibacterial, be considered to be used for the suitable reinforcing agent of nano-emulsion compositions disclosed herein with the parameter under its non-existent situation.

In some embodiments, add the interaction reinforcing agent and produce compositions in nano-emulsion, described compositions is used for deactivation envelope virus, gram-positive bacteria and some gram-negative bacterias, is used for vaccine combination of the present invention.

7. quaternary ammonium compounds

In some embodiments, nano-emulsion of the present invention comprises the chemical compound that contains quaternary ammonium.Exemplary quaternary ammonium compound includes but not limited to alkyl dimethyl benzyl ammonium chloride, DDAC, alkyl dimethyl benzyl and dialkyl dimethyl ammonium chloride, N, N-dimethyl-2-hydroxypropyl ammonium chloride polymer, DDAC, just-alkyl dimethyl benzyl ammonium chloride, just-alkyl dimethyl ethylamino benzonitrile ammonium chloride, dialkyl dimethyl ammonium chloride, just-alkyl dimethyl benzyl ammonium chloride, just-the Zephiramine chloride monohydrate, just-alkyl dimethyl benzyl ammonium chloride, dialkyl dimethyl ammonium chloride, six hydrogen-1,3,5-three (2-ethoxy)-s-triazine, myristyl benzyl dimethyl ammonium chloride (with) QuatRNIUM 14, alkyl two (2-ethoxy) benzyl ammonium chloride, alkyl demethyl benzyl ammonium chloride, alkyl dimethyl 3,4-dichloro-benzenes ammonio methacrylate, alkyl dimethyl benzyl ammonium chloride, alkyl dimethyl benzyl dimethyl benzyl ammonium, alkyl dimethyl dimethyl benzene ammonio methacrylate, alkyl dimethyl ethyl ammonium bromide, alkyl dimethyl ethyl ammonium bromide, alkyl dimethyl ethylamino benzonitrile ammonium chloride, alkyl dimethyl cumene ammonio methacrylate, alkyl trimethyl ammonium chloride, alkyl 1 or 3 benzyls-1-(2-ethoxy)-2-imidazoline chloride, dialkyl methyl benzyl ammonium chloride, dialkyl dimethyl ammonium chloride, DDAC, 2-(2-(right-(diisobutyl) toloxyl) ethyoxyl) ethyl dimethyl benzene ammonio methacrylate, 2-(2-(right-(diisobutyl) phenoxy group) ethyoxyl) ethyl dimethyl benzene ammonio methacrylate, Quaternium 24, two (2-ethoxy) the octyl group hydrochlorinate ammoniums of dodecyl, dodecyl dimethyl benzyl ammonium chloride, dodecyl carbamyl methyl diethylbenzene ammonio methacrylate, seven decyl hydroxyethyl imidazole quinoline chlorides, hexahydroxy-1,3,5-three (2-ethoxy)-s-triazine, the octyl-decyl alkyl dimethyl ammonium chloride, octyl group dodecyl dimethyl ammonium chloride, Octylphenoxy ethoxyethyl group dimethyl benzene ammonio methacrylate, divinyl oxide base two (alkyl-dimethyl ammonium chloride), quaternary ammonium compounds, two cocoa alkyl dimethyls, chloride, trimethoxysilyl quaternary ammonium compound and trimethyldodecane base benzyl ammonium chloride.

8. other components

In some embodiments, nano-emulsion comprises one or more provides character or functional other compositionss of wanting for nano-emulsion.These components add in the time of can incorporating the water or the oil phase of nano-emulsion into and/or can or carry out emulsifying before emulsifying.For example, in some embodiments, nano-emulsion (for example also comprises phenols, triclosan, phenol), (for example, citric acid (for example, 1.5-6%), acetic acid, Fructus Citri Limoniae juice), alkylating agent are (for example for acidizing reagent, sodium hydroxide (for example, 0.3%)), buffer agent (for example, citrate buffer agent, acetate buffer and be used to keep the other buffers of specific pH) and halogen (for example, polyvinylpyrrolidone, sodium hypochlorite, hydrogen peroxide).

The example technique that is used to prepare nano-emulsion (for example, be used for inactivating pathogens and/or produce the nano-emulsion of immunogenic composition of the present invention) is described below.In addition, also show many specific (although being exemplary) pharmaceutical formulations below.

Preparation technique

Can use classical Emulsion formation technology to form nano-emulsion of the present invention.In brief, under high relatively shearing force (for example, using high hydraulic pressure and mechanical force) that oil phase is mixed with water, thus obtain oil-in-water nanometer Emulsion.By with scope at about 1:9 to 5:1, preferably approximately in the 5:1 to 3:1, most preferably the oil phase of the volume by volume of 4:1 is than water, with the oil phase Emulsion that forms mixed with water.Can use any device that can produce the shearing force that is enough to form Emulsion, for example French cell press (French Presses) or high shear force agitator (highshear mixers) are (for example, can be for example from Admix, Inc., Manchester, the high shear force agitator of the FDA approval that NH obtains) mixing oil phase and water.The method that produces this type of Emulsion is described in United States Patent (USP) 5,103, and 497 and 4,895,452, by integrating with this paper with quoting in full of they.

In preferred embodiments, the compositions that is used for method of the present invention comprises the droplet of the oiliness discontinuous phase that is dispersed in aqueous continuous phase (for example water).In preferred embodiments, nano-emulsion of the present invention is stable, and even does not decompose after long term store (for example, surpass a year or for many years).In addition, in some embodiments, nano-emulsion with immunogen (for example, pathogen) after mixing be stable (for example, stable in some embodiments above 3 months, stable in some embodiments above 6 months, stable in some embodiments, stable in some embodiments) above 18 months above 12 months.In preferred embodiments, nano-emulsion of the present invention, when the experimenter is used (for example, by spraying or contact mucomembranous surface, swallow, suction etc.) time be nontoxic and safety.

In some embodiments, the part of Emulsion is the form of lipid conformation, includes but not limited to monolayer, multilamellar and minority layer lipid carrier, micelle and lamella mutually.

Embodiments more of the present invention are used and are comprised alcoholic acid oil phase.For example, in some embodiments, Emulsion of the present invention comprises (i) water and (ii) comprises ethanol as organic solvent with at random sprout the oil phase of reinforcing agent and (iii) make the TYLOXAPOL (preferred 2-5%, more preferably 3%) of surfactant.Said preparation is highly effective for the deactivation of pathogen, also is non-irritating and avirulent (for example, thereby can be used for mucomembranous surface is used) for mammalian subject.

In some other embodiments, Emulsion of the present invention is included in emulsive first Emulsion in second Emulsion, and wherein (a) first Emulsion comprises (i) water; The oil phase that (ii) comprises oil and organic solvent; (iii) surfactant; (b) second Emulsion comprises (i) water; The oil phase that (ii) comprises oil and cation chemical compound; (iii) surfactant.

Exemplary formulation

Following description provides many exemplary Emulsions, comprising being used for compositions BCTP and X ₈W ₆₀The preparation of PC.BCTP comprises the Water-In-Oil nano-emulsion, and wherein oil phase is by soybean oil, three-just-butyl phosphoric acid salt and the preparation of the TRITON X-100 in 80% water.X ₈W ₆₀PC comprises isopyknic BCTP and W ₈₀The mixture of 8P.W ₈₀8P is by the CPC of glyceryl monostearate, refining Generol 122 (for example, GENEROL sterin), TWEEN 60, soybean oil, cation halogen and the liposome sample chemical compound of Oleum menthae preparation.GENEROL family be one group of polyethoxylated Generol 122 (Henkel Corporation, Ambler, Pennsylvania).Table 1B provides and has been used for exemplary emulsion preparations of the present invention.These specific formulation are found in United States Patent (USP) 5,700,679 (NN), 5,618,840,5,549,901 (W ₈₀8P); With 5,547,677, it is separately by integrating with this paper with quoting in full of they.U.S. Patent application series 10/669,865 provides some other emulsion preparations, integrates with this paper by quoting in full with it.

Produce X ₈W ₆₀PC Emulsion prepares W at first respectively ₈₀8P Emulsion and BCTP Emulsion come.Mixture reemulsification with these two kinds of Emulsions is called X with generation then ₈W ₆₀The fresh emulsion composition of PC.The method that produces this type of Emulsion sees United States Patent (USP) 5,103,497 and 4,895,452 (it is separately by integrating with this paper with quoting in full of they).

Table 1B

Above listed compositions be exemplary, the amount that those skilled in the art can change component is to realize being fit to the nano-emulsion compositions of purpose of the present invention.It will be appreciated by those skilled in the art that oil phase can change the ratio of the component of water and discrete oily carrier, surfactant CPC and organic phosphoric acid salt buffer agent, each compositions.

Have the ratio of the water of 4:1 although comprise some compositions of BCTP, but be appreciated that BCTP is mixed with and has more or less water oil.For example, in some embodiments, 3,4,5,6,7,8,9,10 or the part of more a plurality of waters to a part in the oil phase.For W ₈₀The 8P preparation also is like this.Similarly, three (N-butyl) phosphate ester: TRITON X-100: the ratio of soybean oil also can change.

Although having listed, table 1B is used for W ₈₀The specified quantitative of the glycerin mono-fatty acid ester of 8P, polysorbate60, GENEROL 122, hexadecylpyridinium chloride and substrate oil (carrier oil), but these only are exemplary.Preparation has W ₈₀The Emulsion of the character of 8P, described Emulsion have each component or the in fact different components in these components of variable concentrations, and it can realize identical function.For example, Emulsion can have about 80 to about 100g glycerin mono-fatty acid ester in initial oil phase.In other embodiments, Emulsion can have about 15 to about 30g polysorbate60 in initial oil phase.In another embodiment, compositions can comprise about 20 to about 30g GENEROL sterin in initial oil phase.

The avirulence that the individual components of nano-emulsion (in the immunogenic composition for example of the present invention) can be used for inactivating pathogens and gives Emulsion.For example, the active component-TRITON-X100 among the BCTP shows the ability of lower inactivation of viruses when equaling the concentration of 11% BCTP.When oil phase is added to detergent and solvent and significantly reduces same concentrations, the toxicity of these reagent in tissue culture.Although (understanding to mechanism is not that enforcement is essential to the invention not to be bound by any theory, and the present invention is not subject to any specific mechanism), but the hint nano-emulsion strengthens the interaction of its component and pathogen, thereby promotes the deactivation of pathogen and the toxicity of minimizing individual components.In addition, be mixed into a kind of compositions but not in the nano-emulsion structure time, then mixture is not as effective on inactivating pathogens when component is present in the nano-emulsion structure when all components with BCTP.

Many other embodiments are provided with type of preparation below with similar compositions.The different proportion and the mixture of following compositions citation active component.Those skilled in the art are to be understood that the preparation of following citation is exemplary, comprise citation component similar percentage ranges other preparations within the scope of the invention.

In certain embodiments of the invention, nano-emulsion (for example comprises the hexadecylpyridinium chloride (CPC) of the ethanol of the TYLOXAPOL of about 3 to 8 volume %, about 8 volume %, about 1 volume %, about 60 to 70 volume % oil, soybean oil), the water of about 15 to 25 volume % (for example, DiH ₂O or PBS), in a little preparations, be lower than the 1N NaOH of about 1 volume %.Some embodiments in these embodiments comprise PBS.Add 1N NaOH and/or PBS in some embodiments in these embodiments and make the user can advantageously control the pH of preparation, can obtain like this about 7.0 to about 9.0 and the scope of more preferably about pH of 7.1 to 8.5.For example, one embodiment of the invention comprise the soybean oil of the CPC of the ethanol of the TYLOXAPOL of about 3 volume %, about 8 volume %, about 1 volume %, about 64 volume % and the DiH of about 24 volume % ₂O (this paper is called Y3EC).Another similar embodiment comprises ethanol and the soybean oil of the CPC of about 1 volume %, about 64 volume % and the DiH of about 23.5 volume % of the TYLOXAPOL of about 3.5 volume %, about 8 volume % ₂O (this paper is called Y3.5EC).Another embodiment comprises the CPC of the ethanol of the TYLOXAPOL of about 3 volume %, about 8 volume %, about 1 volume %, the 1N NaOH of about 0.067 volume %, and the pH of preparation is about 7.1, the soybean oil of about 64 volume % and the DiH of about 23.93 volume % like this ₂O (this paper is called Y3EC pH 7.1).Another embodiment comprises the CPC of the ethanol of the TYLOXAPOL of about 3 volume %, about 8 volume %, about 1 volume %, the 1N NaOH of about 0.67 volume %, the pH of preparation is about 8.5 and the soybean oil of about 64 volume % and the DiH of about 23.33 volume % like this ₂O (this paper is called Y3EC pH 8.5).Another similar embodiment comprises about 4%TYLOXAPOL, about 8 volume % ethanol, about 1%CPC and the soybean oil of about 64 volume % and the DiH of about 23 volume % ₂O (this paper is called Y4EC).Preparation comprises CPC and the soybean oil of about 64 volume % and the DiH of about 19 volume % of about 8% TYLOXAPOL, about 8% ethanol, about 1 volume % in another embodiment ₂O (this paper is called Y8EC).Other embodiments comprise the soybean oil of the CPC of the ethanol of the TYLOXAPOL of about 8 volume %, about 8 volume %, about 1 volume %, about 64 volume % and the 1x PBS (this paper is called Y8EC PBS) of about 19 volume %.

In some embodiments of the present invention, nano-emulsion comprises the ethanol of about 8 volume % and CPC and the oil (for example, soybean oil) of about 64 volume % and water (for example, the DiH of about 27 volume % of about 1 volume % ₂O or PBS) (this paper is called EC).

In some embodiments, nano-emulsion comprises water (for example, the DiH of the Tributyl phosphate ester (TBP) of the sodium lauryl sulphate (SDS) of about 8 volume %, about 8 volume % and the oil of about 64 volume % (for example, soybean oil) and about 20 volume % ₂O or PBS) (this paper is called S8P).

In some embodiments, the oil that nano-emulsion comprises the hexadecylpyridinium chloride (CPC) of the ethanol of the TYLOXAPOL of the TRITONX-100 of about 1 to 2 volume %, about 1 to 2 volume %, about 7 to 8 volume %, about 1 volume %, about 64 to 57.6 volume % (for example, soybean oil), water (for example, DiH with about 23 volume % ₂O or PBS).

In addition, some in these preparations also comprise about 5mM L-alanine/inosine and about 10mM ammonium chloride.In these preparations some comprise PBS.Adding PBS in some embodiments in these embodiments makes the user advantageously control the pH of preparation.For example, one embodiment of the invention comprise the ethanol of the TYLOXAPOL of the TRITON X-100 of about 2 volume %, about 2 volume %, about 8 volume %, the soybean oil of about 1 volume %CPC, about 64 volume % and the water DiH of about 23 volume % ₂O.In another embodiment, preparation comprises soybean oil and the residue 1x PBS (this paper is called 90% X2Y2EC/GE) of the CPC of the ethanol of the TYLOXAPOL of the TRITON X-100 of about 1.8 volume %, about 1.8 volume %, about 7.2 volume %, about 0.9 volume %, about 5mM L-alanine/inosine and about 10mM ammonium chloride, about 57.6 volume %.

In alternate embodiment, nano-emulsion comprises the DiH of the CPC of the ethanol of the TWEEN 80 of about 5 volume %, about 8 volume %, about 1 volume %, the oil of about 64 volume % (for example, soybean oil) and about 22 volume % ₂(this paper is called W to O ₈₀5EC).

In other embodiments of the present invention, nano-emulsion comprises the DiH of the CPC of the ethanol of the TWEEN20 of about 5 volume %, about 8 volume %, about 1 volume %, the oil of about 64 volume % (for example, soybean oil) and about 22 volume % ₂(this paper is called W to O ₂₀5EC).

In other embodiments of the present invention, the oil that nano-emulsion comprises the CPC of the ethanol of the TRITON X-100 of about 2 to 8 volume %, about 8 volume %, about 1 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 15 to 25 volume % ₂O or PBS).For example, the present invention relates to comprise the soybean oil of the ethanol of the TRITON X-100 of about 2 volume %, about 8 volume %, about 64 volume % and the DiH of about 26 volume % ₂The preparation of O (this paper is called X2E).In other similar embodiments, Emulsion comprises the soybean oil of the ethanol of the TRITON X-100 of about 3 volume %, about 8 volume %, about 64 volume % and the DiH of about 25 volume % ₂O (this paper is called X3E).In other embodiments, preparation comprises the soybean oil of the ethanol of the Triton X-100 of about 4 volume %, about 8 volume %, about 64 volume % and the DiH of about 24 volume % ₂O (this paper is called X4E).In other embodiments, nano-emulsion comprises the soybean oil of the ethanol of the TRITON X-100 of about 5 volume %, about 8 volume %, about 64 volume % and the DiH of about 23 volume % ₂O (this paper is called X5E).In some embodiments, nano-emulsion comprises the soybean oil of the ethanol of the TRITON X-100 of about 6 volume %, about 8 volume %, about 64 volume % and the DiH of about 22 volume % ₂O (this paper is called X6E).In other embodiments of the present invention, nano-emulsion comprises the soybean oil of the ethanol of the TRITON X-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8E).In other embodiments, nano-emulsion comprises the olive oil of the ethanol of the TRITON X-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8E O).In another embodiment, nano-emulsion comprises the soybean oil of the CPC of the ethanol of the TRITON X-100 of 8 volume %, about 8 volume %, about 1 volume %, about 64 volume % and the DiH of about 19 volume % ₂O (this paper is called X8EC).

In alternate embodiment of the present invention, the oil that nano-emulsion comprises the CPC of the TBP of the TYLOXAPOL of the TRITON X-100 of about 1 to 2 volume %, about 1 to 2 volume %, about 6 to 8 volume %, about 0.5 to 1.0 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor) and the water of about 1 to 35 volume % (for example, DiH ₂O or PBS).In addition, some in these nano-emulsioies can comprise the liquid infant formula of the yeast extract of the tryptone meat peptone nutritional solution of about 1 to 5 volume %, about 0.5 to 1.5 volume %, about 5mML-alanine/inosine, about 10mM ammonium chloride and about 20-40 volume %.Comprise in the embodiment of liquid infant formula at some, prescription comprises casein hydrolysate (for example, Neutramigen or Progestimil etc.).In some embodiments in these embodiments, nano-emulsion also comprises the sodium thiosulfate of about 0.1 to 1.0 volume % and the sodium citrate of about 0.1 to 1.0 volume %.Other similar embodiments that comprise these solvents use phosphate buffered saline(PBS) (PBS) as water.For example, an embodiment comprises the soybean oil of the CPC of the TBP of the TYLOXAPOL of the TRITONX-100 of about 2 volume %, about 2 volume %, about 8 volume %, about 1 volume %, about 64 volume % and the DiH of about 23 volume % ₂O (this paper is called X2Y2EC).In other embodiments, preparation of the present invention comprises the DiH of the about 22 volume % of soybean oil master of the sodium citrate of the sodium thiosulfate of the CPC of the TBP of the TYLOXAPOL of the TRITON X-100 of about 2 volume %, about 2 volume %, about 8 volume %, about 1 volume %, about 0.9 volume %, about 0.1 volume %, about 64 volume % ₂O (this paper is called X2Y2PC STS1).In another similar embodiment, nano-emulsion comprises the soybean oil of the TBP of the TYLOXAPOL of the TRITON X-100 of about 1.7 volume %, about 1.7 volume %, about 6.8 volume %, about 0.85% CPC, about 29.2% NEUTRAMIGEN, about 54.4 volume % and the DiH of about 4.9 volume % ₂O (this paper is called 85% X2Y2PC/baby).In another embodiment of the invention, nano-emulsion comprises the CPC of the TBP of the TYLOXAPOL of the TRITONX-100 of about 1.8 volume %, about 1.8 volume %, about 7.2 volume %, about 0.9 volume %, about 5mM L-alanine/inosine, about 10mM ammonium chloride, the soybean oil of about 57.6 volume % and the 0.1 x PBS (this paper is called 90% X2Y2 PC/GE) of residual volume %.In another embodiment, nano-emulsion comprises the CPC of the TBP of the TYLOXAPOL of the TRITON X-100 of about 1.8 volume %, about 1.8 volume %, about 7.2 volume %, about 0.9 volume % and the tryptone meat peptone nutritional solution of about 3 volume %, the soybean oil of about 57.6 volume % and the DiH of about 27.7 volume % ₂O (this paper is called 90% X2Y2PC/TSB).In another embodiment of the invention, nano-emulsion comprises the soybean oil of the yeast extract of the CPC of the TBP of the TYLOXAPOL of the TRITON X-100 of about 1.8 volume %, about 1.8 volume %, about 7.2 volume %, about 0.9 volume %, about 1 volume %, about 57.6 volume % and the DiH of about 29.7 volume % ₂O (this paper is called 90% X2Y2PC/YE).

In some embodiments of the present invention, nano-emulsion comprises the TYLOXAPOL of about 3 volume %, the TBP of about 8 volume %, water (for example, DiH with the CPC of about 1 volume %, the oil of about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and about 15 to 30 volume % ₂O or PBS).In particular of the present invention, nano-emulsion comprises TBP and the Semen sojae atricolor of the CPC of about 1 volume %, about 64 volume % and the DiH of about 24 volume % of the TYLOXAPOL of about 3 volume %, about 8 volume % ₂O (this paper is called Y3PC).

In some embodiments of the present invention, nano-emulsion comprises the TRITON X-100 of about 4 to 8 volume %, the TBP of about 5 to 8 volume %, water (for example, the DiH of the oil of about 30 to 70 volume % (for example, Semen sojae atricolor or olive oil) and about 0 to 30 volume % ₂O or PBS).In addition, some embodiment in these embodiments also comprises cetyldimethylethylambromide bromide ammonium, 500 μ M EDTA, the about 10mM ammonium chloride of the hexadecylpyridinium chloride of the benzalkonium chloride of the CPC of about 1 volume %, about 1 volume %, about 1 volume %, about 1 volume %, approximately 5mM inosine and approximately 5mM L-alanine.For example, in certain embodiments, nano-emulsion comprises the soybean oil of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8P).In another embodiment of the invention, nano-emulsion comprises the TBP of the TRITONX-100 of about 8 volume %, about 8 volume %, the soybean oil of about 1%CPC, about 64 volume % and the DiH of about 19 volume % ₂O (this paper is called X8PC).In another embodiment, nano-emulsion comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 1 volume %, about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called ATB-X1001).In another embodiment, preparation comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 2 volume %, about 50 volume % and the DiH of about 32 volume % ₂O (this paper is called ATB-X002).In some embodiments, Emulsion comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 4 volume %, about 4 volume %, about 0.5 volume %, about 32 volume % and the DiH of about 59.5 volume % ₂O (this paper is called 50% X8PC).In some embodiments, nano-emulsion comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 0.5 volume %, about 64 volume % and the DiH of about 19.5 volume % ₂(this paper is called X8PC to O _1/2).In some embodiments of the present invention, nano-emulsion comprises the soybean oil of the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 2 volume %, about 64 volume % and the DiH of about 18 volume % ₂O (this paper is called X8PC2).In other embodiments, nano-emulsion comprises the TRITON X-100 of about 8 volume %, about 8%TBP, about 1% benzalkonium chloride, the soybean oil of about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called X8P BC).In alternate embodiment of the present invention, nano-emulsion comprises the soybean oil of the hexadecylpyridinium chloride of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 1 volume %, about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called X8P CPB).In another embodiment of the invention, nano-emulsion comprises the soybean oil of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, the cetyldimethylethylambromide bromide ammonium of about 1 volume %, about 50 volume % and the DiH of about 33 volume % ₂O (this paper is called X8P CTAB).In other embodiments, nano-emulsion comprises the CPC of the TBP of the TRITON X-100 of about 8 volume %, about 8 volume %, about 1 volume %, the soybean oil of about 500M EDTA, about 64 volume % and the DiH of about 15.8 volume % ₂O (this paper is called X8PC EDTA).In some embodiments, nano-emulsion comprises the CPC of the TBP of the TRITONX-100 of 8 volume %, about 8 volume %, about 1 volume %, about 10mM ammonium chloride, about 5mM inosine, about 5mM L-alanine, the soybean oil of about 64 volume % and the DiH of about 19 volume % ₂(this paper is called X8PC GE for O or PBS _1x).In another embodiment of the invention, nano-emulsion comprises the TRITON X-100 of about 5 volume %, the approximately soybean oil of CPC, the about 40 volume % of 5%TBP, about 1 volume % and the DiH of about 49 volume % ₂(this paper is called X5P to O ₅C).

In some embodiments of the present invention, nano-emulsion comprises the soybean oil of the ethanol of the TYLOXAPOL of the TRITON X-100 of about 2 volume %, about 6 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X2Y6E).

In other embodiments of the present invention, the oil that nano-emulsion comprises the TRITON X-100 of about 8 volume % and the glycerol of about 8 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 15 to 25 volume % ₂O or PBS).Some nano-emulsion compositions (for example, is used to produce the L-ascorbic acid that immunoreation (for example, as vaccine) comprises about 1 volume %.For example, a specific embodiment comprises the soybean oil of the glycerol of the TRITON X-100 of about 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8G).In another embodiment, nano-emulsion comprises the soybean oil of the glycerol of the TRITON X-100 of about 8 volume %, about 8 volume %, the L-ascorbic acid of about 1 volume %, about 64 volume % and the DiH of about 19 volume % ₂(this paper is called X8GV to O _C).

In other embodiments, the oil that nano-emulsion comprises the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.5 to 0.8 volume %, about 0.5 to 2.0 volume %, about 8 volume %, about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 15 to 25 volume % ₂O or PBS).For example, in a specific embodiment, Emulsion comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.70 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 18.3 volume % ₂(this paper is called X8W60PC to O ₁).In some embodiments, nano-emulsion comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.71 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 18.29 volume % ₂(this paper is called W60 to O _0.7X8PC).In other embodiments, Emulsion comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITONX-100 of about 8 volume %, about 0.7 volume %, about 0.5 volume %, about 8 volume %, about 64 to 70 volume % and the DiH of about 18.8 volume % ₂(this paper is called X8W60PC to O ₂).In other embodiments, Emulsion comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of the TRITON X-100 of about 8 volume %, about 0.71 volume %, about 2 volume %, about 8 volume %, about 64 volume % and the DiH of about 17.3 volume % ₂O.In another embodiment of the invention, Emulsion comprises the soybean oil of the TBP of the CPC of the TWEEN 60 of about 0.71 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 25.29 volume % ₂(this paper is called W60 to O _0.7PC).

In another embodiment of the invention, nano-emulsion comprises the glycerol of the dioctyl sulfosuccinate of about 2 volume %, about 8 volume % or the TBP of about 8 volume %, in addition, the oil of about 60 to 70 volume % (for example, Semen sojae atricolor or olive oil) and water (for example, the DiH of about 20 to 30 volume % ₂O or PBS).For example, in some embodiments, nano-emulsion comprises the dioctyl sulfosuccinate of about 2 volume %, the soybean oil of the glycerol of about 8 volume %, about 64 volume % and the DiH of about 26 volume % ₂O (this paper is called D2G).In another relevant embodiment, nano-emulsion comprises dioctyl sulfosuccinate and the soybean oil of the TBP of about 8 volume %, about 64 volume % and the DiH of about 26 volume % of about 2 volume % ₂O (this paper is called D2P).

In other embodiments of the present invention, nano-emulsion comprises water (for example, the DiH of the glycerol of about 8 to 10 volume % and the oil of the CPC of about 1 to 10 volume %, about 50 to 70 volume % (for example, Semen sojae atricolor or olive oil) and about 15 to 30 volume % ₂O or PBS).In addition, some embodiment in these embodiments, nano-emulsion also comprises the L-ascorbic acid of about 1 volume %.For example, in some embodiments, Emulsion comprises the soybean oil of the CPC of the glycerol of about 8 volume %, about 1 volume %, about 64 volume % and the DiH of about 27 volume % ₂O (this paper is called GC).In some embodiments, nano-emulsion comprises the soybean oil of the CPC of the glycerol of about 10 volume %, about 10 volume %, about 60 volume % and the DiH of about 20 volume % ₂O (this paper is called GC10).In another embodiment of the invention, nano-emulsion comprises the Semen sojae atricolor of the CPC of the glycerol of about 10 volume %, about 1 volume %, the L-ascorbic acid of about 1 volume %, about 64 volume % or the DiH of oil and about 24 volume % ₂(this paper is called GCV to O ₆).

In some embodiments of the present invention, nano-emulsion comprises water (for example, the DiH of the SDS of the glycerol of about 8 to 10 volume %, about 8 to 10 volume %, the oil of about 50 to 70 volume % (for example, Semen sojae atricolor or olive oil) and about 15 to 30 volume % ₂O or PBS).In addition, some embodiment in these embodiments, nano-emulsion also comprises the lecithin of about 1 volume % and right-methyl hydroxybenzoate of about 1 volume %.The exemplary of this type of preparation comprises the soybean oil of the glycerol of SDS, the 8 volume % of about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called S8G).Related preparations comprises the soybean oil of the lecithin of the SDS of the glycerol of about 8 volume %, about 8 volume %, about 1 volume %, the p-methyl hydroxybenzoate of about 1 volume %, about 64 volume % and the DiH of about 18 volume % ₂O (this paper is called S8GL1B1).

In another embodiment of the invention, nano-emulsion comprises the soybean oil of the ethanol of the CPC of the TYLOXAPOL of the TWEEN 80 of about 4 volume %, about 4 volume %, about 1 volume %, about 8 volume %, about 64 volume % and the DiH of about 19 volume % ₂(this paper is called W to O ₈₀4Y4EC).

In some embodiments of the present invention, nano-emulsion comprises the soybean oil of the ethanol of the TYLOXAPOL of the CPC of about 0.01 volume %, about 0.08 volume %, about 10 volume %, about 70 volume % and the DiH of about 19.91 volume % ₂O (Y.08EC.01 this paper be called).

In another embodiment of the invention, nano-emulsion comprises the sodium lauryl sulfate of about 8 volume % and the glycerol of about 8 volume %, the soybean oil of about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called SLS8G).

Above-mentioned specific formulation just illustrate be used for the present invention (for example, deactivation and/or in and pathogen and be used for producing immunoreation (for example, be used for as vaccine) the experimenter) the simple examples of multiple nano-emulsion.The present invention includes many variants of the above-mentioned preparation that is used for method of the present invention and other nano-emulsion.Whether can easily detect candidate's Emulsion suitable to determine them.At first, the component of using method preparation described herein to want is to determine whether forming Emulsion.If can not form Emulsion, discard this material standed for.For example, by 4.5% sodium thiosulfate, 0.5% sodium citrate, 10% just-butanols, 64% soybean oil and 21% DiH ₂The material standed for of O preparation does not form Emulsion.

The second, candidate's Emulsion should form stable Emulsion.If it keeps being enough to allow time of its purposes of wanting (for example, producing immunoreation in the experimenter) with the form of Emulsion, then Emulsion is stable.For example, treat Emulsion storage, transportation etc., wish that compositions keeps the several months to the several years with the form of Emulsion.Unsettled relatively common Emulsion will lose their form in 1 day.For example, by 8% 1-butanols, 5% Tween 10,1% CPC, 64% soybean oil and 22% DiH ₂The candidate set compound of O preparation does not form stable Emulsion.Be shown as stabilized nano Emulsion and included but not limited to the soybean oil of the TBP of the TRITON X-100 of 8 volume %, about 8 volume %, about 64 volume % and the DiH of about 20 volume % ₂O (this paper is called X8P); The oil (for example, soybean oil) of the TWEEN 20 of 5 volume %, the ethanol of about 8 volume %, the CPC of about 1 volume %, about 64 volume % and the DiH of about 22 volume % ₂(this paper is called W to O ₂₀5EC); 0.08% Triton X-100,0.08% glycerol, 0.01% hexadecylpyridinium chloride, 99% butter and 0.83% diH ₂O (this paper is called 1% X8GC butter); 0.8% TritonX-100,0.8% glycerol, 0.1% hexadecylpyridinium chloride, 6.4% soybean oil, 1.9%diH ₂O and 90% butter (this paper is called the 10%X8GC butter); 2%W ₂₀5EC, 1% Natrosol 250LNF and 97% diH ₂(this paper is called 2% W to O ₂₀5EC L GEL); 1% hexadecylpyridinium chloride, 5% Tween 20,8% ethanol, 64% 70 thickness mineral oil and 22% diH ₂(this paper is called W to O ₂₀5EC 70 mineral oil); 1% hexadecylpyridinium chloride, 5% Tween 20,8% ethanol, 64%350 thickness mineral oil and 22% diH ₂(this paper is called W to O ₂₀5EC 350 mineral oil), in some embodiments, nano-emulsion of the present invention is stable above a week, above one month or above 1 year.

The 3rd, candidate's Emulsion should have effect to its purposes of wanting.For example, pathogen that nano-emulsion should deactivation (for example kill or suppress its growth) to the level of wanting (for example, 1log, 2log, 3log, 4log ... minimizing).By using method described herein, people can determine the fitness of the specific anti-purpose pathogen of candidate's Emulsion.Usually, this is included in and (for example uses suitable control sample, negative control is water for example) parallel experiment in pathogen be exposed to Emulsion carry out one or more periods, determine whether deactivation of Emulsion (for example, kill and/or neutralize) microorganism then and determine its degree that reaches.For example, show by 1% ammonium chloride, 5%TWEEN 20,8% ethanol, 64% soybean oil and 22% DiH ₂The candidate set compound of O preparation is not effective Emulsion.Use method described herein to show that following candidate's Emulsion is effective Emulsion: 5%TWEEN 20,5% hexadecylpyridinium chloride, 10% glycerol, 60% soybean oil and 20% diH ₂(this paper is called W to O ₂₀5GC5); 1% hexadecylpyridinium chloride, 5% TWEEN 20,10% glycerol, 64% soybean oil and 20% diH ₂(this paper is called W to O ₂₀5GC); 1% hexadecylpyridinium chloride, 5%TWEEN 20,8% ethanol, 64% olive oil and 22% diH ₂(this paper is called W to O ₂₀The 5EC olive oil); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Semen Lini oil and 22% diH ₂(this paper is called W to O ₂₀The 5EC Semen Lini oil); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Semen Maydis oil and 22% diH ₂(this paper is called W to O ₂₀The 5EC Semen Maydis oil); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Oleum Cocois and 22% diH ₂(this paper is called W to O ₂₀The 5EC Oleum Cocois); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Oleum Gossypii semen and 22% diH ₂(this paper is called W to O ₂₀5EC Oleum Gossypii semen); 8% glucose, 5% TWEEN10,1% hexadecylpyridinium chloride, 64% soybean oil and 22% diH ₂(this paper is called W to O ₂₀The 5C glucose); 8% PEG 200,5%TWEEN 10,1% hexadecylpyridinium chloride, 64% soybean oil and 22% diH ₂(this paper is called W to O ₂₀5C PEG 200); 8% methanol, 5% TWEEN 10,1% hexadecylpyridinium chloride, 64% soybean oil and 22% diH ₂(this paper is called W to O ₂₀5C methanol); 8% PEG 1000,5% TWEEN 10,1% hexadecylpyridinium chloride, 64% soybean oil and 22%diH ₂(this paper is called W to O ₂₀5C PEG 1000); 2% W ₂₀5EC, 2% Natrosol 250H NF and 96% diH ₂(this paper is called 2%W to O ₂₀5EC Natrosol 2 is also referred to as 2%W ₂₀5EC GEL); 2% W ₂₀5EC, 1% Natrosol 250H NF and 97% diH ₂(this paper is called 2% W to O ₂₀5ECNatrosol 1); 2% W ₂₀5EC, 3% Natrosol 250H NF and 95% diH ₂(this paper is called 2% W to O ₂₀5EC Natrosol 3); 2% W ₂₀5EC, 0.5% Natrosol 250H NF and 97.5% diH ₂(this paper is called 2% W to O ₂₀5EC Natrosol 0.5); 2% W ₂₀5EC, 2%Methocel A and 96%diH ₂(this paper is called 2%W to O ₂₀5EC Methocel A); 2%W ₂₀5EC, 2% Methocel K and 96% diH ₂(this paper is called 2% W to O ₂₀5EC Methocel K); 2%Natrosol, 0.1% X8PC, 0.1 x PBS, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride and diH ₂O (this paper is called 0.1%X8PC/GE+2%Natrosol); 2%Natrosol, 0.8% TRITON X-100,0.8% Tributyl phosphate ester, 6.4% soybean oil, 0.1% hexadecylpyridinium chloride, 0.1x PBS, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride and diH ₂O (this paper is called 10%X8PC/GE+2% Natrosol); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% Adeps Sus domestica and 22%diH ₂(this paper is called W to O ₂₀5EC Adeps Sus domestica); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% ethanol, 64% mineral oil and 22% diH ₂(this paper is called W to O ₂₀5EC mineral oil); 0.1% hexadecylpyridinium chloride, 2% nerolidol, 5% TWEEN 20,10% ethanol, 64% soybean oil and 18.9% diH ₂(this paper is called W to O ₂₀5EC _0.1N); 0.1% hexadecylpyridinium chloride, 2% farnesol, 5% TWEEN 20,10% ethanol, 64% soybean oil and 18.9% diH ₂(this paper is called W to O ₂₀5EC _0.1F); 0.1% hexadecylpyridinium chloride, 5% TWEEN 20,10% ethanol, 64% soybean oil and 20.9% diH ₂(this paper is called W to O ₂₀5EC _0.1); 10% hexadecylpyridinium chloride, 8% Tributyl phosphate ester, 8% TRITONX-100,54% soybean oil and 20% diH ₂(this paper is called X8PC to O ₁₀); 5% hexadecylpyridinium chloride, 8%TRITON X-100,8% Tributyl phosphate ester, 59% soybean oil and 20% diH ₂(this paper is called X8PC to O ₅); 0.02% hexadecylpyridinium chloride, 0.1% TWEEN 20,10% ethanol, 70% soybean oil and 19.88% diH ₂(this paper is called W to O ₂₀0.1EC _0.02); 1% hexadecylpyridinium chloride, 5% TWEEN 20,8% glycerol, 64% Mobil 1 and 22% diH ₂(this paper is called W to O ₂₀5GC Mobil 1); 7.2% TRITON X-100,7.2% Tributyl phosphate ester, 0.9% hexadecylpyridinium chloride, 57.6% soybean oil, 0.1 x PBS, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride and 25.87% diH ₂O (this paper is called 90% X8PC/GE); 7.2%TRITON X-100,7.2% Tributyl phosphate ester, 0.9% hexadecylpyridinium chloride, 57.6% soybean oil, 1% EDTA, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride, 0.1xPBS and diH ₂O (this paper is called 90% X8PC/GE EDTA); With 7.2% TRITON X-100,7.2% Tributyl phosphate ester, 0.9% hexadecylpyridinium chloride, 57.6% soybean oil, 1% sodium thiosulfate, 5mM L-alanine, 5mM inosine, 10mM ammonium chloride, 0.1 x PBS; And diH ₂O (this paper is called 90% X8PC/GE STS).

In a preferred embodiment of the invention, nano-emulsion be nontoxic (for example, to people, plant or animal), nonirritating (for example, to people, plant or animal) and non-corrosive (for example, to people, plant or animal or environment), have the potential that resists extensive microorganism (comprising antibacterial, fungus, virus and spore) simultaneously.Though many above-mentioned nano-emulsioies satisfy these conditions, following description provides many preferred nontoxic, nonirritating, non-corrosive, antimicrobial nano-emulsioies (partly being called " avirulence nano-emulsion " hereinafter at Th1) of the present invention.

In some embodiments, the avirulence nano-emulsion comprises the surfactant lipid prepared product (SLP) as the broad-spectrum antimicrobial agent of antibacterium effectively and their spore, envelope virus and fungus.In preferred embodiments, these SLP comprise oil, detergent, solvent and cation halogen compounds, and the ionic mixture of their biocidal activity of several enhancing.And have high zest and/or toxicly be purchased obtainable antibacterial and compare with sporicide, these SLP features are stable, nonirritating and avirulent chemical compounds.

The component that is used for the avirulence nano-emulsion includes but not limited to: detergent (for example, other members of other members of TRITON X-100 (5-15%) or TRITON family, TWEEN 60 (0.5-2%) or TWEEN family or TYLOXAPOL (1-10%)); Solvent (for example, Tributyl phosphate ester (5-15%)); Alcohol (for example, ethanol (5-15%) or glycerol (5-15%)); Oil (for example, soybean oil (40-70%)); Cation halogen compounds (for example, hexadecylpyridinium chloride (0.5-2%), cetyl pyridinium bromide (0.5-2%)) or cetyldimethylethylambromide bromide ammonium (0.5-2%)); Quaternary ammonium compound (for example, benzalkonium chloride (0.5-2%), N-alkyl dimethyl benzyl ammonium chloride (0.5-2%)); Ion (calcium chloride (1mM-40mM), ammonium chloride (1mM-20mM), sodium chloride (5mM-200mM), sodium phosphate (1mM-20mM)); Nucleoside (for example, inosine (50 μ M-20mM)); And aminoacid (for example, L-alanine (50 μ M-20mM)).By for example, in the high shear force agitator, mix and prepared Emulsion in 3 to 10 minutes.Can heat before down mixing 1 hour or can not heat Emulsion at 82 ℃.

Be used for quaternary ammonium compound of the present invention and include but not limited to N-alkyl dimethyl benzyl Saccharin Ammonium salt, 1,3,5-triazine-1,3,5 (2H, 4H, 6H)-three ethanol, 1-ten alkylammoniums, N-decyl-N, the N-dimethyl-, chlorine (or) DDAC, 2-(2-(right-(diisobutyl) toloxyl) ethyoxyl) ethyl dimethyl benzene ammonio methacrylate, 2-(2-(right-(diisobutyl) phenoxy group) ethyoxyl) ethyl dimethyl benzene ammonio methacrylate, alkyl 1 or 3 benzyls-1-(2-ethoxy)-2-imidazolitm chloride quinoline; Alkyl two (2-ethoxy) benzyl ammonium chloride, alkyl demethyl benzyl ammonium chloride, alkyl dimethyl 3,4-dichloro-benzenes ammonio methacrylate (100% C12), alkyl dimethyl 3,4-dichloro-benzenes ammonio methacrylate (50% C14,40% C12,10% C16), alkyl dimethyl 3,4-dichloro-benzenes ammonio methacrylate (55% C14,23% C12,20% C16), alkyl dimethyl benzyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride (100% C14), alkyl dimethyl benzyl ammonium chloride (100% C16), alkyl dimethyl benzyl ammonium chloride (41% C14,28% C12), alkyl dimethyl benzyl ammonium chloride (47% C12,18% C14), alkyl dimethyl benzyl ammonium chloride (55% C16,20% C14), alkyl dimethyl benzyl ammonium chloride (58% C14,28% C16), alkyl dimethyl benzyl ammonium chloride (60% C14,25% C12), alkyl dimethyl benzyl ammonium chloride (61% C11,23% C14), alkyl dimethyl benzyl ammonium chloride (61% C12,23% C14), alkyl dimethyl benzyl ammonium chloride (65% C12,25%C14), alkyl dimethyl benzyl ammonium chloride (67% C12,24% C14), alkyl dimethyl benzyl ammonium chloride (67% C12,25% C14), alkyl dimethyl benzyl ammonium chloride (90% C14,5% C12), alkyl dimethyl benzyl ammonium chloride (93% C14,4% C12), alkyl dimethyl benzyl ammonium chloride (95% C16,5% C18), alkyl dimethyl benzyl ammonium chloride (with) DDAC, alkyl dimethyl benzyl ammonium chloride (as in fatty acid), alkyl dimethyl benzyl ammonium chloride (C12-C16), alkyl dimethyl benzyl ammonium chloride (C12-C18), alkyl dimethyl benzyl and dialkyl dimethyl ammonium chloride, alkyl dimethyl dimethyl benzene ammonio methacrylate, alkyl dimethyl ethyl ammonium bromide (90% C14,5% C16,5% C12), alkyl dimethyl ethyl ammonium bromide (blended alkyl and alkenyl in the fatty acid as soybean oil), alkyl dimethyl ethylamino benzonitrile ammonium chloride, alkyl dimethyl ethylamino benzonitrile ammonium chloride (60% C14), alkyl dimethyl cumene ammonio methacrylate (50% C12,30% C14,17% C16,3% C18), alkyl trimethyl ammonium chloride (58% C18,40% C16,1% C14,1% C12), alkyl trimethyl ammonium chloride (90% C18,10% C16), alkyl dimethyl (ethylbenzyl) ammonium chloride (C12-18), two-(C8-10)-alkyl-dimethyl ammonium chloride, dialkyl dimethyl ammonium chloride, dialkyl dimethyl ammonium chloride, dialkyl dimethyl ammonium chloride, dialkyl methyl benzyl ammonium chloride, DDAC, the diiso decyl alkyl dimethyl ammonium chloride, Quaternium 24, dodecyl two (2-ethoxy) octyl group hydrochlorinate ammonium, dodecyl dimethyl benzyl ammonium chloride, dodecyl carbamyl methyl dimethoxy base benzyl ammonium chloride, seven decyl ethoxy imidazolitm chloride quinolines, six hydrogen-1,3,5-three (2-ethoxy)-s-triazine, myristyl benzyl dimethyl ammonium chloride (with) Quat RNIUM 14, N, N-dimethyl-2-hydroxypropyl ammonium chloride polymer, just-alkyl dimethyl benzyl ammonium chloride, just-alkyl dimethyl ethylamino benzonitrile ammonium chloride, just-the Zephiramine chloride monohydrate, the octyl-decyl alkyl dimethyl ammonium chloride, octyl group dodecyl dimethyl ammonium chloride, Octylphenoxy ethoxyethyl group dimethyl benzene ammonio methacrylate, oxygen diethylene base two (alkyl-dimethyl ammonium chloride), quaternary ammonium compound, two cocoa alkyl dimethyls, chloride, trimethoxy-silylpropyl dimethyl eight decyl ammonium chloride, the trimethoxysilyl quaternary ammonium compound, trimethyldodecane base benzyl ammonium chloride, just-dodecyl dimethyl ethylamino benzonitrile ammonium chloride, decyl dimethyl benzene ammonio methacrylate just-six, just-Zephiramine chloride, just-myristyl dimethyl ethyl benzyl ammonium chloride, decyl dimethyl benzene ammonio methacrylate just-eight.

Generally speaking, being characterized as of preferred avirulence nano-emulsion: their diameters are approximately 200-800nm, although comprise the nano-emulsion of bigger and littler diameter; Electric charge depends on component; They are stable in the long relatively time (for example, as many as 2 years), keep their biocidal activity; Because (to small part because) their the remarkable minimizing detergent and the reason of the toxic oily inclusions of solvent are compared with their individual components, they are nonirritating and nontoxic; They are effective being low to moderate on 0.1% the concentration; They have in 15 minutes the Chinese People's Anti-Japanese Military and Political College most vegetative bacteria (comprising Grain-positive and Gram negative biology), funguses and peplos are arranged and the antimicrobial acivity of nonencapsulated virus (for example, 99.99% kill); Sprout reinforcing agent generation ground with working as to use, they had the spore activity (for example, 99.99% kill) of killing in 1 to 4 hour.

D. animal model

In some embodiments, in the animal model of infectious disease, detect potential nano-emulsion compositions (for example, being used to produce immunoreation (for example, being used for)) as vaccine.Use good (well-developed) animal model of setting up that the effectiveness of measurement vaccine before people experimenter is used and the method for safety are provided.Table 3 shows the exemplary animal model of disease.These animals be purchased obtainable (for example, from Jackson LaboratoriesCharles River; Portage, MI).

The animal model of wax shape bacillus (being closely related with Bacillus anthracis) is used to detect anthrax vaccine of the present invention.Two kinds of antibacterials all are the Grain-positive corynebacteriums that forms spore, by each bacteriogenic disease symptoms mainly owing to the generation of toxin and these toxin to the host's that infects effect (people such as Brown, J.Bact, 75:499 (1958); Burdon and Wende, J.Infect Dis., 107:224 (1960); People such as Burdon, J.Infect.Dis., 117:307 (1967)).The disease symptoms that simulation is caused by Bacillus anthracis is infected in the bacillus of wax shape.Mice dies from the effect of wax shape bacillus toxin rapidly according to reports, and it is the useful model that is used for actute infection.Cavia porcellus produces the skin lesion similar to the epidermis form of anthrax after with wax shape bacillus subcutaneous injection.

Bacillus perfringens in mice and the Cavia porcellus (Clostridium perfringen) infects with the model system that acts on antibiotic medicine in the body (people such as Stevens, Antimicrob.Agents Chemother., 31:312 (1987); People such as Stevens, J.Infect.Dis., 155:220 (1987); People such as Alttemeier, Surgery, 28:621 (1950); People such as Sandusky, Surgery, 28:632 (1950)).Well-known Clostridium tetani (Clostridium tetani) infects in many mammal species and causes disease.Mice, Cavia porcellus and rabbit all are used for experiment (Willis, Topley and Wilson ' sPrinciples of Bacteriology, Virology and Immunity.Wilson, G., A.Miles, and M.T.Parker, eds.pages 442-4751983).

Successfully having caused vibrio cholera in mice, Cavia porcellus and rabbit infects.According to disclosed report, preferably change normal intestinal microbial population and infect in these experiment hosts, to set up., stop feeding animals and realize (people such as Butterton, Infect.Immun., 64:4373 (1996) to suppress the normal bowel flora to suppress normal intestinal microbial population and in some cases by administration of antibiotics; People such as Levine, Microbiol.Rev., 47:510 (1983); People such as Finkelstein, J.Infect.Dis., 114:203 (1964); Freter, J.Exp.Med., 104:411 (1956); And Freter, J.Infect.Dis., 97:57 (1955)).

Successfully having caused shigella flexneri (Shigella flexnerii) in mice, Cavia porcellus infects.The same with the situation of using vibrio infection, preferably change normal intestinal microbial population and infect to help in these experiment hosts, to set up., stop feeding animals and realize (people such as Levine, Microbiol.Rev., 47:510 (1983) to suppress the normal bowel flora to suppress normal intestinal microbial population and in some cases by administration of antibiotics; Freter, J.Exp.Med., 104:411 (1956); People such as Formal, J.Bact, 85:119 (1963); People such as LaBrec, J.Bact88:1503 (1964); People such as Takeuchi, Am.J.Pathol., 47:1011 (1965)).

Mice and rat have been widely used in the experimentation of using Salmonella typhimurium and Salmonella enteritidis (Salmonella enteriditis) (people such as Naughton, 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 rat are to use experimental model (people such as Jacoby, Exp.Gerontol., the 29:89 (1994) of good foundation of the infection of Sendai virus; People such as Massion, Am.J.Respir.Cell Mol.Biol.9:361 (1993); People such as Castleman, Am.J.Path., 129:277 (1987); Castleman, Am.J.Vet.Res., 44:1024 (1983); Mims and Murphy, Am.J.Path., 70:315 (1973)).

Usually the intracranial inoculation by newborn mice realizes that the sindbis virus of mice infects.Randomly, at the weanling mice of sufficient pad (footpad) subcutaneous vaccination (people such as Johnson, J.Infect.Dis., 125:257 (1972); Johnson, Am.J.Path., 46:929 (1965)).

Preferably, after shipping, animal close supported and had a rest with thing in 3 to 5 days, make it before being used to test, adapt to new pass and support environment.Begun in each experiment, killed control animal, obtained tissue to set up baseline parameter.(for example include but not limited to suction (being used for the short time operation) or ketamine/xylazine injection (being used for long-time operation) anesthetized animal of fluorine ether by any suitable method.

E. the algoscopy that is used for the estimation of vaccine

In some embodiments, use a kind of assessment candidate nanoemulsion vaccines in several proper model system.For example, can the cell-mediated immunoreation of external assessment.In addition, can use immunoreation and the immunity that assessment is attacked pathogen in the animal model.Can use any suitable animal model, include but not limited to disclosed animal model in the table 3.

Be to detect in the animal system before the nanoemulsion vaccines, research is enough to the amount of the pathogen of inactivating pathogens to the exposure of nano-emulsion.Allow to carry out immunity in order fully to be neutralized to, pathogen for example bacterial spore needs the longer time to carry out deactivation by nano-emulsion.Use any suitable method (including but not limited to disclosed method among the property of the following describes embodiment) research required time of deactivation.

In addition, in a period of time and under the storage requirement, estimate the stability of the vaccine of Emulsion exploitation especially, permanently effective to guarantee vaccine.Estimate that also other stabilizing materials (for example, dendritic) strengthen the stability and the immunogenic ability of vaccine.

Thereby the given nano-emulsion/pathogen vaccines of preparation cause pathogen inactivated after, optimize vaccine and cause immunoreation and immune ability is provided.The non-limiting example that is used for measuring the method for efficacy of vaccines is described in the following examples 14.For example, the time of application of vaccine and dosage can change, and determine the most effective dosage and time of application table.Come quantitative immunoreactive level by measuring the serum antibody level.In addition, use external test, by measuring H ³The picked-up of-thymidine monitors proliferation activity.Except that propagation, measure Th1 and Th2 cytokine reaction (for example, including but not limited to the level of IL-2, TNF-γ, IFN-γ, IL-4, IL-6, IL-11, IL-12 etc.) with the qualitative assessment immunoreation.

At last, use the effect of animal model assessment nano-emulsion mucosal vaccine.The pathogen of purification is blended in the Emulsion (or contacting Emulsion and pre-infected animals), uses it, determine immunoreation then.Then by with specific pathogen attack animal and afterwards the level of assess disease symptom assess the level of protection.The level of measuring immunity in a period of time is to determine the necessity and the interval of booster immunization.

III. therapeutic agent and preventive

In addition, in preferred embodiments, compositions of the present invention is induced (for example, when the experimenter is used) general and mucosal immunity.Therefore, in some preferred embodiments, the experimenter is used the protection that compositions of the present invention causes the uprising HIV of being exposed to (for example, mucosa exposes).Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, mucosal administration (for example, inoculation) provides anti-HIV to infect the protection of (for example, beginning at mucomembranous surface).Although proved so far the protection that is difficult to stimulate the secretory IgA, sIgA reaction and resists the pathogen of invading at mucomembranous surface (referring to; for example; people such as Mestecky; Mucosal Immunology. the 3rd edition. (Academic Press; San Diego; 2005)), but the invention provides and be used in the compositions and the method for experimenter's moderate stimulation from the mucosal immunity of pathogen (for example, protectiveness IgA reaction).

In some embodiments, the invention provides compositions and (for example, comprise NE and from the immunogenic protein antigen (for example, compositions gp120)) of HIV as mucosal vaccine.Available NE and HIV albumen are (for example, little fragments of peptides, the V3 cyclic peptide of the gp120 in the gp120 of viral source, live vector source and gp160, recombinant mammalian gp120, reorganization degeneration polypeptide, gp120 and gp41) easily produce this material, described material is induced mucosa and general immunity.Produce fast said preparation and provide the vaccine that can be used for using on a large scale (for example, the crowd to cities and towns, rural area, city, state or country uses) by the ability that mucosa (for example, nose or vaginal mucosa) instillation is used it.

In some preferred embodiments, the invention provides and be used to produce immunoreactive compositions, its comprise NE and immunogen (for example purification, isolating or synthetic HIV albumen or derivatives thereof, variant or analog; Or by one or more serotypes of the HIV of nano-emulsion deactivation).When the experimenter was used, compositions of the present invention was in the anti-immunogenic immunoreation of experimenter's moderate stimulation.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, immunoreactive generation (for example, owing to use and comprise that nano-emulsion and combinations of immunogens thing cause) (for example provide immunity wholly or in part to the experimenter, according to disease (for example, sign AIDS), symptom or the patient's condition).Be not subjected to the constraint of any particular theory; after being exposed to immunity compositions of the present invention; to the protection of disease and/or immunity (for example; experimenter's immune system is prevented or (is for example weakened; inhibition) ability of sign, symptom or the patient's condition of disease) (for example comes from adaptability; acquired) immunoreation (for example, be exposed to comprise immunogenic NE of the present invention after by the cell-mediated immunoreation of B and T (for example, showing specificity and the reactive immunoreation that increases) to HIV).Therefore, in some embodiments, the preventative or therapeutic of the compositions and methods of the invention ground is used to prevent or weaken the sign relevant with AIDS, symptom or the patient's condition.

The present invention is not subject to the type or the strain system (for example, comprising NE and immunogen (for example, by the vaccinia subgroup virus of nano-emulsion deactivation)) of employed vaccinia subgroup virus.In fact, can use each vaccinia subgroup virus family member individually or with another family member, be used to produce the compositions of NE of comprising of the present invention and immunogen (for example, being used to produce immunoreation).The vaccinia subgroup virus family member includes but not limited to alastrim virus, vaccinia virus, cowpox, monkeypox, gerbil jird pox, camel pox and other virus.The present invention is not subject to the strain system of employed vaccinia virus.In fact, comprise that multiple vaccinia virus strain is to be used for the present invention, the classical strain that includes but not limited to vaccinia virus (for example is, EM-63, Lister, the healthy committee (New York CityBoard of Health) in city, New York, Elestree and Temple of Heaven strain system), attenuated strain (for example, Ankara), the non-strain system of duplicating, the strain system of improvement (for example, by hereditarily or mechanically transforming the strain system of (for example, with stronger toxicity or the hypotoxicity more of becoming)), Copenhagen strain system (Copenhagen strain), the cowpox Ankara of improvement, the New York vaccinia virus, vaccinia virus _WR(Vaccinia Virus _WR) and vaccinia virus _WR-Luc, or the strain of other serial dilutions of vaccinia virus system.Comprise one or more strain systems that NE and combinations of immunogens thing can comprise the vaccinia subgroup virus of vaccine and/or other types.In addition; comprise NE and combinations of immunogens thing can comprise vaccinia virus one or more strains system and; the immunogen of one or more strain systems of non-vaccinia virus in addition or its immunogenicity epi-position are (for example; antibacterial (for example; Bacillus anthracis) or its immunogenicity epi-position (for example; the reorganization protective antigen) or virus (for example, west nile virus, bird flu virus, Ebola virus, HSV, HPV, HCV, HIV etc.) or its immunogenicity epi-position (for example, gp120)).

In some embodiments, immunogen can comprise the one or more antigens that derive from pathogen (for example, vaccinia subgroup virus).For example, in some embodiments, immunogen is purification, reorganization, synthetic or isolating protein (for example, being added among the NE to produce immunogenic composition).Similarly, immunogenic protein can be the form of proteinic derivant, analog or modification (for example, PEGization) from pathogen.

The present invention is not subject to the type or the strain system of employed bacillus or derives from its immunogenic protein.For example, the strain system of 89 kinds of different Bacillus anthracises of having identified, scope from poisonous Ames with biological war and bioterrorism purposes and Vollum strain system to the optimum Sterne strain system that is used to inoculate (referring to, for example, people such as Easterday., JClinMicrobiol.2005 43 (4): 1995-7).The difference of strain system is to determine their toxicity and the heterogeneic existence and the activity of the generation of antigen and toxin.Any strain system in these strain systems or strain system to be identified or that produce can be used for comprising the immunogenic composition of NE of the present invention.

In some embodiments, immunogen can comprise one or more antigen that derives from pathogen (for example, Bacillus anthracis).For example, in some embodiments, immunogen is purification, reorganization, synthetic or isolating protein (for example, being added among the NE to produce immunogenic composition).Similarly, immunogenic protein can be the form from proteinic derivant, variant, analog or the modification of pathogen.The present invention is not subject to the type of the protein (for example, deriving from the antibacterial of Bacillus) that is used to produce immunogenic composition of the present invention.In fact, can use panimmunity originality albumen, include but not limited to protective antigen (PA), lethal factor (LF), edema factor (EF), PA catabolite (referring to, for example, Farchaus, J. waits the people., Applied; Environmental Microbiol., 64 (3): 982-991 (1998)), with and analog, derivant and adorned form.

For example, the albumen of bacillus of the present invention uses with their native conformation, or more preferably, the modified vaccine use that is used for.These modifications can be that the technical reason relevant with the method for purification is necessary, or they may be used for the albumen (one or more functional characteristics for example, perhaps virose) in deactivation bacillus biologically.Therefore the present invention includes the proteic derivant of bacillus, described derivant can be the protein that for example suddenlys change (for example, having experienced one or more amino acid whose disappearance, interpolation or the metathetical protein that uses the technology that is used for site directed mutagenesis known or any other routine techniques to carry out).

Can in purge process, modify the protein of bacillus of the present invention (for example, rPA) so that this protein stabilization and singulation by chemical method.Prevent that the accumulative method of proteinic oxidation is to use the chemical modification of proteinic sulfydryl.In first step, by for example DTT, beta-mercaptoethanol or glutathion are handled and reduced disulphide bridges with Reducing agent.In second step, by reacting the sulfydryl that seals gained (for example, using iodoacetamide that protein is carried out carboxylic acid amidesization (carboxyamidated)/urea groups methylate (carbamidomethylated)) with alkylating agent.

Can use each bacillus family member to be used to produce the compositions of NE of comprising of the present invention and immunogen (for example, being used to produce immunoreation) individually or with another family member.Comprise one or more strain systems that NE and combinations of immunogens thing can comprise Bacillus anthracis.In addition, comprise NE and combinations of immunogens thing can comprise one or more Bacillus anthracis strains system and, the immunogen that another or multiple non-Bacillus anthracis strain are (for example, virus is west nile virus, bird flu virus, Ebola virus, HSV, HPV, HCV, HIV etc. for example) or its immunogenicity epi-position (for example, gp120)).

The present invention is not subject to employed HIV or from the type (for example, serotype, group or differentiation branch (clade)) of the immunogenic protein of its generation.For example, there are two types HIV:HIV-1 and HIV-2 at present.As if two types of trafficability characteristics contact, transmit by blood and from mother to child, and they cause the AIDS of undistinguishable clinically.As if yet HIV-2 is not easy to be transmitted, the time between primary infection and the disease is longer under the situation of HIV-2.In the world, dominant virus is HIV-1, when people mention HIV and indeterminate point out virus type, then they are meant HIV-1.Uncommon relatively HIV-2 type concentrates on West Africa and seldom sees them in other places.

The varying level that has the HIV classification.With each type packet, each group be divided into hypotype and popular reorganization pattern (circulating recombinant form, CRF).The strain system of HIV-1 can be divided into three groups: " mainly " group M, " (outlier) not in the know " group O and " newly " group N.

In group M, the known hypotype different at least 9 heredity of HIV-1 (or differentiation branch) that exists.These hypotypes are hypotype A, B, C, D, F, G, H, J and K.

In these hypotypes any or have to be identified or the serotype, group or the differentiation that produce to can be used for the immunogenic composition of the NE of comprising of the present invention.

In some embodiments, immunogen can comprise and derives from pathogen (for example, one or more antigens HIV).For example, in some embodiments, immunogen is purification, reorganization, synthetic or isolating protein (for example, being added among the NE to produce the protein of immunogenic composition).Similarly, immunogenic protein can be proteinic derivant, variant, analog or the adorned form from pathogen.The present invention is not subject to the type of the protein (for example, deriving from the protein of HIV) that is used to produce immunogenic composition of the present invention.In fact, panimmunity originality albumen be can use, gp160, gp120, gp41, Tat and Nef included but not limited to; With and analog, derivant and adorned form.

For example, HIV albumen of the present invention uses with their native conformation, or more preferably, modified to be used for vaccine use.These modifications can be that the technical reason relevant with the method for purification is necessary, or they may be used at proteic one or more functional characteristics of deactivation HIV biologically.Therefore the present invention includes the proteinic derivant of HIV, described protein derivatives can be the protein that for example suddenlys change (for example, having experienced one or more amino acid whose disappearance, interpolation or the metathetical protein that uses the technology that is used for site directed mutagenesis known or any other routine techniques to carry out).

For example, the HIV protein that can suddenly change keep simultaneously so that its abiology is active its immunogenicity epi-position (referring to, for example, Clements, Virology 235:48-64,1997).

In addition, can in purge process, modify HIV protein of the present invention so that protein stabilization and singulation by chemical method.Prevent that the accumulative method of the proteic oxidation of HIV is to use the chemical modification of proteinic sulfydryl.In first step, by for example DTT, beta-mercaptoethanol or glutathion are handled and reduced disulphide bridges with Reducing agent.In second step, by with alkylating agent react the sulfydryl that seals gained (for example, can use iodoacetamide to protein carry out carboxylic acid amidesization/urea groups methylates).

Can use serotype, group or the differentiation branch of each HIV to be used to produce the compositions of NE of comprising of the present invention and immunogen (for example, being used to produce immunoreation) individually or with another family member.Comprise one or more serotypes, group or differentiation branch that NE and combinations of immunogens thing can comprise HIV.In addition, comprise one or more serotypes, group or differentiation branch that NE and combinations of immunogens thing can comprise HIV and, the immunogen of one or more strains of non-HIV system (for example, virus for example west nile virus, bird flu virus, Ebola virus, HSV, HPV, HCV, HIV etc. or its immunogenicity epi-position) in addition.

The present invention is not subject to the particular formulations of NE of comprising of the present invention and combinations of immunogens thing.In fact, except NE and immunogen, the NE of comprising of the present invention can comprise one or more different reagent with the combinations of immunogens thing.These reagent or cofactor include but not limited to adjuvant, surfactant, additive, buffer agent, solubilizing agent, chelating agen, oil, salt, therapeutic agent, medicine, bioactivator, antibacterial and antimicrobial (for example, antibiotic, antiviral agent etc.).In some embodiments, NE of comprising of the present invention and combinations of immunogens thing comprise the reagent and/or the cofactor (for example, adjuvant) of the ability of the former induction of immunity reaction of enhance immunity.In some preferred embodiments, the existence of one or more cofactors or reagent has reduced the needed immunogenic amount of induction of immunity reaction (for example, protective immunological reaction (for example, protective immunity)).In some embodiments, the existence of one or more cofactors or reagent can be used for making immunoreation deflection cell (for example, T is cell-mediated) or body fluid (for example, antibody-mediated) immunoreation.The present invention does not limit and is subject to the cofactor that is used for therapeutic agent of the present invention or the type of reagent.

Usually be described in Vaccine Design-the Subunit and AdjuvantApproach at adjuvant, write by Powell and Newman; Plenum Press, New York, 1995.The present invention is not subject to the type (for example, being used to comprise in NE and the combinations of immunogens thing (for example, pharmaceutical composition)) of employed adjuvant.For example, in some embodiments, suitable adjuvant comprises aluminum salt for example gel aluminum hydroxide (Alumen) or aluminum phosphate.In some embodiments, adjuvant can be the salt of calcium, ferrum or zinc, maybe can be the polysaccharide of acidylate tyrosine or acidylate sugar, cation or anionic derivative or the insoluble suspension of polyphosphazene.

In some embodiments, preferred NE of comprising of the present invention and combinations of immunogens thing comprise the adjuvant that one or more induce the reaction of Th1 type.Yet in other embodiments, preferred NE of comprising of the present invention and combinations of immunogens thing comprise the adjuvant that one or more induce the reaction of Th2 type.

Usually, the former immunoreation that creates antagonism of the interaction by antigen and immune cell.Immunoreation can be divided into two classes widely: body fluid and cell-mediated immunoreation (for example, being characterized by the antibody and the cytological effect mechanism of protection routinely respectively).The kind of these reactions is called the reaction of Th1 type (cell-mediated reaction) and Th2 type immunoreation (humoral response).

Immunoreactive stimulation can be caused the direct or indirect reaction of interfering (for example, to immunogenic exposure) by immune cell or component.Can measure immunoreation by many methods, described method comprises activation, propagation or the differentiation of immune cell (for example, B cell, T cell, dendritic cell, APC, macrophage, NK cell, NKT cell etc.); The rise of label and cytokine or downward modulation are expressed; The stimulation of IgA, IgM or IgG titre; Splenomegaly (the splenocyte structure that comprises increase); Hyperplasia in the various organs, blended cellular infiltration.According to immune other reactions, cell and the component estimated at immunostimulation known in the art.

Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, the expression of the compositions and methods of the invention inducing cell factor and secretion are (for example, by macrophage, dendritic cell and CD4+T cellular expression and secretion).Can be local and the regulation and control of the expression of the general ground generation specific cells factor.Known cytokine spectrum can be determined the regulation and control and the effector functions of T cell in the epidemic disease reaction.In some embodiments, can induce the Th1 cytokines, thereby immunostimulatory compositions of the present invention can promote Th1 type antigen specific immune reaction (comprising cytotoxic T cell).Yet in other embodiments, can induce the Th2 cytokines, thereby promote the reaction of Th2 type antigen specific immune.

Cytokine works in instructing t cell responses.Auxiliary (CD4+) T cell is coordinated mammiferous immunoreation by the soluble factor that generation acts on other immune system cells (comprising B cell and other T cells).Most of ripe CD4+T accessory cells are expressed among two cytokine spectrum Th1 or the Th2.Th1 type CD4+T emiocytosis IL-2, IL-3, IFN-γ, GM-CSF and high-caliber TNF-α.Th2 cellular expression IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low-level TNF-α.The Th1 cytokines promotes cell-mediated immunity and is characterised in that immunoglobulins is converted to the humoral immunization of IgG2a (in the mice) and IgG1 (philtrum).The Th1 reaction also can be relevant with delayed-type hypersensitivity and autoimmune disease.The Th2 cytokines is induced elementary humoral immunization and is induced type conversion to IgG1 and IgE.The antibody isotype relevant with the Th1 reaction has neutralization and opsonizing capacity usually, yet relevant with anaphylaxis more with the antibody isotype of Th2 reaction.

Shown that several factors influence immunoreation and deflect to Th1 or the reaction of Th2 type.Characterizing best adjusting control agent is cytokine.IL-12 and IFN-γ are positive Th1 and negative Th2 adjusting control agent.IL-12 promotes the generation of IFN-γ, and IFN-γ provides positive feedback for IL-12.As if IL-4 and IL-10 important for the downward modulation of the output of the foundation of Th2 cytokine spectrum and Th1 cytokine.

Therefore, in some preferred embodiments, the invention provides in the immunoreactive method of experimenter's moderate stimulation Th1 type, it comprises the experimenter used and comprises NE and combinations of immunogens thing.Yet, in other preferred embodiments, the invention provides in the immunoreactive method of experimenter's moderate stimulation Th2 type, it comprises the experimenter used and comprises NE and combinations of immunogens thing.In other preferred embodiments, (for example can use, can use altogether with compositions of the present invention) adjuvant is so that immunoreation deflection Th1 or the immunoreation of Th2 type. and for example, induce the adjuvant of Th2 or reduction Th1 reaction to include but not limited to Alumen, saponin and SB-As4.Induce the adjuvant of Th1 reaction to include but not limited to MPL, MDP, ISCOMS, IL-12, IFN-γ and SB-AS2.

The Th1 type immunogen of several other types can be used for (for example, as adjuvant) the compositions and methods of the invention.These immunogens include but not limited to following immunogen.In some embodiments, use monophosphoryl lipid A (for example, 3-goes-O-acidylate monophosphoryl lipid A (3D-MPL) especially).3D-MPL is by Ribi Immunochem, the adjuvant of knowing that Montana produces.Chemically, its usually with 3-go-mixture of O-acidylate monophosphoryl lipid A and 4,5 or 6 acidylate chains provides.In some embodiments, use two phosphoryl lipid As and its 3-O-deacylated tRNA variant.Can be by the method purification of describing among the GB 2122204B (integrating with this paper) and each immunogen for preparing in these immunogens by quoting in full with it.Described other purification and synthetic liopopolysaccharides (referring to, for example, United States Patent (USP) 6,005,099 and EP 0 729473; People such as Hilgers., 1986, Int.Arch.Allergy.Immunol., 79 (4): 392-6; People such as Hilgers., 1987, Immunology, 60 (1): 141-6; With EP 0 549 074, it integrates with this paper by quoting in full with it separately).In some embodiments, use 3D-MPL with the form (for example, have the small particle size of diameter, be described in EP 0689454, integrate with this paper) of granular preparation by quoting in full with it less than 0.2 μ m.

In some embodiments, saponin is used as immunogen (for example, Th1 type adjuvant) in compositions of the present invention.Saponin is the adjuvant known (referring to, for example, Lacaille-Dubois and Wagner (1996) Phytomedicine the 2nd volume pp 363-386).The example of saponin comprise QuilA (deriving from the bark of tree Chile Gleditsia officinalis (Quillaja Saponaria Molina) in South America) and its fraction (referring to, for example, United States Patent (USP) 5,057,540; Kensil, Crit RevTher Drug Carrier Syst, 1996,12 (1-2): 1-55; With EP 0 362 279, it integrates with this paper by quoting in full with it separately).Also being used for of the present invention is haemolysis saponin QS7, QS17 and the QS21 (fraction of the HPLC purification of Quil A; Referring to, for example, people such as Kensil. (1991) .J.Immunology 146,431-437, United States Patent (USP) 5,057,540; WO96/33739; WO 96/11711 and EP 0 362 279, it integrates with this paper by quoting in full with it separately).Also spendable be QS21 and Polysorbate or cyclodextrin mixture (referring to, for example, WO 99/10008, integrates with this paper by quoting in full with it).

In some embodiments, comprise the immunogenicity oligonucleotide of unmethylated CpG dinucleotide (" CpG ") in the present invention as adjuvant.CpG is the abbreviation that is present in the cytosine-guanosint dinucleotide motif among the DNA.Known in the art, CpG when using be by general and mucosal route adjuvant (referring to, for example, WO 96/02555, EP 468520, people such as Davis., J.Immunol, 1998,160 (2): 870-876; McCluskie and Davis, J.Immunol., 1998,161 (9): 4463-6; With U.S. Patent application 20050238660, it integrates with this paper by quoting in full with it separately).For example, in some embodiments, immunostimulatory sequence purine-purine-C-G-pyrimidine-pyrimidine; Wherein the CG basic sequence is not methylated.

Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, in some embodiments, the existence of one or more CpG oligonucleotide activates the panimmunity subgroup, comprises natural killer cell (it produces IFN-γ) and macrophage.In some embodiments, the CpG oligonucleotide being formulated into compositions of the present invention reacts with induction of immunity.In some embodiments, with the free solution (free solution) of CpG and antigen (for example, be present in antigen in the NE solution (referring to, for example, WO 96/02555; Integrate with this paper by reference)) use altogether together.In some embodiments, with CpG oligonucleotide covalency be conjugated to antigen (referring to, for example, WO 98/16247, integrate with this paper by reference), or with carrier for example aluminium hydroxide prepare (referring to, for example, people such as Brazolot-Millan, Proc.Natl.AcadSci., USA, 1998,95 (26), 15553-8).

In some embodiments, with adjuvant for example complete Freund's adjuvant and incomplete Freund's adjuvant, cytokine (for example, interleukin (for example, IL-2, IFN-γ, IL-4 etc.), M-CSF, tumor necrosis factor etc.), antibacterial ADP ribosylation toxin (the random toxin (CT) of a for example, pertussis toxin, PT (PT) or escherichia coli heat-labile toxin (LT)) the detoxification mutant, LT-K63 (wherein on site 63 lysine displacement wild-type amino acid) especially, LT-R72 (wherein arginine displacement wild-type amino acid on site 72), CT-S109 (wherein at serine displacement wild-type amino acid on the site 109) and PT-K9/G129 (wherein replacing wild-type amino acid and glycine displacement wild-type amino acid on site 129 at lysine on the site 9) (referring to, for example, WO93/13202 and WO92/19265, it integrates with this paper separately by reference) and other immunogenic substance (for example, increasing the material of the effect of compositions of the present invention) use with NE of comprising of the present invention and combinations of immunogens thing.

The other example that is used for adjuvant of the present invention comprises poly-(two (carboxylato benzyl) phosphonitrile (PCPP polymer; Virus Research Institute, USA); The derivant of lipid polysaccharide is monophosphoryl lipid A (MPL for example; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyldipeptide (MDP; Ribi) and threonyl-muramyldipeptide (t-MDP; Ribi); OM-174 (the glycosamine disaccharide relevant with lipid A; OM Pharma SA, Meyrin, Switzerland); With leishmania elongation factor (the leishmania albumen of purification; Corixa Corporation, Seattle, Wash.).

Can in comprising NE and combinations of immunogens thing, add adjuvant, or can with comprise before NE and combinations of immunogens thing mix or use altogether, use carrier for example liposome or slaine (for example, aluminum salt (for example, aluminium hydroxide)) preparation adjuvant.

In some embodiments, comprise NE and combinations of immunogens thing and comprise single adjuvant of planting.In other embodiments, comprise NE and combinations of immunogens thing comprise two or more adjuvants (referring to, for example, WO 94/00153, WO 95/17210, WO 96/33739, WO 98/56414, WO 99/12565, WO 99/11241 and WO 94/00153, it integrates with this paper by quoting in full with it separately).

In some embodiments, NE of comprising of the present invention and combinations of immunogens thing comprise one or more mucoadhesives (referring to, for example, U.S. Patent application 20050281843 is integrated with this paper by quoting in full with it).The present invention is not subject to the type of employed mucoadhesive.In fact, comprise that many kinds of mucoadhesives are used for the present invention, its cross-linked derivant that includes but not limited to gather (acrylic acid) (for example, carbopol and polycarbophil (polycarbophil)), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharide (for example, alginate and chitosan), hydroxypropyl emthylcellulose, agglutinin, pilin (fimbrial protein) and carboxymethyl cellulose.Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, in some embodiments, owing to comparing with persistent period and/or amount under the situation of not using mucoadhesive to immunogenic exposure, experimenter's experience when using mucoadhesive to the persistent period of immunogenic exposure and/or the increase of amount, the use of mucoadhesive (for example, be used for comprising NE and combinations of immunogens thing) the inducing of enhance immunity reaction (for example, using compositions of the present invention) in the experimenter.

In some embodiments, compositions of the present invention can comprise the sterile aqueous prepared product.Acceptable vehicle and solvent include but not limited to water, Ringer's mixture, phosphate buffered saline(PBS) and isotonic sodium chlorrde solution.In addition, aseptic, fixed oil is conventionally used as solvent or suspension media.For this purpose, can use the fixedness mineral oil of any gentleness or non-mineral oil to comprise synthetic list-ordi-glyceride.In addition, fatty acid for example oleic acid be used for injectable prepared product.Be suitable for mucosa, subcutaneous, intramuscular, intraperitoneal, intravenous or be found in Remington ' s Pharmaceutical Sciences, MackPublishing Company, Easton, Pa by the carrier formulation that other approach are used.

Treatability (for example, with the enhance immunity reaction) or preventative (for example, being used for immunity (for example, with prophylactic S or S)) are used NE of comprising of the present invention and combinations of immunogens thing.Can use NE of comprising of the present invention and combinations of immunogens thing to the experimenter by many different route of delivery and method.

For example, can be to the experimenter (for example by several different methods, by mucosa (for example, nasal mucosa, vaginal mucosa etc.)) use compositions of the present invention, described method includes but not limited to: be suspended in the solution, be used for the surface then: be suspended in solution, use the spraying of spraying applicator then from the teeth outwards; Mix with mucoadhesive, be used for (for example, spraying or wiping) then on surface (for example, mucomembranous surface); Place or be immersed on nose and/or the vagina applicator, use then; Use by controlled release mechanism; Form with liposome is used; Or be used for polymer.

In some preferred embodiments, mucosal administration (for example, uses standard technique; Referring to, for example, Remington:The Science and Practice of Pharmacy, MackPublishing Company, Easton, Pa., the 19th edition, 1995 (for example, about the mucosal delivery technology, comprise intranasal, through lung, vagina and rectum technology), and European application 517,565 and people such as Illum., J.Controlled Rel., 1994,29:133-141 is (for example, technology about intranasal administration), it integrates with this paper by quoting in full with it separately) compositions of the present invention.Alternatively, can use standard technique (referring to, for example, Remington:TheScience arid Practice of Pharmacy, Mack Publishing Company, Easton, Pa., the 19th edition, 1995) by skin or transdermal administration compositions of the present invention.The present invention is not subject to the approach of using.

Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action; in some embodiments; mucosal vaccination is a preferred route of administration; has bigger effect of inducing protective immunological reaction (for example, mucosal immunity) at mucomembranous surface (paths that enter of many pathogen) because shown antigenic mucosal administration.In addition, mucosal vaccination, for example intranasal vaccination, not only mucosa immunity-inducing in nasal mucosa, but also mucosa position that can be a long way off for example genitals mucosa inducing mucosal immunity (referring to, for example, Mestecky, Journal of Clinical Immunology, 7:265-276,1987).More advantageously, in other preferred embodiments, except the mucosa immunity-inducing reaction, mucosal vaccination is also induced the general immunity.In some embodiments, non-parenteral (is for example used, the mucosal administration of vaccine) provide effective and conventional enhancing general immunity (for example, induce (for example, therein use repeatedly strengthen) by parenteral or mucosal vaccination with under the situation of keeping intensive general immunity) method.

In some embodiments, using compositions of the present invention by mucosal route (for example, oral/diet or nasal route) can be used to the NE of comprising of the present invention and combinations of immunogens thing to protect or treat experimenter disease-susceptible humans or that suffer disease.Alternative mucosal route comprises intravaginal and internal rectum approach.In a preferred embodiment of the invention, use the nasal route of using, this paper is called " intranasal administration " or " intranasal vaccination ".Intranasally inoculated method is known in this area, comprises the droplet or the Sprayable of vaccine are used into the nasopharynx for the treatment of immune experimenter.In some embodiments, provide the compositions that comprises NE and immunogenic spraying or atomizing.For example enteric coated capsule, the suppository that is used for rectum or vaginal application have also formed part of the present invention to be used for Orally administered Enteral formulations.Also can use compositions of the present invention by the per os approach.In these cases, comprising NE and combinations of immunogens thing can comprise pharmaceutically acceptable excipient and/or comprise alkali buffer agent or enteric coated capsule.Being used for preparation that nose sends can comprise and use glucosan or cyclodextrin and the saponin preparation as adjuvant.

Also can use compositions of the present invention by vaginal approach.In this case, comprise NE and combinations of immunogens thing and can comprise pharmaceutically acceptable excipient and/or emulsifying agent, polymer (for example, CARBOPOL) and the stabilizing agent of other known vaginal creams and suppository.In some embodiments, use compositions of the present invention by the rectum approach.In this case, comprise NE and combinations of immunogens thing and can comprise the excipient that is used to form rectal suppository known in the art and/or wax and polymer.

In some embodiments, select identical route of administration (for example, mucosal administration) to carry out primary vaccination and booster shot.In some embodiments, for immune response stimulating (for example, using NE and the combinations of immunogens thing of comprising of the present invention), (for example, simultaneously or alternatively, in succession) use the multiple approach of using.

For example, in some embodiments, in first or booster shot scheme, experimenter's mucomembranous surface used comprise NE and combinations of immunogens thing.Alternatively, in some embodiments, in first or booster shot scheme, general is used and is comprised NE and combinations of immunogens thing.In some embodiments, in the primary vaccination scheme, use by general, the experimenter is used comprise NE and combinations of immunogens thing by mucosal administration with in strengthened scheme.In some embodiments, in the primary vaccination scheme, use by general and in strengthened scheme by mucosal administration, the experimenter used comprise NE and combinations of immunogens thing.The example of the general approach of using comprises and includes but not limited to parenteral, intramuscular, Intradermal, uses through skin, subcutaneous, intraperitoneal or intravenous.Can be used for preventative and the therapeutic purpose with comprising NE and combinations of immunogens thing.

In some embodiments, send (pulmonary delivery) by lung and use compositions of the present invention.For example, can by suck (for example, thus pass the lung epithelial layer enter blood flow (referring to, for example, Adjei waits people .Pharmaceutical Research 1990; 7:565-569; Adjei waits people Int.J.Pharmaceutics 1990; 63:135-144; Braquet waits people J.Cardiovascular Pharmacology 1989 143-146; Hubbard waits the people. (1989) Annals of Internal Medicine, the 3rd volume, pp.206-212; Smith waits people .J.Clin.Invest.1989; 84:1145-1146; Oswein waits the people. ＂ Aerosolization of Proteins ＂, 1990; Proceedingsof Symposium on Respiratory Drug Delivery II Keystone, Colorado; Debs waits people .J.Immunol.1988; 140:3482-3488; With belong to Platz, wait people's United States Patent (USP) 5,284,656, it integrates with this paper by quoting in full with it separately)) compositions of the present invention is delivered to experimenter (for example, people's) lung.The lung that is used for medicine send with produce the general effect method and composition be described in and belong to Wong, wait people's United States Patent (USP) 5,451,569, incorporate this paper by reference entirely into; Also, integrate with this paper by quoting in full with it referring to the United States Patent (USP) 6,651,655 that belongs to people such as Licalsi)).

Be used to implement the many lung of pharmaceutical agent and/or machinerys that nasal mucosa is sent of being designed for that also comprise of the present invention, include but not limited to aerosol apparatus, metered dose inhaler and Diskus, it all is familiar with to those skilled in the art.Be suitable for implementing some particular instances that are purchased obtainable device of the present invention and be the Ultravent aerosol apparatus (Mallinckrodt Inc., St.Louis, Mo.); Acorn II aerosol apparatus (MarquestMedical Products, Englewood, Colo.); The Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.) and the Spinhaler Diskus (Fisons Corp., Bedford, Mass.).All these type of devices need to use the preparation that is suitable for disperseing therapeutic agent.Usually, each preparation is specific and except commonly used releasing agent, adjuvant, surfactant, carrier and/or other reagent that are used for the treatment of for employed device, also can comprise the use of suitable propellant material.In addition, comprise the use of the carrier of liposome, microcapsule or bead (microsphere), inclusion complex (inclusion complexe) or other types.

Therefore; in some embodiments, by mucosa, intramuscular, intraperitoneal, Intradermal, use through skin, through lung, intravenous, other route of administration subcutaneous or described herein and to comprise NE and combinations of immunogens thing and the NE of comprising of the present invention and combinations of immunogens thing be can be used for protecting and/or treat disease-susceptible humans or the experimenter that suffers disease.The method that the general of vaccine production thing is used can comprise conventional syringe and syringe needle or the device sent through the bombardment that is designed for solid vaccine (referring to, for example, WO 99/27961, integrate with this paper by reference) or needleless pressure fluid injection device (needleless pressure liquid jet device) (referring to, for example, United States Patent (USP) 4,596,556; United States Patent (USP) 5,993,412, it is incorporated into this paper separately by reference) or percutaneous plaster (referring to, for example, WO 97/48440; WO 98/28037, and it integrates with this paper separately by reference).The present invention also can be used for strengthening be used for skin (percutaneous or through subcutaneous, referring to, for example, WO 98/20734; WO 98/28037, and it integrates with this paper separately by reference) immunogenicity of antigens.Therefore, in some embodiments, the invention provides and be used for the delivery apparatus that general is used, vaccine combination of the present invention is housed in advance.

The present invention is not subject to the experimenter's who has used (for example, for immune response stimulating (for example, in order to produce protective immunity (for example, mucosa and/or general immunity))) compositions of the present invention type.In fact, many kinds of experimenters benefit from using of compositions of the present invention.In preferred embodiments, the experimenter is the people.In some embodiments, be the people examination person at any age (for example, adult, child, baby etc.) that maybe may be exposed to microorganism.In some embodiments, people experimenter more may accept the direct exposure of pathogenic microbes or more may show the experimenter (for example, immunosuppressant experimenter) of the S﹠S of disease after exposing pathogen.In some embodiments, the general population is used (for example, with inoculation) compositions of the present invention (for example, with prophylactic generation or diffusion).For example, in some embodiments,, the compositions and methods of the invention are used for inoculation crowd (for example, zone, city, state and/or national crowd) for they self health (for example) with prevention or treatment disease.In some embodiments, the experimenter is inhuman mammal (for example, pig, cattle, goat, horse, sheep or other domestic animal; Or mice, rat, rabbit or other animals).In some embodiments, the compositions and methods of the invention are used to study background (for example, being used to zoologize).

Prepare compositions of the present invention and be used for that for example mucosa, oral, local, parenteral or other approach described herein are used by any approach.Compositions can be that described form includes but not limited to tablet, capsule, powder, granule, lozenge, foam, emulsifiable paste or liquid preparation with any or multiple multi-form.

Can provide topical formulations of the present invention with for example form of ointment, cream or lotion, foam and aerosol, comprise wherein that suitable conventional additives is for example anticorrosion, solvent (for example, to help penetrate) and softening agent.

Topical formulations also can comprise the percutaneous reagent that penetrates of enhanced activity component.Exemplary agents comprise N-(ethoxy) ketopyrrolidine and destroy the binary combination of the chemical compound of cell envelope, with sugar ester and sucrose monooleate acid esters, decyl methyl sulfoxide and the alcohol of sulfoxide or phosphorous oxide combination.

Other exemplary materials that increase skin penetration comprise surfactant or wetting agent, include but not limited to polyoxyethylene groups sorbitol monooleate (polysorbate80), sorbitol monooleate (Span 80), right-iso-octyl polyvinyl-phenol polymer (Triton WR-1330), polyvinyl Sorbitol trioleate (Tween 85), dioctyl aerosol OT (sodiumsulfosuccinate) and sodium sarcosinate (Sarcosyl NL-97) and other pharmaceutically acceptable surfactants.

In certain embodiments of the invention, compositions also comprises one or more alcohol, zinc compound, softening agent, wetting agent, thickening and/or gellant, nertralizer and surfactant.The water that uses in the preparation preferably has the deionized water of neutral pH.Other additives in the topical formulations comprise but are not limited to silicone oil (silicone fluids), dyestuff, aromatic (fragrance), pH regulator agent and vitamin.

Topical formulations also can comprise the conventional carrier of some compatibilitys, for example cream or ointment base and the ethanol or the oleyl alcohol that are used for lotion.Examples of such carriers can account for about 1% to about 98% of preparation.Ointment base can comprise one or more vaseline, mineral oil, ceresine, lanolin alcohol, pantothenylol, glycerol, bisabolol (bisabolol), cocoa butter etc.

In some embodiments, prepare and use pharmaceutical composition of the present invention with foamy form.Pharmaceutical foam includes but not limited to following preparation, example emulsion, microemulsion, cream, gel and liposome.Although similar substantially in essence, these preparations change on the component of end-product and denseness.

Compositions of the present invention can comprise routine in addition and see other adjuvant components in the pharmaceutical composition.Therefore, for example, compositions can comprise other, the compatibility, the pharmaceutically active material for example, pruritus, astringent, local anesthetic or antiinflammatory, the material that maybe can comprise other the compositions of the present invention that is used for physics preparation different dosage form, for example dyestuff, flavoring agent, antiseptic, antioxidant, opacifier, thickening agent and stabilizing agent.Yet, fashionable when adding, the preferred not biologic activity of the component of excessive interference compositions of the present invention of this type of material.Can sterilize to preparation, if want, can with its with or not mix preparation NE and the interactional unfriendly auxiliary reagent of immunogen (auxiliary agent) (for example, lubricant, antiseptic, stabilizing agent, wetting agent, emulsifying agent, the salt that is used to influence osmotic pressure, buffer agent, coloring agent, flavoring agent and/or aromatic substance etc.).In some embodiments, use immunostimulatory compositions of the present invention with the form of pharmaceutically acceptable salt.When using, salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salt can be advantageously used in the preparation of its pharmaceutically acceptable salt.This type of salt includes but not limited to the salt that is equipped with from following processed with acid: hydrochloric acid, hydrobromic acid, sulfonic acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-methyl benzenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid.In addition, this type of salt can be formulated as alkali metal salt or alkali salt, for example sodium of hydroxy-acid group, potassium or calcium salt.

Suitable reducing includes but not limited to acetic acid and salt (1-2%w/v); Citric acid and salt (1-3%w/v); Boric acid and salt (0.5-2.5%w/v); And phosphoric acid and salt (0.8-2%w/v). suitable antiseptic can comprise benzalkonium chloride (0.003-0.03%w/v); Chlorobutanol (0.3-0.9%w/v); Metagin (0.01-0.25%w/v) and thimerosal (0.004-0.02%w/v).

In some embodiments, will comprise NE and combinations of immunogens thing and one or more antibiotic uses altogether.For example, can use comprise NE and combinations of immunogens thing before and/or use afterwards one or more antibiotic.The present invention is not subject to the antibiotic type of using altogether.In fact, can use multiple antibiotic altogether, include but not limited to beta-lactam antibiotic, penicillin (natural penicillin for example, Aminopenicillin, penicillinase-resistant penicillin, the penicillin carboxy class, the urea groups penicillin), cephalosporin (the first generation, the second filial generation and third generation cephalosporin) and other beta-lactams (imipenum for example, the monoamine rhzomorph), beta-lactamase inhibitor, vancomycin, aminoglycoside and spectinomycin, Tetracyclines, chloromycetin, erythromycin, lincomycin, clindamycin, rifampicin, metronidazole, polymyxin, doxycycline (doxycycline), quinolones (for example, ciprofloxacin), sulfa drugs, trimethoprim and quinoline.

Can obtain a large amount of antimicrobials that is used for the treatment of antibacterial, fungus and viral infection at present.For about the general classification of this type of medicine and the comprehensive agreement of their mechanism of action, ask those skilled in the art with reference to Goodman ﹠amp; People such as Gilman ' s ＂ The PharmacologicalBasis of Therapeutics ＂ Hardman edit, and the 9th edition, Pub.McGrawHill, the 43rd to 50 chapter, 1996, (integrating with this paper) by quoting in full with it.Generally speaking, these reagent comprise the inhibition synthetic reagent of cell wall (for example, penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); And imidazole antifungal agents (for example, miconazole, ketoconazole and clotrimazole); Directly act on the reagent (for example, detergent for example polymyxin (polmyxin) and Coli-Mycin S (colistimethate) and antifungal nysfungin and amphotericin B) of the cell wall of destroy microorganisms; Thereby influence the synthetic reagent of ribosomal subunit Profilin matter (for example, chloromycetin, Tetracyclines, erythromycin and clindamycin); Change protein synthesis and cause the reagent (for example, aminoglycoside) of cell death; Influence the reagent (for example, rifomycins and quinolones) of nucleic acid metabolism; Antimetabolite (for example, trimethoprim and sulfa drugs); With the nucleic acid analog of the enzyme that is used to suppress the synthetic necessary virus of DNA, for example zidovudine (zidovudine), gangcyclovir, vidarabine and aciclovir.Can use the various combination of antimicrobial.

The present invention also comprise participate in comprising NE and combinations of immunogens thing and one or more other activity and/or the method for using altogether of immunostimulant (compositions that for example, comprises NE and different immunogens, antibiotic, antioxidant etc.).In fact, another aspect of the present invention provides and is used for by using the method that compositions of the present invention strengthens prior art immunostimulation method (for example, immunization method) and/or pharmaceutical composition altogether.In being total to application process, can simultaneously or one after the other use reagent.In one embodiment, before other activating agents, use compositions described herein.Pharmaceutical preparation and the pattern of using can be any of pharmaceutical preparation described herein and the pattern used.In addition, can use different pattern (for example, approach) or different preparation to use the reagent that two or more are used altogether separately.Treat that other reagent (for example, antibiotic, adjuvant etc.) of using altogether can be any reagent of knowing in this area, include but not limited to the reagent that uses clinically at present.

In some embodiments, by number of ways the experimenter is used and comprise NE and combinations of immunogens thing.For example; benefit from and (for example have at the protective immunological reaction of pathogenic microbes; immunity) experimenter can benefit from and (for example accept mucosal administration; intranasal administration or other mucosal route described herein) and; (for example additionally accept one or more other route of administration; parenteral or pulmonary administration (for example, by aerosol apparatus, inhaler or additive method described herein)).In some implement preferred embodiments, be enough to induce the mucosa and the general immunity of the biology of originating by using of mucosal route at immunogen or immunogen.In other embodiments, be used to provide mucosa and general immunity by using of number of ways.Therefore, although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, but in some embodiments, by multiple route of administration (for example, immunity (for example, mucosa, air flue or the parenteral of NE of comprising of the present invention and combinations of immunogens thing are used)) experimenter who has used compositions of the present invention can have than only by a kind of approach used the experimenter of compositions stronger to immunogenic immunoreation.

Other delivery systems can comprise that time-delay discharges (time-release), slow release or lasting release delivery system.This type systematic can be avoided the repetitive administration of compositions, increases experimenter and doctor's convenience.The release delivery system of many types is obtainable and is known to those skilled in the art.They comprise the system based on polymer, for example poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester), copolymerized oxalate (copolyoxalate), polycaprolactone, polyesteramide (polyesteramide), polyorthoesters, poly butyric and polyanhydride.The microcapsule that comprises the above-mentioned polymer of medicine is described in for example United States Patent (USP) 5,075,109, integrates with this paper by reference.Delivery system also comprises the non-polymer system, and described system is: comprise for example lipid of cholesterol, cholesteryl ester and fatty acid or neutral fat (for example monoglyceride, diester and three esters) of steroid; The hydrogel delivery system; Silicone rubber system (sylastic system); System based on peptide; The wax coating; Use the compressed tablets of conventional binding agent and excipient; The heeling-in body of partial fusion etc.Specific embodiment includes but not limited to: (a) wherein comprise reagent of the present invention with in the substrate form and corrode system (erosional system), for example United States Patent (USP) 4,452,775,4,675,189 and 5, system of describing in 736,152 (they integrate with this paper separately by reference) and (b) the active component diffusion system (diffusional system) of from polymer, permeating wherein with in check speed, for example United States Patent (USP) 3,854,480,5,133,974 and 5, the system of describing in 407,686 (they integrate with this paper separately by reference).In addition, can use the hardware delivery system based on pump, some of them are fit to implant.

In preferred embodiments, NE of comprising of the present invention and combinations of immunogens thing comprise the immunogen of appropriate amount, thus induction of immunity reaction in the experimenter when the experimenter uses.In preferred embodiments, immunoreation is enough to provide the anti-protection (for example, immunoprotection) that is exposed to the microorganism (for example, antibacterial or virus) that immunogen or immunogen originate subsequently for the experimenter.The present invention is not subject to employed immunogenic amount.Implement in the preferred embodiment at some; selection (for example comprises in NE and the combinations of immunogens thing immunogen; by neutral virus of NE or antibacterial, or recombiant protein) the amount amount of immunizing agent (for example, as) do not have the amount of significant harmful side effect as the induction of immunity protective response.Described amount changes according to employed specific immunogen or its combination, includes but not limited to experimenter's species, age and overall state (for example, health) and mode of administration according to many factors and changes between the experimenter.Be used for determining the experimenter used with the method for the immunogenic appropriate amount that causes immunoreation (for example, protective immunological reaction (for example, protective immunity)) the experimenter and know for the art technology people.

In some embodiments; expect that each dosage (for example; (for example comprise NE and combinations of immunogens thing; the experimenter with the induction of immunity reaction (is for example used; protective immunological reaction (for example; protective immunity)) amount) comprise each immunogen (for example, the reorganization and/or the protein of purification) of 0.05-5000 μ g, in some embodiments; each dosage comprises 1-500 μ g; in some embodiments, each dosage will comprise 350-750 μ g, in some embodiments; each dosage will comprise 50-200 μ g; in some embodiments, each dosage will comprise the immunogen (for example, the albumen of reorganization and/or purification) of 25-75 μ g.In some embodiments, each dosage comprises is enough to produce immunoreactive immunogenic amount.Do not need immunogenic effective dose in the dosage quantitative, as long as when the experimenter is used immunogenic amount in the experimenter, produce immunoreation.Can (for example be identified for specific application by the research on standard that use relates to the observation of antibody titer and other reactions among the experimenter by those skilled in the art; with the induction of immunity reaction (for example; protective immunological reaction (for example, protective immunity))) optimised quantity.

In some embodiments, expect that each dosage (for example, (for example comprise NE and combinations of immunogens thing, the experimenter is used with induction of immunity reaction) amount) (for example be by immunogen, antibacterial that is neutralized or virus, or the albumen of reorganization and/or purification) weight calculating 0.001 to 15% or more (for example, 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more).In some embodiments, initial or first application dosage comprises than the more immunogen of booster dose subsequently.

In some embodiments, comprise every dose 10 to 10 when the microorganism of using NE deactivation of the present invention to live (for example, during virus (for example, HIV)), expecting each dosage (for example, the experimenter being used with the induction of immunity reaction)) ⁹The virus of pfu; In some embodiments, each dosage comprises every dose 10 ⁵To 10 ⁸The virus of pfu; In some embodiments, each dosage comprises every dose 10 ³To 10 ⁵The virus of pfu; In some embodiments, each dosage comprises every dose 10 ²To 10 ⁴The virus of pfu; In some embodiments, each dosage comprises the virus of every dose of 10pfu; In some embodiments, each dosage comprises every dose 10 ²The virus of pfu; In some embodiments, each dosage comprises every dose 10 ⁴The virus of pfu.In some embodiments, each dosage comprises above every dose 10 ⁹The virus of pfu.Implement in the preferred embodiment at some, each dosage comprises every dose 10 ³The virus of pfu.

In some embodiments, the microorganism of living when the deactivation of using NE of the present invention is (for example, during antibacterial (for example, the colony of the antibacterial of Bacillus (Bacillus anthracis)), expect each dosage (for example, the experimenter being used with induction of immunity reaction)) comprise every dose 10 to 10 ¹⁰Individual antibacterial; In some embodiments, each dosage comprises every dose 10 ⁵To 10 ⁸Individual antibacterial; In some embodiments, each dosage comprises every dose 10 ³To 10 ⁵Individual antibacterial; In some embodiments, each dosage comprises every dose 10 ²To 10 ⁴Individual antibacterial; In some embodiments, each dosage comprises every dose of 10 antibacterials; In some embodiments, each dosage comprises every dose 10 ²Individual antibacterial; In some embodiments, each dosage comprises every dose 10 ⁴Individual antibacterial.In some embodiments, each dosage comprises every dose and surpasses 10 ¹⁰Individual antibacterial.In some embodiments, each dosage comprises every dose 10 ³Individual antibacterial.

The present invention is not subject to the amount of the NE of the microorganism (for example, virus (for example, the HIV of one or more types)) that is used for deactivation and lives.In some embodiments, use 0.1%-5%NE solution, in some embodiments, use 5%-20% NE solution, in some embodiments, use 20% NE solution, in some embodiments, use NE solution for inactivating pathogenic microbes greater than 20%.In preferred embodiments, use 10% NE solution.

Similarly, the present invention be not subject to microorganism alive in order to be inactivated in NE of the present invention the persistent period of incubation.In some embodiments, microorganism is in NE incubation 1-3 hour.In some embodiments, microorganism is in NE incubation 3-6 hour.In some embodiments, microorganism in NE incubation above 6 hours.In preferred embodiments, microorganism incubation 3 hours in the NE (for example, 10% NE solution) in some embodiments, carries out incubation under 37 ℃.In some embodiments, carry out incubation being higher or lower than under 37 ℃ the temperature.The present invention is not subject to the amount of the microorganism that is used for deactivation yet.The amount of microorganism can be depending on many factors, includes but not limited to the total amount of the immunogenic composition (for example, NE and immunogen) wanted, the concentration of the solution wanted (for example, in dilution with before being used to use), microorganism and NE.Implement in the preferred embodiment at some, the amount that is used for the microorganism of ablation method is such amount, the amount of the immunogen of the experimenter being used with single dose (for example, from the dilution of spissated stock solution) (for example, immunogen described herein) that i.e. this volume production life is wanted.

In some embodiments, with spissated dosage preparation NE and the combinations of immunogens thing of comprising of the present invention, described spissated compositions can be diluted before the experimenter is used.For example, the dilution of spissated combination can be used the experimenter, the experimenter accepts any or multiple given dose provided herein like this.In some embodiments, the dilution of spissated compositions be can prepare, NE and combinations of immunogens thing in the spissated compositions of being present in of 0.5-50% comprised the experimenter is used (for example, with single dose).In some implement preferred embodiments, with single dose the experimenter is used and to comprise 1% NE and the combinations of immunogens thing in the spissated compositions of being present in.Comprise that spissated compositions is used for wherein using numerous experimenters the place (for example, the immunity is clinical, hospital, school etc.) of compositions of the present invention.In some embodiments, of the present inventionly (for example comprise NE and combinations of immunogens thing, spissated compositions) at room temperature stable above 1 week, surpassed for 2 weeks in some embodiments, surpassed for 3 weeks in some embodiments, surpassed for 4 weeks in some embodiments, surpassed for 5 weeks in some embodiments and surpassed for 6 weeks in some embodiments.

Generally speaking, emulsion composition of the present invention will comprise every ml fluid composition at least 0.001% to 100%, preferred 0.01 to 90% Emulsion.The expection preparation comprises every ml fluid composition 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 0.1%, about 0.25%, about 0.5%, about 1.0%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% Emulsion.The scope between any two above-mentioned numerals that should be appreciated that is included in border of the present invention and the scope clearly.Dosage depends on special pathogen and by the experimenter's of immunity situation, must change.

In some embodiments, use for the first time compositions of the present invention (for example, primary vaccination) after, the experimenter can and/or surpass the 1st time, the 2nd time, the 3rd time, the 4th, the 5th, the 6th time, the 7th time, the 8th time, the 9th time, the 10th time and accepts one or many after using for 10 times and strengthen using (for example, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 10 weeks, approximately March, approximately April, approximately June, approximately JIUYUE, about 1 year, about 2 years, about 3 years, about 5 years, about 10 years).Although to the understanding of mechanism is not to implement essential to the invention and the present invention is not subject to any specific mechanism of action, in some embodiments, the immunogenic introducing again makes it possible to produce very strong general immunity in the booster dose in the experimenter.Can use the same preparation that is provided for carrying out primary immune response to strengthen, maybe can use to comprise these immunogenic different preparations and strengthen.Dosage regimen is also incited somebody to action, and to small part, is determined and depended on practitioner's judgement by experimenter's needs.

Dosage unit can add deduct little based on Several Factors (including but not limited to experimenter's body weight, age and health status) pari passu.In addition, can increase or reduce dosage unit and be used for using subsequently (for example, strengthening using).

Comprise combinations of immunogens thing of the present invention be used for wherein causing infect and/or the reagent of disease (for example; searching is at the reagent of the protective immunity of its initiation) character be known situation; and be used for wherein causing infect and/or the character of the reagent of disease be condition of unknown (for example; at urgent sudden illness (for example; pandemic ratio (for example, influenza or other diseases breaks out)) under the situation).For example, the present invention includes compositions of the present invention is treating or (is for example preventing, cause by use and to infect and/or the HIV of disease or the immunity of HIV sample reagent (it neutralize by NE of the present invention)) with the reagent that causes urgent sudden infection and/or disease still to be identified (for example, from the patient separate and/or cultivate but nothing causes the reagent of heredity, biochemistry or other features of the reagent of infection and/or disease) purposes the relevant infection.

The compositions and methods of the invention will be used for different places, comprise the research place.For example, the compositions and methods of the invention also are used for immune research (for example, the sign of adaptive immunity reaction (for example, protective immunological reaction (for example, mucosa or general immunity))).The purposes of compositions provided by the invention and method comprises people and inhuman experimenter and from described experimenter's sample, comprises that also the research of using these experimenters uses.The compositions and methods of the invention also are used to study and optimize nano-emulsion, immunogen and other components and be used to screen new component.Therefore, do not wish that the present invention is defined in any specific experimenter and/or application places.

Can in being used for studying the animal model body of mucosal delivery and other route of delivery, many exploitations detect preparation.Clearly, compositions of the present invention is used to prevent and/or treat by virus, antibacterial, parasite and fungus-caused many kinds of diseases and infection, and is used for causing anti-multiple antigenic immunoreation.As mentioned above, not only can be preventative or therapeutic use described compositions, but also described compositions can be used to prepare antibody (polyclonal antibody and monoclonal antibody) (for example, being used for diagnostic purpose), and be used for the antigenic immune purification of purpose.If want polyclonal antibody, the mammal of available compositions immunoselection of the present invention (for example, mice, rabbit, goat, horse etc.).Usually use described antigen with one or many at 2-6 after week and strengthen animal.Can obtain polyclonal antiserum from the animal of immunity then, use in accordance with known methods then described polyclonal antiserum (referring to, for example, people such as Jurgens, J.Chrom.1985,348:363-370).

In some embodiments, the invention provides and comprise the test kit that comprises NE and combinations of immunogens thing.In some embodiments, test kit also provides the device that is used to use compositions.The present invention is not subject to the type of the device that comprises in the test kit.In some embodiments, design apparatus is used for nose and uses compositions of the present invention (for example, nose applicator (for example, syringe) or nasal inhaler or nasal mist).In some embodiments, test kit comprises with what conc forms (for example, the conc forms that can dilute before the experimenter is used) existed and comprises NE and combinations of immunogens thing.

In some embodiments, all reagent constituents are present in the single container (for example, bottle or test tube).In some embodiments, each reagent constituents places single container (for example, bottle or test tube).In some embodiments, one or more reagent constituents place single container (for example, bottle or test tube), and other components of same test kit place container (for example, bottle or test tube) separately.In some embodiments, test kit comprises buffer.In some embodiments, test kit also comprises operation instructions.

Embodiment

The following example is used to illustrate some preferred embodiment of the present invention and aspect and is not interpreted as limiting its scope.

Embodiment 1

Material and method: the vaccinia virus of nano-emulsion deactivation

Animal.Buy female Balb/c mice of pathogen-free domestic, 5 to 6 ages in week from Charles River laboratory.Look after criticism association (American Associationfor Accreditation of Laboratory Animal Care) standard according to U.S.'s laboratory animal and close foster inoculation group respectively, 5 animals of a cage.(University Committee on Use and Careof Animals UCUCA) relates to all methods of mice according to zoologic the College Board that uses and look after of University of Michigan.

Virus.In development process of the present invention, use two kinds of exemplary vaccinia viruss (VV), VV _WRAnd VV _WR-LucObtain VV from U.S.'s American type culture collection (ATCC) _WR(NIH TC-adaptability (NIH TC-adapted)).Reorganization VV _WR-LucBy p7.5 early stage/late promoter expresses LUC Photinus pyralis LUC Photinus pyralis FL, and be described (referring to, for example, people such as Luker, Virology.2005,341 (2): 284-300).VV _WR-LucIn external or body, do not weakened because the method that this virus is to use does not need to lack any viral gene make up (referring to, for example, Blasco and Moss (1995) .Gene 158 (2), 157-162; People such as Luker, Virology.2005,341 (2): 284-300).

Use have people such as some are improved, Lorenzo (referring to, for example, people such as Lorenzo, Methods Mol Biol.2004; Method 269:15-30) produces all viral stock solutions.Propagative viruses on the vero cells that infects with 0.5 MOI.At the 48th to 72 hour harvesting, from culture supernatant and cell lysate isolated viral.By the fast freeze-thaw cell precipitation, then in Dounce homogenizer in 1mM Tris pH 9.0 homogenate obtain cell lysate.By with the centrifugal cell debris of removing of 2000rpm.By with 25,4% to 40% the saccharose gradient higher slice that 000xg is centrifugal 1 hour obtains the virus stock solution used of purification from clarifying supernatant.Collection comprises the muddy band of virion, and it is diluted among the 1mM TrispH9, and then with 25,000xg concentrated in centrifugal 1 hour.With viral pellet resuspended in 1mM Tris pH 9, then under-80 ℃ as the virus stock solution used refrigerated storage.Titration VV on the cell of dimension Lip river _WRStock solution (referring to, for example, people such as Myc, vaccine .21:3801-3814).VV _WRHaving with natural strain is identical surface protein, but between infection period expressing luciferase.This makes it possible to carry out the appraisal of responsive cytotoxicity and sickness rate and the viral infection in the use imaging technique monitoring animal.By the VV that carries out with algoscopy in ELISA Western blotting or the virus _WRThe comparison of the serological reaction in the animal of immunity shows for VV _WROr VV _WR-LucTitre does not have difference.

Nano-emulsion (NE).From NANOBIO Corporation, Ann Arbor, MI obtains NE (W ₂₀5EC) (referring to belonging to NANOBIO Corporation (Ann Arbor, United States Patent (USP) 6,015,832 MI) are integrated with this paper by quoting in full with it).Make nano-emulsion by using emulsify at a high speed agent emulsifying hexadecylpyridinium chloride (1%), Tween 20 (5%) and ethanol (8%) in the water that contains soybean oil (64%).The droplet of gained have diameter be 300+/-mean particle size of 25nm." generally recognized as safe " surfactant and foodstuff preparation W (GRAS) thought in use by FAD ₂₀5EC.W ₂₀5EC can make economically under pharmaceutical production and quality management (Good Manufacturing Practice) (GMP) and stablize at least 18 months down at 40 ℃.

Preparation based on the vaccine of NE.Show that with data 1 hour incubation of use 10% NE or 0.1% formalin is enough to inactivation of viruses (for example, the minimizing of 6log VV titre) in the vaccinia virus that between development period of the present invention, produces (VV).Based on these results, produce several preparations (for example, compositions) of induction of immunity reaction (for example, bacterin preparation) that are used for to be used for animal immune.Be prepared as follows compositions (for example, being used for immune response stimulating): the VV for NE kills for guaranteeing virus neutralization (for example, inactivation of virus) completely, will comprise every dose 1 x 10 ³Pfu to 5x 10 ⁵The sample of the VV of pfu under 37 ℃ at 10% W ₂₀Incubation is 3 hours among the 5EC NE, is diluted to 1% NE then to be used for intranasal instillation (for example, every dose 1 x 10 ³To 1 x 10 ⁵Pfu).For the bacterin preparation that comprises the virus that formalin kills, at room temperature in 0.1% formalin, carry out formalin (Sigma) deactivation 3 hours of VV.The viral dilution that formalin is killed in saline solution or 1% NE to every dose 10 ³Or 10 ⁵Pfu, immunity is nontoxic concentration for intranasal thereby formalin is reduced to.For each preparation in each experiment,, behind 3 to 4 days incubation, carry out twice next inactivation of virus that uses NE or formalin to carry out in external affirmation that goes down to posterity of culture supernatants then by infecting dimension Lip river cell.The existence that shows viral plaque (viral plaque) is infected in the neither one contrast.In addition, the PCR detection assay of viral DNA of carrying out in the lung of dimension Lip river cell and the animal of accepting processing as described below is to confirm existing of the virus of duplicating alive.

Immunity.Before initial immunity, collect the sample of pre-immune serum from mice.With all animals of isoflurane anesthesia, use pipettor tip (tip) to inoculate (bacterin preparation that for example, uses every nostril 10-15 μ l) then.Use Emulsion lentamente so that swallowing of material is reduced to minimum level.After inoculation, observe animal with regard to disadvantageous reaction.Use (for example, inoculation) 3 weeks of back and when carrying out other use first (for example, initial), second with use (for example, inoculating) for the third time and in blood sample, measure the anti-VV antibody of specificity with the interval in 2 to 3 weeks.

By scratching, in dopey mice, carry out immunity by scratching in the tail base surface.Before carrying out this method, remove hair to expose about 0.5-0.7 square centimeter with shear, the ethanol with 70% is to exposed skin degerming.Use the bifurcated needle surface galling epidermis of sterilization, be used in 1 x 10 among the 10 μ l PBS ⁵The VV alive of pfu dosage _WRFixedly animal to 10 is minute to guarantee that virus is absorbed into skin.

The bioluminescence imaging.As described in other places (referring to, for example, people such as Luker, (2002) .J Virol 76 (23), 12149-12161; Cook and Griffin, (2003) .J Virol77 (9) 5333-5338) uses subcooled CCD photographing unit (IVIS) to carry out the bioluminescence imaging.Analyze the data of quantitative photon flux by the head of mice infected, the purpose district (ROI) of breast and abdominal part.From measuring, all deduct background photon flux from the not mice infected of fluorescein injection.

The collection of blood, bronchoalveolar lavage fluid (BAL) and splenocyte.In the process of the test of in development process of the present invention, carrying out, obtain blood sample from saphena at different time points.Obtain whole sample by cardiac puncture from painless lethal, premorbid mice.At blood after solidifying 30-60 minute under the room temperature, by obtaining serum from blood with centrifugal 5 minutes of 1500xg.Blood serum sample is stored in-20 ℃ until use.

Obtain BAL liquid from the mice that sucks anesthesia by isoflurane.Behind tracheostomize, No. 22 conduits (Angiocath, B-D) the insertion trachea of 1ml syringe will be attached to.The PBS perfusion lung 2 times that comprises 10 μ M DTT and 0.5mg/ml aprotinin with 0.5ml.Reclaim the aspirate (aspirate) of about 1.0ml with syringe.The BAL sample is stored under-20 ℃ until analyzed.

The mechanical separation mouse boosting cell is to obtain the single-cell suspension thing among the PBS.By using ACK buffer (150mM NH ₄Cl, 10mM KHCO ₃, 0.1mM Na ₂EDTA) erythrocyte (RBC) is removed in cracking, and remaining cell is cleaned in PBS 2 times.Measure in order to carry out antigenic specificity propagation or cytokine-expressing, with splenocyte (2-4 x 10 ⁶/ ml) be resuspended in RPMI 1640 culture medium that are supplemented with 5%FBS, 200nM L-glutaminate and penicillin/streptomycin (100U/ml and 100 μ g/ml).

The PCR of viral DNA detects.Use forward primer (SEQ ID NO.1:5 '-ATG ACACGA TTG CCA ATA C 3 ') and reverse primer (SEQ ID NO.2:5 '-CTA GAC TTTGTT TTC TG 3 ') (referring to, for example, people such as Ropp, J.Clin.Microbiol., 1995:2069-2076).These primers be at all vaccinia subgroup viruses (for example, HA gene conservative district VV), and by integrated dna technique (Integrated DNATechnologies) (IDT, Coralville, IA) synthetic.Use Trireagent according to the scheme of manufacturer (MRC, Cincinnati, OH), from dimension Lip river cell or from lung tissue homogenate DNA isolation.Use 0.5 each primer of μ M, each dNTP of 0.2mM, the MgCl of 2.5mM ₂(ROCHE Molecular Biochemicals, Indianapolis IN), carry out pcr amplification with intact cell or the lung DNA of 10 μ g with the Taq archaeal dna polymerase of 0.1U/ μ l.By in the cumulative volume of 20 μ l, 94 ℃ of following incubations 1 minute, carry out 25 circulation then 55 ℃ of annealing down, extend down and carry out the PCR reaction at 72 ℃ 94 ℃ of following degeneration.Be used for electrophoretic Tris borate buffer and be used for using electrophoresis to carry out the PCR product analysis on 1% the agarose gel of the painted ethidium bromide of DNA.(Hercules, CA) photoimaging photographing unit and software are analyzed from BioRad in use.To be used as positive control with the VV DNA (1ng) of the blended purification of lung DNA.(Hercules, CA) photoimaging photographing unit and software carry out gel analysis from BIORAD in use.

The mensuration of specificity antivirus IgA and IgG.Measure the mouse anti vaccinia antibody by ELISA.With comprising at least 5 x 10 among the PBS ⁴The dilution bag of the lysate of the dimension Lip river cell of the infection of the vaccinia virus in pfu/ hole is by the flat NUNC-PolySorp polystyrene board in microtitration 96 holes.Plate is incubated overnight under 4 ℃, fixes 1 hour with 50% ethanol/acetone mixture (EtOH/ acetone) down at-20 ℃ then.After removing fixative, with the PBS clean plate 2 times that comprises 0.001% Tween 20, then at 37 ℃ down with the sealing of 1% defatted milk powder among the PBS that comprises 0.2% Tween 20 1 hour.In containing the PBS of 0.1%BSA, serial dilution mice serum or BAL liquid, Xiang Kongzhong add 100 μ l five equilibriums, with plate 37 ℃ of following incubations 2 hours.With PBS-0.05% Tween 20 clean plates 3 times, the antibody incubation of puting together with anti-mice IgG or anti-mice IgA alkali phosphatase (AP) is 1 hour then, cleans afterwards 3 times.(SIGMA, St.Louis MO) carry out chrominance response to use AP substrate SIGMAFAST according to the scheme of manufacturer.(MOLECULAR DEVICES, Sunnyvale CA) carry out Spectrophotometric reading at 405nm place and 690nm with reference to the wavelength place to use SPECTRAMAX 340 ELISA readers.Terminal point titre and antibody concentration are calculated as and cause absorptance greater than two standard deviations serum dilution of (being higher than the absorptance in the control wells).Put together the concentration of logarithm conversion Calculation IgG antibody of the linear segment of the standard curve that the anti-IgG antibody of AP produces according to use, multiply by the serum dilution gfactor then.The concentration of serum antibody be expressed as meansigma methods+/-labelling mistake (sem).Be used as the contrast of non-specific adsorption from the serum of immature Mus.

Use above-mentioned several improved ELISA that have, measure the activity of targeting to the anti-VV IgG antibody of the alkylating virus epitopes of contrast formalin of pure degeneration.With 1 x 10 ⁵The vaccinia virus bag of the purification in pfu/ hole is incubated overnight under 4 ℃ then by 96 orifice plates.After removing virus removal, handled the hole 1 hour at 4 ℃ down or with 1% formalin solution among the PBS at-20 ℃ with 50% EtOH/ acetone.Clean as mentioned above and closure plate.In the future the pooled serum of personal different bacterin preparation (VV/NE, VV/Fk/NE, VV/Fk) mice immunized and from the Asia that experience is carried out with the vaccinia virus of living (VV lives) cause death (sub-lethal) infect and the serum serial dilution of the mice that survives in 0.1% BSA, 100 μ L five equilibriums are added to EtOH/ acetone and in the hole of formalin fixed.Measure with regard to the mensuration of anti-cowpox IgG as mentioned above.Relatively the optical density (OD) at the 405nm place between the virus antigen of EtOH/ acetone and formalin fixed is worth.By on identical OD 405nm value, the ratio of the IgG titre of EtOH/ acetone contrast formalin is estimated the active difference of anti-vaccinia antibody.

Neutralizing antibody.Use standard standard bacterial plaque minimizing algoscopy (PRA) (referring to, for example, people such as Newman, J.Chem.Microbiol.2003 is 3154-3157) with use reorganization VV _WR-LucThe active inhibition of fluorescein measure neutralizing antibody.By using 10 μ l to comprise in the successive doubling dilution thing of serum-free RPMI culture medium of VV of 200-300pfu, the heat-inactivated mice serum that mixes 10 μ l carries out PRA.37 ℃ of following incubation serum 6 hours, then serum is placed the 0.5ml serum-free medium that covers on the cell monolayer of dimension Lip river.Behind 1 hour incubation, remove virus removal/serum inoculum, the culture medium of newly preparing is placed on the cell monolayer.After 48 to 72 hours, fixed cell, (crystal violet) dyes to it with 0.1% crystal violet.By two independently the observer count bacterial plaque, use NIS calculate in contrast in and titre.

About VV _WR-LucIn and the assessment of titre, with 2 x 10 that comprise of heat-inactivated mice serum in the continuous doubling dilution thing of 10 μ l and 10 μ l ³The serum-free RPMI culture medium of the virus of pfu is mixed.With measure identically based on the neutralization of PRA, sample 37 ℃ of following incubations 6 hours, is resuspended among the serum-free RPMI of 100 μ l, then with dimension Lip river cell incubation 1 hour in 24 orifice plates.After 24-36 hour, the cell that cracking is infected is estimated the activity of viral dependency luciferase by above-mentioned luciferase assay method.NIS among the use PBS suppresses the curve calculation and titre (NT from luciferase in contrast ₅₀).Pin obtains the relation between PRA and the fluorescein enzyme inhibition activity in each sample.

Cowpox specific cell factor expression in the splenocyte.12 weeks were gathered in the crops the spleen from vaccinated mice behind primary vaccination.Obtain splenocyte by the mechanical damage spleen, with splenocyte with 3 x 10 ⁶Individual cell/ml is suspended among the RPMI1640 that is supplemented with 5%FBS, L-glutaminate and penicillin/streptomycin.With cell and every hole 1 x 10 ³Or 1 x 10 ⁴The vaccinia virus of pfu is together 37 ℃ of incubations 72 hours.The harvesting culture supernatants is analyzed it with regard to production of cytokines.With PHA-P (1 μ g/ hole) with the cell incubation as positive control.According to the guidance of manufacturer, use QUANTIKINE M ELISA test kit (R﹠amp; D SYSTEMS Inc, Minneapolis, MN) concentration of the IFN-γ in the mensuration splenocyte supernatant.

The attack of vaccinia virus.The vaccinia virus of use living is attacked mice immunized to estimate the effect of vaccine.Attack preceding 2 days collection blood serum samples in cowpox, animal was weighed on the same day of attacking.The reorganization VV of purification of five equilibrium thaws _WROr VV _WR-Luc(carrying out supersound process and titration before the use) was diluted in it in saline solution on the same day of attacking then.By sucking the isoflurane anesthesia mice, use 20 μ l then corresponding to 10 x LD ₅₀2 x 10 ⁶The VV alive of pfu _WR-LucFloat or use 5 times of scopes in the dilution at 1 x 10 ⁷To 3.2 x 10 ³Between the VV alive that changes _WRDosage is attacked its intranasal.Measure weight and body temperature every day after attack, carried out for 3 weeks.Painless deadly demonstration reduces the mice of 30% initial body weight.Fatal dose (LD ₅₀) and infective dose (ID ₅₀) calculating respectively based on the animal dead rate and based on core temperature and volume alleviate (referring to, for example, Reed and Muench, Am J Hyg 1938; 27:493-7).Anti-the causing death of following calculating attacked (IP _LD) the protection index: IP _LD=log ₁₀Maximal dose-log ₁₀LD ₅₀Contrast.Similarly, following calculating infection (IP _ID) the protection index: IP _ID=log ₁₀The ID of immunity ₅₀-log ₁₀LD ₅₀Contrast.

Statistical analysis.Use ANOVA and the Student's T Test of determining that is used for the p value that the result is carried out statistical analysis.

Embodiment 2

Use the nose immunity of the vaccinia virus of nano-emulsion deactivation to cause the reaction of inducing specific systemic IgG.

For viricidal activity, with a series of NE concentration and VV at external estimation NE _WROr VV _WR-LucMix and 37 ℃ of following incubations 1 to 3 hour.The result of bacterial plaque minimizing (PRA) and luciferase bioluminescence assay has shown the NE concentration dependent deactivation of two kinds of viruses.But 10%NE complete inactivation in 1 hour incubation surpasses 10 ⁶The cowpox of pfu (referring to Figure 1A and B).The going down to posterity subsequently of culture supernatants of cell of VV (using 10% NE deactivation) infection of using by oneself shows there not be the virus of surviving.

VV in the intranasal administration deactivation _WRAfter, by using, further proved the complete inactivation of the virus in the NE preparation in vivo from using the pcr amplification of back mouse lung separated DNA.Do not detect viral DNA in the mice of where managing in office (referring to Fig. 1 C), yet contrast PCR (dopes VV _WRThe lung DNA of DNA) obtain expecting size〉product of 950bp.In addition, the bioluminescence imaging also shows in the body of mice, and from 1 x 10 ⁵Or 1 x 10 ⁶The VV alive of pfu _WR-LucThe strong signal of nose mice infected is compared, and is using 10 ⁵The VV of the NE deactivation of pfu _WR-LucAfter do not have viral infection and do not have the sign (referring to Fig. 1 D) of virus amplification.Therefore, external showing with 10% NE incubation with the interior result of body caused the VV complete inactivation at least in 60 minutes.Therefore, use 10% NE to carry out all inactivation of virus, carried out 3 hours, be diluted to 1%NE then to be used to carry out immunity.

Then, contrived experiment with estimate compositions of the present invention (for example, NE kill VV) whether can produce to by scratch the similar protective immunity seen among the people with the VV inoculation of living, duplicate (referring to, for example, people such as Hammarlund, Nat.Med.2003,9; 1131-1137).Comprise 10 with 6 ⁵Or 10 ³Pfu dosage (be respectively 10 with NE ⁵/ NE and 10 ³/ the VV that NE) kills _WR, with 1% NE (be respectively 10 ⁵/ Fk/NE and 10 ³/ Fk/NE) the virus and the saline solution (10 that kill of blended formalin ⁵/ Fk and 10 ³Preparation intranasal (i.n.) immune mouse of the virus that/ formalin in Fk) kills.Handle control mice with 1% NE individually.3 weeks characterized antibody response (referring to Fig. 2) behind initial vaccine administration.The the 5th and the 9th week with subsequently use booster immunization (Fig. 2 A).Behind booster immunization, using by oneself 10 ⁵/ NE or 10 ⁵Detect significant anti-VV IgG level in the serum of the mice of/Fk/NE inoculation, average anti-VV IgG concentration is respectively 1.5 μ g/ml and 1 μ g/ml.After reinforcement for the second time, the concentration of anti-VV antibody increases in all groups, (the 16th week) use 10 when experiment finishes ³/ NE and 10 ⁵The immunity of/NE produces the highest reaction, and the mean concentration of anti-VV IgG is respectively 44 μ g/ml and 70 μ g/ml, is to use 10 then ⁵The immunity of/Fk/NE (17 μ g/ml).Use 10 ³/ Fk/NE and 10 ⁵/ Fk or 10 ³The immunity of the bacterin preparation of/Fk produces low-level anti-VV antibody all the time, and described antibody does not significantly increase (referring to Fig. 2 A) after reinforcement is used.Use 10 ⁵The relatively demonstration of the single dose of/NE and three doses of immunization protocols, in 12 weeks after immunity, single dose vaccine produces the concentration of significant (about 4 μ g/ml) (though lower than three doses) anti-VV IgG of serum.Therefore, in some embodiments, the invention provides, single dose VV/NE vaccine may be enough to cause immunoreation (for example, mucosa or general immunoreation), and described immunoreation can strengthen (referring to Fig. 2 A illustration) by immunity subsequently.In any control mice, do not detect the anti-VV antibody of specificity.

The analysis showed that of the anti-VV antibody of cross reactivity, (alkylating) virus protein that serum IgG of the mice of the virus immunity that the NE that uses by oneself kills and formalin are crosslinked and react with alcohol fixed (degeneration but not alkylating) virus protein.Use the inoculation of VV/NE vaccine to produce anti-VV IgG, it has than reactivity natural, that non-alkylating epi-position is high 25 times (to similar from the serum of the mice that is exposed to live virus), and these antibody are also discerned the virus protein of formalin fixed effectively.On the contrary, the use by oneself serum of animal of the virus that formalin kills (independent or blended with NE) inoculation does not have increase and reactivity natural VV epi-position.

Embodiment 3

The experimenter who has used the vaccinia virus that nano-emulsion kills has the mucosal immunity at vaccinia virus

By the VV specific secretion IgA TPPA mucosal immunity in the bronchoalveolar lavage fluid (BAL).From using 10 ³/ NE or 10 ⁵Detect anti-VV IgA among the BAL of the animal of/NE immunity.Animal with the preparation that comprises the virus that formalin kills (no matter dilute in saline solution still dilute in NE) inoculation does not produce measurable mucosa reaction, although there is the anti-VV IgG of serum (referring to Fig. 2 B).Therefore, the invention provides, the compositions that comprises the VV that NE kills (for example produces mucosal immunity in the experimenter, as by VV specific secretion IgA antibody among experimenter's the BAL existence proved), yet the compositions that does not comprise the VV that NE kills (for example, formalin kill VV) can not produce the mucosal immunity at VV.

Embodiment 4

Serum and bronchus alveole irrigating solution (BAL) from the experimenter who has used the vaccinia virus that nano-emulsion kills have virucidin

In measuring, the virus neutralization estimates the biological relatedness of anti-VV antibody response.In the serum of more postvaccinal mices once, detect neutralization active (Fig. 3 A).Yet, using 10 ⁵/ NE10 ³/ NE or 10 ⁵After twice immunity of/Fk/NE, the titre of the unanimity in the serum neutralizing antibody appears.These the group in each average 50% in and titre (NT ₅₀) 〉=20.On the contrary, with 10 ³/ Fk/NE, 10 ³/ Fk or 10 ⁵The animal of/Fk inoculation only observes the virus neutralization in minimum serum dilution.Immunity subsequently produces the NT greater than 10 times ₅₀The increase of titre, but only be present in the virus (10 of killing with NE ³/ NE and 10 ⁵/ NE is respectively NT ₅₀=180 and NT ₅₀=500) in Jie Zhong the mice.The 3rd inoculation that use comprises any preparation of the virus that formalin kills does not significantly increase the neutralization of VV.Also using by oneself 10 ³/ NE or 10 ⁵It is active to detect significant neutralization in the BAL liquid of the mice of/NE inoculation, and described neutralization activity is from use 10 ³/ Fk/NE or 10 ⁵Lower among the BAL of/Fk/NE mice immunized (referring to Fig. 3 illustration).Do not exist among the BAL of the mice of the virus immunity that the active formalin that dilutes in saline solution of neutralization kills and contrast, nonvaccinated animal.Therefore, the invention provides, although there is the deactivation (for example neutralization fully) of VV, the nano-emulsion that comprises the VV of deactivation of the present invention (for example keeps important immunogenicity epi-position, by experimenter's immune system (for example, immunity system) identification and reaction).

Embodiment 5

To natural VV _-WRAnd VV _-WR-LucThe comparison of reaction

VV _-WR-LucHaving with natural strain is identical surface protein, but between infection period expressing luciferase albumen.This allows to use imaging technique that the viral infection in the animal is under fire carried out mortality rate estimation and monitoring.During ELISA, Western trace or virus neutralization are measured, VV _-WRThe relatively demonstration VV of the antibody in two kinds of virus strains of animal contrast of immunity _-WRAnd VV _-WR-LucBetween do not have difference.

Embodiment 6

Using of the VV that NE kills produces the VV specific cell immunoreaction

Use cell in vitro factor expression algoscopy is studied the effect based on the vaccine pair cell reaction of NE in splenocyte.With 10 ³With 10 ⁴The cattle on the hoof pox of pfu stimulates the individual culture of mouse boosting cell.By using by oneself 10 ³/ NE or 10 ⁵The vivoexpression of IFN-γ in the splenocyte of the animal of/NE immunity has proved the VV specific cell immunoreaction.On the contrary, in the splenocyte of the immune animal of the virus (even when it is mixed with nano-emulsion) that the formalin of using by oneself kills, do not observe the VV specificity IFN-γ product of increase.The generation of IFN-γ in the cell of the mice that the VV/NE vaccine of using by oneself is handled shows the Th1 polarization of cell effect.In contrast spleen cell cultures thing, do not detect antigenic specificity cytokine-expressing (referring to Fig. 4).

Embodiment 7

The experimenter who has used the VV that NE kills is subjected to the protection at attack that live, infectious VV

In Attack Research, estimate protective immunity by the mucosal immunity generation.With 3 dose 10 ⁵/ NE, 10 ⁵/ Fk/NE or 10 ⁵Three groups of mices of/Fk vaccine nose immunity.Handle control animal with saline solution.In the 12nd week, with mice 10 x LD ₅₀(2 x 10 ⁶Pfu) VV alive _-WR-LucAttack mice.Measure body weight and body temperature every day 2 times, every day is with regard to VV _-WR-LucLuminous taking pictures once.All are 10 years old ⁵The mice of/NE inoculation experiences viral attack and survive (referring to Fig. 5 A).With 10 ⁵/ Fk/NE and 10 ⁵The mice of/Fk inoculation has 40% and 20% survival rate respectively.Although do not obtain protection fully, use 10 ⁵The time (TTD) had prolonged 5 to 7 days before the inoculation of/Fk/NE also will be put down death.Control mice does not have one to live through attack.The bioluminescence imaging that is used to estimate viral infection shows five 10 ⁵But two virus replications with minimum detection limit are arranged in/NE the mice infected, and this does not influence their weight and body temperature, yet other three have and can have more aggressive duplicating (referring to Fig. 5 B) what attack was gone down in back 6 days.Yet these animals do not have a clinical mark ten days with infection.On the contrary, all nonvaccinated contrasts are sick or dead, or in 4 to 7 days of virus attack it are rich in the till death painless of human feelings.As shown in the photon flux data, these animals have a large amount of virus replications and transmission of infection at whole nasopharyngeal meatus, lung and abdominal part.10 ⁵In the mice of/NE inoculation, attack low head (nose) portion that infects the animal that is defined to inoculation in back at intranasal, and do not expand to chest and abdominal part (referring to following table 4).

Table 4

In a word, imaging research shows the diffusion of infection and the negative correlation between the survival.The existence of the autolimiting infection in some immune animals is relevant with the level of neutralizing antibody in the individual animals.

For further studying effect, will use 3 dose 10 based on the vaccine of mucosa NE ⁵The immunity of the intranasal of/NE with use the VV that lives by scratching _WR(10 ⁵/ SC) inoculation relatively.In the 12nd week, use the VV of the work of increasing dosage _WRMice is carried out intranasal to be attacked.Survival data shows that the protective immunity that mucosal immunity produces is equal to by being generally used for the inoculation of scratching (referring to following table 5) of people's variola vaccine.

A is expressed as the ratio of survival mice to all mices

The 1 x LD that b calculates ₅₀Be 5.13 x 10 ⁵The VV of pfu _WR

Table 5

Live through use maximal dose 1 x 10 with mucosa VV/NE vaccine or by all mices of scratching inoculation ⁷The VV of pfu _WR(77 x LD ₅₀) intranasal attack.The anti-protection index (IP that causes death and attack _LD) for based on the vaccine of NE with to scratch all be 1.9.With 15 x LD ₅₀VV _WRAfter the attack, all contrast the animal dead of non-inoculation.In losing weight of survival mice analyzed, also see the high-level protective effect of using the intranasal immunity to obtain.Although mucosal vaccination can not protect mice at the VV that uses high dose fully _WRRespiratory infections (referring to following table 6), but do not have the clinical indication of disease with the animal of NE vaccine immunity, but have 10% or average weight still less alleviate, yet the survival mice in the matched group is at much lower VV _WRDosage under lose 25% body weight.There is the difference of p value＜0.01 in statistical results show between the body weight of mice immunized and control mice.Infection protection index (IP _ID) be 1 for the VV/NE vaccine, be 2.2 for subcutaneous vaccination.

A be expressed as attack the successor when between body weight and the nondecreasing mice of body temperature to the ratios of all mices

The 1 x LD that b calculates ₅₀Be 5.13 x 10 ⁵The VV of pfu _WR

Table 6

Embodiment 8

Material and method: rPA/NE vaccine

Animal.From Charles River laboratory (Wilmington MA) buys pathogen-free domestic, female Balb/c, CBA/J mice (5-6 age in week) and Hartley Cavia porcellus (female, 250g).Look after the criticism association criterion according to U.S.'s laboratory animal and close foster mice and Cavia porcellus.(the University Committee on Use and Care of Animals of zoologic the College Board that uses and look after according to University of Michigan, UCUCA), University of Texas Medical Branch at Galveston, the management of laboratory animal of TX and use committee (Institutional Animal Care and UseCommittee) (IACUC) and Battelle Memorial Institute, Columbus, the standard operating instructions of OH are carried out all methods that relate to animal.

Reagent.Form with the proteinic lyophilized formulations of purification; from List BiologicalLaboratories; Inc. (Campbell, CA) and BEI armory (BEI ResourceRepository) (ATCC) obtain recombinant anthrax bacillus protective antigen (rPA) and lethal factor (rLF).After in aseptic MILLI-Q water (5mg/ml), rebuilding, five equilibrium is stored down at-80 ℃.By integrated dna technique (IDT, Coralville, IA) the synthetic polynucleotide (ODN) 5 that comprises the multiple 20-mer of non-methylated CpG '-TCC ATG ACG TTC CGT ACG TT-3 ' (SEQ ID NO.:3) (referring to, for example, people such as Moldoveanu, 1999.15:1469-1476).From SIGMA-ALDRI CHCorporation (St.Louis, MO) buy the escherichia coli monophosphoryl lipid A (MPLA, #L-6638), PHA-P, BSA, DTT and be used for other chemical drugss of buffer.Respectively from GIBCO (Grand Island, NY) and HYCLONE (Logan UT) buys phosphate buffered saline(PBS) (PBS), cell culture medium and hyclone (FBS).(the α chain is specific to buy antibody, goat anti-mouse IgG (#A-3562) and the goat anti-mouse IgA that bovine serum albumin (BSA), alkali phosphatase (AP) put together from SIGMA, #A-4937) with from BETHYL (#A90-115P, Montgomery TX) buys goat anti-mouse IgE HPR conjugate.(New Jersey NY) buys cell proliferation reagent box (XTT) from ROCHE DIAGNOSTICS.

The rPA/ adjuvant formulation.From NANOBIO Corporat ion, Ann Arbor, MI obtains nano-emulsion (preparation W ₂₀5EC).By (for example using the emulsify at a high speed device, according to belonging to NANOBIOCorporation (Ann Arbor, MI) United States Patent (USP) 6,015,832 (incorporating this paper entirely into by quoting in full with it) prepared by two step methods), make this nano-emulsion with hexadecylpyridinium chloride (CPC, 1%), Tween 20 (5%) and ethanol (8%) in hot pressing soybean oil (64%) emulsified water.Except CPC, " generally recognized as safe " surfactant and foodstuff preparation W (GRAS) thought in use by FAD ₂₀5EC.W ₂₀5EC can produce under pharmaceutical production and quality management (Good Manufacturing Practice) (GMP) economically, and it need not any special storage requirement 40 ℃ of following stablizing at least 18 months.By dynamic light scattering (DLS), (PSS NICOMP Particle SizingSystems, Santa Barbara CA) determine the diameter of nano-emulsion to use NICOMP 380 ZLS.Average droplet size is all the time less than 400nm.

The use saline solution prepared the rPA/ nano-emulsion as diluent by the rPA protein solution is mixed with NE in 30 to 60 minutes before immunity.Use with concentration be that the rPA of 0.1%, 0.5%, 1% and 2% blended 20 μ g dosage of nano-emulsion carries out mouse immune research.About using the immunity of immunostimulant, 20 μ g rPA are mixed in saline solution with 5 μ g MPLA or 10 μ g CpG oligonucleotide.After carrying out described adsorption method people such as (, 2004 Vaccine 22:2843-2852) See Little, and preparation rPA aluminium hydroxide preparation (Alu, # A-8222, SIGMA).Use and 1%NE and carry out the Cavia porcellus immune Research as 10 μ g, the 50 μ g of the salt solution mix of adjuvant and the rPA of 100 μ g dosage.The immunity volume is 10 μ l/ nostrils (to mice) and 50 μ l/ nostrils (for Cavia porcellus).

The PAGE of reorganization PA analyzes.Use is used for electrophoretic X CELL SURELOCKMini-Cell platform, 10% NU PAGE NOVEX Bis-Tris gel (INVITROGEN, #NP0301BOX) go up in the saline solution or carry out PAGE with the blended 0.5 μ g rPA albumen of 1%NE and analyze.Carrying out silver according to INVITROGEN SILVERXPRESS (# LC6100) method dyes.Use molecular weight marker MARK12 (INVITROGEN; # LC5677) determines the proteic size of rPA.

Antigenic external picked-up.Use FLUOROTAG FITCConjugation test kit (#FITC1, SIGMA) the fluorescently-labeled rPA albumen of preparation according to the scheme of manufacturer.With dendritic cells in mouse (Jaws II) under 37 ℃ in PBS the PA-FITC conjugate or with nano-emulsion in blended PA-FITC incubation 30 minutes.Select 0.001% NE concentration to guarantee complete viability with the cell of the form growth of monolayer culture thing.Behind incubation, clean cell 3 times with PBS, use 1.25% formalin fixed cell among the PBS then.Pass through the picked-up of fluorescence microscope analysis of cells then.Be inverted the OLYMPUS IX70 microscope photographing microphotograph that reflected fluorescent light is observed adnexa (inverted reflected fluorescence observation attachment) with being furnished with IXFLA.Use SPOT basis and SPOT advanced procedures to handle video.

Immunization method.For each experiment, use 1 time or 2 experimental vaccine (3 weeks at interval) intranasal (i.n.) immunity mices in groups.With regard to adverse effect monitoring animal, in the period that reached for 12 cycles with the interval measurement antibody response in 3 to 4 weeks.At first, then they are inverted to get hold of and carry out immunity by using the isoflurane anesthesia mice.Use pipettor tip (10 μ l/ nostril) that the rPA/NE mixture is used for the nostril, allow animal suck material then.Use the Hartley of vaccine administration (50 μ l/ nostril) intranasal (i.n.) inoculation once or twice Cavia porcellus at interval 4 weeks, in the period that reached for 22 weeks, measure reaction with 3 to 4 weekly intervals.

Blood collecting, bronchoalveolar lavage fluid (BAL) and splenocyte.In process of the test, obtain blood sample from saphena at different time points.Obtain whole sample by cardiac puncture from painless lethal, premorbid mice.At blood after solidifying 30-60 minute under the room temperature, by obtaining serum from blood with centrifugal 5 minutes of 1500xg.Blood serum sample is stored in-20 ℃ until analysis.

Obtain BAL liquid from the mice that sucks anesthesia by isoflurane.Behind tracheostomize, No. 22 conduits (ANGIOCATH, B-D) the insertion trachea of syringe will be attached to.Comprise the PBS perfusion lung 2 times of 10 μ M DTT and 0.5mg/ml aprotinin (protease inhibitor) with 0.5ml, reclaim the aspirate of about 1ml then.The BAL sample is stored under-20 ℃ to treat further research.

The mechanical separation mouse boosting cell is to obtain the single-cell suspension thing among the PBS.By using ACK buffer (150mM NH ₄Cl, 10mM KHCO ₃, 0.1mM Na ₂EDTA) erythrocyte (RBC) is removed in cracking, and remaining cell is cleaned in PBS 2 times.Measure in order to carry out antigenic specificity propagation or cytokine-expressing, with splenocyte (2-4 x 10 ⁶/ ml) be resuspended in RPMI 1640 culture medium that are supplemented with 2%FBS, 200nML-glutamine and penicillin/streptomycin (100U/ml and 100 μ g/ml).

Anti-PA IgG and IgA determine.Determine the level of mouse anti PA specific IgG and IgA by ELISA.(50mM sodium carbonate, 50mM sodium bicarbonate, pH9.6) the 5 μ g/ml in (100 μ l) rPA bag is incubated overnight under 4 ℃ then by microtitration plate (NUNC) with the coating buffer.After removing protein solution,, carried out 30 minutes with the PBS closure plate that comprises 1% milk powder.The suction lock solution is used plate immediately or is descended storage up to needs at 4 ℃ it.Serial dilution serum or BAL sample among 0.1% BSA in PBS, with 100 μ l/ hole five equilibriums in the plate of rPA bag quilt 37 ℃ of following incubations 1 hour.With PBS-0.05% Tween 20 clean plates 3 times, antibody (ROCKLAND) incubation of puting together with anti-mice IgG or anti-mice IgA alkali phosphatase (AP) is 1 hour then, cleans afterwards 3 times, then with AP substrate SIGMAFAST (SIGMA) incubation.Use 1N NaOH to stop chrominance response according to the scheme of manufacturer, use SPECTRAMAX 340 ELISA readers (MOLECULAR DEVICES, Sunnyvale, CA) reading at 405nm place and 690nm with reference to the wavelength place.Record terminal point titre under the situation of BAL liquid, (that is, is used goat F (ab ') according to obtaining for each assay plate ₂Anti-mice IgG is as the mice IgG and the IgA immunoglobulin of trapping agent and concentration known, detect acquisition with anti-IgG or anti-IgA-AP conjugate then) standard curve calculate whole antibody concentration (referring to people such as Rhie, 2003 PNAS 100:10925-10930).Measure the anti-PA IgG of Cavia porcellus by identical method (except the anti-Cavia porcellus Cavia porcellus of rabbit IgG alkali phosphatase (AP) conjugate being used for detect (ROCKLAND)).Antibody concentration be expressed as the average of terminal point titre+/-sem (standard error of meansigma methods).

The dot blotting of IgE detects.With saline solution rPA solution (2 μ l, 5 μ g/ml) be adsorbed to the NYTRAN film (hole of 0.2 μ m, Schleicher and Schuell, Keene, NH), at room temperature air-dry then 30 minutes.With the PBS closing membrane that contains 1% milk powder 30 minutes, clean 3 times with PBS then, air-dry afterwards.For the detection of IgE, will be diluted among the PBS that contains 0.1%BSA with 1:10,1:20,1:40 and 1:80 from the pooled serum of the animal of all groups.Duplicate each dilution sample (2 μ l) is placed on the antigen speckle, and at room temperature incubation is 30 minutes: after in PBS, cleaning 3 times, and the antibody incubation of the anti-mice IgE horseradish peroxidase (HRP) that dot blotting is diluted with 1:1000-put together.After cleaning 5 times, with HPR substrate incubation as seen until trace with dot blotting with PBS.

Lethal toxin (LeTx) cytotoxicity and neutralizing antibody algoscopy.Use serial dilution thing to carry out neutralizing antibody mensuration with LeTx (containing 0.1 μ g/ml rPA and 0.1 μ g/ml rLF among the PBS) 1 hour serum of incubation.In RAW264.7 (20,000-30,000 cells/well), add antibody-toxin mixture then, then at 37 ℃ of following incubation 4-6 hours.Use the XTT algoscopy to estimate the viability of cell.Cause according to the cell survival rate curve calculation 50% the Cytotoxic protection of anti-LeTx serum titer (in and concentration NC ₅₀), it is expressed as the meansigma methods of individual serum.

The spore of living is attacked.(Columbus, OH) (Galveston, BSL4 TX) and BSL3 laboratory are attacked experiment with University of Texas Medical Branch at Battelle Memorial Institute respectively.According to Battelle research number: 556-G607602 carries out Intradermal (i.d.) and attacks.In brief, counting Bacillus anthracis (Ames strain system) spore is attacked its dilution then to be used for the i.d. spore.The spissated stock solution of AmesBattelle Lot B22 is diluted to 5 x 10 of expection in sterilized water ³The concentration of individual colony forming unit/ml (cfu/ml).On the same day of research, with the target dosage i.d. attack Cavia porcellus of about 500 spores (0.1ml).The counting of attacking the back spore shows that actual number is 1380, and this is corresponding to i.d.1000 LD ₅₀After attack,, carried out 14 days with regard to the symptom or the dead every day of clinical disease observing Cavia porcellus 2 times.The dead note of record is to the nearest observation period.To experience and attack back all Animal Anesthesia of survival, then the 14th day painless kill animals after attack to carry out terminal blood collecting (terminalblood collection).Carrying out intranasal (i.n.) according to JWP-004-0012 Nasal Challenge SOP scheme attacks.In brief, counting Bacillus anthracis (Ames strain system) spore, then with its at calcic not but among the magniferous PBS dilution to be used for the attack of intranasal spore.By intranasal administration 1.2 x 10 ⁶Or 1.2 x10 ⁷(this is respectively corresponding to intranasal 10 x LD for individual spore ₅₀With 100 x LD ₅₀Dosage) attack the Cavia porcellus of anesthesia.Observe after as above attacking the described attack of carrying out Cavia porcellus for Intradermal.

Proliferation assay.(Mannheim Germany), measures the propagation of mouse boosting cell by the mensuration of 5-bromo-2-deoxyuridine (BrdU) integration for CELL PROLIFERATION ELISA, ROCHE Molecular Biochemicals to use cell proliferation ELISA.Under the situation that rPA (5 μ g/ml) or PHA-P mitogen (2 μ g/ml) exist,, used the BrdU pulse then 24 hours with cell incubation 48 hours.According to manufacturers instruction, use the propagation with reference to wavelength place measurement cell of SPECTRA MAX 340ELISA reader at 370nm and 492nm.

The analysis of cell in vitro factor expression.With new isolating mouse boosting cell with 2 x 10 ⁶Individual cell/0.5ml (RPMI 1640,2% FBS) inoculation, and with rPA (5 μ g/ml) or PHA-P mitogen (2 μ g/ml) incubation 72 hours.The harvesting culture supernatants is analyzed it with regard to the existence of cytokine.According to manufacturers instruction, use QUANTIKINEELISA test kit (R﹠amp; D SYSTEMS, Inc., Minneapolis MN) carries out the mensuration of IL-2, IL-4, IFN-γ and TNF-α cytokine.

Statistical analysis.Be expressed as the standard error of meansigma methods ± meansigma methods from the data of Individual testwas.By the ANOVA variance analysis, use Xue Shengshi t check and Fisher definitely to check (Fisher exact test) to determine significance,statistical.On 95% confidence level (two-tailed test), carrying out all checks.It is significant that p value＜0.05 is considered to statistics.

Embodiment 9

The physical property of blended rPA/NE vaccine.

RPA and nano-emulsion (NE) mixed display are not changed antigenic protein structure, because it keeps single isolating band on non-degeneration PAGE, this band is corresponding to complete, the full length protein (referring to Fig. 6 A) of the molecular weight with 83kD.NE shows the stability that improves rPA, this prevented the viewed degraded gradually that causes owing to deacylated tRNA amine in the antigen of incubation in buffer solution (referring to, for example, people such as Gupta, 2003.FEBS Letters554:505-510; People such as Zomber, 2005 Journal of Biological Chemistry280:39897-39906).The proteic adding of rPA do not change nano-emulsion size (359+/-109nm), apparent or stable, as independent and with the microphotograph (referring to Fig. 6 B) of the blended NE of antigen in as shown in.

Embodiment 10

Nano-emulsion increases dendritic cell inflammatory reaction is not induced in the picked-up of rPA

Using back 24 hours to the 4th day, and do not showing the sign of inflammatory reaction with independent NE or with the combination pathology of the nasal mucosa of the blended NE intranasal of rPA mice immunized.On the contrary, the in vitro study proof is mixed rPA with NE has increased the picked-up (referring to Fig. 7) of protein in Jaws II dendritic cell significantly.Therefore, in some embodiments, the invention provides, intravital NE adjuvanticity relates to antigen by the increase (for example, under the situation of not inducing inflammation) of antigen presenting cell to the picked-up of nasal mucosa.

Embodiment 11

The anti-PA antibody of rPA/NE immune induction serum

In CBA/J and Balb/c mice, measure the influence of nanometer adjuvant antagonist reaction.With with the blended 20 μ g rPA intranasal of the NE of 0.1%, 0.5%, 1% or 2% concentration immunity CBA/J mice. in the animal of all inoculations, obtained the rapid induction of anti-PA antibody in the serum, and partly depended on the concentration of nano-emulsion.(the terminal point titre is 10 for the serum that 5th week the produced high titre anti-PA IgG of all CBA/J mices after only carrying out 2 vaccine administrations (the 1st and the 3rd week) ⁴To 10 ⁵In the scope).Though the further mensuration in the 8th to 12 week shows the lower titre of existence in the animal of 0.1% and 0.5% NE immunity, does not have the difference on the statistics between with the titre in the animal of 1% or 2% rPA/NE immunity.On the contrary, in the animal of the rPA intranasal immunity in the saline solution, find the positive mice (referring to Fig. 8 A) of serum-free.

The pattern of IgG hypotype antibody shows that with respect to IgG1 IgG2 and IgG2b account for main body.Therefore, in some embodiments, the experimenter is used the Th1 polarization (referring to Fig. 8 A, illustration) of reacting with the blended rPA induction of immunity of NE.For further characterizing the immunoreation that produces by the intranasal nano-emulsion, with with the blended 20 μ g rPA of 1% NE (rPA/NE) immunity Balb/c mice, then the immunity with itself and use and MPL A (rPA/MPL A), unmethylated CpG ODN (rPA/CpG) or the blended 20 μ g rPA of aluminium hydroxide (rPA/Alu) compare (referring to, for example, people such as Peterson, 2006.Infection and Immunity74:1016-1024; People such as Pittman, 2001. vaccine 20:972-978; People such as Pittman, 2002. vaccine 20:2107-2115; People such as Reuveny, 2001.Infect Immun69:2888-2893).After using each preparation 2 times, be seropositive with all mices of rPA/NE immunity, anti-PA IgG terminal point titre is at least 10 ⁵With 10 in this result and rPA/MPL A, rPA/CpG and the rPA/Alu immune group ²To 10 ³Titre in the scope compares (referring to Fig. 8 B).In animal, do not detect anti-PA antibody with the rPA nose immunity in the saline solution.

Also with regard to the serum analysis that exists of anti-PA IgE antibody, be presented at in the rPA/Alu intramuscular mice immunized but in any other group, do not have the anti-PA of IgE (dilution factor with 1:80 at least is detectable in dot blotting) (referring to Fig. 8 B, illustration).This with based on the report of the vaccine-induced Th2 reaction of the adjuvant of Alumen (referring to, for example, people such as Johansson, 2004.Vaccine22:2873-2880; Lindblad, 2004.Vaccine 22:3658-3668) unanimity.

Embodiment 12

Intranasal rPA/NE inoculation produces mucosal immunity

Determine the nose immunity whether mucosa immunity-inducing (for example, be protected from respiratory infections mucosal immunity (referring to, for example, Davis, 2001 Advanced Drug Delivery Reviews51:21-42; Zuercher, 2003.Viral Immunology 16:279-289)).In bronchial perfusate (BAL) sample of the Balb/c mice that the rPA/NE that uses by oneself inoculates, observe the anti-PA specific secretion IgA antibody (referring to Fig. 9 A) of significant level.Detect parallel pattern (referring to Fig. 9 B) with regard to the anti-PA IgG among the BAL with higher antibody concentration.But the animal that has the titre of secretory IgA, sIgA in BAL also has the anti-PA IgA of the serum of detection level.Therefore, the invention provides, comprise the vaccine-induced significant mucosal immunoreaction of the rPA among the NE by intranasal administration (but not intramuscular immunity).Use have or do not have antigenic NE after, in the histopathological examination of the nasal mucosa of animal, do not observe inflammatory reaction, show that nano-emulsion is not a proinflammatory.

Embodiment 13

The rPA/NE vaccine produces the neutralizing antibody of anti-anthrax toxin in mice

In order to estimate whether can produce the toxin neutralizing antibody based on the vaccine of mucosa nano-emulsion, just in and the ability of anthrax lethal toxin (LeTx) detect the serum of the mice immunized of controlling oneself.The serum of the rPA/NE mice immunized of using by oneself is in effectively and LeTx and prevent the RAW264.7 cell death, its NC ₅₀10 ³On the contrary, the use by oneself serum of rPA/MPL A, rPA/CpG or rPA/Alu mice immunized has NC ₅₀＜10.The serum of the rPA mice immunized in Shi Yan (Naive) control serum or the saline solution of using by oneself does not suppress LeTx cytotoxicity (referring to Figure 10) on any concentration.

Embodiment 14

The rPA/NE immunity produces the Th1 cell effect

In proliferation assay (referring to Fig. 5) and by to measuring the cell effect of PA antigenic specificity from analysis (referring to following table 7) with the excretory cytokine of splenocyte of rPA stimulated in vitro.As shown in Figure 11, rPA stimulates the propagation available from the splenocyte of using the rPA/NE mice immunized.Using by oneself independent rPA or in the splenocyte of the animal of the rPA immunity of CpG ODN, do not detect antigenic specificity propagation.

When comparing with contrast (non-stimulation) cell, the activatory splenocyte of PA shows a large amount of INF-of generation γ, TNF-α and IL-2, but can not produce IL-4.Therefore, in some embodiments, the invention provides, use the immunity (for example, intranasal administration) of rPA/NE to produce the specific T polar cell effect of h1 type (referring to table 7).On the contrary, induce the remarkable propagation and the secretion of Th1 and Th2 cytokine with the spleen cell cultures thing of PHA incubation.

Table 7

Embodiment 15

RPA/NE vaccine protection Cavia porcellus avoids Intradermal spore alive and attacks

Use three groups of Cavia porcelluss of rPA intranasal vaccination with blended 10, the 50 and 100 μ g dosage of 1% NE.Observe the IgG reaction after the single inoculation, the IgG reaction continues to increase after using for the second time (the 4th week), thereby produces greater than 1 x 10 ⁵The terminal point antibody titer.Carry out 6 months to estimate the persistent period of immunity with the relief animal.The immunity of nose in three treated animals produces at least 6 months persistent and has high antibody titer (〉 10 ⁴) lasting immunoreation (referring to Figure 12 A).At 6th month, with 1000 x LD ₅₀The spore subcutaneous (i.d.) of Ames strain system is attacked animal.Survival data shows the protection that the anti-i.d. of the mucosal vaccination generation 100% of any Cavia porcellus that carries out in three kinds of concentration using the rPA among the NE attacks, however neither one control animal survival (referring to Figure 12 B).Among the LeTx before attacking and mensuration be presented in the 10 μ g rPA/NE group and have 3 x 10 ²Average serum NC ₅₀, in the group of 50 μ g and 100 μ g rPA/NE immunity, have about 1 x 10 ³NC ₅₀(referring to Figure 12 B, illustration).

Embodiment 16

The rPA/NE vaccine is protected from the intranasal spore and attacks

Also in sucking challenge trial, detect the protective effect of intranasal immunity.With the three groups of Cavia porcelluss of preparation immunity that comprise with the rPA of blended 10, the 50 and 100 μ g of 1%NE.Immunity produces 100% serum conversion and significant anti-PA IgG reaction in the animal of immunity.The reinforcement in the 4th week causes the anti-PA IgG in the serum to increase fast, is higher than 1 x 10 thereby produce in all groups ⁵The terminal point antibody titer.Has 1-2 x 10 with measuring in the group that is presented at all inoculations among the LeTx before attacking ³Average N C ₅₀Titre (referring to Figure 13 A).In the 7th week, with 10 x LD ₅₀(1.2x 10 ⁶Individual spore) or 100 x LD ₅₀(1.2 x 10 ⁷Individual spore) Bacillus anthracis Ames strain is that the spore intranasal is attacked animal.Though the survival of neither one contrast Cavia porcellus uses the intranasal immunity of each preparation of rPA/NE to produce protective immunity.The rPA/NE immunity is at 10 x LD ₅₀Attack the back and produce 70% survival rate, at 100 x LD ₅₀Attack the back and produce 40% survival rate (respectively referring to Figure 13 B and 13C).10 x LD ₅₀The result who attacks provides, the protective immunity that the generation of mucosa rPA/NE vaccine can be suitable with the intramuscular inoculation that uses rPA and Alumen (referring to, for example, people such as Patton, 2006.ASM Bodefense Meeting:Abstract 232).Although do not have difference in the overall survival with the Cavia porcellus of rPA/NE inoculation on a plurality of rPA concentration, in the animal of immunity, there is the prolongation (referring to following table 8) of time (TTD) before the average death of significant dose dependent.

Table 8

Embodiment 17

Material and method

Animal.From Charles River laboratory (Wilmington, MA) buy pathogen-free domestic, female Ba1b/c mice (5 to 6 age in week) and Hartley Cavia porcellus (female, 250g).Look after the criticism association criterion according to U.S.'s laboratory animal and close foster connect mice (5 animals of a cage) and Cavia porcellus (1 animal of each cage) respectively.All methods that relate to mice according to zoologic the College Board that uses and look after (UCUCA) of University of Michigan.

Reagent.The Recombinant HIV gp120 that from Dr.Joseph Sodorski (respectively at Harvard Medical School and University of Michigan) obtains yeast, produces by Dr.David Markovitz _BaLAnd gp120 _SF162Serotype albumen.The five equilibrium of the protein solution in the 5mg/ml Sterile Saline is stored until use down at-80 ℃.Obtain synthetic V3 cyclic peptide (BaL) from Dr.StevenKing (University of Michigan).By integrated dna technique (IDT, Coralville, IA) synthetic comprise the multiple 20-mer oligonucleotide of non-methylated CpG (ODN) 5 '-TCC ATG ACG TTC CGT ACG TT-3 ' (SEQ IDNO.:4) (referring to, for example, people such as Moldoveanu, Vaccine1998; 16 (11-12): 1216-24).From SIGMA-ALDRICH Corporation (St.Louis, MO) buy salmonella minnesota (S.minnesota) monophosphoryl lipid A (MPLA, #L-6638), PHA-P, BSA, DTT and be used for other chemical drugss of buffer.Respectively from GIBCO (Grand Island, NY) and HYCLONE (Logan UT) buys phosphate buffered saline(PBS) (PBS), cell culture medium and hyclone (FBS).(the α chain is specific, #A-4937) with from the anti-Cavia porcellus IgG of ROCKLAND (#606-408) purchase rabbit to buy antibody, goat anti-mouse IgG (#A-3562) and the goat anti-mouse IgA that alkali phosphatase (AP) puts together from SIGMA.

The preparation of gp120/ adjuvant formulation.From NANOBIO Corporation, Ann Arbor, MI obtains to be used for the oil-in-water nanometer Emulsion (NE) of these researchs.By using the emulsify at a high speed device, with hot pressing soybean oil (64%) emulsifying hexadecylpyridinium chloride (CPC in water, 1%), non-from surfactant (5%) and ethanol (8%) and by two step methods (referring to the United States Patent (USP) 6 that belongs to NANOBIOCorporation.Ann Arbor.MI., 015,832, for all purposes, incorporate this paper entirely into by quoting in full with it) prepare.Except CPC, use and to be thought that by FAD " generally recognized as safe " surfactant and foodstuff (GRAS) prepare this nano-emulsion.By dynamic light scattering (DLS) use NICOMP 380 ZLS (PSS NICOMP ParticleSizing Systems, Santa Barbara, CA) determine the average droplet size of NE (approximately 300+/-25nm).

By the gp120 protein solution is mixed with NE, use saline solution to prepare the gp120/NE preparation as diluent.Use with concentration be that the gp120 of 0.1%, 0.5%, 1% blended 20 μ g dosage of NE concentration carries out mouse immune research.For the immunity of using immunostimulant, add 5 μ g MPL A or 10 μ g CpG ODN among the gp120 of the gp120 of 20 μ g in 1%/NE or 20 μ g in saline solution.That uses 50 μ g dosage carries out the Cavia porcellus immune Research with 1%NE with as the gp120 of the salt solution mix of diluent.

Immunization method.Use 2 (using 3 times under the situation once in a while) intranasal (i.n.) to use the gp120/NE preparation and come immune Ba1b/c mice with the interval in 3 weeks.By gp120/NE mixture (every nostril 10 μ l) being used for carrying out immunity lentamente through the nostril of the mice of isoflurane anesthesia.In delivery process, animal stood upside down to pick up until droplet to suck fully.In matched group, with the gp120 in the saline solution with independent NE or independent saline solution immune mouse.Use the 50 μ l saline solution or the injection of 20 μ g gp120 among the 1%NE at 2 doses (with intervals in 3 weeks) to carry out intramuscular administration (i.m.).Use ketamine injection (40mg/kg) anesthesia Hartley Cavia porcellus (every group of 3 animals), come the intranasal immune guinea pig by interval twice intranasal administration gp120/NE mixture (every nostril 50 μ l) then with 3 weeks.

The collection of blood, bronchoalveolar lavage fluid and vaginal lotion and splenocyte sample.In experimentation, obtain blood sample from saphena, or obtain blood sample from painless lethal, premorbid mice by cardiac puncture at different time points.By obtaining serum from the blood (under room temperature, solidify and placed 30-60 minute) that solidifies with centrifugal 5 minutes of 1500xg.Under 56 ℃, carry out coming in 1 hour the blood serum sample of heat inactivation collection, store the serum product down until analysis at-20 ℃ then.

Obtain mouse bronchial bronchoalveolar lavage fluid (BAL) from the mice that sucks anesthesia by isoflurane.With the 0.5ml PBS perfusion lung that contains 10 μ M DTT and 0.5mg/ml aprotinin 2 times, reclaim the aspirate of about 1ml then.Store the BAL sample down until analysis at-20 ℃.

By come to collect the vaginal lotion sample with the 100 μ l PBS infusion vaginal canals that contain 10 μ M DTT and 0.5mg/ml aprotinin from the mice of anesthesia.With 10, the centrifugal sample of 000xg 5 minutes is preserved supernatant down until analysis at-20 ℃ then under 4 ℃.

From spleen mechanical separation mouse boosting cell to obtain the single-cell suspension thing the PBS.By using ACK buffer (150mM NH ₄Cl, 10mM KHCO ₃, 0.1mM Na ₂EDTA) erythrocyte (RBC) is removed in cracking, and remaining cell is cleaned in PBS 2 times.For antigenic specificity propagation or cytokine-expressing mensuration, with splenocyte (2-4 x 10 ⁶Individual cell/ml) is resuspended to RPMI 1640 culture medium that are supplemented with 2% FBS, L-glutaminate and penicillin/streptomycin (100U/ml and 100 μ g/ml).

The mensuration of anti-gp120 IgG and IgA antibody.Determine the level of mouse anti gp120 specific IgG and IgA by ELISA.With 5g/ml (the 100 μ l) gp120 in the coating buffer (50mM sodium carbonate, 50mM sodium bicarbonate, pH 9.6) _BaLOr gp12 _SF162Serotype envelope protein bag is by microtitration plate (MAXISORP; NALGE NUNC International, Rochester NY), is incubated overnight under 4 ℃ then.After removing protein solution,, carried out 30 minutes at 37 ℃ with PBS-1% milk power solution closure plate.The suction lock solution is used plate immediately or is descended storage up to needs at 4 ℃ it.Serial dilution serum or BAL liquid among 0.1% the BSA in PBS, with 100 μ l/ hole five equilibriums in the plate of gp120 bag quilt in 37 ℃ of following incubations 1 hour.According to PBS-0.05% Tween 20 clean plates 3 times of the scheme of manufacturer, the antibody incubation of puting together with anti-mice IgG or anti-mice IgA alkali phosphatase (AP) is 1 hour then, cleans afterwards 3 times, then with AP substrate SIGMAFAST (SIGMA, St.Louis, MO) incubation together.(MOLECULAR DEVICES, Sunnyvale CA) carry out Spectrophotometric reading at 405nm place and 690nm with reference to wavelength to use SPECTRA MAX 340 ELISA readers.Stop the inverse that antibody titer is defined as the last dilution factor (the absorptance negative control at 405nm place is high 2 times on this dilution factor) of serial serum dilution.Measure the anti-gp120I gG of Cavia porcellus by identical method (being used for detecting (ROCKLAND)) except resisting Cavia porcellus IgG alkali phosphatase (AP) conjugate.Antibody concentration be expressed as meansigma methods+/-standard deviation (s.d.) of terminal point titre.

In the HIV-1 single-wheel and measure (single-round neutralization assay).It is BaL, SF162 and MN and former generation HIV-1 separated strain SS1196.01, BG1168.01, QH0692.42 that 8 strain system groups that are used for the clade B HIV-1 of this research comprise laboratory strains, 3988.25 and 5768.04 (Li05).After the virus neutralization is measured as described first round viral infection TZM-b1 cell, the function of the minimizing that luciferase reporter gene is expressed (referring to, for example, Montefiori, editor.Evaluating neutralizing antibodyagainst HIV, SIV and SHIV in luciferase reporter gene assays.New York, NY:John Wiley ﹠amp; Sons, 2004).The TZM-b1 cell is carried out genetic engineering modified, thereby make it express CD4 and CCR5 and comprise the LUC Photinus pyralis LUC Photinus pyralis FL that is used under the control of HIV-1LTR and the reporter gene of the integration of escherichia coli beta galactosidase.With former generation HIV-1 separated strain (TCID ₅₀, 100 to 200) with the serial dilution thing of serum 37 ℃ of following incubations 1 hour.Then, virus/serum is added the flat culture plate in 96 holes that comprises adherent TZM-b1 cell.Contrast comprises cell+virus (virus control) and has only cell (background contrast).(WI) described, after 48 hours, use BRIGHT GLO substrate solution is measured bioluminescence for PROMEGA, Madison as provider.In and titre (NT ₅₀) be to compare with the RLU of virus control cell in subtracting background relative light unit (RLU) back, RLU reduces by 50% o'clock residing dilution factor.

Proliferation assay.Use is purchased obtainable labelling and detection kit (CellProliferation ELISA, ROCHE Molecular Biochemicals, Mannheim, Germany), the mensuration of the integration by 5-bromo-2-deoxyuridine (BrdU) is measured the propagation of mouse boosting cell.In order to estimate antigenic specificity propagation, with cell (2 x 10 ⁶Individual cell/ml) in independent culture medium and at gp120 _BaLUnder the situation that (5 μ g/ml) exists, or, carried out 48 hours, carried out 24 hours with the BrdU pulse then in contrast with PHA-P (2 μ g/ml) incubation.According to manufacturers instruction, (MOLECULAR DEVICES, Sunnyvale is CA) in the propagation with reference to wavelength place measurement cell of 370nm place and 492nm to use SPECTRA MAX 340 ELISA readers.

The analysis of cytokine vivoexpression.With 4 x 10 ⁶Individual cell/ml (RPMI 1640,2% FBS) inoculates mouse boosting cell, then with itself and gp120 _BaL, gp120 _SF162(5 μ g/ml) or with V3 cyclic peptide (20nM) 37 ℃ of following incubations 72 hours.The harvesting culture supernatants is analyzed it with regard to the existence of cytokine.According to manufacturers instruction, use QUANTIKINE ELISA test kit (R﹠amp; D SYSTEMS, Inc., Minneapolis MN) carries out the mensuration of cytokine.

Statistical analysis.Use ANOVA, student t check to carry out result's statistical analysis, be used for determining of p value.

Embodiment 18

Use with the proteic nose immunity of the blended Recombinant HIV gp120 of nano-emulsion and in serum, induced effective I gG reaction.

In order to determine that whether NE has the activity of adjuvant in using the proteic mucosal immunity of Recombinant HIV gp120, use antigenic gp120 _BaLOr gp120 _SF162The immune Balb/c mice of serotype intranasal (i.n.).Use in the saline solution or with the gp120 of 0.1%, 0.5% and 1% blended 20 μ g of a plurality of NE concentration _BaLEstimate the concentration effect of NE.Collect blood 2 the 6th weeks of immunity back with the 3rd the 12nd week of immunity back, by ELISA described blood is analyzed with regard to the gp120-specific antibody.Use gp120 _BaLAll mices of any immunity in the/NE prepared product only are seropositive after 2 immunity.Anti-gp120 _BaLThe concentration dependent of IgG reaction and display NE, gp120 _BaLHave minimum titre and gp120 among/the 0.1%NE _BaLHave the highest titre in the/1%NE immune group and (be respectively 1.3 x 10 ⁴With 2.6 x 10 ⁵Average titer).Use gp120 _BaL/ saline solution mice immunized does not have detectable anti-gp120 _BaLAntibody.Use 0.5% or the intranasal immunity of 1%NE after, the anti-gp120IgG titre of serum can with using gp120 _BaLThe antibody response that produces after the intramuscular injection of (in the saline solution or blended with 1%NE) is suitable, or even than its height.The 3rd intranasal immunity does not significantly increase antibody titer (referring to Figure 14 A) in arbitrary immune group.Therefore, the invention provides, only need twice intranasal administration gp120 _BaLIn mice, start effective systemic IgG reaction with the NE adjuvant.

NE is enough to as strong mucosal adjuvants.Immunoreation that NE is produced and known immunologic stimulant, the CpG ODN of firstization and the effect of MPL A do not compare.Use gp120 with the blended 20 μ g of 1%NE _SF162(gp120 _SF162/ NE) come the intranasal immune mouse, then with use and CpG ODN (gp120 _SF162/ CpG) or with MPL A (gp120 _SF162/ MPL A) immunity of phase antigens mixed compares.In order to study the effect of combination NE and immunologic stimulant, use gp120 _SF162/ NE and additionally use CpG (gp120 _SF162/ NE+CpG) or MPLA (gp120 _SF162/ NE+MPL A) immune mouse.With use gp120 _BaLImmune similar, use gp120 _SF162/ NE mice immunized responds high anti-gp120 _SF162The IgG titre.The combination of NE and MPL A (but not with CpG) causes the suitable increase of average antibody titre, and (2 to 3 times to independent use gp120 _SF162The immunity of/NE), however difference does not have significance,statistical (p〉0.05).On the contrary, use the immunity with CpG antigens mixed or independent MPL A only to produce faint immunoreation (referring to Figure 14 B).

Embodiment 19

Antibody and other gp120 serotype cross reactions of a kind of serotype of the anti-gp120 that produces.

The experiment of carrying out between development period of the present invention has determined to use the intranasal immunity of proteic any serotype of gp120 to produce the IgG antibody of height cross reaction.For example, the anti-gp120 of generation _BaLOr gp120 _SF162IgG antibody and the cross reaction of allos serotype, the activity of cross reaction with to combine quite (referring to Figure 14 C and 14D) from the body envelope protein.Therefore, the invention provides, use the mucosal immunity of gp120 serotype can induce suitable immunoreation.Therefore, in some embodiments, the NE adjuvant can produce the IgG storehouse (for example, ginseng is at the protective immunity of dissimilar HIV-1) that can discern the immunogenic variable and conserved epitope of gp120 (referring to, for example, Mascola, Curr Mol Med 2003; 3 (3): 209-16).

Embodiment 20

The intranasal administration of pg120/NE is created in detectable anti-gp120 specificity IgA antibody in bronchus and the vaginal mucosa surface.

Analyze BAL liquid, vaginal lotion and serum to estimate mucosa reaction.Use gp120 _SF162/ NE mice immunized has the gp120 of significant level in BAL liquid _SF162-specific secretion IgA and IgG antibody (referring to Figure 15 A and 15B).Similar to serum, IgA and IgG antibody all show and allos gp120 _BaLThe immunogen cross reaction.Also arrive anti-gp120 at serum and mucosa position probing at a distance _BaLIgA antibody (for example, (referring to Figure 15 C)) as in the vaginal lotion sample, measuring.The immunity of the gp120 of any type in the use saline solution can not produce can detected mucosa IgA and IgG reaction in BAL, serum and vaginal secretions.Therefore, the invention provides, can respond use the antigen sent with the NE adjuvant (for example, intranasal gp120) immune and partly (for example, in bronchial mucosa) and position a long way off (for example, vaginal secretions) induce significant mucosa reaction.

Embodiment 21

Cell-mediated immunoreation.

The sign of the generation by antigen specific T-cell proliferating determining and T assisted class cytokines is come external appraisal cell immune response.At the gp120 that uses by oneself _BaLDetect the antigenic specificity breeder reaction in the splenocyte that stimulates again of the animal of/NE immunity, but using gp120 _BaLThere is not this reaction (referring to Figure 16 A) in/saline solution mice immunized or the control mice (handling with saline solution or NE separately).Use gp120 _BaLThe intranasal immunity of/NE produces strong cell-mediated immunoreation, as measured (referring to Figure 16 B) of generation by spleen IFN-γ.Use gp120 _BaLOr gp120 _SF162The stimulated in vitro of serotype produces the high IFN-gamma reaction from the gp120 of body (BaL) and allos (SF162) type.Use the oligopeptide fragment of V3 ring also to obtain significantly inducing of IFN-γ, this show existence for participate in viral combination and neutral advantage epi-position (dominant epitope) be specific CTL (referring to, for example, people such as Kwong, Nature 1998; 393 (6686): 648-59; People such as Takahashi, Science1992; 255 (5042): 333-6).Antigenic specificity is induced or IFN-γ has proved that with the expression that lacks detectable IL-4 the Th1 of cell immune response polarizes.From control mice or with the gp120 in the saline solution _BaLDo not detect the remarkable expression of cytokine in the splenocyte of mice immunized.

Embodiment 22

Use the immune induction HIV-1 neutralizing antibody of gp120/NE

Active in order to characterize by the potential neutralization of the inductive gp120-specific antibody of mucosal immunity, Cavia porcellus is used two doses and the blended gp120 of 1% NE _SF162Immune in individual animals, the generation significantly (though change) the anti-gp120 IgG of serum antibody horizontal (referring to Figure 17 A).As viewed in mice, anti-gp120 IgG of Cavia porcellus and the cross reaction of allos gp120 immunogenic.With regard to the active immune serum that detects from Cavia porcellus of the neutralization of anti-HIV-1.In having 8 kinds of viruses group of (comprising 3 kinds of laboratory strains systems and 5 former generation HIV separated strains), estimate the range of neutralization reaction.In serum, detect at HIV from the highest animal of response _SF162Senior middle school and titre (NT from the strain of body M preferendum ₅₀) (NT ₅₀=225) (referring to Figure 17 B).Yet, also in two kinds of other animals, detect the active (NT of significant neutralization ₅₀50), although anti-gp120 IgG level is much lower.

Allos M preferendum strain HIV _BaLNeutralization in all Cavia porcelluss quite, NT ₅₀Greater than 50.For T preferendum HIV _MNThe laboratory strains system of virus does not observe neutralization.Use has neutralized effectively all 5 from the serum of vaccinated Cavia porcellus and has been tried former generation HIV separated strain.Active suitable for the neutralization of former generation HIV separated strain with two laboratory strains systems.Depend on serum, BG1168.1, SS1196.11 and 3988.25 NT ₅₀Value is in 50 to 100 scope.Separated strain QH0692.42 and 5768.4 is effectively neutralized NT ₅₀Value is greater than 100.

All publications and the patent mentioned in the above-mentioned description are integrated with this paper by reference.The various modifications of the compositions of description of the invention and method and variation will be conspicuous to those skilled in the art, not deviate from scope and spirit of the present invention.Although describe the present invention, should be appreciated that invention required for protection should exceedingly not be defined in this type of specific embodiment in conjunction with specific preferred embodiment.In fact, it is evident that for various equivalent modifications that the various modifications meanings that are used to carry out described method of the present invention within the scope of the invention.

Claims

1. induce the immunoreactive method at vaccinia subgroup virus in the experimenter, this method comprises

A) provide and comprise nano-emulsion and combinations of immunogens thing, wherein said immunogen comprises by the vaccinia subgroup virus of described nano-emulsion deactivation; With

B) make under the immunoreactive condition of described experimenter's generation at described vaccinia subgroup virus, described experimenter's applying said compositions.

2. the process of claim 1 wherein that the described mucomembranous surface that comprises described experimenter of using contacts with described compositions.

3. the method for claim 2, wherein said mucomembranous surface comprises nasal mucosa.

4. the process of claim 1 wherein that described induction of immunity is reflected at the immunity of inducing among the described experimenter at described vaccinia subgroup virus.

5. the method for claim 4, wherein said immunity comprises the general immunity.

6. the method for claim 4, wherein said immunity comprises mucosal immunity.

7. the process of claim 1 wherein that described immunoreation comprises the expression of the INF-γ that increases among the described experimenter.

8. the process of claim 1 wherein that described immunoreation comprises the systemic IgG reaction at the vaccinia subgroup virus of described deactivation.

9. the process of claim 1 wherein that described immunoreation comprises the mucosa IgA reaction at the vaccinia subgroup virus of described deactivation.

10. the process of claim 1 wherein and make 10-10 ³The described inactivation of viruses of pfu is present under the condition in the dosage that described experimenter is used, and described experimenter is used by the described vaccinia subgroup virus of described Emulsion deactivation.

11. the process of claim 1 wherein 10% nano-emulsion solution is used for the described vaccinia virus of deactivation.

12. the process of claim 1 wherein that described nano-emulsion comprises W ₂₀5EC.

13. the process of claim 1 wherein that the described experimenter of described immunoprotection avoids showing the S or S of the disease that is caused by described vaccinia subgroup virus.

14. the process of claim 1 wherein that the described experimenter of described immunoprotection avoids being exposed to subsequently the attack of vaccinia subgroup virus alive.

15. the process of claim 1 wherein that described compositions also comprises adjuvant.

16. the process of claim 1 wherein that described experimenter is the people.

17. the process of claim 1 wherein that described vaccinia subgroup virus is a vaccinia virus.

18. the method for claim 13 wherein protects described experimenter to avoid showing the S﹠S of variola.

19. be used for the compositions of immune response stimulating, it comprises nano-emulsion and by the vaccinia subgroup virus of described nano-emulsion deactivation, wherein prepares described compositions to make it to induce the immunity to described vaccinia subgroup virus in the experimenter.

20. the compositions of claim 19, wherein said nano-emulsion comprises W ₂₀SEC.

21. the compositions of claim 19, wherein when described experimenter was used, described compositions provided 10-10 for described experimenter ³The virus of the described deactivation of pfu.

22. the compositions 19 of claim, wherein the dosage of the described compositions that described experimenter is used comprises 1% nano-emulsion solution.

23. the compositions of claim 19, the vaccinia subgroup virus of wherein said deactivation are heat-staple in described nano-emulsion.

24. the compositions of claim 23, wherein said vaccinia subgroup virus are stable above 4 weeks in described nano-emulsion.

25. the compositions of claim 19 wherein before the experimenter is used, is diluted described compositions.

26. the compositions of claim 23, wherein said experimenter is the people.

27. the compositions of claim 19, wherein said immunity are the general immunity.

28. the compositions of claim 19, wherein said immunity is a mucosal immunity.

29. the compositions of claim 19, wherein said compositions also comprises adjuvant.

30. the compositions of claim 19, wherein said vaccinia subgroup virus is a vaccinia virus.

31. the compositions of claim 19, wherein said vaccinia subgroup virus are selected from alastrim virus, cowpox, monkeypox, gerbil jird pox and camel pox.

32. comprise compositions that is used for immune response stimulating and the test kit that is used for the description of applying said compositions, described compositions comprises nano-emulsion and by the vaccinia subgroup virus of described nano-emulsion deactivation, wherein prepares described compositions to make it to induce the immunity at described vaccinia subgroup virus in the experimenter.

33. the test kit of claim 32, wherein said vaccinia subgroup virus is a vaccinia virus.

34. the test kit of claim 32, wherein said vaccinia subgroup virus are selected from alastrim virus, cowpox, monkeypox, gerbil jird pox and camel pox.

35. induce the immunoreactive method at Bacillus anthracis in the experimenter, this method comprises

A) provide and comprise nano-emulsion and combinations of immunogens thing, wherein said immunogen comprises the reorganization protective antigen (rPA) of Bacillus anthracis; With

B) make under the immunoreactive condition of described experimenter's generation at described Bacillus anthracis, described experimenter's applying said compositions.

36. the method for claim 35, the wherein said mucomembranous surface that comprises described experimenter of using contacts with described compositions.

37. the method for claim 36, wherein said mucomembranous surface comprises nasal mucosa.

38. the method for claim 35, wherein said induction of immunity are reflected at the immunity of inducing among the described experimenter at described Bacillus anthracis.

39. the method for claim 38, wherein said immunity comprises the general immunity.

40. the method for claim 38, wherein said immunity comprises mucosal immunity.

41. the method for claim 35, wherein said immunoreation comprise the expression of the INF-γ that increases among the described experimenter.

42. the method for claim 35, wherein said immunoreation comprise the systemic IgG reaction to described Bacillus anthracis.

43. the method for claim 35, wherein said immunoreation comprise the mucosa IgA reaction at described Bacillus anthracis.

44. the method for claim 35, wherein said compositions comprise the described rPA of 25-75 μ g.

45. the method for claim 35, the wherein said compositions of stating comprises 10% nano-emulsion solution.

46. the method for claim 35, the described experimenter of wherein said immunoprotection avoids showing the S or S of the disease that is caused by Bacillus anthracis.

47. the method for claim 35, the described experimenter of wherein said immunoprotection avoids being exposed to subsequently the attack of Bacillus anthracis alive.

48. the method for claim 35, wherein said compositions also comprises adjuvant.

49. the method for claim 48, wherein said adjuvant comprises the CpG oligonucleotide.

50. the method for claim 35, wherein said experimenter is the people.

51. the method for claim 35, the described examination person of wherein said immunoprotection avoids showing the S﹠S of anthrax.

52. be used for the compositions of immune response stimulating, it comprises the reorganization protective antigen of nano-emulsion and Bacillus anthracis, wherein prepares described compositions to make it to induce the immunity at described Bacillus anthracis in the experimenter.

53. the compositions of claim 52, wherein said nano-emulsion comprises W ₂₀5EC.

54. the compositions of claim 52, wherein when described experimenter was used, described compositions provided the described reorganization protective antigen of 25-75 μ g for described experimenter.

55. the compositions of claim 52, wherein the dosage of the described compositions that described experimenter is used comprises 1% nano-emulsion solution.

56. the compositions of claim 52, wherein said reorganization protective antigen is heat-staple in described nano-emulsion.

57. the compositions of claim 56, wherein said reorganization protective antigen are stable above 4 weeks in described nano-emulsion.

58. the compositions of claim 52 wherein before the experimenter is used, is diluted described compositions.

59. the compositions of claim 52, wherein said experimenter is the people.

60. the compositions of claim 52, wherein said immunity are the general immunity.

61. the compositions of claim 52, wherein said immunity is a mucosal immunity.

62. the compositions of claim 52, wherein said compositions also comprises adjuvant.

63. the compositions of claim 62, wherein said adjuvant comprises the CpG oligonucleotide.

64. comprise compositions that is used for immune response stimulating and the test kit that is used for the description of applying said compositions; described compositions comprises the reorganization protective antigen of nano-emulsion and Bacillus anthracis, wherein prepares described compositions to make it to induce the immunity at described Bacillus anthracis in the experimenter.

65. the test kit of claim 64, it also comprises the device that is used for applying said compositions.

66. the test kit of claim 65, wherein said device are selected from nose applicator, syringe, nasal inhaler and nasal spray device.

67. induce the immunoreactive method at HIV in the experimenter, this method comprises:

A) provide and comprise nano-emulsion and combinations of immunogens thing, wherein said immunogen comprises reorganization gp120; With

B) make under the immunoreactive condition of described experimenter's generation at described HIV, described experimenter's applying said compositions.

68. the method for claim 67, the wherein said mucomembranous surface that comprises described experimenter of using contacts with described compositions.

69. the method for claim 68, wherein said mucomembranous surface comprises nasal mucosa.

70. the method for claim 67, wherein said induction of immunity are reflected at the immunity of inducing among the described experimenter at described HIV.

71. the method for claim 70, wherein said immunity comprises the general immunity.

72. the method for claim 70, wherein said immunity comprises mucosal immunity.

73. the method for claim 67, wherein said immunoreation comprise the expression of the INF-γ that increases among the described experimenter.

74. the method for claim 67, wherein said immunoreation comprise the systemic IgG reaction at described HIV.

75. the method for claim 67, wherein said immunoreation comprise the mucosa IgA reaction at described HIV.

76. the method for claim 67, wherein said compositions comprise the described reorganization gp120 of 15-75 μ g.

77. the method for claim 67, wherein said compositions comprise 10% nano-emulsion solution.

78. the method for claim 67, the described experimenter of wherein said immunoprotection avoids showing the S or S of the disease that is caused by HIV.

79. the method for claim 67, the described experimenter of wherein said immunoprotection avoids being exposed to subsequently the attack of HIV alive.

80. the method for claim 67, wherein said compositions also comprises adjuvant.

81. the method for claim 80, wherein said adjuvant comprises the CpG oligonucleotide.

82. the method for claim 80, wherein said adjuvant comprises monophosphoryl lipid A.

83. the method for claim 67, wherein said experimenter is the people.

84. the method for claim 67, the described experimenter of wherein said immunoprotection avoids showing the S or S of AIDS.

85. be used for the compositions of immune response stimulating, it comprises nano-emulsion and reorganization gp120, wherein prepares described compositions to make it to induce the immunity at HIV in the experimenter.

86. the compositions of claim 85, wherein said nano-emulsion comprises W ₂₀5EC.

87. the compositions of claim 85, wherein said nano-emulsion comprises X8P.

88. the compositions of claim 85, wherein when described experimenter was used, described compositions provided the described reorganization gp120 of 15-75 μ g for described experimenter.

89. the compositions of claim 85, wherein the dosage of the described compositions that described experimenter is used comprises 1% nano-emulsion solution.

90. the compositions of claim 85, wherein said reorganization gp120 is heat-staple in described nano-emulsion.

91. the compositions of claim 85 wherein before the experimenter is used, is diluted described compositions.

92. the compositions of claim 85, wherein said experimenter is the people.

93. the compositions of claim 85, wherein said immunity are the general immunity.

94. the compositions of claim 85, wherein said immunity is a mucosal immunity.

95. the compositions of claim 85, wherein said compositions also comprises adjuvant.

96. the compositions of claim 95, wherein said adjuvant comprises the CpG oligonucleotide.

97. the compositions of claim 95, wherein said adjuvant comprises monophosphoryl lipid A.

98. comprise compositions that is used for immune response stimulating and the test kit that is used for the description of applying said compositions, described compositions comprises nano-emulsion and reorganization gp120, wherein prepares described compositions to make it to induce the immunity at HIV in the experimenter.

99. the test kit of claim 98, it also comprises the device that is used for applying said compositions.

100. the test kit of claim 99, wherein said device are selected from nose applicator, syringe, nasal inhaler and nasal spray device.