HK1173661B - Nanoemulsion vaccines - Google Patents

Description

Nanoemulsion vaccines

This application claims priority from U.S. provisional patent application serial No. 61/187,529, filed on 16/6/2009, which is incorporated herein by reference in its entirety.

Technical Field

The present invention provides methods and compositions for immune response stimulation. In particular, the invention provides immunogenic compositions and uses thereof to induce targeting to Paramyxoviridae: (paramyxoviridae) Of (e.g., Paramyxovirinae (sub) of (a))Paramyxovirinae) Viruses (e.g. paramyxovirus: (Paramyxovirus) Genus mumps: ( Rubulavirus) And/or morbillivirus (morbillivirus)Morbillivirus) And/or Pneumovirinae subfamily (Pneumovirinae) A virus (e.g., respiratory syncytial virus))) and a method of immune response (e.g., immunization (e.g., protective immunization)). The compositions and methods of the invention are particularly useful in clinical (e.g., therapeutic and prophylactic medicine (e.g., vaccination)) and research applications.

Background

Immunization is a major feature used to improve human health. Despite the availability of multiple successful vaccines against many common diseases, infectious diseases remain a major cause of health problems and death. Significant problems inherent in existing vaccines include the need for repeated immunizations, and the ineffectiveness of current vaccine delivery systems for a broad spectrum of diseases.

To develop vaccines against pathogens that have been refractory to vaccine development and/or to overcome the disadvantages of commercially available vaccines (e.g., due to adverse consequences, expense, complexity, and/or underutilization), new methods of antigen presentation must be developed that allow for less immunization, more efficient use, and/or fewer side effects for the vaccine.

Summary of The Invention

The present invention provides methods and compositions for immune response stimulation. In particular, the invention provides immunogenic compositions and methods of use thereof to induce an immune response (e.g., immunity (e.g., protective immunity)) against a pathogenic virus of the paramyxoviridae family (e.g., a paramyxoviridae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus)). The compositions and methods of the invention are particularly useful in clinical (e.g., therapeutic and prophylactic medicine (e.g., vaccination)) and research applications.

In some embodiments, the present invention provides immunogenic compositions comprising a nanoemulsion (nanoemusion) inactivated immunogen, e.g., a pathogenic virus of the paramyxoviridae family, e.g., a paramyxoviridae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus)), the nanoemulsion comprising an aqueous phase, an oil phase, and a solvent. In some embodiments, the immunogen comprisesIn some embodiments, the virus is a paramyxoviridae virus (e.g., paramyxovirus, mumps virus, and/or measles virus)₈₀5EC, although the invention is not limited thereto. For example, in some embodiments, the nanoemulsion is selected from one of the nanoemulsion formulations described herein. In some embodiments, the composition comprises 1-50% nanoemulsion solutions, although greater and lesser amounts are also useful in the present invention. For example, in some embodiments, the immunogenic composition comprises about 1.0% -10%, about 10% -20%, about 20% -30%, about 30% -40%, about 40% -50%, about 50% -60% or more nanoemulsion solutions. In some embodiments, the immunogenic composition comprises an about 10% nanoemulsion solution. In some embodiments, the immunogenic composition comprises an about 15% nanoemulsion solution. In some embodiments, the immunogenic composition comprises an about 20% nanoemulsion solution. In some embodiments, the immunogenic composition comprises an about 12% nanoemulsion solution. In some embodiments, the immunogenic composition comprises an about 8% nanoemulsion solution. In some embodiments, the immunogenic composition comprises an about 5% nanoemulsion solution. In some embodiments, the immunogenic composition comprises an about 2% nanoemulsion solution. In some embodiments, the immunogenic composition comprises an about 1% nanoemulsion solution. In some embodiments, an immunogenic composition (e.g., administered to a subject to generate an immune response in a subject) comprises 2x10 ⁶Plaque Forming Units (PFU) of inactivated pathogenic viruses of the paramyxoviridae family (e.g., RSV), although more (e.g., about 4x 10) can be utilized⁶ PFU、8x10⁶ PFU、1x10⁷ PFU、2x10⁷ PFU、4x10⁷ PFU、8x10⁷ PFU、1x10⁸ PFU、1x10⁹PFU or more PFU of RSV inactivated by nanoemulsion) and less (e.g., about 1x 10)⁶ PFU、5x10⁵ PFU、1x10⁵ PFU、5x10⁴ PFU、1x10⁴ PFU、5x10³ PFU、1x10³An amount of PFU or less of a virus of the family paramyxoviridae (e.g., RSV)) inactivated by a nanoemulsion. In some embodiments, the compositions are stable (e.g., at room temperature (e.g., up to 12 hours, 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year or more.) in some embodiments, the immunogenic composition comprises a pharmaceutically acceptable carrier the invention is not limited to any particular pharmaceutically acceptable carrier indeed, any suitable carrier can be utilized including, but not limited to, those described herein A peptide, polypeptide, nucleic acid, polysaccharide, or membrane component). In some embodiments, the immunogen and the nanoemulsion are combined in a single container.

In some embodiments, the invention provides methods of inducing an immune response against a pathogenic virus of the paramyxoviridae family, such as Respiratory Syncytial Virus (RSV), in a subject, comprising: providing an immunogenic composition comprising a nanoemulsion and an immunogen, wherein the immunogen comprises a virus of the paramyxoviridae family (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a virus of the pneumovirinae family (e.g., respiratory syncytial virus)); and administering the composition to the subject under conditions such that the subject generates an immune response against the virus.The present invention is not limited by the route selected for administration of the compositions of the present invention. In some embodiments, administering the immunogenic composition comprises contacting a mucosal surface of the subject with the composition. In some embodiments, the mucosal surface comprises nasal mucosa. In some embodiments, inducing an immune response induces immunity to a virus of the paramyxoviridae family (e.g., RSV) in the subject. In some embodiments, the immunization comprises systemic immunization. In some embodiments, the immunization comprises mucosal immunization. In some embodiments, the immune response comprises increased expression of IFN- γ in the subject. In some embodiments, the immune response comprises increased expression of IL-17 in the subject. In some embodiments, the immune response comprises an increased absence of expression of Th2 type cytokines (e.g., IL-4, IL-5, and IL-13). In some embodiments, the immune response comprises a systemic IgG response against an inactivated virus of the paramyxoviridae family (e.g., RSV). In some embodiments, a virus of the family paramyxoviridae (e.g., RSV) inactivated by a nanoemulsion is administered to a subject under conditions such that 10-10 ³Plaque Forming Unit (PFU) inactivated virus is present in the dose administered to the subject, although more may be utilized (e.g., about 10)⁴、10⁵、10⁶、10⁷、10⁸ Or more) and less (e.g., about 1-10 or less) PFU of a virus of the family paramyxoviridae (e.g., RSV) inactivated by nanoemulsion. In some embodiments, a 15% nanoemulsion solution is used to inactivate viruses. In some embodiments, the nanoemulsion comprises W₈₀5 EC. In some embodiments, the subject is immunoprotected from displaying signs or symptoms of disease caused by a virus of the paramyxoviridae family (e.g., RSV). In some embodiments, immunization protects a subject from challenge by subsequent exposure to a live virus of the paramyxoviridae family (e.g., RSV). In some embodiments, the composition further comprises an adjuvant. In some embodiments, the subject is a human.

In some embodiments, inducing an immune response induces a virus against the paramyxoviridae family in a subject(e.g., a virus of the Paramyxoviridae sub-family (e.g., Paramyxoviridae, Rubulaviridae, and/or morbillivirus) and/or a virus of the Pneumovirinae sub-family (e.g., respiratory syncytial virus))). In some embodiments, inducing immunity against a virus of the paramyxoviridae family (e.g., RSV) comprises systemic immunity. In some embodiments, the immunization comprises mucosal immunization. In some embodiments, the immune response comprises increased expression of IFN- γ in the subject. In some embodiments, the immune response comprises increased expression of IL-17 or other types of Th1 cytokines in the subject. In some embodiments, the immune response comprises a systemic IgG response to the immunogen. In some embodiments, the immune response comprises a mucosal IgA response to the immunogen. In some embodiments, each dose comprises an amount of nanoemulsion inactivated virus of the family paramyxoviridae (e.g., RSV) sufficient to generate an immune response against the virus. An effective amount of a virus of the paramyxoviridae family (e.g., RSV) is a dose that need not be quantified, so long as the amount of the virus of the paramyxoviridae family (e.g., RSV) generates an immune response in a subject when administered to the subject. In some embodiments, when the nanoemulsion of the present invention is used to inactivate a live virus of the family paramyxoviridae (e.g., RSV), each dose (e.g., administered to a subject to induce an immune response)) is expected to comprise 10-10 ¹⁰pfu virus/dose; in some embodiments, each dose comprises 10⁵ - 10⁸ pfu virus/dose; in some embodiments, each dose comprises 10³ - 10⁵ pfu virus/dose; in some embodiments, each dose comprises 10² - 10⁴ pfu virus/dose; in some embodiments, each dose comprises 10 pfu virus/dose; in some embodiments, each dose comprises 10² pfu virus/dose; and in some embodiments, each dose comprises 10⁴ pfu virus/dose. In some embodiments, each dose comprises more than 10¹⁰ pfu virus/dose. In some preferred embodiments, each dose comprises 10³ pfu virus/dose.

The present invention is not limited to any particular nanoemulsion composition. Indeed, various nanoemulsion compositions useful in the present invention are described herein. Similarly, the present invention is not limited to the specific oils present in the nanoemulsion. A variety of oils are contemplated, including but not limited to soybean, avocado, squalene, olive, canola, corn, rapeseed, safflower, sunflower, fish, spices, and water-insoluble vitamins. The present invention is also not limited to a specific solvent. A variety of solvents are contemplated, including but not limited to alcohols (e.g., including but not limited to methanol, ethanol, propanol, and octanol), glycerol, polyethylene glycol, and organophosphate-based solvents. Including oils, solvents, and other nanoemulsion components, are described in further detail below.

In some embodiments, the emulsion further comprises a surfactant. The present invention is not limited to a specific surfactant. A variety of surfactants are contemplated, including but not limited to nonionic and ionic surfactants (e.g., TRITON X-100; TWEEN 20; and TYLOXAPOL).

In certain embodiments, the emulsion further comprises a cationic halogen-containing compound. The present invention is not limited to a specific cationic halogen-containing compound. A variety of cationic halogen-containing compounds are contemplated, including but not limited to cetyl pyridinium halidesCetyl trimethyl ammonium halide, cetyl dimethylethyl ammonium halide, cetyl dimethylbenzyl ammonium halide, cetyl tributyl ammonium halideDodecyl trimethyl ammonium halide and tetradecyl trimethyl ammonium halide. The present invention is not limited to a specific halide. A variety of halides are contemplated, including but not limited to halides selected from chloride, fluoride, bromide, and iodide.

In yet a further embodiment, the emulsion further comprises a quaternary ammonium-containing compound. The present invention is not limited to a specific quaternary ammonium-containing compound. A variety of quaternary ammonium-containing compounds are contemplated, including but not limited to alkyl dimethyl benzyl ammonium chloride, dialkyl dimethyl benzyl ammonium chloride, n-alkyl dimethyl ethyl benzyl ammonium chloride, dialkyl dimethyl ammonium chloride, and n-alkyl dimethyl benzyl ammonium chloride.

In some embodiments, the present invention provides vaccines comprising an immunogenic composition comprising a virus of the paramyxoviridae family (e.g., RSV) inactivated by a nanoemulsion. In some embodiments, the present invention provides a kit comprising a vaccine comprising an emulsion comprising an aqueous phase, an oil phase and a solvent, and an immunogenic composition comprising a virus of the paramyxoviridae family (e.g., RSV) inactivated by a nanoemulsion. In some embodiments, the kit further comprises instructions for using the kit for vaccinating a subject against a virus of the paramyxoviridae family (e.g., RSV).

In yet a further embodiment, the present invention provides a method of inducing immunity to one or more viruses of the family paramyxoviridae (e.g., RSV), comprising providing an emulsion comprising an aqueous phase, an oil phase, and a solvent; and one or more viruses of the paramyxoviridae family (e.g., RSV); combining the emulsion with one or more viruses of the family paramyxoviridae (e.g., RSV) to produce a vaccine composition; and administering the vaccine composition to the subject. In some embodiments, administering comprises contacting the vaccine composition with a mucosal surface of the subject. For example, in some embodiments, administering comprises intranasal administering. In some preferred embodiments, administration occurs under conditions such that the subject generates immunity (e.g., via generation of a humoral immune response against one or more immunogens) against one or more viruses of the paramyxoviridae family (e.g., RSV).

The invention is not limited by the nature of the immune response generated (e.g., following administration of an immunogenic composition comprising a virus of the paramyxoviridae family (e.g., RSV) inactivated by nanoemulsion).Indeed, a variety of immune responses may be generated and measured in a subject administered a composition comprising a nanoemulsion and a virus of the paramyxoviridae family inactivated by the nanoemulsion of the present invention (e.g., RSV), including but not limited to activation, proliferation, or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, Antigen Presenting Cells (APCs), macrophages, Natural Killer (NK) cells, etc.); up-or down-regulated expression of markers and cytokines; stimulation of IgA, IgM, and/or IgG titers; splenomegaly (e.g., increased splenocyte formation); hyperplasia in various organs, mixed cellular infiltration, and/or other responses of the immune system (e.g., cellular), which can be assessed in terms of immune stimulation as known in the art. In some embodiments, administering comprises contacting a mucosal surface of the subject with the composition. The invention is not limited by the mucosal surface in contact. In some preferred embodiments, the mucosal surface comprises nasal mucosa. In some embodiments, the mucosal surface comprises vaginal mucosa. In some embodiments, the administering comprises parenteral administration. The present invention is not limited by the route selected for administration of the compositions of the present invention. In some embodiments, inducing an immune response induces immunity to one or more viruses of the paramyxoviridae family (e.g., RSV) in a subject. In some embodiments, the immunization comprises systemic immunization. In some embodiments, the immunization comprises mucosal immunization. In some embodiments, the immune response comprises increased expression of IFN- γ and/or IL-17 in the subject. In some embodiments, the immune response comprises a systemic IgG response. In some embodiments, the immune response comprises a mucosal IgA response. In some embodiments, the composition comprises a 15% nanoemulsion solution. However, the present invention is not limited to this amount (e.g., percentage) of nanoemulsion. For example, in some embodiments, the composition comprises less than 10% nanoemulsion (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%). In some embodiments, the composition comprises more than 10% nanoemulsion (e.g., 12%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60% or more). In some embodiments, of the invention The composition comprises any of the nanoemulsions described herein. In some embodiments, the nanoemulsion comprises W₂₀5 EC. In some preferred embodiments, the nanoemulsion comprises W₈₀5 EC. In some embodiments, the nanoemulsion is X8P. In some embodiments, the subject is immunoprotected from displaying signs or symptoms of disease caused by a virus of the paramyxoviridae family (e.g., RSV).

In some embodiments, immunization protects a subject from challenge by subsequent exposure to a live virus of the paramyxoviridae family (e.g., RSV). In some embodiments, the composition further comprises an adjuvant. The invention is not limited by the type of adjuvant utilized. In some embodiments, the adjuvant is a CpG oligonucleotide. In some embodiments, the adjuvant is monophosphoryl lipid a. Many other adjuvants useful in the present invention are described herein. In some embodiments, the subject is a human. In some embodiments, the subject is immunoprotected from exhibiting signs or symptoms of infection by a virus of the paramyxoviridae family (e.g., RSV). In some embodiments, immunization reduces the risk of infection upon one or more exposures to a virus of the paramyxoviridae family (e.g., RSV).

Description of the drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects and embodiments of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the description of specific embodiments presented herein.

Figure 1 shows the killing of Respiratory Syncytial Virus (RSV) by nanoemulsion.

Figure 2 shows the induction of RSV-specific antibodies after immunization with nanoemulsion inactivated RSV (NE-RSV).

Figure 3 shows that administration of NE-RSV to a subject results in an enhanced RSV-specific CD 8T cell response.

Figure 4 shows that administration of NE-RSV to a subject enhances antiviral cytokines in BAL fluid from the airways of RSV-challenged mice.

Figure 5 shows that vaccination of mice with NE-RSV enhances IL-17 production in the lungs after challenge with live RSV.

Figure 6 shows that administration of NE-RSV to a subject provides improved clearance and induces a protective response upon subsequent live virus challenge.

FIG. 7 shows the expression of various genes in mice administered NE-RSV compared to controls.

FIG. 8 shows periodic acid-Schiff (PAS) staining of lung histological sections in mice administered NE-RSV compared to controls.

Figure 9 shows cytokine expression in mice administered NE-RSV compared to controls.

Figure 10 shows that significant RSV-specific antibody responses produced total Ig systemically in mice following vaccination with NE-RSV (a) and bronchoalveolar lavage of vaccinated mice 2 days after challenge with live virus.

General description of the invention

The present invention provides methods and compositions for immune response stimulation. In particular, the invention provides immunogenic compositions and methods of use thereof to induce an immune response (e.g., immunity (e.g., protective immunity)) against a pathogenic virus of the paramyxoviridae family (e.g., a paramyxoviridae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus)). The compositions and methods of the invention are particularly useful in clinical (e.g., therapeutic and prophylactic medicine (e.g., vaccination)) and research applications.

Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, treatment with NE of the invention (e.g., neutralization of a virus of the paramyxoviridae family (e.g., a paramyxovirinae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus))) preserves an important antigenic epitope of the virus (e.g., recognizable by the immune system of the subject) (e.g., simultaneously neutralizing and/or eradicating the infectious potential of the virus) thereby stabilizing its hydrophobic and hydrophilic components in the oil and water interfaces of the emulsion (e.g., thereby providing one or more immunogens against which the subject can mount an immune response (e.g., stabilizing the antigen)). In other embodiments, because the NE formulations penetrate the mucosa through the pores, they can carry the immunogen to a submucosal location of the dendritic cells (e.g., to initiate and/or stimulate an immune response). Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, combining NE and a virus of the paramyxoviridae family, such as a virus of the paramyxoviridae family (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a virus of the pneumovirinae family (e.g., respiratory syncytial virus)), stabilizes the viral immunogen and provides the correct immunogenic material for generating an immune response.

Dendritic cells greedily engulf the oil droplets of the Nanoemulsion (NE), and this can provide a means of neutralizing viral immunogens, such as antigenic proteins or peptide fragments thereof generated following inactivation of a virus of the paramyxoviridae family (e.g., paramyxovirinae (e.g., paramyxovirus, mumps, and/or measles) and/or pneumovirinae (e.g., respiratory syncytial virus)) of the nanoemulsion, for antigen presentation. While other vaccines rely on inflammatory toxins or other immune stimuli for adjuvant activity (see, e.g., Holmgren and Czerkinsky, Nature med. 2005, 11; 45-53), NEs has not been shown to be inflammatory when placed on the skin or mucosa in studies in animals and humans. Thus, while an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, however, a composition of the invention comprising NE (e.g., a composition comprising NE and one or more viruses of the paramyxoviridae family (e.g., a paramyxoviridae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus)) can act as a "physical" adjuvant to the immune system (e.g., to transport and/or present immunogenic compositions (e.g., peptides and/or antigens of a paramyxoviridae virus).

Cellular and humoral immunity play a role in protection against multiple pathogens, and both can be induced by the NE formulations of the present invention. In some embodiments, administration (e.g., mucosal administration) of a composition of the invention (e.g., NE inactivation of pneumovirinae virus (e.g., RSV)) to a subject results in the induction of both a humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocyte) immune response (e.g., against pneumovirinae virus (e.g., RSV)). In some embodiments, a composition of the invention (e.g., NE inactivated pneumovirinae virus (e.g., RSV)) is used as a vaccine (e.g., RSV vaccine).

Furthermore, in some embodiments, a composition of the invention (e.g., a composition comprising NE and a virus of the paramyxoviridae family, e.g., a virus of the paramyxoviridae family (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a virus of the pneumovirinae family (e.g., respiratory syncytial virus)) induces (e.g., when administered to a subject) systemic and mucosal immunity. Thus, in some preferred embodiments, administration of a composition of the invention to a subject results in protection against exposure (e.g., lethal mucosal exposure) to a virus of the paramyxoviridae family (e.g., a paramyxoviridae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus))). Mucosal administration (e.g., vaccination) provides protection against viral infection (e.g., that initiated at a mucosal surface), although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action. Although it has proven difficult to stimulate secretory IgA responses and protection against pathogens invading at Mucosal surfaces to date (see, e.g., Mestecky et al, Mucosal immunology. 3ed edn. (Academic Press, San Diego, 2005)), the present invention provides compositions and methods for stimulating Mucosal immunity (e.g., protective IgA responses) against viruses of the paramyxoviridae family, e.g., paramyxovirinae (e.g., paramyxovirus, mumps virus, and/or measles virus) and/or pneumovirinae (e.g., respiratory syncytial virus)).

Definition of

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

As used herein, the term "microorganism" refers to any species or type of microorganism, including, but not limited to, bacteria, viruses, archaea, fungi, protozoa, mycoplasma, prions, and parasites. The term microorganism encompasses those organisms which are themselves and spontaneously pathogenic to another organism (e.g. animals including humans and plants), and those organisms which produce an agent which is pathogenic to another organism, although the organism itself is not directly pathogenic or infectious to the other organism.

As used herein, the term "pathogen" and grammatical equivalents refers to an organism (e.g., biological agent) that causes a disease condition (e.g., infection, pathological condition, disease, etc.) in another organism (e.g., animals and plants) including a microorganism, by direct infection of the other organism or by production of an agent that causes the disease in another organism (e.g., a bacterium that produces a pathogenic toxin, etc.). "pathogens" include, but are not limited to, viruses, bacteria, archaea, fungi, protozoa, mycoplasmas, prions, and parasites.

The term "bacteria" refers to all prokaryotes, the packageIncluding those within all of the gates in the prokaryote kingdom. The term is intended to encompass all microorganisms considered to be bacteria, including the genus cladosporium (Mycoplasma) The genus of Chlamydia (A)Chlamydia) Actinomycetes genus (A)Actinomyces) Streptomyces (I), (II)Streptomyces) And genus Rickettsia: (Rickettsia). All forms of bacteria are included within this definition, including cocci, bacilli, spirochetes, spheroplasts, protoplasts, and the like.

As used herein, the term "fungus" is used in relation to eukaryotes, such as molds and yeasts, including bimodal fungi.

As used herein, unless otherwise specified herein, the terms "disease" and "pathological condition" are used interchangeably to describe a deviation from a condition considered normal or average for members of a species or group (e.g., humans), and which is harmful to the affected individual under conditions that are not harmful to the majority of individuals of that species or group. Such a deviation may manifest as a condition, sign and/or symptom (e.g., diarrhea, nausea, fever, pain, blisters, boils, rash, immunosuppression, inflammation, etc.) associated with any impairment of the normal state of the subject or any of its organs or tissues, which interrupts or alters the achievement of normal function. A disease or pathological condition may be caused by or result from contact with a microorganism (e.g., a pathogen or other infectious agent (e.g., a virus or bacterium)), may be responsive to environmental factors (e.g., malnutrition, industrial hazards, and/or climate), may be responsive to inherent deficiencies of the organism (e.g., genetic abnormalities), or a combination of these and other factors.

As used herein, the term "host" or "subject" refers to an individual to be treated (e.g., administered) by the compositions and methods of the invention. Subjects include, but are not limited to, mammals (e.g., mice, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably include humans. In the context of the present invention, the term "subject" generally refers to an individual to whom or to whom one or more compositions of the present invention (e.g., a composition for inducing an immune response) is to be administered.

As used herein, the terms "inactivation" and grammatical equivalents, when used in reference to a microorganism (e.g., a pathogen (e.g., a virus)), refer to the killing, elimination, neutralization, and/or reduction of the ability of the microorganism (e.g., a pathogen (e.g., a virus)) to kill, eliminate, neutralize, and/or infect and/or cause a pathological response and/or disease in a host. For example, in some embodiments, the present invention provides compositions comprising Nanoemulsion (NE) -inactivated Respiratory Syncytial Virus (RSV). Thus, as referred to herein, a composition comprising "NE inactivated RSV", "NE killed RSV", NE neutralized RSV "," NE-RSV ", or grammatical equivalents refers to a composition that, when administered to a subject, is characterized by the absence or significantly reduced presence of RSV replication in the host (e.g., over a period of time (e.g., over a period of days, weeks, months, or longer).

As used herein, the term "fused" means an emulsion capable of fusing with a membrane of a microbial agent (e.g., a bacteria, bacterial spore, or viral capsid). Specific examples of the fusion emulsion are described herein.

As used herein, the term "lysogenic" refers to an emulsion (e.g., a nanoemulsion) that is capable of disrupting the membrane of a microbial agent (e.g., a virus (e.g., a viral envelope) or a bacterium or bacterial spore). In a preferred embodiment of the invention, the presence of the lysogen and the fusing agent in the same composition produces an enhanced inactivation as compared to either agent alone. Methods and compositions for using such improved antimicrobial compositions (e.g., for inducing an immune response (e.g., for use as a vaccine) are described in detail herein.

As used herein, the term "emulsion" includes typical oil-in-water or water-in-oil dispersions or droplets, as well as other lipid structures that may form due to hydrophobic forces that drive non-polar residues (e.g., long hydrocarbon chains) away from water and polar headgroups toward water when a water immiscible oil phase is mixed with a water phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar and multilamellar lipid vesicles, micelles and lamellar phases. Similarly, as used herein, the term "nanoemulsion" refers to oil-in-water dispersions comprising small lipid structures. For example, in a preferred embodiment, the nanoemulsion comprises an oil phase having droplets with an average particle size of about 0.1-5 microns (e.g., 150 +/-25 nm in diameter), although smaller and larger particle sizes are contemplated. The terms "emulsion" and "nanoemulsion" are often used interchangeably herein to refer to the nanoemulsions of the present invention.

As used herein, the terms "contacting," "contacted," "exposed," and "exposed," when used in reference to a nanoemulsion and a live microorganism, refer to bringing one or more nanoemulsions into contact with a microorganism (e.g., a pathogen), such that the nanoemulsions inactivate the microorganism or pathogenic agent, if present. The present invention is not limited by the amount or type of nanoemulsion used for microbial inactivation. Various nanoemulsions useful in the present invention are described herein and elsewhere (e.g., the nanoemulsions described in U.S. patent applications 20020045667 and 20040043041, and U.S. patent nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which is incorporated herein by reference in its entirety for all purposes). The ratio and amount of nanoemulsion (e.g., sufficient to inactivate a microorganism (e.g., viral inactivation)) and microorganism (e.g., sufficient to provide an antigenic composition (e.g., a composition capable of inducing an immune response)) are contemplated in the present invention, including but not limited to those described herein.

The term "surfactant" refers to any molecule having a polar head group that strongly prefers solvation by water and a hydrophobic tail that is not well solvated by water. The term "cationic surfactant" refers to a surfactant having a cationic head group. The term "anionic surfactant" refers to a surfactant having an anionic head group.

The terms "hydrophilic-lipophilic balance index" and "HLB index" refer to the chemistry used to associate surfactant moleculesIndex of chemical structure and its surface activity. The HLB index may be calculated by a variety of empirical formulas, as described, for example, by Meyers, which is incorporated herein by reference (see, e.g., Meyers,Surfactant Science and Technology ，VCH Publishers Inc., New York, pages 231-245 (1992)). As used herein, the HLB index of a surfactant is the HLB index specified for that surfactant in McCutcheon, Vol.1: Emulsifiers and Detergents North American Edition, 1996, incorporated herein by reference. The HLB index ranges from 0 to about 70 or more for commercial surfactants. Hydrophilic surfactants with high solubility and solubilization properties in water are at the high end of the scale, while surfactants with low solubility in water, which are good solubilizers of water in oil, are at the low end of the scale.

Contemplated interaction enhancers include, but are not limited to, chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA), ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), etc.) and certain biological agents (e.g., bovine serum albumin (abumin) (BSA), etc.).

The term "buffer" or "buffering agent" refers to a material that, when added to a solution, causes the solution to resist a change in pH.

The terms "reducing agent" and "electron donor" refer to a material that donates an electron to a second material to reduce the oxidation state of one or more atoms of the second material.

The term "monovalent salt" refers to any salt in which the metal (e.g., Na, K, or Li) has a net 1+ charge in solution (i.e., one more proton than electron).

The term "divalent salt" refers to any salt in which the metal (e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.

The term "chelating agent" or "chelating agent" refers to any material having more than one atom containing a non-lone pair of electrons available for bonding to a metal ion.

The term "solution" refers to an aqueous or non-aqueous mixture.

As used herein, the term "composition for inducing an immune response", "immunogenic composition" or grammatical equivalents refers to a composition that, upon administration to a subject (e.g., 1, 2, 3, or more times (e.g., separated by weeks, months, or years)), stimulates, generates, and/or elicits an immune response in the subject (e.g., results in total or partial immunity against a microorganism (e.g., a pathogen) capable of causing a disease). In a preferred embodiment of the invention, the composition comprises a nanoemulsion and an immunogen. In a further preferred embodiment, the composition comprising the nanoemulsion and immunogen comprises one or more other compounds or agents, including but not limited to therapeutic agents, physiologically tolerable liquids, gels, carriers, diluents, adjuvants, excipients, salicylates, steroids, immunosuppressive agents, immunostimulating agents, antibodies, cytokines, antibiotics, binders, fillers, preservatives, stabilizers, emulsifiers and/or buffers. The immune response can be an innate (e.g., non-specific) immune response or a learned (e.g., acquired) immune response (e.g., one that reduces infectivity, pathogenesis, or onset of pathogenesis in the subject (e.g., caused by exposure to a pathogenic microorganism) or one that prevents infectivity, pathogenesis, or onset of pathogenesis in the subject (e.g., caused by exposure to a pathogenic microorganism)). Thus, in some preferred embodiments, the composition comprising the nanoemulsion and immunogen is administered to a subject as a vaccine (e.g., to prevent or attenuate a disease (e.g., by providing the subject with overall or partial immunity to the disease or overall or partial attenuation (e.g., inhibition) of the signs, symptoms, or conditions of the disease).

As used herein, the term "adjuvant" refers to any substance that can stimulate an immune response (e.g., a mucosal immune response). Some adjuvants can cause cellular activation of the immune system (e.g., adjuvants can cause immune cells to produce and secrete cytokines). Examples of adjuvants that can cause cellular activation of the immune system include, but are not limited to, saponins purified from the bark of the quillaja saponaria (q. saponaria), such as QS21 (glycolipids eluted in the 21 st peak by HPLC fractionation; Aquila Biopharmaceuticals, inc., Worcester, Mass.); poly (carboxyphenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA); lipopolysaccharides such as derivatives of monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi), and threonyl muramyl dipeptide (t-MDP; Ribi); OM-174 (glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania (Leishmania) elongation factors (purified Leishmania proteins; Corixa Corporation, Seattle, Wash.). Conventional adjuvants are well known in the art and include, for example, aluminum phosphate or hydroxide salts ("alum"). In some embodiments, a composition of the invention (e.g., comprising nanoemulsion inactivated RSV) is administered with one or more adjuvants (e.g., to bias the immune response toward a Th1 or Th2 type response).

As used herein, the term "amount effective to induce an immune response" (e.g., a composition for inducing an immune response) refers to the dosage level required to stimulate, generate, and/or elicit an immune response in a subject (e.g., when administered to a subject). An effective amount may be administered in one or more administrations (e.g., via the same or different routes), applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

As used herein, the term "under such conditions such that the subject generates an immune response" refers to any qualitative or quantitative induction, generation and/or stimulation of an immune response (e.g., innate or acquired).

As used herein, the term "immune response" refers to a response by the immune system of a subject. For example, immune responses include, but are not limited to, Toll receptor activation, lymphokine (e.g., cytokine (e.g., Th1 or Th2 type cytokine) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or detectable changes (e.g., increases) in B cell activation (e.g., antibody production and/or secretion). Additional examples of immune responses include binding of an immunogen (e.g., an antigen (e.g., an immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production) and/or a T helper lymphocyte response, and/or a delayed-type hypersensitivity (DTH) response to an antigen from which the immunogenic polypeptide is derived, expansion of cells of the immune system (e.g., T cells, B cells (e.g., any developmental stage (e.g., plasma cells), such as growth of a cell population, and increased processing and presentation of antigen by antigen presenting cells). As used herein, "immune response" refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascades), cell-mediated immune responses (e.g., responses mediated by T cells of the immune system (e.g., antigen-specific T cells) and non-specific cells), and humoral immune responses (e.g., responses mediated by B cells (e.g., via antibody production and secretion into plasma, lymph, and/or interstitial fluid).

As used herein, the term "immunity" refers to protection from a disease (e.g., prevention or attenuation (e.g., inhibition) of signs, symptoms, or conditions of a disease) upon exposure to a microorganism (e.g., pathogen) capable of causing the disease. The immunity can be innate (e.g., a non-adaptive (e.g., non-adaptive) immune response that exists in the absence of prior exposure to the antigen), and/or adaptive (e.g., an immune response that is mediated by B and T cells after prior exposure to the antigen (e.g., that exhibits increased specificity and reactivity to the antigen)).

As used herein, the term "immunogen" refers to an agent (e.g., a microorganism (e.g., a bacterium, virus, or fungus) and/or a portion or component thereof (e.g., a protein antigen)) that is capable of eliciting an immune response in a subject. In a preferred embodiment, the immunogen elicits immunity against an immunogen, such as a microorganism (e.g., a pathogen or pathogen product), when administered in combination with the nanoemulsion of the present invention.

As used herein, the term "pathogen product" refers to any component or product derived from a pathogen, including but not limited to polypeptides, peptides, proteins, nucleic acids, membrane fractions, and polysaccharides.

As used herein, the term "enhanced immunity" refers to an increase in the level of adaptive and/or adaptive immunity to a given immunogen (e.g., a microorganism (e.g., a pathogen)) in a subject following administration of a composition (e.g., a composition for inducing an immune response of the invention) relative to the level of adaptive and/or adaptive immunity in a subject to which a composition has not been administered (e.g., a composition for inducing an immune response of the invention).

As used herein, the term "purified" or "to be purified" refers to the removal of contaminants or unwanted compounds from a sample or composition. As used herein, the term "substantially purified" refers to the removal of about 70-90%, up to 100%, of contaminants or unwanted compounds from a sample or composition.

As used herein, the term "administering" refers to the act of administering a composition of the invention (e.g., a composition for inducing an immune response (e.g., a composition comprising a nanoemulsion and an immunogen)) to a subject. Exemplary routes of administration for humans include, but are not limited to, through the eye (ocular), mouth (oral), skin (transdermal), nose (nasal), lung (inhaled), oral mucosa (buccal), ear, rectum, by injection (e.g., intravenous, subcutaneous, intraperitoneal, etc.), topically, and the like.

As used herein, the term "co-administration" refers to the administration of at least 2 agents (e.g., a composition comprising a nanoemulsion and an immunogen and one or more other agents-e.g., an adjuvant) or treatments to a subject. In some embodiments, the co-administration of 2 or more agents or treatments is concurrent. In other embodiments, the first agent/treatment is administered before the second agent/treatment. In some embodiments, co-administration may be via the same or different routes of administration. One skilled in the art will appreciate that the formulation and/or route of administration of the various agents or treatments used may vary. Suitable dosages for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or treatments are co-administered, the respective agent or treatment is administered at a lower dosage than is appropriate for its separate administration. Thus, co-administration is particularly desirable in embodiments where co-administration of an agent or treatment reduces the necessary dosage of one or more potentially harmful (e.g., toxic) agents, and/or when co-administration of 2 or more agents results in sensitization of the subject to the beneficial effects of one of the agents via co-administration of the other agent. In other embodiments, co-administration is preferred to elicit an immune response in a subject against 2 or more different immunogens (e.g., microorganisms (e.g., pathogens)) at or near the same time (e.g., when the subject is unlikely to be available for subsequent administration of the second, third, or more compositions for inducing an immune response).

As used herein, the term "topically" refers to the application of a composition of the present invention (e.g., a composition comprising a nanoemulsion and an immunogen) to the surface of skin and/or mucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory, vaginal or nasal mucosa, and other tissues and cells lining hollow organs or body cavities).

In some embodiments, the compositions of the present invention are administered in the form of topical emulsions, injectable compositions, ingestible solutions, and the like. When the route is topical, the form may be, for example, a spray (e.g., a nasal spray), a cream, or other viscous solution (e.g., a composition of nanoemulsion and immunogen contained in polyethylene glycol).

As used herein, the term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a composition that, when administered to a subject, produces substantially no adverse reaction (e.g., toxic, allergic, or immune reaction).

As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), polyethylene glycols, and the like. The composition may also include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants have been described and are known in the art (see, e.g., Martin, Remington's Pharmaceutical Sciences, 15 th edition, Mack pub. co., Easton, Pa, (1975), incorporated herein by reference).

As used herein, the term "pharmaceutically acceptable salt" refers to any salt of the composition of the invention that is physiologically tolerated in the target subject (e.g., obtained by reaction with an acid or base). The "salts" of the compositions of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, p-toluenesulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acids and the like. Other acids, such as oxalic, while not per se pharmaceutically acceptable, may be used to prepare salts useful as intermediates in obtaining the compositions of the invention and their pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and NW' s₄ ⁺Wherein W is C_1-4Alkyl groups, and the like.

Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate (flucoheptanoate), glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate (palmoate), pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include salts with suitable cations such as Na ⁺、NH₄ ⁺And NW₄ ⁺(wherein W is C_1-4Alkyl) and the like. For therapeutic use, salts of the compounds of the present invention are expected to be pharmaceutically acceptable. However, salts of acids and bases which are not pharmaceutically acceptable may also be useful, for example, in the preparation or purification of pharmaceutically acceptable compounds.

For therapeutic use, salts of the compositions of the present invention are contemplated to be pharmaceutically acceptable. However, salts of acids and bases which are not pharmaceutically acceptable may also be useful, for example, in the preparation or purification of pharmaceutically acceptable compositions.

As used herein, the term "at risk for a disease" refers to a subject predisposed to experiencing a particular disease. Such predisposition may be genetic (e.g., a particular genetic predisposition to experience a disease, such as a genetic disease), or due to other factors (e.g., age, environmental conditions, exposure to harmful compounds present in the environment, etc.). Thus, it is not intended that the present invention be limited to any particular risk (e.g., a subject may be "at risk for disease" simply by exposure to and interaction with other people) nor is it intended that the present invention be limited to any particular disease.

As used herein, "nasal application" means application through the nose into the nasal or sinus passages or both. Application may be accomplished, for example, by drops, sprays, mists, coatings or mixtures thereof applied to the nasal and sinus passages.

As used herein, the term "kit" refers to any delivery system for delivering a material. In the context of immunogenic agents (e.g., compositions comprising nanoemulsions and immunogens), such delivery systems include systems that allow for the storage, transport, or delivery of the immunogenic agent and/or support materials (e.g., written instructions regarding the use of the materials, etc.) from one location to another. For example, the kit includes one or more shells (e.g., a cassette) containing the relevant immunogenic agent (e.g., nanoemulsion) and/or support material. As used herein, the term "fragmented kit" refers to a delivery system comprising 2 or more separate containers, each containing a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising a nanoemulsion and an immunogen for a particular use, while a second container contains a second agent (e.g., an antibiotic or spray applicator). Indeed, any delivery system comprising 2 or more separate containers each containing a sub-portion of the total kit components is encompassed within the term "divided kit". In contrast, a "combination kit" refers to a delivery system containing all of the components of an immunogenic agent required for a particular use in a single container (e.g., in a single cartridge housing each of the required components). The term "kit" includes both divided and combined kits.

Detailed Description

Respiratory Syncytial Virus (RSV) infects almost all infants up to 2 years of age and is the leading cause of bronchiolitis in children worldwide. It is estimated by the CDC that up to 125,000 pediatric hospitalizations in the united states per year are due to RSV, costing more than $300,000,000 per year (1). Despite the generation of RSV-specific adaptive immune responses, RSV does not confer protective immunity and recurrent infections throughout life are common (2, 3). Although RSV is particularly harmful in very young infants where the airways are small and easily blocked, RSV also widely becomes recognized as an important pathogen in transplant recipients, patients with Chronic Obstructive Pulmonary Disease (COPD), the elderly, and other patients with chronic lung disease, particularly asthma. Recent data suggests that mortality from 1990-2000 for all age groups has been about 30/100,000, with an average mortality rate in the United states of 17,000 (4, 5) per year. These numbers can be severely underestimated as it has not yet been thoroughly examined in a consistent manner in adults. Thus, RSV not only causes severely exacerbated lung disease in young and elderly people, but is also directly associated with significant mortality. Although anti-RSV antibodies are available and appear to alleviate severe disease, they are only performed upon prophylactic (prophylactically) administration, and there are few other options for combating RSV infection in a susceptible patient population (6-10).

In the late twentieth sixties, attempts to vaccinate children with alum-precipitated formalin inactivated RSV vaccine formulations failed and caused severely exacerbated disease upon re-infection with live RSV. The clinical manifestations appear to be the result of enhanced Th2 disease, mucus production, and eosinophilia, which were not observed in unvaccinated children. These same symptoms can occur in severely infected subpopulations of infants.

Several epidemiological studies link severe RSV response to the later development of hyperreactive airway disease even years after the infection has been eliminated (3). Sigurs et al found that hospitalized infants due to RSV bronchiolitis were at higher risk of developing asthma and wheezing episodes at 1, 3 and 7 years of age compared to healthy controls (11-13). RSV has also been associated with asthma exacerbations and can cause prolonged episodes of disease (14). An interesting study suggests a causal link, since treatment of infants with severe RSV disease with RSV immunoglobulin significantly reduces their later risk of developing childhood asthma and lung dysfunction (15). One clinically relevant feature of RSV disease that predisposes children and adults to chronic disease is the inability to acquire protective immunity due to altered immune responses. More recent studies have suggested that patient populations with impaired lung function (especially patients) are also at risk for serious complications due to RSV infection, which are nearly as prevalent as those associated with influenza infection (16-20). These complications can be compounded by the possibility that RSV has the ability to persist in the lungs for long periods of time even after the acute disease has been eliminated. This has been postulated to be associated with the development of altered immune environments that are less effective at clearing the virus. Thus, effective vaccines that can be used in children and adults have the potential for broad application across populations, and can provide significant protection from the onset and exacerbation of chronic lung disease.

Thus, in some embodiments, the present invention provides methods and compositions for specific immune response stimulation. In particular, the present invention provides immunogenic nanoemulsion compositions and methods of using the same to induce an immune response (e.g., immunity (e.g., protective immunity)) against a pathogenic virus of the paramyxoviridae family (e.g., a paramyxovirinae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus)). The compositions and methods of the invention are particularly useful in clinical (e.g., therapeutic and prophylactic medicine (e.g., vaccination)) and research applications. Exemplary immunogenic compositions (e.g., vaccine compositions) and methods of administering the compositions are described in more detail below.

In some embodiments, the present invention provides compositions for inducing an immune response comprising a nanoemulsion and one or more immunogens (e.g., inactivated pathogens or pathogen products (e.g., inactivated viruses (e.g., inactivated respiratory syncytial virus))). The present invention is not limited to any particular nanoemulsion. Indeed, a variety of nanoemulsions are useful in the present invention, including but not limited to those described herein and elsewhere (e.g., the nanoemulsions described in U.S. patent applications 20020045667 and 20040043041, and U.S. patent nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which is incorporated herein by reference in its entirety for all purposes).

The immunogens (e.g., pathogens or pathogen products (e.g., inactivated viruses (e.g., inactivated respiratory syncytial virus)))) and nanoemulsions of the present invention can be combined in any suitable amount and delivered to a subject using a variety of delivery methods. Any suitable pharmaceutical formulation may be utilized, including but not limited to those disclosed herein. Suitable formulations may be tested for immunogenicity using any suitable method. For example, in some embodiments, immunogenicity is studied by quantifying antibody titers and specific T cell responses. The nanoemulsion compositions of the present invention can also be tested in animal models of infectious disease states. Suitable animal models, pathogens, and assays for immunogenicity include, but are not limited to, those described below.

In some embodiments, the present invention provides for the development of immunity (e.g., immunity against a virus of the paramyxoviridae family, e.g., a paramyxovirinae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or a pneumovirinae virus (e.g., respiratory syncytial virus))) in a subject following mucosal administration (e.g., mucosal vaccination) of a composition comprising a Nanoemulsion (NE) -inactivated virus of the paramyxoviridae family, e.g., RSV) identified and characterized during development of the present invention. As described in examples 1-2, NE is mixed with RSV, resulting in a formulation (e.g., NE-killed RSV composition) that is stable at room temperature (e.g., in some embodiments, for more than 2 weeks, more preferably more than 3 weeks, even more preferably more than 4 weeks, and most preferably more than 5 weeks) and can be used to induce an immune response against RSV in a subject (e.g., can be used alone or as an adjuvant to induce an anti-RSV immune response).

Mucosal administration of a composition comprising NE and RSV (e.g., NE-killed RSV) to a subject results in a high titer antibody response and specific Th1 cellular immunity (see, e.g., examples 1-4). Further, the animals are protected from subsequent challenge with RSV (see, e.g., example 4). Furthermore, in sharp contrast to alum-precipitated formalin-inactivated RSV vaccine formulations (e.g., described above), NE-inactivated RSV of the invention result in a strong Th1 immune (e.g., as demonstrated by enhanced IFN- γ and IL-17 expression (see, e.g., example 4) response, and do not enhance and/or elevate expression of Th2 cytokines (e.g., IL-4, IL-5, or IL-13) associated with a Th 2-type response 4 weeks post-administration mice administered even a single dose of a composition comprising NE-killed RSV develop serum concentrations of anti-RSV IgG that continue to increase at 8 weeks post-administration and are significantly elevated post-booster administration (see, e.g., examples 2-4) Such as mucosal and systemic immunity)). In some embodiments, subsequent administrations to the subject (e.g., one or more booster administrations after the initial administration) provide induction of an enhanced immune response against RSV in the subject. Thus, the present invention demonstrates that administration of a composition comprising NE-killed RSV to a subject provides protective immunity against RSV infection.

Both cellular and humoral immunity may play a role in protection against RSV, and both are induced by NE formulations (see, e.g., examples 1-4). RSV-specific antibody titers are considered important for protective immune estimation in human subjects and in vaccinated animal models.

Data generated during development of embodiments of the invention demonstrate that NE is effective in killing RSV and generates a non-infectious vaccination composition (e.g., for use as a vaccine) suitable for use in inducing an immune response against RSV in a subject. Upon administration to a subject, the immunogenic composition induces specific anti-RSV serum antibody titers and initiates important antiviral cellular immune responses (e.g., induces increased antiviral cytokine production and development of RSV-specific CD8+ cytotoxic T cells). The immunogenic compositions of the invention also provide improved viral clearance upon challenge with live RSV. Thus, in some embodiments, the compositions and methods of the invention provide the ability to generate a suitable innate immune response (e.g., resulting from exposure to antigens maintained in recognizable form in NE that mimics the antigens provided by active infection), thereby providing a more suitable vaccine strategy (e.g., as compared to formalin-killed RSV).

Thus, in some embodiments, administration (e.g., mucosal administration) of a composition of the invention (e.g., NE-killed RSV) to a subject provides for the induction of both a humoral (e.g., development of specific antibodies) and cellular (e.g., cytotoxic T lymphocytes) immune response (e.g., against RSV). In some preferred embodiments, the compositions of the invention (e.g., NE-killed RSV) are used as vaccines.

Production of antibodies

An immunogenic composition comprising Nanoemulsion (NE) -inactivated paramyxoviridae viruses, such as paramyxoviridae viruses (e.g., paramyxovirus, mumps virus, and/or measles virus) and/or pneumovirinae viruses (e.g., respiratory syncytial virus)), can be used to immunize an animal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, the pneumovirinae virus (e.g., respiratory syncytial virus) antigen may be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, keyhole limpetHemocyanin, or other carriers described herein. Depending on the host species, various adjuvants may be used to increase the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, polyols, polyanions, peptides, nanoemulsions described herein, keyhole limpet @) Hemocyanin and dinitrophenol). Among adjuvants used in humans, BCG (bacillus calmette-guerin) and Corynebacterium parvum (Corynebacterium parvum) are particularly useful.

Monoclonal antibodies that specifically bind to antigens of viruses of the pneumovirinae subfamily (e.g., respiratory syncytial virus) can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV hybridoma technology (see, e.g., Kohler et al, Nature 256, 495497, 1985; Kozbor et al, J. Immunol. Methods 81, 3142, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 20262030, 1983; Cole et al, mol. cell. biol. 62, 109120, 1984).

In addition, splicing of mouse antibody genes to human antibody genes to obtain molecules with appropriate antigen specificity and biological activity can be used using techniques developed for the generation of "chimeric antibodies" (see, e.g., Morrison et al, Proc. Natl. Acad. Sci. 81, 68516855, 1984; Neuberger et al, Nature 312, 604608, 1984; Takeda et al, Nature 314, 452454, 1985). Monoclonal and other antibodies may also be "humanized" to prevent a patient from developing an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy, or may require changes in a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by site-directed mutagenesis of individual residues or by grafting entire complementarity determining regions in place of residues that differ from those in the human sequence.

Alternatively, humanized antibodies may be produced using recombinant methods, as described below. Antibodies that specifically bind to a particular antigen may contain partially or fully humanized antigen binding sites, as disclosed in U.S. Pat. No. 5,565,332.

Alternatively, the techniques described for single chain antibody production may be modified using methods known in the art to produce single chain antibodies that specifically bind to a particular antigen. Antibodies with related specificity but with different idiotype compositions can be generated from randomly combined immunoglobulin libraries by chain shuffling (see, e.g., Burton, proc. natl. acad. sci. 88, 1112023, 1991).

Single chain antibodies can also be constructed using DNA amplification methods such as PCR, using hybridoma cDNA as a template (see, e.g., Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11). Single chain antibodies may be mono-or bispecific, and may be bivalent or tetravalent. The construction of tetravalent, bispecific single chain antibodies is taught, for example, in Coloma & Morrison, 1997, nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single chain antibodies is taught, for example, in Malender & Voss, 1994, J. biol. chem. 269, 199-206.

Nucleotide sequences encoding single-chain antibodies can be constructed using manual or automated nucleotide synthesis, cloned into expression constructs using standard recombinant DNA methods, and introduced into cells to express the coding sequence, as described below. Alternatively, single chain antibodies can be generated directly using, for example, filamentous phage technology (see, e.g., Verhaar et al, 1995, int. J. Cancer 61, 497-501; Nichols et al, 1993, J. Immunol. meth. 165, 81-91).

Antibodies that specifically bind to a particular antigen can also be produced by inducing in vivo production in a lymphocyte population or by screening immunoglobulin libraries or groups of subjects for highly specific binding agents, as disclosed in the literature (see, e.g., Orlandi et al, Proc. Natl. Acad. Sci. 86, 38333837, 1989; Winter et al, Nature 349, 293299, 1991).

Chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, may also be prepared. Antibodies can be purified by methods well known in the art. For example, antibodies can be affinity purified by passing through a column to which the relevant antigen binds. The bound antibody can then be eluted from the column using a buffer with a high salt concentration.

Nano-emulsion

The nanoemulsion vaccine compositions of the present invention are not limited to any particular nanoemulsion. Any number of suitable nanoemulsion compositions may be used in the vaccine compositions of the present invention, including but not limited to, Hamouda et al, j. infection dis, 180:1939 (1999); hamouda and Baker, j. appl. microbiol, 89:397 (2000); and those disclosed in Donovan et al, anti. chem. chemither, 11:41 (2000), and those shown in tables 1 and 2 and fig. 4 and 9. Preferred nanoemulsions of the invention are those that are effective in killing or inactivating pathogens and are non-toxic to animals. Thus, it is preferred that the emulsion formulation utilize a non-toxic solvent, such as ethanol, and achieve more effective kill at lower concentrations of the emulsion. In a preferred embodiment, the nanoemulsion utilized in the method of the present invention is stable and does not decompose even after long storage periods (e.g., one or more years). In addition, it is preferred that the emulsion maintain stability even after exposure to high temperatures and freezing. This is particularly useful if they are to be applied in extreme conditions, for example on a battlefield. In some embodiments, one of the nanoemulsions described in table 1 is utilized.

In some preferred embodiments, the emulsion comprises (i) an aqueous phase; (ii) an oil phase; and at least one additional compound. In some embodiments of the invention, these additional compounds are mixed into the water or oil phase of the composition. In other embodiments, these additional compounds are mixed into a previously emulsified composition of oil and water phases. In certain of these embodiments, one or more additional compounds are mixed into the existing emulsion composition immediately prior to its use. In other embodiments, one or more additional compounds are mixed into the existing emulsion composition immediately prior to use of the composition.

Additional compounds suitable for use in the compositions of the present invention include, but are not limited to, one or more organic and more specifically organophosphate-based solvents, surfactants and detergents, quaternary ammonium-containing compounds, cationic halogen-containing compounds, germination enhancers, interaction enhancers, and pharmaceutically acceptable compounds. Some exemplary embodiments of various compounds contemplated for use in the compositions of the present invention are presented below.

Some embodiments of the present invention employ an oil phase comprising ethanol. For example, in some embodiments, the emulsion of the invention contains (i) an aqueous phase and (ii) an oily phase containing ethanol as organic solvent and optionally a germination enhancer, and (iii) TYLOXAPOL as surfactant (preferably 2-5%, more preferably 3%). Such formulations are highly effective against microorganisms and are also non-irritating and non-toxic to mammalian users (and thus may come into contact with mucous membranes).

In some other embodiments, the emulsion of the present invention comprises a first emulsion emulsified within a second emulsion, wherein (a) the first emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and an organic solvent; and (iii) a surfactant; and (b) the second emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and a cationic-containing compound; and (iii) a surfactant.

The following description provides a number of exemplary emulsions, including compositions X8P and X for use in compositions₈W₆₀Formulations of PC. X8P comprises a water-in-oil nanoemulsion in which the oil phase was made from soybean oil, tri-n-butyl phosphate and TRITON X-100 dissolved in 80% water. X₈W₆₀PC comprises X8P and W₈₀An equal volume of 8P mixture. W₈₀8P is a liposome-like compound made from glyceryl monostearate, refined soya sterol (e.g. GENEROL sterol), TWEEN 60, soybean oil, cationic halogen-containing CPC and peppermint oil. The GENEROL family is a group of polyethoxylated soy sterols (Henkel Corporation, Ambler, Pennsylvania). Emulsion formulations for certain embodiments of the present invention are given in table 1. These particular formulations may be described in U.S. patent No. 5,700,679 (NN), which is incorporated herein by reference in its entirety; 5,618,840, respectively; 5,549,901 (W) ₈₀8P); and 5,547,677.

X8W₆₀PC emulsion is prepared by first preparing W separately₈₀8P emulsion and X8P emulsion. The mixture of these 2 emulsions was then re-emulsified to give a mixture called X8W₆₀Fresh emulsion composition of PC. Methods of producing such emulsions are described in U.S. Pat. Nos. 5,103,497 and 4,895,452 (incorporated herein by reference in their entirety). These compounds have broad spectrum antimicrobial activity and are capable of inactivating vegetative bacteria by membrane disruption.

The compositions listed above are exemplary only, and one skilled in the art will be able to vary the amount of components to achieve a nanoemulsion composition suitable for the purposes of the present invention. One skilled in the art will appreciate that the ratio of oil phase to water, as well as the individual oil carriers, the surfactant CPC and the organophosphate buffer, the components of each composition may be different.

While certain compositions comprising X8P have a water to oil ratio of 4:1, it is understood that X8P can be formulated with more or less aqueous phase. For example, in some embodiments, 3,4, 5, 6, 7, 8, 9, 10, or more parts of aqueous phase are present for each part of oil phase. This is for W₈₀The 8P formulation was also correct. Class ISimilarly, the ratio of tri-n-butyl phosphate to TRITON X-100 to soybean oil may also be varied.

Although Table 1 lists the values for W₈₀8P glyceryl monooleate, polysorbate 60, GENEROL 122, cetyl pyridinium chlorideAnd specific amounts of carrier oil, but these are exemplary only. Can be formulated to have W₈₀An emulsion of 8P nature with different concentrations of each of these components or indeed different components that will perform the same function. For example, the emulsion may have from about 80 to about 100g of glycerol monooleate in the starting oil phase. In other embodiments, the emulsion may have about 15 to about 30 g of polysorbate 60 in the starting oil phase. In another embodiment, the composition may comprise about 20 to about 30 g of GENEROL sterol in the starting oil phase.

The nanoemulsion structure of certain embodiments of the emulsions of the present invention may play a role in its biocidal activity as well as contributing to the non-toxicity of these emulsions. For example, the active component in X8P, TRITON-X100, showed less biocidal activity against the virus at a concentration equivalent to 11% X8P. The addition of an oil phase to the detergent and solvent significantly reduces the toxicity of these agents at the same concentrations in tissue culture. While not being bound by any theory (understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism), it is suggested that the nanoemulsion enhances the interaction of its components with the pathogen, thereby promoting inactivation of the pathogen and reducing the toxicity of the individual components. It should be noted that when all of the components of X8P are combined in one composition but not in the nanoemulsion structure, the mixture is not as effective as an antimicrobial agent when the components are in the nanoemulsion structure.

Numerous additional embodiments presented in the formulation categories having similar compositions are presented below. The following compositions describe various ratios and mixtures of active ingredients. One skilled in the art will recognize that the formulations described below are exemplary and that additional formulations comprising similar ranges of percentages of the components are within the scope of the invention.

In certain embodiments of the invention, the formulations of the invention comprise about 3-8% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume cetylpyridinium chloride(CPC), about 60-70% by volume oil (e.g., soybean oil), about 15-25% by volume aqueous phase (e.g., DiH)₂O or PBS), and in some formulations less than about 1 vol% 1N NaOH. Some of these embodiments comprise PBS. It is contemplated that the addition of 1N NaOH and/or PBS in some of these embodiments allows the user to advantageously control the pH of the formulation such that the pH ranges from about 4.0 to about 10.0, and more preferably to about 7.1 to 8.5. For example, one embodiment of the present invention comprises about 3% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, and about 24% by volume DiH ₂O (designated herein as Y3 EC). Another similar embodiment comprises about 3.5% by volume TYLOXAPOL, about 8% by volume ethanol, and about 1% by volume CPC, about 64% by volume soybean oil, and about 23.5% by volume DiH₂O (designated herein as Y3.5EC). Another embodiment comprises about 3% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 0.067% by volume 1N NaOH, such that the pH of the formulation is about 7.1, about 64% by volume soybean oil, and about 23.93% by volume DiH₂O (designated herein as Y3EC pH 7.1). Another embodiment comprises about 3% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 0.67% by volume 1N NaOH, such that the pH of the formulation is about 8.5, and about 64% by volume soybean oil, and about 23.33% by volume DiH₂O (designated herein as Y3EC pH 8.5). Another similar embodiment comprises about 4% TYLOXAPOL, about 8% ethanol by volume, about 1% CPC, and about 64% soybean oil by volume, and about 23% DiH by volume₂O (designated herein as Y4 EC). In another embodiment, the formulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1% by volume CPC, about 64% by volume soybean oil, and about 19% by volume DiH ₂O (designated herein as Y8 EC). A further embodiment comprises about 8% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, about 19% by volume 1x PBS (designated herein as Y8EC PBS).

In some embodiments of the invention, the formulations of the invention comprise about 8% ethanol by volume, and about 1% CPC by volume, and about 64% oil (e.g., soybean oil) by volume, and about 27% aqueous phase (e.g., DiH) by volume₂O or PBS) (designated herein as EC).

In the present invention, some embodiments comprise about 8% by volume Sodium Dodecyl Sulfate (SDS), about 8% by volume tributyl phosphate (TBP), and about 64% by volume oil (e.g., soybean oil), and about 20% by volume aqueous phase (e.g., DiH)₂O or PBS) (designated herein as S8P).

In certain embodiments of the invention, the formulations of the present invention comprise about 1-2% by volume TRITON X-100, about 1-2% by volume TYLOXAPOL, about 7-8% by volume ethanol, about 1% by volume cetylpyridinium chloride(CPC), about 64-57.6% by volume oil (e.g., soybean oil), and about 23% by volume water phase (e.g., DiH)₂O or PBS). In addition, some of these formulations further comprise about 5 mM L-alanine/inosine and about 10 mM ammonium chloride. Some of these formulations contained PBS. It is contemplated that the addition of PBS in some of these embodiments allows the user to advantageously control the pH of the formulation. For example, one embodiment of the present invention comprises about 2% by volume TRITON X-100, about 2% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, and about 23% by volume aqueous phase DiH ₂And O. In another embodiment, the formulation comprises about 1.8% by volume TRITON X-100, about 1.8% by volume TYLOXAPOL, about 7.2% by volumeEthanol, about 0.9 vol% CPC, about 5 mM L-alanine/inosine, and about 10 mM ammonium chloride, about 57.6 vol% soybean oil, and the remainder 1X PBS (designated herein as 90% X2Y2 EC/GE).

In a preferred embodiment of the invention, the formulation comprises about 5% by volume TWEEN 80, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume oil (e.g., soybean oil), and about 22% by volume DiH₂O (designated herein as W)₈₀5EC）。

In still other embodiments of the invention, the formulation comprises about 5% TWEEN 20 by volume, about 8% ethanol by volume, about 1% CPC by volume, about 64% oil (e.g., soybean oil) by volume, and about 22% DiH by volume₂O (designated herein as W)₂₀5EC）。

In still other embodiments of the present invention, the formulation comprises about 2-8% by volume TRITON X-100, about 8% by volume ethanol, about 1% by volume CPC, about 60-70% by volume oil (e.g., soy or olive oil), and about 15-25% by volume aqueous phase (e.g., DiH)₂O or PBS). For example, the present invention contemplates a composition comprising about 2% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 26% by volume DiH ₂Formulation of O (designated herein as X2E). In other similar embodiments, the formulation comprises about 3% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 25% by volume DiH₂O (designated herein as X3E). In yet a further embodiment, the formulation comprises about 4% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 24% by volume DiH₂O (designated herein as X4E). In still other embodiments, the formulation comprises about 5% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 23% by volume DiH₂O (designated herein as X5E). Another embodiment of the present invention comprises about 6% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 22% by volume DiH₂O (book)Designated herein as X6E). In still a further embodiment of the present invention, the formulation comprises about 8% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X8E). In still a further embodiment of the present invention, the formulation comprises about 8% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume olive oil, and about 20% by volume DiH ₂O (designated herein as X8E O). In another embodiment 8% by volume TRITON X-100, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, and about 19% by volume DiH₂O (designated herein as X8 EC).

In an alternative embodiment of the invention, the formulation comprises about 1-2% by volume TRITON X-100, about 1-2% by volume TYLOXAPOL, about 6-8% by volume TBP, about 0.5-1.0% by volume CPC, about 60-70% by volume oil (e.g., soybean oil), and about 1-35% by volume aqueous phase (e.g., DiH)₂O or PBS). In addition, some of these formulations may comprise about 1-5% by volume tryptic soy broth, about 0.5-1.5% by volume yeast extract, about 5 mM L-alanine/inosine, about 10 mM ammonium chloride, and about 20-40% by volume liquid infant formula (formula). In some embodiments comprising liquid infant formula, the formula comprises casein hydrolysate (e.g., Neutramigen or Progestimil, etc.). In some of these embodiments, the formulation of the present invention further comprises about 0.1 to 1.0% by volume sodium thiosulfate, and about 0.1 to 1.0% by volume sodium citrate. Other similar embodiments comprising these essential components employ Phosphate Buffered Saline (PBS) as the aqueous phase. For example, one embodiment comprises about 2% by volume TRITON X-100, about 2% by volume TYLOXAPOL, about 8% by volume TBP, about 1% by volume CPC, about 64% by volume soybean oil, and about 23% by volume DiH ₂O (designated herein as X2Y2 EC). In still other embodiments, the formulations of the present invention comprise about 2% by volume TRITON X-100, about 2% by volume TYLOXAPOL, about 8% by volume TBP, about 1% by volume CPC, about 0.9% by volume sodium thiosulfate, about 0.1% by volume citric acidSodium, about 64% by volume soybean oil, and about 22% by volume DiH₂O (designated herein as X2Y2PC STS 1). In another similar embodiment, the formulation comprises about 1.7% by volume TRITON X-100, about 1.7% by volume TYLOXAPOL, about 6.8% by volume TBP, about 0.85% CPC, about 29.2% neutamigen, about 54.4% by volume soybean oil, and about 4.9% by volume DiH₂O (designated herein as 85% X2Y2 PC/infant). In another embodiment of the invention, the formulation comprises about 1.8% by volume TRITON X-100, about 1.8% by volume TYLOXAPOL, about 7.2% by volume TBP, about 0.9% by volume CPC, about 5 mM L-alanine/inosine, about 10 mM ammonium chloride, about 57.6% by volume soybean oil, and the remaining% by volume 0.1X PBS (designated herein as 90% X2Y2 PC/GE). In another embodiment, the formulation comprises about 1.8% by volume TRITON X-100, about 1.8% by volume TYLOXAPOL, about 7.2% by volume TBP, about 0.9% by volume CPC, about 3% by volume tryptic Soy broth, about 57.6% by volume Soybean oil, and about 27.7% by volume DiH ₂O (designated herein as 90% X2Y2 PC/TSB). In another embodiment of the invention, the formulation comprises about 1.8% TRITON X-100 by volume, about 1.8% TYLOXAPOL by volume, about 7.2% TBP by volume, about 0.9% CPC by volume, about 1% yeast extract by volume, about 57.6% soybean oil by volume, and about 29.7% DiH by volume₂O (designated herein as 90% X2Y2 PC/YE).

In some embodiments of the invention, the formulations of the invention comprise about 3% by volume TYLOXAPOL, about 8% by volume TBP, and about 1% by volume CPC, about 60-70% by volume oil (e.g., soy or olive oil), and about 15-30% by volume aqueous phase (e.g., DiH)₂O or PBS). In a particular embodiment of the invention, the formulation of the invention comprises about 3% by volume TYLOXAPOL, about 8% by volume TBP, and about 1% by volume CPC, about 64% by volume soy, and about 24% by volume DiH₂O (designated herein as Y3 PC).

In some embodiments of the invention, the formulations of the invention comprise about 4-8% by volume TRITON X-100, about 5-8% by volume TBP, about 30-70% by volume oil (for exampleSuch as soy or olive oil), and about 0-30 vol% of an aqueous phase (e.g., DiH)₂O or PBS). Additionally, certain of these embodiments further comprise about 1% by volume CPC, about 1% by volume benzalkonium chloride, about 1% by volume cetylpyridinium bromide About 1 vol% hexadecyldimethylethylammonium bromide, 500 μ M EDTA, about 10 mM ammonium chloride, about 5 mM inosine, and about 5 mM L-alanine. For example, in certain of these embodiments, the formulations of the present invention comprise about 8% by volume TRITON X-100, about 8% by volume TBP, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X8P). In another embodiment of the invention, the formulation of the invention comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 1% CPC, about 64% by volume soybean oil, and about 19% by volume DiH₂O (designated herein as X8 PC). In another embodiment, the formulation comprises about 8% TRITON X-100 by volume, about 8% TBP by volume, about 1% CPC by volume, about 50% soybean oil by volume, and about 33% DiH by volume₂O (designated herein as ATB-X1001). In another embodiment, the formulation comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 2% by volume CPC, about 50% by volume soybean oil, and about 32% by volume DiH₂O (designated herein as ATB-X002). Another embodiment of the present invention comprises about 4% by volume TRITON X-100, about 4% by volume TBP, about 0.5% by volume CPC, about 32% by volume soybean oil, and about 59.5% by volume DiH ₂O (designated herein as 50% X8 PC). Another related embodiment comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 0.5% by volume CPC, about 64% by volume soybean oil, and about 19.5% by volume DiH₂O (designated herein as X8PC_1/2). In some embodiments of the invention, the formulations of the invention comprise about 8% by volume TRITON X-100, about 8% by volume TBP, about 2% by volume CPC, about 64% by volume soybean oil, and about 18% by volume DiH₂O (designated herein as X8PC 2). In other embodimentsIn one embodiment, the formulation of the present invention comprises about 8% by volume TRITON X-100, about 8% TBP, about 1% benzalkonium chloride, about 50% by volume soybean oil, and about 33% by volume DiH₂O (designated herein as X8P BC). In an alternative embodiment of the invention, the formulation comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 1% by volume cetylpyridinium bromideAbout 50% by volume soybean oil, and about 33% by volume DiH₂O (designated herein as X8P CPB). In another exemplary embodiment of the invention, the formulation comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 1% by volume cetyldimethylethylammonium bromide, about 50% by volume soybean oil, and about 33% by volume DiH ₂O (designated herein as X8P CTAB). In still further embodiments, the invention comprises about 8 vol.% TRITON X-100, about 8 vol.% TBP, about 1 vol.% CPC, about 500 μ M EDTA, about 64 vol.% soybean oil, and about 15.8 vol.% DiH₂O (designated herein as X8PC EDTA). An otherwise similar embodiment comprises 8% by volume TRITON X-100, about 8% by volume TBP, about 1% by volume CPC, about 10 mM ammonium chloride, about 5 mM inosine, about 5 mM L-alanine, about 64% by volume soybean oil, and about 19% by volume DiH₂O or PBS (designated herein as X8PC GE)_1x). In another embodiment of the invention, the formulation of the present invention further comprises about 5% by volume TRITON X-100, about 5% TBP, about 1% by volume CPC, about 40% by volume soybean oil, and about 49% by volume DiH₂O (designated herein as X5P₅C）。

In some embodiments of the invention, the formulations of the invention comprise about 2% by volume TRITON X-100, about 6% by volume TBP, about 8% by volume ethanol, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X2Y 6E).

In a further embodiment of the invention, the formulation comprises about 8% by volume TRITON X-100, and about 8% by volume About 60-70% by volume of an oil (e.g., soy or olive oil), and about 15-25% by volume of an aqueous phase (e.g., DiH)₂O or PBS). Certain related embodiments further comprise about 1% by volume L-ascorbic acid. For example, one particular embodiment comprises about 8% by volume TRITON X-100, about 8% by volume glycerol, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X8G). In another embodiment, the formulation of the present invention comprises about 8% by volume TRITON X-100, about 8% by volume glycerin, about 1% by volume L-ascorbic acid, about 64% by volume soybean oil, and about 19% by volume DiH₂O (designated herein as X8GV_c）。

In yet a further embodiment, the formulations of the present invention comprise about 8% by volume TRITON X-100, about 0.5-0.8% by volume TWEEN 60, about 0.5-2.0% by volume CPC, about 8% by volume TBP, about 60-70% by volume oil (e.g., soy or olive oil), and about 15-25% by volume aqueous phase (e.g., DiH)₂O or PBS). For example, in one particular embodiment, the formulation comprises about 8% by volume TRITON X-100, about 0.70% by volume TWEEN 60, about 1% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 18.3% by volume DiH ₂O (designated herein as X8W60PC₁). Another related embodiment comprises about 8% by volume TRITON X-100, about 0.71% by volume TWEEN 60, about 1% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 18.29% by volume DiH₂O (designated herein as W60_0.7X8 PC). In still other embodiments, the formulations of the present invention comprise about 8% by volume TRITON X-100, about 0.7% by volume TWEEN 60, about 0.5% by volume CPC, about 8% by volume TBP, about 64% to 70% by volume soybean oil, and about 18.8% by volume DiH₂O (designated herein as X8W60PC₂). In still other embodiments, the invention comprises about 8% by volume TRITON X-100, about 0.71% by volume TWEEN 60, about 2% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 17.3% by volume DiH₂And O. In another embodiment of the present inventionIn one embodiment, the formulation comprises about 0.71% by volume TWEEN 60, about 1% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 25.29% by volume DiH₂O (designated herein as W60_0.7PC）。

In another embodiment of the invention, the formulation of the invention comprises about 2% dioctyl sulfosuccinate by volume, about 8% glycerin by volume, or about 8% TBP by volume, plus about 60-70% oil (e.g., soy or olive oil) by volume, and about 23% aqueous phase (e.g., DiH) by volume ₂O or PBS). For example, one embodiment of the present invention comprises about 2% dioctyl sulfosuccinate by volume, about 8% glycerin by volume, about 64% soybean oil by volume, and about 26% DiH by volume₂O (designated herein as D2G). In another related embodiment, the formulation of the present invention comprises about 2% dioctyl sulfosuccinate by volume, and about 8% TBP by volume, about 64% soybean oil by volume, and about 26% DiH by volume₂O (designated herein as D2P).

In still other embodiments of the present invention, the formulations of the present invention comprise about 8-10% by volume glycerin, and about 1-10% by volume CPC, about 50-70% by volume oil (e.g., soy or olive oil), and about 15-30% by volume water phase (e.g., DiH)₂O or PBS). Additionally, in certain of these embodiments, the composition further comprises about 1% by volume of L-ascorbic acid. For example, one particular embodiment comprises about 8% by volume of glycerin, about 1% by volume of CPC, about 64% by volume of soybean oil, and about 27% by volume of DiH₂O (designated herein as GC). An additional related embodiment comprises about 10% by volume of glycerin, about 10% by volume of CPC, about 60% by volume of soybean oil, and about 20% by volume of DiH ₂O (designated herein as GC 10). In another embodiment of the invention, a formulation of the invention comprises about 10% by volume glycerin, about 1% by volume CPC, about 1% by volume L-ascorbic acid, about 60% by volume soybean or oil, and about 24% by volume DiH₂O (designated herein as GCV)_c）。

In some embodiments of the invention, the formulations of the invention comprise about 8-10% by volume glycerol, about 8-10% by volume SDS, about 50-70% by volume oil (e.g., soybean or olive oil), and about 15-30% by volume aqueous phase (e.g., DiH)₂O or PBS). Additionally, in certain of these embodiments, the composition further comprises about 1% by volume lecithin and about 1% by volume methylparaben. An exemplary embodiment of such a formulation comprises about 8% SDS, 8% glycerol, about 64% soybean oil, and about 20% DiH by volume₂O (designated herein as S8G). A related formulation comprises about 8% by volume of glycerin, about 8% by volume of SDS, about 1% by volume of lecithin, about 1% by volume of methylparaben, about 64% by volume of soybean oil, and about 18% by volume of DiH₂O (designated herein as S8GL1B 1).

In another embodiment of the invention, a formulation of the invention comprises about 4% TWEEN 80 by volume, about 4% TYLOXAPOL by volume, about 1% CPC by volume, about 8% ethanol by volume, about 64% soybean oil by volume, and about 19% DiH by volume ₂O (designated herein as W)₈₀4Y4EC）。

In some embodiments of the invention, the formulations of the invention comprise about 0.01% by volume CPC, about 0.08% by volume TYLOXAPOL, about 10% by volume ethanol, about 70% by volume soybean oil, and about 19.91% by volume DiH₂O (designated herein as y.08ec.01).

In another embodiment of the invention, the formulation of the invention comprises about 8% by volume sodium lauryl sulfate, about 8% by volume glycerin, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as SLS 8G).

The specific formulations described above are merely illustrative of the various compositions useful in the present invention. The present invention contemplates many variations of the above-described formulations, as well as additional nanoemulsions, to be useful in the methods of the invention. To determine whether a candidate emulsion is suitable for use with the present invention, one canTo analyze 3 criteria. Candidate emulsions can be readily tested to determine if they are suitable using the methods and criteria described herein. First, the desired ingredients are prepared using the methods described herein to determine if an emulsion can be formed. If an emulsion cannot be formed, the candidate is eliminated. For example, from 4.5% sodium thiosulfate, 0.5% sodium citrate, 10% n-butanol, 64% soybean oil and 21% DiH ₂O do not form emulsions.

Second, in a preferred embodiment, the candidate emulsion should form a stable emulsion. An emulsion is stable if it is maintained in emulsion form for a sufficient period of time to allow its intended use. For example, for emulsions to be stored, transported, etc., it may be desirable for the composition to remain in emulsion form for months to years. A relatively unstable typical emulsion will lose its form within one day. For example, 8% 1-butanol, 5% TWEEN 10, 1% CPC, 64% soybean oil, and 22% DiH₂Candidate compositions made with O do not form stable emulsions. The following candidate emulsions were shown to be stable using the methods described herein: 0.08% TRITON X-100, 0.08% glycerin, 0.01% cetylpyridinium chloride99% butter and 0.83% diH₂O (designated herein as 1% X8GC butter); 0.8% TRITON X-100, 0.8% glycerin, 0.1% cetylpyridinium chloride6.4% of soybean oil and 1.9% of diH₂O and 90% butter (designated herein as 10% X8GC butter); 2% W₂₀5EC, 1% Natrosol 250L NF, and 97% diH₂O (designated herein as 2% W)₂₀5EC L GEL); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% 70% viscous mineral oil and 22% diH ₂O (designated herein as W)₂₀5EC 70 mineral oil); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% 350% viscous mineral oil and 22% diH₂O (designated herein as W)₂₀5EC 350 mineral oil).

Third, the candidate emulsion should have efficacy for its intended use. For example, an antibacterial emulsion should kill or incapacitate pathogens to detectable levels. As shown herein, certain emulsions of the present invention have efficacy against specific microorganisms, but not others. Using the methods described herein, one can determine the suitability of a particular candidate emulsion for a desired microorganism. Generally, this involves exposing the microorganisms to the emulsion in a parallel experiment with a suitable control sample (e.g., a negative control such as water) for one or more time periods and determining whether the emulsion kills or disables the microorganisms, and to what extent. For example, from 1% ammonium chloride, 5% TWEEN 20, 8% ethanol, 64% soybean oil, and 22% DiH₂O the candidate composition made was shown not to be an effective emulsion. The following candidate emulsions were shown to be effective using the methods described herein: 5% TWEEN 20, 5% cetyl pyridinium chloride10% glycerol, 60% soybean oil and 20% diH ₂O (designated herein as W)₂₀5GC 5); 1% cetyl pyridinium chloride5% TWEEN 20, 10% glycerol, 64% soybean oil and 20% diH₂O (designated herein as W)₂₀5 GC); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% olive oil and 22% diH₂O (designated herein as W)₂₀5EC olive oil); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% linseed oil and 22% diH₂O (designated herein as W)₂₀5EC linseed oil); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% corn oil and 22% diH₂O (designated herein as W)₂₀5EC corn oil); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% coconut oil and 22% diH₂O (designated herein as W)₂₀5EC coconut oil); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% cottonseed oil and 22% diH₂O (designated herein as W)₂₀5EC cottonseed oil); 8% glucose, 5% TWEEN 10, 1% cetylpyridinium chloride64% soybean oil and 22% diH₂O (designated herein as W)₂₀5C glucose); 8% PEG 200, 5% TWEEN 10, 1% cetylpyridinium chloride64% soybean oil and 22% diH₂O (designated herein as W)₂₀5C PEG 200); 8% methanol, 5% TWEEN 10, 1% cetylpyridinium chloride 64% soybean oil and 22% diH₂O (designated herein as W)₂₀5C methanol); 8% PEG 1000, 5% TWEEN 10, 1% cetylpyridinium chloride、64% Soybean oil and 22% diH₂O (designated herein as W)₂₀5C PEG 1000）；2％W₂₀5EC, 2% Natrosol 250H NF, and 96% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 2, also known as 2% W₂₀5EC GEL）；2％W₂₀5EC, 1% Natrosol 250H NF, and 97% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 1）；2％W₂₀5EC, 3% Natrosol 250H NF, and 95% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 3）；2％W₂₀5EC, 0.5% Natrosol 250H NF, and 97.5% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 0.5）；2％W₂₀5EC, 2% Methocel A and 96% diH₂O (designated herein as 2% W)₂₀5EC Methocel A）；2％W₂₀5EC, 2% Methocel K and 96% diH₂O (designated herein as 2% W)₂₀5EC Methocel K); 2% Natrosol, 0.1% X8PC, 0.1 XPBS, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride and diH₂O (designated herein as 0.1% X8PC/GE + 2% Natrosol); 2% of Natrosol, 0.8% of TRITON X-100, 0.8% of tributyl phosphate, 6.4% of soybean oil, 0.1% of cetylpyridinium chloride0.1 XPBS, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride and diH₂O (designated herein as 10% X8PC/GE + 2% Natrosol); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% lard and 22% diH ₂O (designated herein as W)₂₀5EC lard); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% mineral oil and 22% diH₂O (designated herein as W)₂₀5EC mineral oil); 0.1% cetylpyridinium chloride2% Nerolidol, 5% TWEEN 20, 10% ethanol, 64% soybean oil and 18.9% diH₂O (designated herein as W)₂₀5EC_0.1N); 0.1% cetylpyridinium chloride2% farnesol, 5% TWEEN 20, 10% ethanol, 64% soybean oil and 18.9% diH₂O (designated herein as W)₂₀5EC_0.1F) (ii) a 0.1% cetylpyridinium chloride5% TWEEN 20, 10% ethanol, 64% soybean oil and 20.9% diH₂O (designated herein as W)₂₀5EC_0.1) (ii) a 10% cetyl pyridinium chloride8% tributyl phosphate, 8% TRITON X-100, 54% soybean oil and 20% DIH₂O (designated herein as X8PC₁₀) (ii) a 5% cetyl pyridinium chloride8% TRITON X-100, 8% tributyl phosphate, 59% soybean oil and 20% DIH₂O (designated herein as X8PC₅) (ii) a 0.02% cetylpyridinium chloride0.1% TWEEN 20, 10% ethanol, 70% soybean oil and 19.88% diH₂O (designated herein as W)₂₀0.1EC_0.02) (ii) a 1% cetyl pyridinium chloride5% TWEEN 20, 8% glycerol, 64% Mobil 1 and 22% diH₂O (designated herein as W) ₂₀5GC Mobil 1); 7.2% TRITON X-100, 7.2% tributyl phosphate, 0.9% cetylpyridinium chloride57.6% Soybean oil, 0.1 XPBS, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride and 25.87% diH₂O (designated herein as 90% X8 PC/GE); 7.2% TRITON X-100, 7.2% tributyl phosphate, 0.9% cetylpyridinium chloride57.6% Soybean oil, 1% EDTA, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride, 0.1 XPBS, and diH₂O (designated herein as 90% X8PC/GE EDTA); and 7.2% TRITON X-100, 7.2% tributyl phosphate, 0.9% cetylpyridinium chloride57.6% Soybean oil, 1% sodium thiosulfate, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride, 0.1 XPBS, and diH₂O (designated herein as 90% X8PC/GE STS).

1. Aqueous phase

In some embodiments, the emulsion comprises an aqueous phase. In certain preferred embodiments, the emulsion comprises 5-50, preferably 10-40, more preferably 15-30 vol% aqueous phase based on the total volume of the emulsion (although other concentrations are also contemplated). In a preferred embodiment, the aqueous phase comprises water at a pH of about 4 to 10, preferably about 6 to 8. The water is preferably deionized (hereinafter "DiH) ₂O "). In some embodiments, the aqueous phase comprises Phosphate Buffered Saline (PBS). In some preferred embodiments, the aqueous phase is sterile and pyrogen free.

2. Oil phase

In some embodiments, the emulsion comprises an oil phase. In certain preferred embodiments, the oil phase (e.g., carrier oil) of the emulsions of the present invention comprises 30-90, preferably 60-80, and more preferably 60-70 volume% oil, based on the total volume of the emulsion (although higher and lower concentrations are also useful in the emulsions described herein).

The oil in the nanoemulsion vaccine of the present invention may be any cosmetically or pharmaceutically acceptable oil. The oil may be volatile or non-volatile, and may be selected from the group consisting of animal oils, vegetable oils, natural oils, synthetic oils, hydrocarbon oils, silicone oils, semi-synthetic derivatives thereof, and combinations thereof.

Suitable oils include, but are not limited to, mineral oil, squalene oil, flavor oils (flavor oils), silicone oils, essential oils, water insoluble vitamins, isopropyl stearate, butyl stearate, octyl palmitate, cetyl palmitate, behenic acidTridecyl acid, diisopropyl adipate, dioctyl sebacate, Menthyl anthranilate (Menthy anthranilate), cetyl octanoate, octyl salicylate, isopropyl myristate, neopenty glycol dicarbate capsules, ceraminyls, decyl oleate, diisopropyl adipate, C lactate _12-15Alkyl esters, cetyl lactate, lauryl lactate, isostearyl neopentanoate, myristyl lactate, isohexadecyl stearate, octyldodecyl stearate, hydrocarbon oils, isoparaffins, liquid paraffin, isododecane, petrolatum, Argan (Argan) oil, canola oil, chili oil (Chile oil), coconut oil, corn oil, cottonseed oil, linseed oil, grapeseed oil, mustard oil, olive oil, palm kernel oil, peanut oil, pine nut oil, poppyseed oil, pumpkin oil, rice bran oil, safflower oil, tea oil, Truffle oil (Truffle oil), vegetable oil, apricot (kernel) oil, jojoba oil (simmondsia chinensis seed oil), grape seed oil, Macadamia oil (Macadamia oil), wheat germ oil, almond oil, rapeseed oil, gourd oil, soybean oil, sesame oil, hazelnut oil, corn oil, sunflower oil, corn, Hemp oil, Bois oil, macadamia nut (Kuki nut) oil, avocado oil, walnut oil, fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise oil, celery seed oil, cumin oil, nutmeg oil, leaf oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, sage leaf oil, eucalyptus leaf oil Lemon grass leaf oil, cajeput leaf oil, oregano leaf oil, patchouli oil, peppermint leaf oil, pine needle oil, rosemary leaf oil, spearmint leaf oil, tea tree leaf oil, thyme leaf oil, wintergreen leaf oil, flower oil, chamomile oil, sage oil, clove oil, geranium oil, hyssop flower oil, jasmine oil, lavender flower oil, manuka (manuka) flower oil, marhomam flower oil, orange flower oil, rose oil, ylang-ylang oil, bark oil, chinese cinnamon bark oil, sassafras bark oil, wood oil, camphorwood oil, cedar wood oil, rose wood oil, sandalwood oil), rhizome (ginger) wood oil, resin oil, frankincense oil, myrrh oil, fruit bark oil, bergamot bark oil, grapefruit bark oil, lemon bark oil, lime bark oil, orange bark oil, tangerine bark oil, root oil, valerian oil, oleic acid, linoleic acid, valerian alcohol, octadecanol derivatives, and any combination thereof.

The oil may further comprise a silicone component, such as a volatile silicone component, which may be the sole oil in the silicone component or may be combined with other silicone and non-silicone, volatile and non-volatile oils. Suitable siloxane components include, but are not limited to, methylphenylpolysiloxane, simethicone, dimethylsiloxane, phenyltrimethylsiloxane (or an organically modified form thereof), alkylated derivatives of polymerized siloxanes, hexadecyldimethylsiloxane, lauryl trimethylsiloxane, hydroxylated derivatives of polymerized siloxanes such as dimethiconol, volatile silicone oils, cyclic and linear siloxanes, cyclomethicones, derivatives of cyclomethicones, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, volatile linear dimethylpolysiloxane, isohexadecane, isoeicosane, isotetracosane, polyisobutylene, isooctane, isododecane, semisynthetic derivatives thereof, and combinations thereof.

The volatile oil may be an organic solvent, or a volatile oil other than an organic solvent may be present. Suitable volatile oils include, but are not limited to, terpenes, monoterpenes, sesquiterpenes, carminatives, azulenes, menthol, camphor,Ketone, thymol, nerol, linalool, methyl ethyl ketone, methyl propyl ketone, methyl,Limonene, geraniol, perillyl alcohol, nerolidol, farnesol, ylang-ylang, bisabolol, farnesene, ascaridole, chenopodium oil, citronellal, citral, citronellol, chamomileYarrow and guaiacumChamomile, a semisynthetic derivative or combination thereof.

In one aspect of the invention, the volatile oil in the silicone component is different from the oil in the oil phase.

In some embodiments, the oil phase comprises 3-15 and preferably 5-10 volume% organic solvent based on the total volume of the emulsion. While the present invention is not limited to any particular mechanism, it is contemplated that the organophosphate-based solvent employed in the emulsion acts to remove or rupture lipids in the membranes of pathogens. Thus, any solvent that removes sterols or phospholipids from the microbial membranes is useful in the methods of the invention. Suitable organic solvents include, but are not limited to, organophosphate-based solvents or alcohols. In some preferred embodiments, a non-toxic alcohol (e.g., ethanol) is used as the solvent. The oil phase and any additional compounds provided in the oil phase are preferably sterile and pyrogen free.

3. Surfactants and detergents

In some embodiments, the emulsion further comprises a surfactant or detergent. In some preferred embodiments, the emulsion comprises about 3-15% and preferably about 10% of one or more surfactants or detergents (although other concentrations are also contemplated). While the present invention is not limited to any particular mechanism, it is contemplated that the surfactant, when present in the emulsion, helps stabilize the emulsion. Nonionic (non-anionic) and ionic surfactants are contemplated. In addition, surfactants from the BRIJ family of surfactants are useful in the compositions of the present invention. The surfactant may be provided in the water or oil phase. Surfactants suitable for use with the emulsion include a variety of anionic and nonionic surfactants, as well as other emulsifying compounds capable of promoting the formation of oil-in-water emulsions. In general, the emulsifying compounds are relatively hydrophilic, and blends of emulsifying compounds can be used to achieve the necessary quality. In some formulations, nonionic surfactants have advantages over ionic emulsifiers because they are substantially more compatible with a wide pH range and often form emulsions that are more stable than ionic (e.g., soap-type) emulsifiers.

The surfactant in the nanoemulsion vaccine of the present invention may be a pharmaceutically acceptable ionic surfactant, a pharmaceutically acceptable non-ionic surfactant, a pharmaceutically acceptable cationic surfactant, a pharmaceutically acceptable anionic surfactant, or a pharmaceutically acceptable zwitterionic surfactant.

Exemplary useful Surfactants are described in Applied Surfactants: Principles and applications, Tharwat F. Tadros, Copyright8 2005 WILEY-VCH Verlag GmbH &Co, KGaA, Weinheim ISBN: 3-527-30629-3), which is expressly incorporated by reference.

Further, the surfactant can be a pharmaceutically acceptable ionic polymer surfactant, a pharmaceutically acceptable non-ionic polymer surfactant, a pharmaceutically acceptable cationic polymer surfactant, a pharmaceutically acceptable anionic polymer surfactant, or a pharmaceutically acceptable zwitterionic polymer surfactant. Examples of polymeric surfactants include, but are not limited to, graft copolymers of poly (methyl methacrylate) backbones with multiple (at least one) polyethylene oxide (PEO) side chains, polyhydroxystearic acid, alkoxylated alkylphenol formaldehyde condensates, polyalkylene glycol modified polyesters with fatty acid hydrophobes, polyesters, semi-synthetic derivatives thereof, or combinations thereof.

Surfactants or surfactants are amphiphilic molecules consisting of a non-polar hydrophobic moiety attached to a polar or ionic hydrophilic moiety, typically a straight or branched hydrocarbon or fluorocarbon chain containing 8 to 18 carbon atoms. The hydrophilic moiety may be non-ionic, ionic or zwitterionic. The hydrocarbon chains interact weakly with water molecules in an aqueous environment, while the polar or ionic headgroups interact strongly with water molecules via dipole or ion-dipole interactions. Surfactants are classified into anionic, cationic, zwitterionic, nonionic and polymeric surfactants based on the nature of the hydrophilic group.

Suitable surfactants include, but are not limited to, ethoxylated nonylphenols containing 9 to 10 ethylene glycol units, ethoxylated undecanols containing 8 ethylene glycol units, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (20) sorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, ethoxylated hydrogenated castor oil, sodium lauryl sulfate, diblock copolymers of ethylene oxide (ethylenoxide) and propylene oxide (propyleneoxide), ethylene oxide-propylene oxide block copolymers, and tetrafunctional block copolymers based on ethylene oxide and propylene oxide, monoglycerides, and mixtures thereof, Glyceryl caprate, Glyceryl caprylate, Glyceryl cocoate (Glyceryl cocoate), Glyceryl erucate, Glyceryl hydroxystearate, Glyceryl isostearate, Glyceryl lanolin, Glyceryl laurate, Glyceryl linoleate, Glyceryl myristate, Glyceryl oleate, Glyceryl PABA, Glyceryl palmitate, Glyceryl ricinoleate, Glyceryl stearate, Glyceryl thioglycolate, Glyceryl dilaurate, Glyceryl dioleate, Glyceryl dimyristate, Glyceryl distearate, Glyceryl sesquioleate (Glyceryl sesquioleate), Glyceryl stearate lactate, polyoxyethylene cetyl/stearyl ether, polyoxyethylene cholesterol ether, polyoxyethylene laurate or dilaurate, polyoxyethylene stearate or distearate, polyoxyethylene fatty ether, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, polyoxyethylene myristyl ether, Glyceryl cocoate, Glyceryl monostearate, Glyceryl distearate, Glyceryl sesquioleate, Glyceryl monostearate, Glyceryl, Steroids, cholesterol, beta-sitosterol, bisabolol, fatty acid esters of alcohols, isopropyl myristate, isopropyl n-butyl aliphatic ester, isopropyl n-hexanoate, isopropyl n-decanoate, isopropyl palmitate, octyldodecyl myristate, alkoxylated alcohols, alkoxylated acids, alkoxylated amides, alkoxylated sugar derivatives, alkoxylated derivatives of natural oils and waxes, polyoxyethylene polyoxypropylene block copolymers, nonoxynol-14, PEG-8 laurate, PEG-6 cocamide (Cocoamide), PEG-20 methyl glucose sesquistearate, PEG40 lanolin, PEG-40 castor oil, PEG-40 hydrogenated castor oil, polyoxyethylene fatty ethers, glyceryl diesters, polyoxyethylene stearyl ethers, polyoxyethylene myristyl ethers, and polyoxyethylene lauryl ethers, glyceryl dilaurate, glyceryl stearate, sodium lauryl sulfate, glyceryl stearate, and sodium lauryl ethers, Glyceryl dimyristate, glyceryl distearate, semisynthetic derivatives thereof, or mixtures thereof.

Additional suitable surfactants include, but are not limited to, nonionic lipids, such as glyceryl laurate, glyceryl myristate, glyceryl dilaurate, glyceryl dimyristate, semi-synthetic derivatives thereof, and mixtures thereof.

In further embodiments, the surfactant is a polyoxyethylene fatty ether having a polyoxyethylene head group ranging from about 2 to about 100 groups, or has the structure R₅ --（OCH₂ CH₂）_yAlkoxylated alcohol of-OH, wherein R₅Is a branched or unbranched alkyl group having from about 6 to about 22 carbon atoms, and y is from about 4 to about 100, and preferably from about 10 to about 100. Preferably, the alkoxylated alcohol is wherein R₅Is a lauryl group and y has an average value of 23.

In various embodiments, the surfactant is an alkoxylated alcohol, which is an ethoxylated derivative of lanolin alcohol. Preferably, the ethoxylated derivative of lanolin alcohol is laneth-10, which is a polyethylene glycol ether of lanolin alcohol having an average ethoxylation value of 10.

Nonionic surfactants include, but are not limited to, ethoxylated surfactants, ethoxylated alcohols, ethoxylated alkylphenols, ethoxylated fatty acids, ethoxylated monoalkanolamides, ethoxylated sorbitan esters, ethoxylated fatty amino groups, ethylene oxide-propylene oxide copolymers, bis (polyethylene glycol bis [ imidazolyl carbonyl ] carbonyl) ]) Nonoxynol-9, bis (polyethylene glycol bis [ imidazolecarbonyl)]）、Brij^® 35、Brij^® 56、Brij^® 72、Brij^® 76、Brij^® 92V、Brij^® 97、Brij^® 58P、Cremophor^®EL, decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine, N-decyl α -D-glucopyranoside, decyl β -D-maltopyranoside, N-lauroyl-N-methylglucamide, N-dodecyl α -D-maltoside, N-dodecyl β -D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monotetradecyl ether, N-hexadecyl β -D-maltoside, hexaethylene glycol monododecyl ether, hexaethylene glycol monotexadecyl ether, hexaethylene glycol monostearyl ether, hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-L, methyl-6-O- (N-heptylcarbamoyl) -alpha-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monocetyl ether, octaethylene glycol monostearyl ether, octaethylene glycol monotetradecyl ether, octyl-beta-D-glucopyranoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monocetyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monostearyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 isooctyl ether, Polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate Acid ester, polyoxyethylene 8 stearate, polyoxyethylene bis (imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, saponin from quillaja, Span^® 20、Span^® 40、Span^® 60、Span^® 65、Span^® 80、Span^®85. Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol, Type TMN-10, Tergitol, Type TMN-6, tetradecyl-beta-D-maltoside, tetraethyleneglycol monodecyl ether, tetraethyleneglycol monotetradecyl ether, triethylene glycol monodecyl ether, triethylene glycol monocetyl ether, triethylene glycol monooctyl ether, triethylene glycol monotecyl ether, CF-21, Type, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton^® X-100、Triton^® X-114、Triton^® X-165、Triton^® X-305、Triton^® X-405、Triton^® X-45、Triton^® X-705-70、TWEEN^® 20、TWEEN^® 21、TWEEN^® 40、TWEEN^® 60、TWEEN^® 61、TWEEN^® 65、TWEEN^® 80、TWEEN^® 81、TWEEN^®85. Tyloxapol, n-undecyl beta-D-glucopyranoside, semisynthetic derivatives thereof, or combinations thereof.

Further, the nonionic surfactant can be a poloxamer. Poloxamers are polymers made from blocks of polyoxyethylene, followed by blocks of polyoxypropylene, followed by blocks of polyoxyethylene. The average number of polyoxyethylene and polyoxypropylene units varies based on the number associated with the polymer. For example, the minimum polymer poloxamer 101 consists of a block with an average of 2 polyoxyethylene units, a block with an average of 16 polyoxypropylene units, followed by a block with an average of 2 polyoxyethylene units. Poloxamers range from colorless liquids and pastes to white solids. Poloxamers are used in the formulation of skin cleansers, bath products, shampoos, conditioners, mouthwashes, eye make-up removers and other skin and hair products in cosmetic and personal care products. Examples of poloxamers include, but are not limited to, poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407, poloxamer 105 benzoate, and poloxamer 182 dibenzoate.

Suitable cationic surfactants include, but are not limited to, quaternary ammonium compounds, alkyltrimethylammonium chloride compounds, dialkyldimethylammonium chloride compounds, cationic halogen-containing compounds such as cetylpyridinium chlorideBenzalkonium chloride, benzyldimethylhexadecylammonium chloride, benzyldimethyltetradecylammonium chloride, benzyldodecyldimethylammonium bromide, benzyltrimethyltetrachloroammonium iodide, dimethyldioctadecylammonium bromide, dodecylethyldimethylammonium bromide, dodecyltrimethylammonium bromide, ethylhexadecyldimethylammonium bromide, Girard's reagent T, hexadecyltrimethylammonium bromide, N ', N ' -polyoxyethylene (10) -N-tallow-1, 3-diaminopropane, ammoniumbranonozol, trimethyl (tetradecyl) ammonium bromide, 1,3, 5-triazine-1, 3,5 (2H, 4H, 6H) -triethanol, 1-deacaminium, N-decyl-N, n-dimethyl chloride, didecyl dimethyl ammonium chloride, 2- (2- (p- (diisobutyl)Tolyloxy (cresyloxy)) ethoxy) ethyldimethylbenzyl ammonium chloride, 2- (2- (p- (diisobutyl) phenoxy) ethoxy) ethyldimethylbenzyl ammonium chloride, alkyl 1 or 3 benzyl-1- (2-hydroxyethyl) -2-imidazolinium chloride Alkyl bis (2-hydroxyethyl) benzyl ammonium chloride, alkyl demethylbenzyl ammonium chloride, alkyl dimethyl 3, 4-dichlorobenzyl ammonium chloride (100% C12), alkyl dimethyl 3, 4-dichlorobenzyl ammonium chloride (50% C14, 40% C12, 10% C16), alkyl dimethyl 3, 4-dichlorobenzyl ammonium chloride (55% C14, 23% C12, 20% C16), alkyl dimethylbenzyl ammonium chloride (100% C14), alkyl dimethylbenzyl ammonium chloride (100% C16), alkyl dimethylbenzyl ammonium chloride (41% C14, 28% C12), alkyl dimethylbenzyl ammonium chloride (47% C12, 18% C14), alkyl dimethylbenzyl ammonium chloride (55% C16, 20% C14), alkyl dimethylbenzyl ammonium chloride (58% C14, 28% C16), alkyl dimethylbenzyl 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, alkyl didecyl dimethyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride (C12-16), alkyl dimethyl benzyl ammonium chloride (C12-18), alkyl dimethyl benzyl ammonium chloride, dialkyl dimethyl benzyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride, alkyl dimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12), alkyl dimethyl ethyl ammonium bromide (e.g., mixed alkyl and alkenyl in the fatty acids of soybean oil), alkyl dimethyl ethyl benzyl ammonium chloride, alkyl dimethyl ethyl Benzylammonium chloride (60% C14), alkyldimethylisopropylbenzylammonium chloride (50% C12, 30% C14, 17% C16, 3% C18), alkyltrimethylammonium chloride (58% C18, 40% C16, 1% C14, 1% C12), alkyltrimethylammonium chloride (90% C18, 10% C16), alkyldimethyl (ethylbenzyl) ammonium chloride (C12-18), di- (C8-10) -alkyldimethylammonium chloride, dialkyldimethylammonium chloride, dialkylmethylbenzylammonium chloride, didecyldimethylammonium chloride, diisodecyldimethylammonium chloride, dioctyldimethylammonium chloride, dodecylbis (2-hydroxyethyl) octylammonium chloride, dodecyldimethylbenzylammonium chloride, dodecylcarbamoylmethyldimethylammonium chloride, heptadecyldimethylammonium chlorideHexahydro-1, 3, 5-tris (2-hydroxyethyl) -s-triazine, tetradecyldimethylbenzylammonium chloride (and) Quat RNIUM 14, N-dimethyl-2-hydroxypropylammonium chloride polymer, N-tetradecyldimethylbenzylammonium chloride monohydrate, octyldecyldimethylammonium chloride, octyldodecyldimethylammonium chloride, octylphenoxyethoxyethyldimethylbenzylammonium chloride, oxydiethylenebis (alkyldimethylammonium chloride), quaternary ammonium compounds, dicocoalkyldimethylammonium chloride, trimethoxysilylpropyldimethyloctadecylammonium chloride, trimethoxysilylquats, trimethyldodecylbenzylammonium chloride, semi-synthetic derivatives thereof and combinations thereof.

Exemplary cationic halogen-containing compounds include, but are not limited to, cetyl pyridinium halidesCetyl trimethyl ammonium halide, cetyl dimethylethyl ammonium halide, cetyl dimethylbenzyl ammonium halide, cetyl tributyl ammonium halideTwelve, twelveAlkyl trimethyl ammonium halides, or tetradecyl trimethyl ammonium halides. In some particular embodiments, suitable cationic halogen-containing compounds include, but are not limited to, cetylpyridinium chloride(CPC), cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide(CPB), cetyltrimethylammonium bromide (CTAB), cetyldimethylethylammonium bromide, cetyltributylammonium bromideDodecyl trimethyl ammonium bromide, and tetradecyl trimethyl ammonium bromide. In a particularly preferred embodiment, the cationic halogen-containing compound is CPC, although the compositions of the present invention are not limited to formulations having a particular cationic-containing compound.

Suitable anionic surfactants include, but are not limited to, carboxylates, sulfates, sulfonates, phosphates, chenodeoxycholic acid sodium salt, cholic acid, bull or sheep bile, dehydrocholic acid, deoxycholic acid methyl ester, digitonin, digitoxin aglycone, N-dimethyldodecylamine N-oxide, docusate sodium salt, glycochenodeoxycholic acid sodium salt, synthetic glycocholic acid hydrate, synthetic glycocholic acid sodium salt hydrate, glycodeoxycholic acid monohydrate, glycodeoxycholic acid sodium salt, glycolithocholic acid 3-sulfate disodium salt, glycolithocholic acid ethyl ester, N-lauroylsarcosine salt, N-lauroylsarcosine solution, lithium lauryl sulfate, Lithium dodecyl sulfate, Lugol solution, Niaproof 4, Type 4, sodium 1-octanesulfonate, sodium 1-butanesulfonate, sodium 1-decanesulfonate, sodium 1-dodecanesulfonate, anhydrous sodium 1-heptanesulfonate, 1-nonanesulfonic acid Sodium, sodium 1-propanesulfonate monohydrate, sodium 2-bromoethanesulfonate, sodium cholate hydrate, sodium cholate, sodium deoxycholate monohydrate, sodium dodecylsulfate, anhydrous sodium hexanesulfonate, sodium octylsulfate, anhydrous sodium pentanesulfonate, sodium taurocholate, sodium taurochenodeoxycholate, sodium taurodeoxycholate monohydrate, sodium tauroshinocholic acid sodium salt hydrate, taurolithocholic acid 3-disodium sulfate, sodium tauroursodeoxycholate, Trizma^®Dodecyl sulfate, TWEEN^®80. Ursodeoxycholic acid, semisynthetic derivatives thereof, and combinations thereof.

Suitable zwitterionic surfactants include, but are not limited to, N-alkyl betaine, lauryl amido (amido) propyl dimethyl betaine, alkyl dimethyl glycine acetate, N-alkyl aminopropionic acid salt, CHAPS, minimum 98% (TLC), CHAPS, SigmaUltra, minimum 98% (TLC), CHAPS for electrophoresis, minimum 98% (TLC), CHAPSO, minimum 98%, CHAPSO, SigmaUltra, CHAPSO for electrophoresis, 3- (decyldimethylammonium) propanesulfonic acid inner salt, 3-dodecyldimethylammonium) propanesulfonic acid inner salt, SigmaUltra, 3- (dodecyldimethylammonium) propanesulfonic acid inner salt, 3- (N, N-dimethylmyristylammonium) propanesulfonic acid salt, 3- (N, N-dimethyloctadecylammonium) propanesulfonic acid salt, 3- (N, N-dimethyloctylammonium) propanesulfonic acid inner salt, 3- (N, N-dimethylpalmitylammonium) propanesulfonic acid salt, semisynthetic derivatives thereof, and combinations thereof.

The present invention is not limited to the surfactants disclosed herein. Additional surfactants and Detergents useful in the compositions of the present invention may be identified by reference works (e.g., including but not limited to McCutheon, Vol. 1: Emulsions and Detergents-North American Edition, 2000) and commercial sources.

4. Cationic halogen-containing compounds

In some embodiments, the emulsion further comprises a cationic halogen-containing compound. In some preferred embodiments, the emulsion comprises from about 0.5 to 1.0% by weight or more of the cationic halogen-containing agentCompound, based on the total weight of the emulsion (although other concentrations are also contemplated). In a preferred embodiment, the cationic halogen-containing compound is preferably premixed with the oil phase; however, it is to be understood that the cationic halogen-containing compound may be provided in combination with the emulsion composition in different formulations. Suitable cationic halogen-containing compounds may be selected from compounds containing chloride, fluoride, bromide and iodide ions. In a preferred embodiment, suitable cationic halogen-containing compounds include, but are not limited to, cetyl pyridinium halidesCetyl trimethyl ammonium halide, cetyl dimethylethyl ammonium halide, cetyl dimethylbenzyl ammonium halide, cetyl tributyl ammonium halide Dodecyl trimethyl ammonium halide, or tetradecyl trimethyl ammonium halide. In some particular embodiments, suitable cationic halogen-containing compounds include, but are not limited to, cetylpyridinium chloride(CPC), cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide(CPB), and cetyltrimethylammonium bromide (CTAB), cetyldimethylethylammonium bromide, cetyltributylammonium bromideDodecyl trimethyl ammonium bromide, and tetradecyl trimethyl ammonium bromide. In a particularly preferred embodiment, the cationic halogen-containing compound is CPC, although the compositions of the present invention are not limited to formulations having any particular cationic-containing compound.

5. Germination enhancer

In other embodiments of the invention, the nanoemulsion further comprises a germination enhancer. In some preferred embodiments, the emulsion comprises from about 1 mM to 15 mM, and more preferably from about 5 mM to 10 mM of one or more germination enhancing compounds (although other concentrations are also contemplated). In a preferred embodiment, the germination enhancing compound is provided in the aqueous phase prior to formation of the emulsion. The present invention contemplates that when the germination enhancer is added to the nanoemulsion composition, the sporicidal properties of the nanoemulsion are enhanced. The present invention further contemplates that such germination enhancers initiate sporicidal activity near neutral pH (pH 6-8, and preferably 7). Such a neutral pH emulsion can be obtained, for example, by dilution with Phosphate Buffered Saline (PBS) or by preparing a neutral emulsion. The sporicidal activity of the nanoemulsion preferably occurs when the spores initiate germination.

In a particular embodiment, the emulsions utilized in the vaccines of the present invention have been demonstrated to have sporicidal activity. Although the present invention is not limited to any particular mechanism, and an understanding of the mechanism is not necessary to practice the present invention, it is believed that the fused component of the emulsion acts to initiate germination, and that the fused component of the emulsion acts to lyse newly germinated spores before reversion to the vegetative form is complete. These components of the emulsion thus act synergistically to render the spores susceptible to disruption by the emulsion. The addition of the germination enhancer further promotes the anti-sporicidal activity of the emulsion, for example by accelerating the rate at which sporicidal activity occurs.

Germination of bacterial endospores and fungal spores is associated with increased metabolism and reduced resistance to heat and chemical reactants. For germination to occur, the spores must sense the environment sufficiently to support vegetative and reproductive life. The amino acid L-alanine stimulates bacterial spore germination (see, e.g., Hills, J. Gen. micro. 4:38 (1950); and Halvorson and Church, Bacteriol Rev. 21:112 (1957)). L-alanine and L-proline have also been reported to initiate fungal spore germination (Yanagita, Arch Mikrobiol 26:329 (1957)). Simple alpha-amino acids such as glycine and L-alanine occupy central positions in metabolism. Conversion of alpha-amino acids Amino-or deamination yields glycogenic or ketogenic carbohydrates and nitrogen required for metabolism and growth. For example, transamination or deamination of L-alanine yields pyruvate, which is the end product of glycolytic metabolism (Embden-Meyerhof pathway). Obtaining acetyl coenzyme A, NADH, H by pyruvate oxidation of pyruvate dehydrogenase complex⁺And CO₂. Acetyl-coa is the starting substrate for the tricarboxylic acid cycle (Kreb's cycle), which in turn supplies the mitochondrial electron transport chain. Acetyl-coa is also the ultimate carbon source for fatty acid synthesis as well as for sterol synthesis. Simple alpha-amino acids can provide the nitrogen, CO required for germination and subsequent metabolic activity₂Glycogenic and/or ketogenic equivalents.

In certain embodiments, suitable germination enhancing agents of the present invention include, but are not limited to, the L-enantiomer of amino acids including glycine, as well as alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and alkyl esters thereof. Additional information on the effect of amino acids on germination can be found in U.S. Pat. No. 5,510,104; incorporated herein by reference in its entirety. In some embodiments, glucose, fructose, asparagine, sodium chloride (NaCl), ammonium chloride (NH), may also be used ₄Cl), calcium chloride (CaCl)₂) And potassium chloride (KCl). In a particularly preferred embodiment of the invention, the formulation comprises L-alanine, CaCl as germination enhancer₂Inosine and NH₄And (4) Cl. In some embodiments, the composition further comprises one or more common forms of growth media (e.g., tryptic soy broth, etc.), which may or may not additionally comprise itself a germination enhancer and a buffer.

The above compounds are merely exemplary germination enhancers, and it should be understood that other known germination enhancers will be useful in the nanoemulsions utilized in some embodiments of the present invention. Candidate germination enhancers should meet 2 criteria for inclusion in the compositions of the invention: it should be capable of binding to the emulsions disclosed herein, and when incorporated into the emulsions disclosed herein, it should increase the germination rate of the target spores. One skilled in the art can determine whether a particular agent has the desired function of acting as a germination enhancer by applying such an agent in combination with a nanoemulsion disclosed herein to a target and comparing the inactivation of the target when contacted with the mixture to the inactivation of a similar target by the composition of the invention without the agent. Any agent that increases germination and thereby reduces or inhibits growth of an organism is considered a suitable enhancer for use in the nanoemulsion compositions disclosed herein.

In still other embodiments, the addition of a germination enhancer (or growth medium) to the neutral emulsion composition results in a composition for use in the vaccine compositions of the present invention that is useful in inactivating bacterial spores plus enveloped viruses, gram negative bacteria, and gram positive bacteria.

6. Interaction enhancers

In still other embodiments, the nanoemulsion comprises a compound capable of increasing the interaction of the composition with a target pathogen (e.g., the cell wall of a gram-negative bacterium, such as vibrio (r) (r))Vibrio) Salmonella genus (A), (B)Salmonella) Shigella (A), (B), (C)Shigella) And Pseudomonas (Pseudomonas) Or "interaction enhancer"). In a preferred embodiment, the interaction enhancer is preferably pre-mixed with the oil phase; however, in other embodiments, the interaction enhancer is provided in combination with the composition after emulsification. In certain preferred embodiments, the interaction enhancer is a chelating agent, such as ethylenediaminetetraacetic acid (EDTA), ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA) in a buffer, such as tris buffer. It should be understood that the chelating agents are merely exemplary interaction enhancing compounds. Indeed, other agents that increase the interaction of the nanoemulsion used in some embodiments of the present invention with microbial agents and/or pathogens are contemplated. In a particularly preferred embodiment, the interaction enhancer is at a concentration of about 50 to about 250 μ M. The skill of the art The skilled person will be able to determine whether a particular agent has the function of acting as an interaction enhancer by applying such an agent in combination with a composition of the invention to a target and comparing the inactivation of the target when contacted with the mixture with the inactivation of a similar target by a composition of the invention without the agent. Any agent that increases the interaction of the emulsion with the bacteria and thereby reduces or inhibits the growth of the bacteria, as compared to that parameter in the absence thereof, is considered an interaction enhancer.

In some embodiments, the addition of an interaction enhancer to a nanoemulsion results in a composition for use in a vaccine composition of the invention that is useful in inactivating enveloped viruses, some gram-positive bacteria, and some gram-negative bacteria.

7. Quaternary ammonium compounds

In some embodiments, the nanoemulsion of the present invention comprises a quaternary ammonium-containing compound. Exemplary quaternary ammonium compounds include, but are not limited to, alkyl dimethyl benzyl ammonium chloride, didecyl dimethyl ammonium chloride, alkyl dimethyl benzyl and dialkyl dimethyl ammonium chloride, N-dimethyl-2-hydroxypropyl ammonium chloride polymer, didecyl dimethyl ammonium chloride, N-alkyl dimethyl benzyl ammonium chloride, N-alkyl dimethyl ethyl benzyl ammonium chloride, dialkyl dimethyl ammonium chloride, N-alkyl dimethyl benzyl ammonium chloride, N-tetradecyl dimethyl benzyl ammonium chloride monohydrate, N-alkyl dimethyl benzyl ammonium chloride, dialkyl dimethyl ammonium chloride, hexahydro-1, 3, 5-tris (2-hydroxyethyl) -s-triazine, tetradecyl dimethyl benzyl ammonium chloride (and) Quat RNIUM 14, alkyl bis (2-hydroxyethyl) benzyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride, and mixtures thereof, Alkyl demethylbenzyl ammonium chloride, alkyl dimethyl 3, 4-dichlorobenzyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride, alkyl dimethyl ethyl ammonium bromide, alkyl dimethyl ethyl benzyl ammonium chloride, alkyl dimethyl isopropyl benzyl ammonium chloride, alkyl trimethyl ammonium chloride, alkyl 1 or 3 benzyl-1- (2-hydroxyethyl) -2-imidazoline chloride Dialkyl methyl benzyl ammonium chloride, dialkyl dimethyl ammonium chloride, didecyl dimethyl ammonium chloride, 2- (2- (p- (diisobutyl) tolyloxy (cresyloxy)) ethoxy) ethyl dimethyl benzyl ammonium chloride, 2- (2- (p- (diisobutyl) phenoxy) ethoxy) ethyl dimethyl benzyl ammonium chloride, dioctyl dimethyl ammonium chloride, dodecyl bis (2-hydroxyethyl) octyl ammonium hydrochloride, dodecyl dimethyl benzyl ammonium chloride, dodecyl carbamoyl methyl dimethyl (dineth) benzyl ammonium chloride, heptadecyl hydroxyethyl imidazoline chlorideHexahydro-1, 3, 5-tris (2-hydroxyethyl) -s-triazine, octyldecyl dimethyl ammonium chloride, octyldodecyl dimethyl ammonium chloride, octylphenoxy ethoxy ethyl dimethyl benzyl ammonium chloride, oxydiethylene bis (alkyl dimethyl ammonium chloride), quaternary ammonium compounds, dicocoalkyl dimethyl chloride, trimethoxysilyl quats, and trimethyldodecyl benzyl ammonium chloride.

8. Other Components

In some embodiments, the nanoemulsion comprises one or more additional components that provide the nanoemulsion with desired properties or functionality. These components may be incorporated into the aqueous or oil phase of the nanoemulsion and/or may be added before or after emulsification. For example, in some embodiments, the nanoemulsion further comprises phenols (e.g., trichlorophenol, phenylphenol), acidifying agents (e.g., citric acid (e.g., 1.5-6%), acetic acid, lemon juice), alkylating agents (e.g., sodium hydroxide (e.g., 0.3%)), buffers (e.g., citrate buffers, acetate buffers, and other buffers used to maintain a particular pH), and halogens (e.g., polyvinylpyrrolidone, sodium hypochlorite, hydrogen peroxide).

Exemplary techniques for preparing nanoemulsions (e.g., for inactivating pathogens and/or production of immunogenic compositions of the invention) are described below. In addition, a number of specificities are set forth below, although exemplary formulation formulations are also set forth below.

Preparation technology

The nanoemulsions of the present invention can be formed using standard emulsion forming techniques. Briefly, the oil phase is mixed with the aqueous phase under relatively high shear forces (e.g., using high hydraulic and mechanical forces) to obtain an oil-in-water nanoemulsion. An emulsion is formed by blending the oil phase with the aqueous phase on a volume to volume basis ranging from about 1:9 to 5:1, preferably from about 5:1 to 3:1, most preferably 4:1, of the oil phase to the aqueous phase. The oil and aqueous phases may be blended using any device capable of generating shear forces sufficient to form an emulsion, such as a french press or a high shear mixer (e.g., FDA approved high shear mixers such as are available from Admix, inc., Manchester, NH). Methods of producing such emulsions are described in U.S. patent nos. 5,103,497 and 4,895,452, which are incorporated herein by reference in their entirety.

In a preferred embodiment, the composition used in the process of the invention comprises droplets of an oil discontinuous phase dispersed in an aqueous continuous phase, such as water. In a preferred embodiment, the nanoemulsion of the present invention is stable and does not disintegrate even after long storage periods (e.g., more than one or more years). In addition, in some embodiments, the nanoemulsion is stable (e.g., in some embodiments, for more than 3 months, in some embodiments, for more than 6 months, in some embodiments, for more than 12 months, in some embodiments, for more than 18 months) upon combination with an immunogen (e.g., a pathogen). In preferred embodiments, the nanoemulsion of the present invention is non-toxic and safe when administered (e.g., via spraying or contact with a mucosal surface, swallowing, inhalation, etc.) to a subject.

In some embodiments, portions of the emulsion may be in the form of lipid structures including, but not limited to, unilamellar, multilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.

Some embodiments of the present invention employ an oil phase comprising ethanol. For example, in some embodiments, the emulsion of the invention contains (i) an aqueous phase and (ii) an oily phase containing ethanol as organic solvent and optionally a germination enhancer, and (iii) TYLOXAPOL as surfactant (preferably 2-5%, more preferably 3%). Such formulations are highly effective for pathogen inactivation, and are also non-irritating and non-toxic to mammalian subjects (e.g., and thus can be applied to mucosal surfaces).

In some other embodiments, the emulsion of the present invention comprises a first emulsion emulsified within a second emulsion, wherein (a) the first emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and an organic solvent; and (iii) a surfactant; and (b) the second emulsion comprises (i) an aqueous phase; and (ii) an oil phase comprising an oil and a cationic-containing compound; and (iii) a surfactant.

Exemplary formulations

The following description provides a number of exemplary emulsions, including BCTP and X for use in the compositions ₈W₆₀Formulations of PC. BCTP comprises a water-in-oil nanoemulsion in which the oily phase is made of soybean oil, tri-n-butyl phosphate and TRITON X-100 dissolved in 80% water. X₈W₆₀PC comprises BCTP and W₈₀An equal volume of 8P mixture. W₈₀8P is a liposome-like compound made from glyceryl monostearate, refined soya sterol (e.g. GENEROL sterol), TWEEN 60, soybean oil, cationic halogen-containing CPC and peppermint oil. The GENEROL family is a group of polyethoxylated soy sterols (Henkel Corporation, Ambler, Pennsylvania). Exemplary emulsion formulations useful in the present invention are provided in table 1B. These particular formulations may be found in U.S. patent nos. 5,700,679 (NN); 5,618,840, respectively; 5,549,901 (W)₈₀8P); and 5,547,677, each of which is incorporated herein by reference in its entirety. Certain other emulsion formulations are presented in U.S. patent application serial No. 10/669,865, which is incorporated herein by reference in its entirety.

X₈W₆₀PC emulsion is prepared by first preparing W separately₈₀8P emulsion and BCTP emulsion. The mixture of these 2 emulsions is then re-emulsified to give what is known as X₈W₆₀Fresh emulsion composition of PC. Methods of producing such emulsions are described in U.S. patent nos. 5,103,497 and 4,895,452 (each of which is incorporated herein by reference in its entirety).

TABLE 1B

The compositions listed above are exemplary only, and one skilled in the art will be able to vary the amount of components to achieve a nanoemulsion composition suitable for the purposes of the present invention. One skilled in the art will appreciate that the ratio of oil phase to water, as well as the individual oil carriers, the surfactant CPC and the organophosphate buffer, the components of each composition may be different.

While certain compositions comprising BCTP have a water to oil ratio of 4:1, it is understood that BCTP can be formulated with more or less aqueous phase. For example, in some embodiments, 3, 4, 5, 6, 7, 8, 9, 10, or more parts of aqueous phase are present for each part of oil phase. This is for W₈₀The 8P formulation was also correct. Similarly, the ratio of tri-n-butyl phosphate to TRITON X-100 to soybean oil can also be varied.

Although Table 1B lists the values for W₈₀8P glyceryl monooleate, polysorbate 60, GENEROL 122, cetyl pyridinium chlorideAnd specific amounts of carrier oil, but these are exemplary only. Can be formulated to have W₈₀An emulsion of 8P nature with different concentrations of each of these components or indeed different components that will perform the same function. For example, the emulsion may have from about 80 to about 100g of glycerol monooleate in the starting oil phase. In other embodiments In one embodiment, the emulsion may have about 15 to about 30 g of polysorbate 60 in the starting oil phase. In another embodiment, the composition may comprise about 20 to about 30 g of GENEROL sterol in the starting oil phase.

The individual components of the nanoemulsion (e.g., in the immunogenic compositions of the invention) can act to inactivate pathogens as well as contribute to the non-toxicity of the emulsion. For example, the active component in BCTP, TRITON-X100, showed less ability to inactivate viruses at concentrations equivalent to 11% BCTP. The addition of an oil phase to the detergent and solvent significantly reduces the toxicity of these agents at the same concentrations in tissue culture. While not being bound by any theory (understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism), it is suggested that the nanoemulsion enhances the interaction of its components with the pathogen, thereby promoting inactivation of the pathogen and reducing the toxicity of the individual components. Furthermore, when all components of BCTP are combined in one composition but not in a nanoemulsion configuration, the mixture is not as effective at inactivating pathogens as when the components are in a nanoemulsion configuration.

Numerous additional embodiments presented in the formulation categories having similar compositions are presented below. The following compositions describe various ratios and mixtures of active ingredients. One skilled in the art will recognize that the formulations described below are exemplary and that additional formulations comprising similar ranges of percentages of the components are within the scope of the invention.

In certain embodiments of the invention, the nanoemulsion comprises about 3-8% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume cetylpyridinium chloride(CPC), about 60-70% by volume oil (e.g., soybean oil), about 15-25% by volume aqueous phase (e.g., DiH)₂O or PBS), and in some formulations less than about 1 vol% 1N NaOH. Some of these embodiments comprise PBS. The addition of 1N NaOH and/or PBS in some of these embodiments is contemplatedThe user is allowed to advantageously control the pH of the formulation such that the pH ranges from about 7.0 to about 9.0, and more preferably from about 7.1 to 8.5. For example, one embodiment of the present invention comprises about 3% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, and about 24% by volume DiH₂O (designated herein as Y3 EC). Another similar embodiment comprises about 3.5% by volume TYLOXAPOL, about 8% by volume ethanol, and about 1% by volume CPC, about 64% by volume soybean oil, and about 23.5% by volume DiH₂O (designated herein as Y3.5EC). Another embodiment comprises about 3% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 0.067% by volume 1N NaOH, such that the pH of the formulation is about 7.1, about 64% by volume soybean oil, and about 23.93% by volume DiH ₂O (designated herein as Y3EC pH 7.1). Another embodiment comprises about 3% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 0.67% by volume 1N NaOH, such that the pH of the formulation is about 8.5, and about 64% by volume soybean oil, and about 23.33% by volume DiH₂O (designated herein as Y3EC pH 8.5). Another similar embodiment comprises about 4% TYLOXAPOL, about 8% ethanol by volume, about 1% CPC, and about 64% soybean oil by volume, and about 23% DiH by volume₂O (designated herein as Y4 EC). In another embodiment, the formulation comprises about 8% TYLOXAPOL, about 8% ethanol, about 1% CPC by volume, and about 64% soybean oil by volume, and about 19% DiH by volume₂O (designated herein as Y8 EC). A further embodiment comprises about 8% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, about 19% by volume 1x PBS (designated herein as Y8EC PBS).

In some embodiments of the invention, the nanoemulsion comprises about 8% ethanol by volume, and about 1% CPC by volume, and about 64% oil by volume (e.g., soybean oil), and about 27% aqueous phase by volume (e.g., DiH) ₂O or PBS) (designated herein as EC).

In some embodiments of the present invention, the substrate is,the nanoemulsion comprises about 8% by volume Sodium Dodecyl Sulfate (SDS), about 8% by volume tributyl phosphate (TBP), and about 64% by volume oil (e.g., soybean oil), and about 20% by volume aqueous phase (e.g., DiH)₂O or PBS) (designated herein as S8P).

In some embodiments, the nanoemulsion comprises from about 1-2% by volume TRITON X-100, from about 1-2% by volume TYLOXAPOL, from about 7-8% by volume ethanol, about 1% by volume cetylpyridinium chloride(CPC), about 64-57.6% by volume oil (e.g., soybean oil), and about 23% by volume water phase (e.g., DiH)₂O or PBS). In addition, some of these formulations further comprise about 5 mM L-alanine/inosine and about 10 mM ammonium chloride. Some of these formulations contained PBS. It is contemplated that the addition of PBS in some of these embodiments allows the user to advantageously control the pH of the formulation. For example, one embodiment of the present invention comprises about 2% by volume TRITON X-100, about 2% by volume TYLOXAPOL, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, and about 23% by volume aqueous phase DiH ₂And O. In another embodiment, the formulation comprises about 1.8% by volume TRITON X-100, about 1.8% by volume TYLOXAPOL, about 7.2% by volume ethanol, about 0.9% by volume CPC, about 5 mM L-alanine/inosine, and about 10 mM ammonium chloride, about 57.6% by volume soybean oil, and the remainder 1X PBS (designated herein as 90% X2Y2 EC/GE).

In some embodiments, the nanoemulsion comprises about 5% by volume TWEEN 80, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume oil (e.g., soybean oil), and about 22% by volume DiH₂O (designated herein as W)₈₀5EC）。

In still other embodiments of the present invention, the nanoemulsion comprises about 5% by volume TWEEN 20, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume oil (e.g., soybean oil), and about 22% by volumeDiH (g)₂O (designated herein as W)₂₀5EC）。

In still other embodiments of the present invention, the nanoemulsion comprises about 2-8% by volume TRITON X-100, about 8% by volume ethanol, about 1% by volume CPC, about 60-70% by volume oil (e.g., soybean or olive oil), and about 15-25% by volume aqueous phase (e.g., DiH)₂O or PBS). For example, the present invention contemplates a composition comprising about 2% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 26% by volume DiH ₂Formulation of O (designated herein as X2E). In other similar embodiments, the nanoemulsion comprises about 3% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 25% by volume DiH₂O (designated herein as X3E). In yet a further embodiment, the formulation comprises about 4% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 24% by volume DiH₂O (designated herein as X4E). In still other embodiments, the nanoemulsion comprises about 5% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 23% by volume DiH₂O (designated herein as X5E). In some embodiments, the nanoemulsion comprises about 6% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 22% by volume DiH₂O (designated herein as X6E). In still a further embodiment of the present invention, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X8E). In a still further embodiment, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume ethanol, about 64% by volume olive oil, and about 20% by volume DiH ₂O (designated herein as X8E O). In another embodiment, the nanoemulsion comprises 8% by volume TRITON X-100, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume soybean oil, and about 19% by volume DiH₂O (designated herein as X8 EC).

In an alternative embodiment of the invention, the nanoemulsion comprises from about 1-2% by volume TRITON X-100, from about 1-2% by volume TYLOXAPOL, from about 6-8% by volume TBP, from about 0.5-1.0% by volume CPC, from about 60-70% by volume oil (e.g., soybean oil), and from about 1-35% by volume aqueous phase (e.g., DiH)₂O or PBS). Additionally, some of these nanoemulsions may comprise about 1-5% by volume tryptic soy broth, about 0.5-1.5% by volume yeast extract, about 5 mM L-alanine/inosine, about 10 mM ammonium chloride, and about 20-40% by volume liquid infant formula. In some embodiments comprising liquid infant formula, the formula comprises casein hydrolysate (e.g., Neutramigen or Progestimil, etc.). In some of these embodiments, the nanoemulsion further comprises from about 0.1-1.0% by volume sodium thiosulfate, and from about 0.1-1.0% by volume sodium citrate. Other similar embodiments comprising these essential components employ Phosphate Buffered Saline (PBS) as the aqueous phase. For example, one embodiment comprises about 2% by volume TRITON X-100, about 2% by volume TYLOXAPOL, about 8% by volume TBP, about 1% by volume CPC, about 64% by volume soybean oil, and about 23% by volume DiH ₂O (designated herein as X2Y2 EC). In still other embodiments, the formulations of the present invention comprise about 2% by volume TRITON X-100, about 2% by volume TYLOXAPOL, about 8% by volume TBP, about 1% by volume CPC, about 0.9% by volume sodium thiosulfate, about 0.1% by volume sodium citrate, about 64% by volume soybean oil, and about 22% by volume DiH₂O (designated herein as X2Y2PC STS 1). In another similar embodiment, the nanoemulsion comprises about 1.7% by volume TRITON X-100, about 1.7% by volume TYLOXAPOL, about 6.8% by volume TBP, about 0.85% CPC, about 29.2% netreamagigen, about 54.4% by volume soybean oil, and about 4.9% by volume DiH₂O (designated herein as 85% X2Y2 PC/infant). In another embodiment of the invention, the nanoemulsion comprises about 1.8% by volume TRITON X-100, about 1.8% by volume TYLOXAPOL, about 7.2% by volume TBP, about 0.9% by volume CPC, about 5 mM L-alanine/inosine, about 10 mM ammonium chloride, about 57.6% by volume soybean oil, and the remaining% by volume 0.1X PBS (designated herein as 90X PBS)% X2Y2 PC/GE). In another embodiment, the nanoemulsion comprises about 1.8% by volume TRITON X-100, about 1.8% by volume TYLOXAPOL, about 7.2% by volume TBP, about 0.9% by volume CPC, about 3% by volume tryptic soy broth, about 57.6% by volume soybean oil, and about 27.7% by volume DiH ₂O (designated herein as 90% X2Y2 PC/TSB). In another embodiment of the invention, the nanoemulsion comprises about 1.8% TRITON X-100 by volume, about 1.8% TYLOXAPOL by volume, about 7.2% TBP by volume, about 0.9% CPC by volume, about 1% yeast extract by volume, about 57.6% soybean oil by volume, and about 29.7% DiH by volume₂O (designated herein as 90% X2Y2 PC/YE).

In some embodiments of the invention, the nanoemulsion comprises about 3% by volume TYLOXAPOL, about 8% by volume TBP, and about 1% by volume CPC, about 60-70% by volume oil (e.g., soy or olive oil), and about 15-30% by volume aqueous phase (e.g., DiH)₂O or PBS). In a particular embodiment of the invention, the nanoemulsion comprises about 3% by volume TYLOXAPOL, about 8% by volume TBP, and about 1% by volume CPC, about 64% by volume soybean, and about 24% by volume DiH₂O (designated herein as Y3 PC).

In some embodiments of the invention, the nanoemulsion comprises from about 4-8% by volume TRITON X-100, from about 5-8% by volume TBP, from about 30-70% by volume oil (e.g., soy or olive oil), and from about 0-30% by volume aqueous phase (e.g., DiH)₂O or PBS). Additionally, certain of these embodiments further comprise about 1% by volume CPC, about 1% by volume benzalkonium chloride, about 1% by volume cetylpyridinium bromide About 1 vol% hexadecyldimethylethylammonium bromide, 500 μ M EDTA, about 10 mM ammonium chloride, about 5 mM inosine, and about 5 mM L-alanine. For example, in certain preferred embodiments, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X8P). In another embodiment of the invention, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 1% CPC, about 64% by volume soybean oil, and about 19% by volume DiH₂O (designated herein as X8 PC). In another embodiment, the nanoemulsion comprises about 8% TRITON X-100 by volume, about 8% TBP by volume, about 1% CPC by volume, about 50% soybean oil by volume, and about 33% DiH by volume₂O (designated herein as ATB-X1001). In another embodiment, the formulation comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 2% by volume CPC, about 50% by volume soybean oil, and about 32% by volume DiH₂O (designated herein as ATB-X002). In some embodiments, the nanoemulsion comprises about 4% TRITON X-100 by volume, about 4% TBP by volume, about 0.5% CPC by volume, about 32% soybean oil by volume, and about 59.5% DiH by volume ₂O (designated herein as 50% X8 PC). In some embodiments, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 0.5% by volume CPC, about 64% by volume soybean oil, and about 19.5% by volume DiH₂O (designated herein as X8PC_1/2). In some embodiments of the invention, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 2% by volume CPC, about 64% by volume soybean oil, and about 18% by volume DiH₂O (designated herein as X8PC 2). In other embodiments, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% TBP, about 1% benzalkonium chloride, about 50% by volume soybean oil, and about 33% by volume DiH₂O (designated herein as X8P BC). In an alternative embodiment of the invention, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 1% by volume cetylpyridinium bromideAbout 50% by volume soybean oil, and about 33% by volume DiH₂O (designated herein as X8P CPB). In another exemplary embodiment of the invention, the nanoemulsion comprises about 8% by volume of TRITON X-100, about 8% by volume TBP, about 1% by volume cetyldimethylethyl ammonium bromide, about 50% by volume soybean oil, and about 33% by volume DiH ₂O (designated herein as X8P CTAB). In yet a further embodiment, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume TBP, about 1% by volume CPC, about 500 μ M EDTA, about 64% by volume soybean oil, and about 15.8% by volume DiH₂O (designated herein as X8PC EDTA). In some embodiments, the nanoemulsion comprises 8% by volume TRITON X-100, about 8% by volume TBP, about 1% by volume CPC, about 10 mM ammonium chloride, about 5 mM inosine, about 5 mM L-alanine, about 64% by volume soybean oil, and about 19% by volume DiH₂O or PBS (designated herein as X8PC GE)_1x). In another embodiment of the present invention, the nanoemulsion comprises about 5% by volume TRITON X-100, about 5% TBP, about 1% by volume CPC, about 40% by volume soybean oil, and about 49% by volume DiH₂O (designated herein as X5P₅C）。

In some embodiments of the invention, the nanoemulsion comprises about 2% by volume TRITON X-100, about 6% by volume TBP, about 8% by volume ethanol, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X2Y 6E).

In a further embodiment of the invention, the nanoemulsion comprises about 8% by volume TRITON X-100, and about 8% by volume glycerin, about 60-70% by volume oil (e.g., soybean or olive oil), and about 15-25% by volume aqueous phase (e.g., DiH) ₂O or PBS). Certain nanoemulsion compositions (e.g., for generating an immune response (e.g., for use as a vaccine) comprise about 1% by volume L-ascorbic acid, for example, one particular embodiment comprises about 8% by volume TRITON X-100, about 8% by volume glycerol, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X8G). In another embodiment, the nanoemulsion comprises about 8% by volume TRITON X-100, about 8% by volume glycerol, about 1% by volume L-ascorbic acid, about 64% by volume soybean oil, and about 19% by volume DiH₂O (designated herein as X8GV_c）。

In yet a further embodiment, the nanoemulsion comprises about 8% by volume TRITON X-100, about 0.5-0.8% by volume TWEEN 60, about 0.5-2.0% by volume CPC, about 8% by volume TBP, about 60-70% by volume oil (e.g., soybean or olive oil), and about 15-25% by volume aqueous phase (e.g., DiH)₂O or PBS). For example, in one particular embodiment, the nanoemulsion comprises about 8% by volume TRITON X-100, about 0.70% by volume TWEEN 60, about 1% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 18.3% by volume DiH ₂O (designated herein as X8W60PC₁). In some embodiments, the nanoemulsion comprises about 8% by volume TRITON X-100, about 0.71% by volume TWEEN 60, about 1% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 18.29% by volume DiH₂O (designated herein as W60_0.7X8 PC). In still other embodiments, the nanoemulsion comprises about 8% by volume TRITON X-100, about 0.7% by volume TWEEN 60, about 0.5% by volume CPC, about 8% by volume TBP, about 64-70% by volume soybean oil, and about 18.8% by volume DiH₂O (designated herein as X8W60PC₂). In still other embodiments, the nanoemulsion comprises about 8% by volume TRITON X-100, about 0.71% by volume TWEEN 60, about 2% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 17.3% by volume DiH₂And O. In another embodiment of the invention, the nanoemulsion comprises about 0.71% by volume TWEEN 60, about 1% by volume CPC, about 8% by volume TBP, about 64% by volume soybean oil, and about 25.29% by volume DiH₂O (designated herein as W60_0.7PC）。

In another embodiment of the invention, the nanoemulsion comprises dioctyl sulfosuccinate at about 2% by volume, glycerin at about 8% by volume, or TBP at about 8% by volume, plus oil (e.g., soy or olive oil) at about 60-70% by volume, and an aqueous phase (e.g., DiH) at about 23% by volume ₂O or PBS). For example, in some instancesIn an embodiment, the nanoemulsion comprises dioctyl sulfosuccinate of about 2% by volume, glycerin of about 8% by volume, soybean oil of about 64% by volume, and DiH of about 26% by volume₂O (designated herein as D2G). In another related embodiment, the nanoemulsion comprises dioctyl sulfosuccinate at about 2% by volume, and TBP at about 8% by volume, soybean oil at about 64% by volume, and DiH at about 26% by volume₂O (designated herein as D2P).

In still other embodiments of the present invention, the nanoemulsion comprises from about 8-10% by volume of glycerin, and from about 1-10% by volume of CPC, from about 50-70% by volume of an oil (e.g., soybean or olive oil), and from about 15-30% by volume of an aqueous phase (e.g., DiH)₂O or PBS). Additionally, in certain of these embodiments, the nanoemulsion further comprises about 1% by volume of L-ascorbic acid. For example, in some embodiments, the nanoemulsion comprises about 8% glycerol by volume, about 1% CPC by volume, about 64% soybean oil by volume, and about 27% DiH by volume₂O (designated herein as GC). In some embodiments, the nanoemulsion comprises about 10% glycerol by volume, about 10% CPC by volume, about 60% soybean oil by volume, and about 20% DiH by volume ₂O (designated herein as GC 10). In another embodiment of the present invention, the nanoemulsion comprises about 10% by volume of glycerin, about 1% by volume of CPC, about 1% by volume of L-ascorbic acid, about 60% by volume of soybean or oil, and about 24% by volume of DiH₂O (designated herein as GCV)_c）。

In some embodiments of the invention, the nanoemulsion comprises about 8-10% by volume glycerol, about 8-10% by volume SDS, about 50-70% by volume oil (e.g., soy or olive oil), and about 15-30% by volume aqueous phase (e.g., DiH)₂O or PBS). Additionally, in certain of these embodiments, the nanoemulsion further comprises about 1% by volume lecithin and about 1% by volume methylparaben. An exemplary embodiment of such a formulation comprises about 8% SDS, 8% glycerol, about 64% soybean oil, and about 20% DiH by volume₂O（Designated herein as S8G). A related formulation comprises about 8% by volume of glycerin, about 8% by volume of SDS, about 1% by volume of lecithin, about 1% by volume of methylparaben, about 64% by volume of soybean oil, and about 18% by volume of DiH₂O (designated herein as S8GL1B 1).

In another embodiment of the invention, the nanoemulsion comprises about 4% by volume TWEEN 80, about 4% by volume TYLOXAPOL, about 1% by volume CPC, about 8% by volume ethanol, about 64% by volume soybean oil, and about 19% by volume DiH ₂O (designated herein as W)₈₀4Y4EC）。

In some embodiments of the invention, the nanoemulsion comprises about 0.01% by volume CPC, about 0.08% by volume TYLOXAPOL, about 10% by volume ethanol, about 70% by volume soybean oil, and about 19.91% by volume DiH₂O (designated herein as y.08ec.01).

In another embodiment of the present invention, the nanoemulsion comprises sodium lauryl sulfate in about 8 vol%, glycerin in about 8 vol%, soybean oil in about 64 vol%, and DiH in about 20 vol%₂O (designated herein as SLS 8G).

The specific formulations described above are merely examples illustrating a variety of nanoemulsions useful in the present invention (e.g., to inactivate and/or neutralize pathogens, and to generate an immune response in a subject (e.g., for use as a vaccine)). The present invention contemplates many variations of the above-described formulations, as well as additional nanoemulsions, to be useful in the methods of the invention. Candidate emulsions can be readily tested to determine if they are suitable. First, the desired ingredients are prepared using the methods described herein to determine if an emulsion can be formed. If an emulsion cannot be formed, the candidate is eliminated. For example, from 4.5% sodium thiosulfate, 0.5% sodium citrate, 10% n-butanol, 64% soybean oil and 21% DiH ₂O do not form emulsions.

Second, the candidate emulsion should form a stable emulsion. If the emulsion is in the form of an emulsion, the foot is maintainedFor a sufficient period of time to allow its intended use (e.g., for generating an immune response in a subject), it is stable. For example, for emulsions to be stored, transported, etc., it may be desirable for the composition to remain in emulsion form for months to years. A relatively unstable typical emulsion will lose its form within one day. For example, 8% 1-butanol, 5% Tween 10, 1% CPC, 64% soybean oil and 22% DiH₂Candidate compositions made with O do not form stable emulsions. Nanoemulsions that have been shown to be stable include, but are not limited to, 8% by volume TRITON X-100, about 8% by volume TBP, about 64% by volume soybean oil, and about 20% by volume DiH₂O (designated herein as X8P); 5% by volume TWEEN 20, about 8% by volume ethanol, about 1% by volume CPC, about 64% by volume oil (e.g. soybean oil), and about 22% by volume DiH₂O (designated herein as W)₂₀5 EC); 0.08% TRITON X-100, 0.08% glycerin, 0.01% cetylpyridinium chloride99% butter and 0.83% diH₂O (designated herein as 1% X8GC butter); 0.8% TRITON X-100, 0.8% glycerin, 0.1% cetylpyridinium chloride 6.4% of soybean oil and 1.9% of diH₂O and 90% butter (designated herein as 10% X8GC butter); 2% W₂₀5EC, 1% Natrosol 250L NF, and 97% diH₂O (designated herein as 2% W)₂₀5EC L GEL); 1% cetyl pyridinium chloride5% TWEEN 20, 8% ethanol, 64% 70% viscous mineral oil and 22% diH₂O (designated herein as W)₂₀5EC 70 mineral oil); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% 350% viscous mineral oil and 22% diH₂O (designated herein asW₂₀5EC 350 mineral oil). In some embodiments, the nanoemulsion of the present invention is stable for more than 1 week, more than 1 month, or more than 1 year.

Third, the candidate emulsion should have efficacy for its intended use. For example, the nanoemulsion should inactivate (e.g., kill or inhibit its growth) the pathogen to a desired level (e.g., 1 log, 2 log, 3 log, 4 log,. reduction). Using the methods described herein, one can determine the suitability of a particular candidate emulsion for a desired pathogen. Generally, this involves exposing the pathogen to the emulsion in a parallel experiment with a suitable control sample (e.g., a negative control such as water) for one or more time periods and determining whether and to what extent the emulsion inactivates (e.g., kills and/or neutralizes) the microorganism. For example, from 1% ammonium chloride, 5% Tween 20, 8% ethanol, 64% soybean oil and 22% DiH ₂O the candidate composition made was shown not to be an effective emulsion. The following candidate emulsions were shown to be effective using the methods described herein: 5% Tween 20, 5% cetyl pyridinium chloride10% glycerol, 60% soybean oil and 20% diH₂O (designated herein as W)₂₀5GC 5); 1% cetyl pyridinium chloride5% Tween 20, 10% glycerol, 64% soybean oil and 20% diH₂O (designated herein as W)₂₀5 GC); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% olive oil and 22% diH₂O (designated herein as W)₂₀5EC olive oil); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% linseed oil and 22% diH₂O (referred to herein asIs given as W₂₀5EC linseed oil); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% corn oil and 22% diH₂O (designated herein as W)₂₀5EC corn oil); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% coconut oil and 22% diH₂O (designated herein as W)₂₀5EC coconut oil); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% cottonseed oil and 22% diH₂O (designated herein as W)₂₀5EC cottonseed oil); 8% glucose, 5% Tween 10, 1% cetylpyridinium chloride 64% soybean oil and 22% diH₂O (designated herein as W)₂₀5C glucose); 8% PEG 200, 5% Tween 10, 1% cetylpyridinium chloride64% soybean oil and 22% diH₂O (designated herein as W)₂₀5C PEG 200); 8% methanol, 5% Tween 10, 1% cetylpyridinium chloride64% soybean oil and 22% diH₂O (designated herein as W)₂₀5C methanol); 8% PEG 1000, 5% Tween 10, 1% cetylpyridinium chloride64% soybean oil and 22% diH₂O (designated herein as W)₂₀5C PEG 1000）；2％W₂₀5EC, 2% Natrosol 250H NF, and 96% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 2, also known as 2% W₂₀5EC GEL）；2％W₂₀5EC, 1% Natrosol 250H NF, and 97% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 1）；2％W₂₀5EC, 3% Natrosol 250H NF, and 95% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 3）；2％W₂₀5EC, 0.5% Natrosol 250H NF, and 97.5% diH₂O (designated herein as 2% W)₂₀5EC Natrosol 0.5）；2％W₂₀5EC, 2% Methocel A and 96% diH₂O (designated herein as 2% W)₂₀5EC Methocel A）；2％W₂₀5EC, 2% Methocel K and 96% diH₂O (designated herein as 2% W)₂₀5EC Methocel K); 2% Natrosol, 0.1% X8PC, 0.1 XPBS, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride and diH₂O (designated herein as 0.1% X8PC/GE + 2% Natrosol); 2% of Natrosol, 0.8% of Triton X-100, 0.8% of tributyl phosphate, 6.4% of soybean oil, 0.1% of cetylpyridinium chloride 0.1 XPBS, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride and diH₂O (designated herein as 10% X8PC/GE + 2% Natrosol); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% lard and 22% diH₂O (designated herein as W)₂₀5EC lard); 1% cetyl pyridinium chloride5% Tween 20, 8% ethanol, 64% mineral oil and 22% diH₂O (designated herein as W)₂₀5EC mineral oil); 0.1% cetylpyridinium chloride2% Neolidol, 5% Tween 20, 10% ethanol, 64% Soybean oil and 18.9% diH₂O (herein)Is designated as W₂₀5EC_0.1N); 0.1% cetylpyridinium chloride2% farnesol, 5% Tween 20, 10% ethanol, 64% soybean oil and 18.9% diH₂O (designated herein as W)₂₀5EC_0.1F) (ii) a 0.1% cetylpyridinium chloride5% Tween 20, 10% ethanol, 64% soybean oil and 20.9% diH₂O (designated herein as W)₂₀5EC_0.1) (ii) a 10% cetyl pyridinium chloride8% tributyl phosphate, 8% Triton X-100, 54% Soybean oil and 20% diH₂O (designated herein as X8PC₁₀) (ii) a 5% cetyl pyridinium chloride8% Triton X-100, 8% tributyl phosphate, 59% Soybean oil and 20% diH₂O (designated herein as X8PC₅) (ii) a 0.02% cetylpyridinium chloride 0.1% Tween 20, 10% ethanol, 70% soybean oil and 19.88% diH₂O (designated herein as W)₂₀0.1EC_0.02) (ii) a 1% cetyl pyridinium chloride5% Tween 20, 8% glycerol, 64% Mobil 1 and 22% diH₂O (designated herein as W)₂₀5GC Mobil 1); 7.2% Triton X-100, 7.2% tributyl phosphate, 0.9% cetylpyridinium chloride57.6% Soybean oil, 0.1 XPBS, 5 mM L-alanine, 5 mM inosine10 mM ammonium chloride and 25.87% diH₂O (designated herein as 90% X8 PC/GE); 7.2% Triton X-100, 7.2% tributyl phosphate, 0.9% cetylpyridinium chloride57.6% Soybean oil, 1% EDTA, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride, 0.1 XPBS, and diH₂O (designated herein as 90% X8PC/GE EDTA); and 7.2% Triton X-100, 7.2% tributyl phosphate, 0.9% cetylpyridinium chloride57.6% Soybean oil, 1% sodium thiosulfate, 5 mM L-alanine, 5 mM inosine, 10 mM ammonium chloride, 0.1 XPBS, and diH₂O (designated herein as 90% X8PC/GE STS).

In a preferred embodiment of the invention, the nanoemulsion is non-toxic (e.g., for humans, plants or animals), non-irritating (e.g., for humans, plants or animals), and non-corrosive (e.g., for humans, plants or animals or the environment), while having efficacy against a wide range of microorganisms, including bacteria, fungi, viruses and spores. While many of the above-described nanoemulsions meet these qualifications, the following description provides many of the preferred non-toxic, non-irritating, non-corrosive, antimicrobial nanoemulsions of the present invention (hereinafter referred to in this section as "non-toxic nanoemulsions").

In some embodiments, the non-toxic nanoemulsion comprises surfactant lipid formulations (SLPs) for use as broad-spectrum antimicrobial agents, which are effective against bacteria and their spores, enveloped viruses, and fungi. In a preferred embodiment, these SLPs comprise a mixture of oil, detergent, solvent and a cationic halogen-containing compound plus several ions that enhance their biocidal activity. These SLPs are characterized by compounds that are stable, non-irritating and non-toxic compared to the highly irritating and/or toxic commercially available bactericidal and sporicidal agents.

Used in nontoxic nano emulsionIngredients used in (a) include, but are not limited to: detergents (e.g., TRITON X-100 (5-15%) or other member of the TRITON family, TWEEN 60 (0.5-2%) or other member of the TWEEN family, or TYLOXAPOL (1-10%)); solvents (e.g., tributyl phosphate (5-15%)); alcohols (e.g., ethanol (5-15%) or glycerol (5-15%)); oil (e.g., soybean oil (40-70%)); cationic halogen-containing compounds (e.g. cetylpyridinium chloride)(0.5-2%), hexadecyl pyridine bromide(0.5-2%)), or hexadecyldimethylethylammonium bromide (0.5-2%)); quaternary ammonium compounds (e.g., benzalkonium chloride (0.5-2%), N-alkyldimethylbenzyl ammonium chloride (0.5-2%)); ions (calcium chloride (1 mM-40 mM), ammonium chloride (1 mM-20 mM), sodium chloride (5 mM-200 mM), sodium phosphate (1 mM-20 mM)); nucleosides (e.g., inosine (50. mu.M-20 mM)); and amino acids (e.g., L-alanine (50. mu.M-20 mM)). The emulsion is prepared by mixing in a high shear mixer for 3-10 minutes. The emulsion may or may not be heated prior to mixing at 82 ℃ for 1 hour.

Quaternary ammonium compounds for use in the present invention include, but are not limited to, N-alkyldimethylbenzylsaccharinate ammonium; 1,3, 5-triazine-1, 3,5 (2H, 4H, 6H) -triethanol; 1-decaaminium, N-decyl-N, N-dimethyl chloride (or) didecyl dimethyl ammonium chloride; 2- (2- (p- (diisobutyl) tolyloxy (crossxy)) ethoxy) ethyldimethylbenzyl ammonium chloride; 2- (2- (p- (diisobutyl) phenoxy) ethoxy) ethyldimethylbenzyl ammonium chloride; alkyl 1 or 3 benzyl-1- (2-hydroxyethyl) -2-imidazolinium chlorides(ii) a Alkyl bis (2-hydroxyethyl) benzyl ammonium chloride; alkyl demethyl benzyl ammonium chloride; alkyl dimethyl 3, 4-dichlorobenzyl ammonium chloride (100% C12); alkyl bisMethyl 3, 4-dichlorobenzyl ammonium chloride (50% C14, 40% C12, 10% C16); alkyl dimethyl 3, 4-dichlorobenzyl ammonium chloride (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 (and) didecyl dimethyl ammonium chloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids); alkyl dimethyl benzyl ammonium chloride (C12-16); alkyl dimethyl benzyl ammonium chloride (C12-18); alkyl dimethyl benzyl and dialkyl dimethyl benzyl ammonium chlorides; alkyl dimethyl benzyl ammonium chloride; alkyl dimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyl dimethyl ethyl ammonium bromide (e.g., mixed alkyl and alkenyl in the fatty acids of soybean oil); alkyl dimethyl ethyl benzyl ammonium chloride; alkyl dimethyl ethyl benzyl ammonium chloride (60% C14); alkyl dimethyl isopropyl benzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3% C18); alkyltrimethylammonium chlorides (58% C18, 40% C16, 1% C14, 1% C12); alkyltrimethylammonium chlorides (90% C18, 10% C16); alkyldimethyl (ethylbenzyl) ammonium chloride (C12-18); di- (C8-10) -alkyl dimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkyl methyl benzyl ammonium chloride; didecyl bis Methyl ammonium chloride; diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammonium chloride; dodecyl bis (2-hydroxyethyl) octyl ammonium hydrochloride; dodecyl dimethyl benzyl ammonium chloride; dodecyl carbamoylmethyl dimethyl (dinethyl) benzyl ammonium chloride; heptadecyl hydroxyethyl imidazoline chloride(ii) a Hexahydro-1, 3, 5-tris (2-hydroxyethyl) -s-triazine; tetradecyldimethylbenzylammonium chloride (and) Quat RNIUM 14; n, N-dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethyl benzyl ammonium chloride; n-alkyl dimethyl ethyl benzyl ammonium chloride; n-tetradecyldimethylbenzylammonium chloride monohydrate; octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammonium chloride; octyl phenoxy ethoxy ethyl dimethyl benzyl ammonium chloride; oxydiethylene bis (alkyl dimethyl ammonium chloride); quaternary ammonium compounds, dicocoalkyl dimethyl chloride; trimethoxysilylpropyl dimethyloctadecyl ammonium chloride; trimethoxysilyl quats, trimethyldodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethyl benzyl ammonium chloride; n-hexadecyldimethylbenzyl ammonium chloride; n-tetradecyldimethylbenzylammonium chloride; n-tetradecyldimethylethylbenzyl ammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride.

In general, preferred non-toxic nanoemulsions are characterized by the following: they are about 200-800 nm in diameter, although larger and smaller diameter nanoemulsions are contemplated; the charge depends on the composition; they are stable for a relatively long period of time (e.g. up to 2 years), preserving their biocidal activity; they are non-irritating and non-toxic compared to their individual components, at least in part due to the oil component which significantly reduces the toxicity of detergents and solvents; they are effective at concentrations as low as 0.1%; they have antimicrobial activity (e.g., 99.99% kill) against most vegetative bacteria (including gram-positive and gram-negative organisms), fungi, and enveloped and non-enveloped viruses within 15 minutes; and when produced with germination enhancers, they have sporicidal activity (e.g., 99.99% kill) within 1-4 hours.

D. Animal model

In some embodiments, potential nanoemulsion compositions (e.g., for generating immune responses (e.g., for use as vaccines) are tested in animal models of infectious diseases.

Bacillus cereus (B.cereus)Bacillus cereus) (with Bacillus anthracis: (B.), (Bacillus anthracis) Closely related) animal models are used to test the anthrax vaccine of the present invention. The 2 bacteria are spore-forming gram-positive bacilli and the disease syndrome produced by each is largely due to toxin production and the effects of these toxins on the infected host (Brown et al, J. Bact., 75:499 (1958); Burdon and Wende, J. Infect Dis., 107:224 (1960); Burdon et al, J. Infect. Dis., 117:307 (1967)). Bacillus cereus infection mimics the disease syndrome caused by Bacillus anthracis. Mice are reported to rapidly succumb to the action of bacillus cereus toxin and are a useful model for acute infection. Guinea pigs develop skin lesions following subcutaneous infection with bacillus cereus, which resemble the cutaneous form of anthrax.

Clostridium perfringens (c) in mice and guinea pigsClostridium perfringens) Infection has been used as a model system for in vivo testing of antibiotic drugs (Stevens et al, antimicrob, Agents Chemother, 31:312 (1987); stevens et al, j. infect. dis., 155:220 (1987); alttemeier et al, Surgery, 28:621 (1950); sandusky et al, Surgery, 28:632 (1950)). Clostridium tetani (C.tetani) Clostridium tetani) Infections and diseases are caused in a variety of mammalian species. Mice, guinea pigs, and rabbits have all been experimentally generatedUse (Willis, Top and Wilson's Principles of Bacteriology, Virology and immunity. Wilson, G., A. Miles and M.T. Parker, eds., pp.442-475 1983).

Vibrio cholerae (Vibrio cholerae) Infection has been successfully initiated in mice, guinea pigs and rabbits. According to published reports, it is preferred to alter the normal intestinal bacterial flora for infection to be established in these experimental hosts. This is accomplished by administering antibiotics to inhibit the normal gut flora and, in some cases, withholding food from these animals (Butterton et al, Infect. Immun., 64:4373 (1996); Levine et al, Microbiol. Rev., 47:510 (1983); Finkelstein et al, J. Infect. Dis., 114:203 (1964); Freater, J. exp. Med., 104:411 (1956); and Freater, J. Infect. Dis., 97:57 (1955)).

Shigella flexneri: (Shigella flexnerii) Infection has been successfully initiated in mice and guinea pigs. As with Vibrio infection, it is preferred to alter the normal intestinal bacterial flora to aid in the establishment of infection in these experimental hosts. This is accomplished by administering antibiotics to inhibit normal gut flora and, in some cases, withholding food from these animals (Levine et al, microbiol. Rev., 47:510 (1983); Freater, J. exp. Med., 104:411 (1956); Formal et al, J. Bact., 85:119 (1963); LaBrec et al, J. Bact. 88:1503 (1964); Takeuchi et al, Am. J. Pathol., 47:1011 (1965)).

Mice and rats have been widely used to utilize Salmonella typhimurium (S.) (Salmonella typhimurium) And Salmonella enteritidis: (Salmonella enteriditis) In the experimental study of (Naughton et al, J. appl. Bact., 81:651 (1996); carter and Collins, j. exp. med., 139:1189 (1974); collins, input. Immun., 5:191 (1972); collins and Carter, feed. Immun., 6:451 (1972)).

The mouse and rat are prepared from XIANTAI (a mixture of XIANTAI and XIANTAI)Sendai) Well established experimental models of viral infection (Jacoby et al, exp. Geronto)l., 29:89 (1994); massion et al, Am. J. Respir. Cell mol. biol. 9:361 (1993); castleman et al, Am. J. Path, 129:277 (1987); castleman, Am. J. ve. Res., 44:1024 (1983); mims and Murphy, Am. J. Path, 70:315 (1973)).

Mouse sindbis: (Sindbis) Viral infection is usually accomplished by intracerebral inoculation of neonatal mice. Alternatively, weaned mice are inoculated subcutaneously in the paw pad (Johnson et al, J. Infect. Dis., 125:257 (1972); Johnson, Am. J. Path., 46:929 (1965)).

Animals are preferably housed (house) for 3-5 days to recover from transport and to adapt to the new housing environment prior to use in the experiment. At the beginning of each experiment, control animals were sacrificed and tissues harvested to establish baseline parameters. Animals are anesthetized by any suitable method, such as, but not limited to, inhalation of isoflurane (isoflurane) for short procedures or ketamine/xylazine injection for longer procedures.

E. Assays for vaccine evaluation

In some embodiments, candidate nanoemulsion vaccines are evaluated using one of several suitable model systems. For example, cell-mediated immune responses can be evaluated in vitro. In addition, animal models can be used to evaluate in vivo immune responses and immunity to pathogen challenge. Any suitable animal model may be utilized including, but not limited to, those disclosed in table 3.

Before testing nanoemulsion vaccines in animal systems, the exposure of pathogens to an amount of nanoemulsion sufficient to inactivate the pathogen is studied. It is expected that pathogens such as bacterial spores require a longer period of time for inactivation by the nanoemulsion to neutralize sufficiently to allow immunization. The time period required for inactivation may be studied using any suitable method, including but not limited to those described in the illustrative examples below.

In addition, the stability of the emulsion developed vaccine, particularly over a range of time and storage conditions, was evaluated to ensure that the vaccine was effective for a long period of time. The ability of other stabilizing materials (e.g., dendrimers) to enhance vaccine stability and immunogenicity was also evaluated.

Once a given nanoemulsion/pathogen vaccine has been formulated to result in pathogen inactivation, the ability of the vaccine to elicit an immune response and provide immunity is optimized. Non-limiting examples of methods for determining the effectiveness of a vaccine are described in examples 1-4 below. For example, the timing and dosage of the vaccine can be varied, and the most effective dose and schedule of administration determined. The level of immune response is quantified by measuring serum antibody levels. In addition, in vitro assays were performed by measuring H ³Thymidine uptake is used to monitor proliferative activity. In addition to proliferation, Th1 and Th2 cytokine responses (e.g., including but not limited to, IL-2, TNF-gamma, IFN-gamma, IL-4, IL-6, IL-11, IL-12, etc. levels) were measured to quantitatively assess immune responses.

Finally, animal models were used to evaluate the effect of nanoemulsion mucosal vaccines. The purified pathogen is mixed in an emulsion (or the emulsion is contacted with a pre-infected animal), administered, and the immune response is measured. The level of protection is then assessed by challenging the animal with a particular pathogen and then assessing the level of disease symptoms. The level of immunity over time was measured to determine the necessity and interval of booster immunizations.

Treatment and prevention of

Furthermore, in preferred embodiments, the compositions of the invention induce (e.g., when administered to a subject) systemic and mucosal immunity. Thus, in some preferred embodiments, administration of a composition of the invention to a subject results in protection against exposure (e.g., mucosal exposure) to RSV. Mucosal administration (e.g., vaccination) provides protection against RSV infection (e.g., one initiated at a mucosal surface), although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action. Although it has proven difficult to stimulate secretory IgA responses and protection against pathogens invading at Mucosal surfaces to date (see, e.g., Mestecky et al, Mucosal immunology, 3 ed. (Academic Press, San Diego, 2005)), in some embodiments, the present invention provides compositions and methods for stimulating Mucosal immunity (e.g., a protective IgA response) from a pathogen in a subject.

In some embodiments, this material can be readily produced with NE and M2, F proteins, and/or other proteins/peptides (e.g., virus-derived proteins, live virus vector-derived proteins, recombinant denatured proteins/antigens, small peptide segment proteins/antigens, and induce mucosal and systemic immunity).

In some preferred embodiments, the present invention provides compositions for generating an immune response comprising NE and an immunogen (e.g., a purified, isolated or synthetic protein or derivative, variant or analog thereof; or one or more serotypes of RSV inactivated by nanoemulsion). When administered to a subject, the compositions of the invention stimulate an immune response against the immunogen in the subject. Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, generation of an immune response (e.g., resulting from administration of a composition comprising a nanoemulsion and an immunogen) provides overall or partial immunity to a subject (e.g., from signs, symptoms, or conditions of a disease (e.g., RSV)). Without wishing to be bound by any particular theory, protection from and/or immunity to a disease (e.g., the ability of the subject's immune system to prevent or attenuate (e.g., suppress) signs, symptoms, or conditions of the disease) following exposure to an immunogenic composition of the invention is due to an adaptive (e.g., acquired) immune response (e.g., an immune response mediated by B and T cells (e.g., an immune response that exhibits increased specificity and reactivity against RSV) following exposure to an immunogen-containing NE of the invention.

In some embodiments, NE comprising an immunogen (e.g., recombinant RSV protein) is administered alone. In some embodiments, a composition comprising NE and an immunogen (e.g., recombinant RSV protein) comprises one or more additional agents (e.g., a pharmaceutically acceptable carrier; an adjuvant, an excipient, etc.). In some embodiments, the compositions for stimulating an immune response of the present invention are administered in a manner that induces a humoral immune response. In some embodiments, the compositions of the invention for stimulating an immune response are administered in a manner that induces a cellular (e.g., cytotoxic T lymphocyte) immune response rather than a humoral response. In some embodiments, the compositions of the invention comprising NE and an immunogen induce cellular and humoral immune responses.

The invention is not limited by the type or strain of virus of the paramyxoviridae family (e.g., paramyxoviridae virus (e.g., paramyxovirus, mumps virus, and/or morbillivirus) and/or pneumovirinae virus (e.g., respiratory syncytial virus))) used in the composition comprising NE and an immunogen (e.g., RSV inactivated by nanoemulsion). Indeed, each paramyxoviridae member, alone or in combination with another family member, may be used to generate a composition of the invention comprising NE and an immunogen (e.g. for generating an immune response). In some embodiments, the virus is RSV strain a2 (available from ATCC, Manassas, VA, ATCC accession No. VR-1540). In some embodiments, the virus is RSV strain B (B WV/14617/85, ATCC accession No. VR-1400). In some embodiments, the virus is RSV strain 9320 (ATCC accession No. VR-955). In some embodiments, the virus is RSV strain 18537 (ATCC accession No. VR-1580). In some embodiments, the virus is RSV strain Long (ATCC accession number VR-26). In some embodiments, the virus is RSV strain Line 19 (see, e.g., Lukacs et al, immunology and Infection, 169, 977-986 (2006)). Thus, in some embodiments, the virus (e.g., RSV) strain utilized is a modified (e.g., genetically modified (e.g., naturally modified via natural selection or modified using recombinant genetic techniques)) strain that displays greater pathogenic capability (e.g., causing more severe RSV-induced disease (e.g., comprising enhanced airway hyperreactivity and/or mucus overproduction)). In some embodiments, any member of a member of the family paramyxoviridae is used in the immunoreactive composition of the invention, including, but not limited to, paramyxovirus, mumps virus, measles virus, and respiratory syncytial virus, among others. The invention is not limited by the virus strain used. Indeed, a variety of viral strains are contemplated to be useful in the present invention, including but not limited to classical strains, attenuated strains, non-replicating strains, modified strains (e.g., genetically or mechanically modified strains (e.g., to become more or less virulent)), or other serially diluted strains of the virus. The composition comprising NE and immunogen may comprise one or more strains of RSV and/or other types of viruses of the paramyxoviridae family. In addition, a composition comprising NE and an immunogen may comprise one or more strains of RSV plus one or more strains of a non-RSV viral immunogen.

In some embodiments, the immunogen may comprise one or more antigens derived from a pathogen (e.g., RSV). For example, in some embodiments, the immunogen is a purified, recombinant, synthetic, or otherwise isolated protein (e.g., added to NE to produce an immunogenic composition). Similarly, the immunogenic protein may be a derivative, analog or otherwise modified (e.g., pegylated) form of a protein from a pathogen.

The present invention is not limited by the particular formulation of the composition of the present invention comprising NE and the immunogen. Indeed, a composition of the invention comprising NE and an immunogen may comprise one or more different agents plus NE and immunogen. Such agents or cofactors include, but are not limited to, adjuvants, surfactants, additives, buffers, solubilizers, chelating agents, oils, salts, therapeutic agents, drugs, bioactive agents, antibacterial agents, and antimicrobial agents (e.g., antibiotics, antiviral agents, etc.). In some embodiments, the compositions of the invention comprising NE and an immunogen comprise an agent and/or cofactor (e.g., adjuvant) that enhances the ability of the immunogen to induce an immune response. In some preferred embodiments, the presence of one or more cofactors or agents reduces the amount of immunogen required for induction of an immune response, e.g., a protective immune response (e.g., protective immunization). In some embodiments, the presence of one or more cofactors or agents may be used to bias an immune response towards a cellular (e.g., T cell-mediated) or humoral (e.g., antibody-mediated) immune response. The invention is not limited by the type of cofactor or agent used in the therapeutic agents of the present invention.

Adjuvants are generally described in Vaccine Design- -the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995. The invention is not limited by the type of adjuvant utilized, e.g., for use in a composition (e.g., a pharmaceutical composition) comprising NE and an immunogen. For example, in some embodiments, suitable adjuvants include aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate. In some embodiments, the adjuvant may be a calcium, iron or zinc salt, or may be an insoluble suspension of acylated tyrosine or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes.

In some embodiments, it is preferred that the composition comprising NE and immunogen of the present invention comprises one or more adjuvants which induce a Th1 type response. However, in other embodiments it will be preferred that the composition comprising NE and immunogen of the present invention comprises one or more adjuvants which induce a Th2 type response.

In general, an immune response to an antigen is generated by the interaction of the antigen with cells of the immune system. Immune responses can be broadly classified into 2 categories: humoral and cell-mediated immune responses (e.g., traditionally characterized by antibody and cellular effector protection mechanisms, respectively). These classes of responses have been termed Th 1-type responses (cell-mediated responses) and Th 2-type immune responses (humoral responses).

Stimulation of the immune response may result from a direct or indirect response of cells or components of the immune system to an intervention (e.g., exposure to an immunogen). Immune responses can be measured in a number of ways, including activation, proliferation, or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells, etc.); up-or down-regulation of expression of markers and cytokines; stimulation of IgA, IgM, or IgG titers; splenomegaly (including increased splenocyte formation); proliferation and mixed cell infiltration in various organs. Other responses, cells, and components of the immune system that can be evaluated in terms of immune stimulation are known in the art.

Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, the compositions and methods of the invention induce the expression and secretion of cytokines (e.g., by macrophages, dendritic cells, and CD4+ T cells). Modulation of expression of a particular cytokine may occur locally or systemically. It is known that cytokine profiles may determine T cell regulation and effector functions in immune responses. In some embodiments, a Th1 type cytokine may be induced, and thus the immunostimulatory composition of the invention may promote a Th1 type antigen-specific immune response, including cytotoxic T cells (e.g., to avoid an unwanted Th2 type immune response (e.g., production of a Th2 type cytokine (e.g., IL-13) is associated with enhancing the severity of the disease (e.g., IL-13 induction of mucus formation)).

Cytokines play a role in directing T cell responses. Helper (CD 4 +) T cells coordinate the immune response of mammals through the production of soluble factors that act on other cells of the immune system, including B and other T cells. Most mature CD4+ T helper cells express one of 2 cytokine profiles: th1 or Th 2. Th1 type CD4+ T cells secrete IL-2, IL-3, IFN-gamma, GM-CSF and high levels of TNF-alpha. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF and low levels of TNF- α. Th 1-type cytokines promote cell-mediated immunity, and humoral immunity characterized by immunoglobulin class switching to IgG2a in mice and to IgG1 in humans. Th1 responses may also be associated with delayed type hypersensitivity and autoimmune diseases. Th 2-type cytokines mainly induce humoral immunity and induce class switching to IgG1 and IgE. Antibody isotypes associated with Th1 responses generally have neutralizing and opsonizing abilities, while those associated with Th2 responses are more associated with allergic responses.

Several factors have been shown to affect the bias of immune responses towards either Th1 or Th2 type responses. The best characterized modulator is a cytokine. IL-12 and IFN-gamma are positive Th1 and negative Th2 modulators. IL-12 promotes IFN-gamma production, and IFN-gamma provides positive feedback for IL-12. IL-4 and IL-10 appear to be important for the establishment of a Th2 cytokine profile and for down-regulation of Th1 cytokine production.

Accordingly, in a preferred embodiment, the present invention provides a method of stimulating a Th 1-type immune response in a subject comprising administering to the subject a composition comprising NE and an immunogen. However, in other embodiments, the invention provides methods of stimulating a Th 2-type immune response (e.g., if a balance of T cell-mediated responses is desired) in a subject, comprising administering to the subject a composition comprising NE and an immunogen. In further preferred embodiments, adjuvants may be used (e.g. may be co-administered with the compositions of the invention) to bias the immune response towards a Th1 or Th2 type immune response. For example, adjuvants that induce a Th2 or weak Th1 response include, but are not limited to, alum, saponin, and SB-As 4. Adjuvants that induce Th1 responses include, but are not limited to, MPL, MDP, ISCOMS, IL-12, IFN- γ, and SB-AS 2.

Several other types of Th 1-type immunogens can be used (e.g., as adjuvants) in the compositions and methods of the present invention. These include, but are not limited to, the following. In some embodiments, monophosphoryl lipid A (e.g., in particular 3-de-O-acylated monophosphoryl lipid A (3D-MPL)) is used. 3D-MPL is a well-known adjuvant manufactured by Ribi Immunochem, Montana. Chemically, it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A having 4, 5 or 6 acylated chains. In some embodiments, diphosphoryl lipid a and 3-O-deacylated variants thereof are used. Each of these immunogens can be purified and prepared by the methods described in GB 2122204B, which is incorporated herein by reference in its entirety. Other purified and synthetic lipopolysaccharides have been described (see, e.g., U.S. Pat. Nos. 6,005,099 and EP 0729473; Hilgers et al, 1986, int. Arch. allergy. Immunol., 79 (4): 392-6; Hilgers et al, 1987, Immunology, 60 (1): 141-6; and EP 0549074, each of which is incorporated herein by reference in its entirety). In some embodiments, 3D-MPL is used in the form of a particulate formulation (e.g., having a small particle size of less than 0.2 μm in diameter, as described in EP 0689454, which is incorporated herein by reference in its entirety).

In some embodiments, saponins are used as immunogens (e.g., Th 1-type adjuvants) in the compositions of the present invention. Saponins are well known adjuvants (see, e.g., Lacaille-Dubois and Wagner (1996) phytomedine, Vol.2, pp.363-386). Examples of saponins include Quil A (bark derived from the south America tree Quillaja Saponaria Molina), and fractions thereof (see, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev the Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0362279, each of which is incorporated herein by reference in its entirety). Also contemplated as useful in the present invention are lysosaponins QS7, QS17 and QS21 (HPLC purified fractions of Quil A; see, e.g., Kensil et al (1991) J. Immunology 146,431-437, U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0362279, each of which is incorporated herein by reference in its entirety). Also contemplated to be useful are combinations of QS21 and polysorbates or cyclodextrins (see, e.g., WO 99/10008, incorporated herein by reference in its entirety.

In some embodiments, immunogenic oligonucleotides containing unmethylated CpG dinucleotides ("CpG") are used as adjuvants in the present invention. CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. CpG, when administered by systemic and mucosal routes, is known in the art as an adjuvant (see, e.g., WO 96/02555, EP 468520, Davis et al, J.Immunol, 1998, 160 (2): 870-876; McCluskie and Davis, J.Immunol., 1998, 161 (9): 4463-6; and U.S. patent application No. 20050238660, each of which is incorporated herein by reference in its entirety). For example, in some embodiments, the immunostimulatory sequence is a purine-C-G-pyrimidine; wherein the CG motif is not methylated.

Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, the presence of one or more CpG oligonucleotides activates various immune subpopulations, including natural killer cells (which produce IFN- γ) and macrophages. In some embodiments, CpG oligonucleotides are formulated into compositions of the invention for inducing an immune response. In some embodiments, a free solution of CpG is co-administered with an antigen (e.g., present in a NE solution (see, e.g., WO 96/02555; incorporated herein by reference.) in some embodiments, a CpG oligonucleotide is covalently conjugated to an antigen (see, e.g., WO 98/16247, incorporated herein by reference), or formulated with a carrier such as aluminum hydroxide (see, e.g., Brazolot-Millan et al, Proc. Natl. Acadsi., USA, 1998, 95 (26), 15553-8).

In some embodiments, adjuvants such as complete and incomplete freund' S adjuvants, cytokines (e.g., interleukins (e.g., IL-2, IFN- γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.), detoxified mutants of bacterial ADP-ribosylating toxins, such as Cholera Toxin (CT), Pertussis Toxin (PT), or e.coli (e.coli) heat Labile Toxin (LT), particularly LT-K63 (in which a lysine is substituted for the wild-type amino acid at position 63), LT-R72 (in which an arginine is substituted for the wild-type amino acid at position 72), CT-S109 (in which a serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (in which a lysine is substituted for the wild-type amino acid at position 9 and a glycine is substituted for position 129) (see, for example, WO93/13202 and WO92/19265, each of which is incorporated herein by reference), and other immunogenic substances (e.g., those that enhance the effectiveness of the compositions of the invention) are used with the NE and immunogen-containing compositions of the invention.

Further examples of adjuvants useful in the present invention include poly (di (carboxyphenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA), lipopolysaccharides such as derivatives of monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl muramyl dipeptide (t-MDP; Ribi), OM-174 (glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland), and Leishmania (Leishmania) elongation factor (purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

The adjuvant may be added to the composition comprising NE and the immunogen or the adjuvant may be formulated with a carrier such as a liposome or a metal salt (e.g., an aluminum salt (e.g., aluminum hydroxide)) prior to combination or co-administration with the composition comprising NE and the immunogen.

In some embodiments, the composition comprising NE and the immunogen comprises a single adjuvant. In other embodiments, the composition comprising NE and the immunogen comprises 2 or more adjuvants (see, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241; and WO 94/00153, each of which is incorporated herein by reference in its entirety).

In some embodiments, the compositions of the invention comprising NE and an immunogen comprise one or more mucoadhesives (see, e.g., U.S. patent application No. 20050281843, which is incorporated herein by reference in its entirety). The present invention is not limited by the type of mucoadhesive utilized. Indeed, a variety of mucoadhesives are contemplated to be useful in the present invention, including but not limited to cross-linked derivatives of poly (acrylic acid) (e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides (e.g., alginate and chitosan), hydroxypropyl methylcellulose, lectins, pilin, and carboxymethyl cellulose. Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, the use of a mucoadhesive (e.g., in a composition comprising NE and an immunogen) enhances induction of an immune response in a subject (e.g., administered a composition of the invention) due to an increase in the duration and/or amount of exposure to the immunogen experienced by the subject when using the mucoadhesive as compared to the duration and/or amount of exposure to the immunogen in the absence of use of the mucoadhesive.

In some embodiments, the compositions of the present invention may comprise a sterile aqueous formulation. Acceptable carriers and solvents include, but are not limited to, water, ringer's solution, phosphate buffered saline, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed mineral or non-mineral oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Suitable carrier formulations for mucosal, subcutaneous, intramuscular, intraperitoneal, intravenous or via other routes of administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa..

The compositions of the invention comprising NE and an immunogen may be used therapeutically (e.g. to enhance an immune response) or as a prophylactic agent (e.g. for vaccination (e.g. to prevent signs or symptoms of disease)). The compositions of the invention comprising NE and immunogen may be administered to a subject via a number of different delivery routes and methods.

For example, the compositions of the present invention can be administered to a subject (e.g., mucosa (e.g., nasal mucosa, vaginal mucosa, etc.)) by a variety of methods, including but not limited to: suspended in a solution and applied to a surface; suspended in a solution and sprayed onto a surface using a spray applicator; mixed with a mucoadhesive and applied (e.g., sprayed or wiped) onto a surface (e.g., a mucosal surface); placed on or impregnated into a nasal and/or vaginal applicator and applied; applied by a controlled release mechanism; use as liposomes; or in polymers.

In some preferred embodiments, The compositions of The invention are administered mucosally (e.g., using standard techniques; see, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19 th edition, 1995 (e.g., for mucosal delivery techniques, including intranasal, pulmonary, vaginal, and rectal techniques), and European publication No. 517,565 and Illum et al, J. Controlled Rel., 1994, 29:133-141 (e.g., techniques for intranasal administration), each of which is incorporated herein by reference in its entirety). Alternatively, The compositions of The present invention may be applied dermally or transdermally using standard techniques (see, e.g., Remington: The Science arm Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19 th edition, 1995). The present invention is not limited by the route of administration.

Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments mucosal vaccination is the preferred route of administration, as mucosal administration of antigens has been shown to have greater efficacy in inducing protective immune responses (e.g., mucosal immunity) at mucosal surfaces-the entry route for many pathogens. In addition, mucosal vaccination, such as intranasal vaccination, can induce mucosal immunity not only in the nasal mucosa, but also in remote mucosal sites such as the genital mucosa (see, e.g., Mestecky, Journal of Clinical Immunology, 7:265-276, 1987). More advantageously, in a further preferred embodiment, the mucosal vaccination induces systemic immunity in addition to the induction of a mucosal immune response. In some embodiments, non-parenteral administration (e.g., mucosal administration of a vaccine) provides an effective and convenient means of boosting systemic immunity (e.g., induced by parenteral or mucosal vaccination (e.g., in cases where multiple boosting is used to maintain robust systemic immunity)).

In some embodiments, the compositions of the invention comprising NE and an immunogen may be used to protect or treat a subject susceptible to or suffering from a disease by administering the compositions of the invention via a mucosal route (e.g., oral/dietary or nasal route). Alternative mucosal routes include intravaginal and intrarectal routes. In a preferred embodiment of the invention, the nasal route of administration is used, referred to herein as "intranasal administration" or "intranasal vaccination". Methods of intranasal vaccination are well known in the art and include the administration of the vaccine in droplet or spray form into the nasopharynx of the subject to be vaccinated. In some embodiments, an aerosolized or aerosolized composition comprising NE and an immunogen is provided. Enteral formulations for oral administration, such as gastro-resistant capsules, suppositories for rectal or vaginal administration also form part of the invention. The compositions of the present invention may also be administered via the oral route. In these circumstances, the composition comprising NE and immunogen may comprise a pharmaceutically acceptable excipient and/or comprise an alkaline buffer or enteric capsule. Formulations for nasal delivery may include those with dextran or cyclodextrin (cyclodextran) and saponin as adjuvants.

The compositions of the invention may also be administered via the vaginal route. In such a case, the composition comprising NE and immunogen may comprise pharmaceutically acceptable excipients and/or emulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers for vaginal creams and suppositories. In some embodiments, the compositions of the present invention are administered via the rectal route. In such cases, the composition comprising NE and immunogen may comprise excipients and/or waxes and polymers known in the art for forming rectal suppositories.

In some embodiments, the same route of administration (e.g., mucosal administration) is selected for priming and boosting. In some embodiments, multiple routes of administration (e.g., simultaneously or alternatively sequentially) are utilized to stimulate an immune response (e.g., using a composition of the invention comprising NE and an immunogen).

For example, in some embodiments, a composition comprising NE and an immunogen is administered to a mucosal surface of a subject in a prime or boost vaccination regimen. Alternatively, in some embodiments, the composition comprising NE and the immunogen is administered systemically in a prime or boost vaccination regimen. In some embodiments, the composition comprising NE and immunogen is administered to the subject via mucosal administration in a priming vaccination regimen and via systemic administration in a boosting regimen. In some embodiments, the composition comprising NE and immunogen is administered to the subject via mucosal administration in a priming vaccination regimen via a systemic administration and a boosting regimen. Examples of systemic routes of administration include, but are not limited to, parenteral, intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal, or intravenous administration. Compositions comprising NE and an immunogen may be used for prophylactic and therapeutic purposes.

In some embodiments, the compositions of the invention are administered by pulmonary delivery. For example, compositions of the invention may be delivered to the lungs of a subject (e.g., a human) via inhalation (e.g., thereby passing through the epithelial lining of the lungs to the bloodstream (see, e.g., Adjei et al, Pharmaceutical Research 1990; 7: 565-569; Adjei et al, int. J. pharmaceuticals 1990; 63: 135-144; Braquet et al, J. Cardiovasular Pharmacology 1989143-146; Huard et al (1989) Annals of interstitial Medicine, Vol. III, pp. 206-212; Smith et al, J. Clin. invest. 1989; 84: 1145-1146; Oswein et al, "Aerosolvozation of Proteins", 1990; Proceedings of sympository on Drug Delivery, II Keoto, Coloro et al; Demuso et al, J. incorporated herein by reference to the entire US patent No. 3582; incorporated herein by reference for systemic Drug Delivery; see, e.g. 3, et al, incorporated herein by reference, for systemic Drug Delivery, 2. A. 7. A. Delivery of the invention is also incorporated herein incorporated by reference to the general methods of A. A U.S. patent No. 6,651,655 to licaalsi et al)).

Further contemplated for use in the practice of the present invention are a wide range of mechanical devices designed for pulmonary and/or nasal mucosal delivery of pharmaceutical agents, including but not limited to sprinklers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available equipment suitable for the practice of the present invention are Ultravent sprinklers (Mallinckrodt inc., St. Louis, Mo.); acorn II sprinklers (Marquest Medical Products, Englewood, Colo.); ventolin metered dose inhalers (Glaxo inc., Research Triangle Park, N.C.); and Spinhaler powder inhalers (Fisons Corp., Bedford, Mass.). All such devices require the use of a formulation suitable for the dispensing of the therapeutic agent. Generally, each formulation is specific to the type of device employed and may involve the use of a suitable propellant material, plus usual diluents, adjuvants, surfactants, carriers and/or other agents useful in the treatment. Also, the use of liposomes, microcapsules or spheroids, inclusion complexes or other types of carriers is contemplated.

Thus, in some embodiments, the compositions of the invention comprising NE and an immunogen may be used to protect or treat a subject susceptible to or suffering from a disease by administering the composition comprising NE and the immunogen via mucosal, intramuscular, intraperitoneal, intradermal, transdermal, pulmonary, intravenous, subcutaneous, or other routes of administration described herein. Methods of systemic administration of vaccine formulations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (see, e.g., WO 99/27961, incorporated herein by reference), or needleless pressurized liquid jet devices (see, e.g., U.S. patent No. 4,596,556; U.S. patent No. 5,993,412, each of which is incorporated herein by reference), or transdermal patches (see, e.g., WO 97/48440; WO 98/28037, each of which is incorporated herein by reference). The present invention may also be used to enhance the immunogenicity of antigens applied to the skin (transdermal or transdermal delivery, see, e.g., WO 98/20734; WO 98/28037, each of which is incorporated herein by reference). Thus, in some embodiments, the present invention provides a delivery device for systemic administration that is pre-filled with the vaccine composition of the present invention.

The invention is not limited by the type of subject to whom the compositions of the invention are administered, e.g., to stimulate an immune response, e.g., to generate protective immunity (e.g., mucosal and/or systemic immunity). Indeed, a wide variety of subjects are expected to benefit from administration of the compositions of the invention. In a preferred embodiment, the subject is a human. In some embodiments, the human subject is of any age (e.g., adult, child, infant, etc.), who has been or may become exposed to a microorganism (e.g., RSV). In some embodiments, a human subject is one that is more likely to receive direct exposure to a pathogenic microorganism or to exhibit signs and symptoms of disease after exposure to a pathogen (e.g., immunosuppressed subjects). In some embodiments, the compositions of the present invention are administered to (e.g., vaccinated with) the public (e.g., to prevent the occurrence or spread of disease). For example, in some embodiments, the compositions and methods of the invention are used to vaccinate a population of humans (e.g., a population of regions, cities, states, and/or countries) for their own health (e.g., to prevent or treat disease). In some embodiments, the subject is a non-human mammal (e.g., a pig, cow, goat, horse, sheep or other livestock; or a mouse, rat, guinea pig or other animal). In some embodiments, the compositions and methods of the invention are used in a research setting (e.g., with a research animal).

The compositions of the invention may be formulated for administration by any route, for example mucosal, oral, topical, parenteral, or other routes described herein. The composition may be in any one or more of a variety of forms including, but not limited to, tablets, capsules, powders, granules, lozenges, foams, creams, or liquid formulations.

The topical formulations of the present invention may be presented as, for example, ointments, creams or lotions, foams and aerosols, and may contain suitable conventional additives such as preservatives, solvents (e.g., to aid penetration) and emollients in ointments and creams.

Topical formulations may also include agents that enhance the penetration of the active ingredient through the skin. Exemplary agents include binary combinations of N- (hydroxyethyl) pyrrolidone and cell envelope disordering compounds, sugar esters in combination with sulfoxides or phosphine oxides, and sucrose monooleate, decyl methyl sulfoxide, and alcohols.

Other exemplary materials that increase skin penetration include surfactants or wetting agents, including but not limited to polyoxyethylene sorbitan monooleate (polysorbate 80); sorbitan monooleate (Span 80); p-isooctylpolyoxyethylene-phenol polymer (Triton WR-1330); polyoxyethylene sorbitan trioleate (Tween 85); dioctyl sodium sulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and other pharmaceutically acceptable surfactants.

In certain embodiments of the present invention, the composition may further comprise one or more alcohols, zinc-containing compounds, emollients, humectants, thickening and/or gelling agents, neutralizing agents and surfactants. The water used in the formulation is preferably deionized water having a neutral pH. Additional additives in topical formulations include, but are not limited to, silicone fluids, dyes, fragrances, pH adjusters, and vitamins.

The topical formulations may also contain compatible conventional carriers such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present in about 1% up to about 98% of the formulation. The ointment base may comprise one or more of petrolatum, mineral oil, ozokerite wax, lanolin alcohol, panthenol, glycerin, bisabolol, cocoa butter, and the like.

In some embodiments, the pharmaceutical compositions of the present invention may be formulated and used as a foam. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies, and liposomes. Although substantially similar in properties, these formulations differ in the composition and consistency of the final product.

The compositions of the present invention may additionally contain other additional components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically active materials, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, preferably do not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulation may be sterile and, if desired, mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and/or fragrances, etc.) that do not deleteriously interact with the NE and immunogen of the formulation. In some embodiments, the immunostimulatory composition of the invention is administered in the form of a pharmaceutically acceptable salt. When used, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulfonic acids. Also, such salts may be prepared as alkali metal or alkaline earth salts, for example sodium, potassium or calcium salts of carboxylic acid groups.

Suitable buffering agents include, but are not limited to, acetic acid and salts (1-2% w/v); citric acid and salts (1-3% w/v); boric acid and salts (0.5-2.5% w/v); and phosphoric acid and salts (0.8-2% w/v). Suitable preservatives may include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

In some embodiments, the composition comprising NE and the immunogen is co-administered with one or more antibiotics. For example, one or more antibiotics may be administered with, before, and/or after administration of the composition comprising NE and the immunogen. The invention is not limited by the type of antibiotic co-administered. Indeed, multiple antibiotics may be co-administered, including but not limited to beta-lactam antibiotics, penicillins (e.g., natural penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxypenicillins, ureidopenicillins), cephalosporins (first, second and third generation cephalosporins), and other beta-lactams (e.g., imipenems, monobactams), beta-lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinolines.

There are a large number of antimicrobial agents currently available for use in the treatment of bacterial, fungal and viral infections. For extensive papers on The general class of such drugs and their mechanism of action, The skilled person refers to Goodman & Gilman's "The Pharmacological Basis of Therapeutics" editor Hardman et al, 9 th edition, pub. McGraw Hill, chapters 43 to 50, 1996, which is incorporated herein by reference in its entirety. Typically, these agents include agents that inhibit cell wall synthesis (e.g., penicillins, cephalosporins, cycloserines, vancomycin, bacitracin); and imidazole antifungal agents (e.g., miconazole, ketoconazole, and clotrimazole); agents that act directly on the cell membrane of the disrupted microorganism (e.g., detergents such as polymyxin (polmyxin) and colistin M and the antifungal agents nystatin and amphotericin B); agents that affect the ribosomal subunit to inhibit protein synthesis (e.g., chloramphenicol, tetracycline, erythromycin (erthromycin), and clindamycin); agents that alter protein synthesis and cause cell death (e.g., aminoglycosides); agents that affect nucleic acid metabolism (e.g., rifamycin and quinolones); antimetabolites (e.g., trimethoprim and sulfonamides); and nucleic acid analogs such as zidovudine, ganciclovir (gancyclovir), vidarabine and acyclovir, which act to inhibit viral enzymes essential for DNA synthesis. Various combinations of antimicrobial agents may be employed.

The invention also includes methods involving co-administration of a composition comprising NE and an immunogen with one or more additional active and/or immunostimulatory agents (e.g., a composition comprising NE and a different immunogen, an antibiotic, an antioxidant, etc.). Indeed, a further aspect of the invention is to provide a method for enhancing prior art immunostimulatory methods (e.g. vaccination) and/or pharmaceutical compositions by co-administering the compositions of the invention. In a co-administration procedure, the agents may be administered simultaneously or sequentially. In one embodiment, the compositions described herein are administered prior to one or more other active agents. The pharmaceutical formulation and mode of administration may be any of those described herein. In addition, 2 or more co-administered agents may each be administered using a different mode (e.g., route) or different formulation. The additional agent(s) (e.g., antibiotic, adjuvant, etc.) to be co-administered may be any of those well known in the art, including but not limited to those currently in clinical use.

In some embodiments, the composition comprising NE and immunogen is administered to the subject via more than one route. For example, a subject who would benefit from having a protective immune response (e.g., immunity) against a pathogenic microorganism may benefit from receiving mucosal administration (e.g., nasal administration or other mucosal routes described herein), and additionally from one or more other routes of administration (e.g., parenteral or pulmonary administration (e.g., via a nebulizer, inhaler, or other methods described herein) Parenterally) a subject administered a composition of the invention may have a stronger immune response against the immunogen than a subject administered the composition via only one route.

Other delivery systems may include time-release, delayed release, or sustained release delivery systems. Such a system may avoid repeated administration of the composition, thereby increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer-based systems such as poly (lactide-co-glycolide), copolyoxalates, polycaprolactone, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the aforementioned drugs containing polymers are described, for example, in U.S. Pat. No. 5,075,109, incorporated herein by reference. The delivery system further comprises a non-polymeric system which is: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-and triglycerides; a hydrogel release system; the sylastic system; a peptide-based system; coating with wax; tableting agents using conventional binders and excipients; a partially fused implant; and the like. Specific examples include, but are not limited to: (a) erosion systems in which the agents of the invention are contained in a matrix, such as those described in U.S. Pat. nos. 4,452,775, 4,675,189, and 5,736,152, each of which is incorporated herein by reference in its entirety, and (b) diffusion systems in which the active component permeates from the polymer at a controlled rate, such as those described in U.S. Pat. nos. 3,854,480, 5,133,974, and 5,407,686, each of which is incorporated herein by reference in its entirety. In addition, pump-based hardware delivery systems may be used, some of which are suitable for implantation.

In a preferred embodiment, the composition comprising NE and the immunogen of the present invention comprises a suitable amount of the immunogen to induce an immune response in a subject when administered to the subject. In preferred embodiments, the immune response is sufficient to provide protection (e.g., immunoprotection) to the subject against subsequent exposure to the immunogen or a microorganism (e.g., bacteria or virus) from which the immunogen is derived. The present invention is not limited by the amount of immunogen used. In some preferred embodiments, the amount of immunogen (e.g., viral or bacterial, or recombinant protein neutralized by NE) in a composition comprising NE and immunogen (e.g., for use as an immunizing dose) is selected as an amount that induces an immune protective response without significant adverse side effects. The amount will vary depending on the particular immunogen employed or combination thereof, and may vary from subject to subject, depending on a number of factors, including but not limited to the species, age, and general condition (e.g., health) of the subject, and the mode of administration. Procedures for determining the appropriate amount of immunogen to administer to a subject to elicit an immune response (e.g., a protective immune response (e.g., protective immunity)) in the subject are well known to those skilled in the art.

In some embodiments, it is contemplated that each dose (e.g., of a composition comprising NE and immunogen (e.g., administered to a subject to induce an immune response (e.g., a protective immunity)) comprises 0.05 to 5000 μ g of each immunogen (e.g., recombinant and/or purified protein), in some embodiments, each dose will comprise 1 to 500 μ g, in some embodiments, each dose will comprise 350 to 750 μ g, in some embodiments, 50 to 200 μ g, in some embodiments, each dose will comprise 25 to 75 μ g of immunogen (e.g., recombinant and/or purified protein). So long as the amount of immunogen, when administered to a subject, generates an immune response in the subject. The optimal amount for a particular administration, e.g., to induce an immune response (e.g., a protective immune response (e.g., protective immunity)), can be determined by one of skill in the art using standard studies involving observation of antibody titers and other responses in a subject.

In some embodiments, each dose (e.g., of a composition comprising NE and immunogen (e.g., administered to a subject to induce an immune response)) is expected to be 0.001-15% or more (e.g., 0.001-10%, 0.5-5%, 1-3%, 2%, 6%, 10%, 15% or more) immunogen (e.g., a neutralized bacterium or virus, or a recombinant and/or purified protein) by weight. In some embodiments, the initial or priming dose contains more immunogen than the subsequent booster dose.

In some embodiments, when the NE of the invention is used to inactivate a living microorganism (e.g. a virus (e.g. RSV)), each dose (e.g. administered to a subject to induce an immune response)) is expected to comprise 10-10⁹pfu virus/dose; in some embodiments, each dose comprises 10⁵ - 10⁸ pfu virus/dose; in some embodiments, each dose comprises 10³ - 10⁵ pfu virus/dose; in some embodiments, each dose comprises 10² - 10⁴ pfu virus/dose; in some embodiments, each dose comprises 10pfu virus/dose; in some embodiments, each dose comprises 10² pfu virus/dose; and in some embodiments, each dose comprises 10 ⁴ pfu virus/dose. In some embodiments, each dose comprises more than 10⁹ pfu virus/dose. In some preferred embodiments, each dose comprises 10³ pfu virus/dose.

The present invention is not limited by the amount of NE used to inactivate a living microorganism, such as a virus (e.g., one or more types of RSV). In some embodiments, a 0.1% to 5% NE solution is used, in some embodiments, a 5% to 20% NE solution is used, in some embodiments, a 20% NE solution is used, and in some embodiments, a more than 20% NE solution is used, to inactivate pathogenic microorganisms. In a preferred embodiment, a 15% NE solution is used.

Similarly, the present invention is not limited by the duration of incubation of live microorganisms in the NE of the present invention to become inactivated. In some embodiments, the microorganism is incubated in NE for 1-3 hours. In some embodiments, the microorganism is incubated in NE for 3-6 hours. In some embodiments, the microorganism is incubated in NE for more than 6 hours. In a preferred embodiment, the microorganism is incubated in NE (e.g., a 10% NE solution) for 3 hours. In some embodiments, the incubation is performed at 37 ℃. In some embodiments, the incubation is performed at a temperature greater than or less than 37 ℃. The present invention is also not limited by the amount of microorganisms used for inactivation. The amount of microorganism can depend on a number of factors, including but not limited to the total amount of immunogenic composition (e.g., NE and immunogen) desired, the concentration of solution desired (e.g., prior to dilution for administration), the microorganism, and NE. In some preferred embodiments, the amount of microorganism used in the inactivation procedure is that amount which produces the amount of immunogen (e.g., as described herein) to be administered to the subject in a single dose (e.g., diluted from a concentrated stock solution).

In some embodiments, the compositions of the invention comprising NE and immunogen are formulated in concentrated doses, which may be diluted prior to administration to a subject. For example, a dilution of the concentrated composition can be administered to a subject such that the subject receives any one or more of the specific doses provided herein. In some embodiments, dilution of the concentrate composition can be performed such that the subject administers (e.g., in a single dose) a composition comprising 0.5-50% NE and immunogen present in the concentrate composition. In some preferred embodiments, the subject administers a composition comprising 1% NE and immunogen present in a concentrated composition in a single dose. The concentrated compositions are expected to be useful in settings where a large number of subjects may administer the compositions of the invention (e.g., immunization clinics, hospitals, schools, etc.). In some embodiments, the compositions (e.g., concentrated compositions) of the invention comprising NE and immunogen are stable at room temperature for more than 1 week, in some embodiments, more than 2 weeks, in some embodiments, more than 3 weeks, in some embodiments, more than 4 weeks, in some embodiments, more than 5 weeks, and in some embodiments, more than 6 weeks.

Generally, the emulsion compositions of the present invention will comprise at least 0.001% to 100%, preferably 0.01 to 90% emulsion per ml of liquid composition. It is contemplated that the formulation may comprise about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.025%, about 0.05%, about 0.075%, about 0.1%, 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 per ml of liquid composition. It should be understood that ranges between any 2 of the numbers listed above are specifically contemplated to be encompassed within the bounds and bounds of the present invention. Some variation in dosage will necessarily occur depending on the particular pathogen and the condition of the subject to be immunized.

In some embodiments, following an initial administration (e.g., initial vaccination) of a composition of the invention, the subject may receive one or more booster administrations (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 10 weeks, about 3 months, about 4 months, about 6 months, about 9 months, about 1 year, about 2 years, about 3 years, about 5 years, about 10 years) following the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and/or more than the tenth administration. Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, reintroduction of the immunogen in a booster dose enables robust systemic immunity in a subject. The boosting may be with the same formulation administered for the primary immune response, or may be with a different formulation containing the immunogen. The dosage regimen will also be at least partially determined by the needs of the subject and will depend on the judgment of the practitioner.

Dosage units may be scaled up or down based on several factors, including but not limited to the weight, age, and health of the subject. In addition, the dosage unit may be increased or decreased for subsequent administrations (e.g., booster administrations).

The composition of the invention comprising an immunogen is useful in the following cases: wherein the nature of the infectious and/or pathogenic agent (e.g. for which it is sought to elicit protective immunity) is known, and wherein the nature of the infectious and/or pathogenic agent is unknown (e.g. in emerging diseases (e.g. with a pandemic proportion (e.g. influenza or other outbreaks of disease))). For example, the invention contemplates the use of the compositions of the invention in the treatment or prevention (e.g., via immunization with RSV or RSV-like agents that are infectious and/or pathogenic via neutralization of the NEs of the invention) of infections associated with emerging infectious and/or pathogenic agents still to be identified (e.g., isolated and/or cultured from a diseased individual, but without genetic, biochemical or other characterization of the infectious and/or pathogenic agents).

It is contemplated that the compositions and methods of the present invention will be useful in a variety of contexts, including research contexts. For example, the compositions and methods of the invention are also useful in the study of the immune system, such as the characterization of adaptive immune responses, such as protective immune responses (e.g., mucosal or systemic immunity). The use of the compositions and methods provided by the invention includes human and non-human subjects and samples from these subjects, and also includes research applications using these subjects. The compositions and methods of the invention are also useful in the study and optimization of nanoemulsions, immunogens and other compositions, and for screening for new compositions. Thus, it is not intended that the present invention be limited to any particular subject and/or context of use.

The formulations can be tested in vivo in developing a number of animal models for studying mucosal and other delivery pathways. As is apparent, the compositions of the present invention are useful for the prevention and/or treatment of a wide variety of diseases and infections caused by viruses, bacteria, parasites and fungi, as well as for eliciting an immune response against a variety of antigens. The compositions may not only be used prophylactically or therapeutically, as described above, but the compositions may also be used for the preparation of antibodies, both polyclonal and monoclonal (e.g. for diagnostic purposes), and for the immunopurification of an antigen of interest. If polyclonal antibodies are desired, the selected mammal (e.g., mouse, rabbit, goat, horse, etc.) can be immunized with the compositions of the invention. Animals are usually boosted 2-6 weeks after one or more administrations with antigen. Polyclonal antisera can then be obtained from the immunized animal and used according to known procedures (see, e.g., Jurgens et al, J. Chrom. 1985, 348: 363-370).

In some embodiments, the invention provides kits comprising a composition comprising NE and an immunogen. In some embodiments, the kit further provides a device for administering the composition. The invention is not limited by the type of equipment included in the kit. In some embodiments, the device is configured for nasal application of a composition of the invention (e.g., a nasal applicator (e.g., a syringe) or a nasal inhaler or nasal fine particle nebulizer (mister)). In some embodiments, the kit comprises a composition comprising NE and immunogen in concentrated form (e.g., which may be diluted prior to administration to a subject).

In some embodiments, all kit components are present in a single container (e.g., vial or tube). In some embodiments, each kit component is in a single container (e.g., vial or tube). In some embodiments, one or more kit components are in a single container (e.g., vial or tube) while other components of the same kit are in separate containers (e.g., vial or tube). In some embodiments, the kit comprises a buffer. In some embodiments, the kit further comprises instructions for use.

Examples

The following examples serve to illustrate certain preferred embodiments and aspects of the invention and should not be construed as limiting its scope.

In the following experimental disclosure, the following abbreviations are used: eq (equivalent); μ (micrometers); m (molar); μ M (micromolar); mM (millimolar); n (normal); mol (mole); mmol (millimole); μ mol (micromolar); nmol (nanomole); g (grams); mg (milligrams); μ g (μ g); ng (nanogram); l (liter); ml (milliliters); μ l (microliter); cm (centimeters); mm (millimeters); μ m (micrometers); nM (nanomolar); deg.C (degrees Celsius); and PBS (phosphate buffered saline).

Example 1

Compositions comprising nanoemulsion inactivated respiratory syncytial virus and methods of using the same

Materials and methods

A mouse. Balb/c mice were purchased from Jackson Laboratories. All animal work was performed according to the University of Michigan Committee on Use and Care of Animals policy.

Viral plaque assay. Right lung leaves from infected mice were harvested and ground with sand using a mortar and pestle. Samples from lungs were either spun 2x, or obtained after incubation with nanoemulsion, and supernatants serially diluted onto 90% confluent monolayers of Vero cells. The samples were incubated at 37 ℃ for 2 hours with gentle rotation, after which the infected supernatant was removed and replaced with 0.9% methylcellulose. After 5 days of incubation at 37 ℃, the methylcellulose was removed, replaced with methanol, and incubated at-80 ℃ for 1 hour. After methanol removal, samples were stored at-80 ℃ until plaque development. Plaque was developed using a modified ELISA protocol. Briefly, cells were blocked with 25% Blotto (milk powder diluted in phosphate buffered saline) for 1 hour at 37 ℃, washed and incubated with goat anti-human RSV polyclonal Ab (Chemicon International) for 1 hour at 37 ℃. The cells were washed again and incubated with horseradish peroxidase conjugated anti-goat/sheep IgG (Serotec) for 1 hour. Cells were washed and incubated with chloronaphthol at room temperature and plaques were counted.

And (3) preparing the RSV nano emulsion. RSV (RSV strain Line 19 (see, e.g., Lukacs et al, immunology and Infection, 169, 977-986 (2006)) (2X 10)⁶pfu) with 15% W₈₀5EC nanoemulsion was incubated for 60 minutes together, and the time to completely inactivate the virus was determined in the experiments performed during the development of the embodiments of the present invention. RSV vaccine formulations were freshly prepared for each immunization. Each animal received 10 μ l of emulsion or emulsion + RSV into the left nostril at day 0 and day 28. This means that a total of 1X 10 was used per vaccination/mouse⁴ pfu。

Bronchoalveolar lavage cytokine measurements. Bronchoalveolar lavage (BAL) was performed on infected mice using 1 ml sterile PBS. The cell suspension was centrifuged and the supernatant was collected for cytokine analysis and measured by Bioplex using a kit purchased from R & D system.

Lung dispersion and RSV re-challenge in vitro. Lungs were removed after 1 ml PBS-EDTA lavage and dispersed with collagenase (0.2%, Type IV, Sigma) for 45 minutes in a rotating water bath (37C). The dispersed cells were then counted. Single cell suspensions were then incubated at 2 x 10⁶Concentrations of/ml were plated and incubated with RSV (MOI-0.5). Cell-free supernatants were collected after 36 hours and assessed for cytokine levels produced by Bioplex. This assay allows us to assess the overall response in the lungs of vaccinated versus unvaccinated groups of mice.

Example 2

Nanoemulsion effective inactivation of RSV

To test the ability of emulsions to inactivate RSV, RSV (10)⁶Particle Forming Units (PFU) at different concentrations (0% -20%) And incubated with the nanoemulsion for various times (1-3 hours) (see figure 1). The number of infectious viruses was determined via plaque assay using Vero cells. Nanoemulsion incubated viruses were used to infect sub-confluent Vero cells. RSV plaques were visualized using immunohistochemical techniques. Detection of absence of active virus as assessed by standard plaque assay at as little as 3 hours of incubation of 1% nanoemulsion (see figure 1). Thus, the present invention provides a nanoemulsion that is effective at a concentration of 2% in as little as 1 hour, or at a complete kill of RSV in as little as 1% in 3 hours. Thus, in some embodiments, the present invention provides nanoemulsions that are effective in reducing and/or completely inactivating RSV infectivity.

Example 3

Nanoemulsion immunization enhances immunity upon RSV challenge

Next it was determined whether the nanoemulsion could be used as an immunopotentiating agent to induce an immune response important for protection against viral infections. To examine this aspect, animals were immunized with the immunization protocol, containing intranasal sensitization of virus inactivated with nanoemulsion by boosting at day 0 and at day 28 (nanoemulsion (15%) -RSV cocktail (10 μ l total, 5 μ l/nostril)) or nanoemulsion alone without RSV alone as a control group. Animals were subsequently challenged with live, infectious RSV at day 56 (8 weeks) and evaluated for evidence of protective immunity. One objective is to monitor RSV-specific antibody production during the course of an immunization protocol. The reciprocal titer of RSV-specific antibodies in serum was determined via enzyme linked immunosorbent assay (ELISA) against RSV protein extract. Blood was harvested and sera were collected at specific time points post-immunization, including days 0, 1 week, 4 weeks, and 8 weeks (at RSV challenge), and total serum IgG specific for RSV was assessed. As shown in figure 2, anti-RSV IgG titers were undetectable at 1 week post-immunization, increased before boosting after 4 weeks, and significantly increased by 8 weeks post-initial immunization. Accordingly, the present invention provides for immunization (e.g., intranasal immunization) of a subject with nanoemulsion inactivated RSV to induce an anti-RSV immune response in the subject. In some embodiments, the anti-RSV immune response is induced in the subject within 4 weeks after nanoemulsion-inactivated RSV is administered to the subject. In some embodiments, an anti-RSV immune response is induced in the subject upon a second nanoemulsion-inactivated RSV administration to the subject (e.g., after a "booster" administration). In some embodiments, the present invention provides compositions comprising nanoemulsion inactivated RSV useful for generating an anti-RSV specific immune response in a subject administered the composition.

Example 4

RSV-nanoemulsion immunization induces cytotoxicity and Th1 type antiviral immune response

Because of the serious problems associated with other vaccine types for RSV (e.g., the generation of disease that worsens severely upon infection following formalin inactivated RSV administration to a subject), it is next determined what type of immune response is generated upon nanoemulsion inactivated RSV administration to a subject.

Administration of NE-RSV enhances antiviral cytokines in BAL fluid from the airways of RSV-challenged mice. Wash with 1 ml PBS of airways and determine cytokine levels in lung via multiplex analysis of BAL fluid from lung at day 8 post challenge (Bioplex, R & D system) when T cell cytokine peaks.

As shown in figure 3, subjects administered (e.g., nasally) nanoemulsion inactivated RSV exhibited an increase in the number of M2 peptide-specific cytotoxic CD8+ cytotoxic T cells compared to the nanoemulsion control immunized group. The number of RSV M82-90 specific CD 8T cells was determined by flow cytometry analysis of enzymatically digested lungs at day 4 post challenge using specific MHC class I tetramers that specifically recognize TCRs for the immunodominant peptide of M82-90. Furthermore, evaluation of the antiviral environment in the airways using BAL fluid indicated increased IFN- γ and IL-17 production in the subject, but no increase in the pathogenic Th2 cytokines IL-4, IL-5 and IL-13 during the viral challenge phase (see figure 4). As described above, Th 2-type cytokines were identified as having causal effects in previous vaccine trials performed with formalin inactivated RSV. In addition, the Th2 cytokine interleukin-13 (IL-13) is a mediator of lung mucus secretion (see Hershey, G.K. 2003. J. Allergy Clin. Immunol. 111: 677-690, Walter et al, 2001J. Immunol. 167: 4668-4675; Zhu et al, 1999J. Clin. investig. 103: 779-788), and RSV-specific T cells expressing IL-13 are found in RSV bronchiolitis (DeWaal, 2003J. Med. Virol. 70: 309-318). Thus, in some embodiments, the invention provides immunogenic compositions comprising NE-inactivated RSV and methods of using the same to generate an immune response against RSV in a subject without enhanced mucus production, airway narrowing, airway hyperresponsiveness, air retention capture, hypoxia (hpoxia), and/or partial lung collapse (e.g., due to enhanced expression of Th2 type cytokines (e.g., IL-13)).

To further evaluate cytokine response in subjects administered NE-RSV, lungs were isolated from uninfected animals or from those vaccinated and challenged and compared to unvaccinated and RSV challenged animals. Lungs were removed and collagenase dispersed as single cell suspension followed by in vitro re-challenge with virus. Total lung leukocytes were isolated from the lungs of mice that were not infected with control (UC), vaccinated and challenged (vaccine), or control RSV challenged (unvaccinated). Cells at 2 x 10⁶The concentration/ml was cultured for 36 hours in the presence of live RSV (MOI = 0.5). Cytokine concentrations were measured in the supernatant via bioplex multiplex analysis.

It was observed that although IL-17 production was again significantly upregulated and IFN increased, the Th2 cytokine IL-4 was unchanged (see figure 5). Thus, the present invention provides that vaccination with NE-RSV does not pre-prime mice (presensitize) to a more pathogenic response (e.g., in contrast to results obtained with formalin inactivated RSV). Although an understanding of the mechanism is not necessary to practice the present invention, and the present invention is not limited to any particular mechanism of action, in some embodiments, the increase in IFN- γ and IL-17 reflects a more antiviral immune environment induced by nanoemulsion inactivated RSV immunization protocols.

It is important for the outcome of any immunization protocol to determine whether there is an increase in viral clearance following immunization and exposure to live virus. Mice used 10 in 15% NE⁶PFU RSV (NE-RSV) or 15% NE (NE) alone was immunized intranasally 2 times with 4 weeks intervals as controls. Mice were then challenged 4 weeks after the second vaccination (8 weeks total) and viable viral particle numbers were determined via plaque assay of the right lung. When the subjects were evaluated by plaque assay to determine the viable virus count present in the lungs of vaccinated and subsequently virus-infected subjects, nanoemulsion-RSV immunized subjects showed significantly increased clearance (reduced viral plaque) compared to non-vaccinated subjects (see figure 6). Thus, the present invention provides that administration of nanoemulsion inactivated RSV (NE-RSV) establishes a protective response in a subject.

Example 5

RSV-nanoemulsion immunization and allergic asthma

Epidemiological studies have suggested a link between early severe RSV infection and the subsequent development of allergic asthma. Thus, to determine whether vaccination with NE-RSV followed by live virus challenge will affect subsequent responses to the allergic asthma model, mice were vaccinated 2 times with NE-RSV and 10 times with NE-RSV ⁵PFU RSV was challenged intranasally and sensitized to cockroach allergens. In this model, mice received intraperitoneal/subcutaneous administration of clinical skin test grade cockroach allergen (100 μ g) emulsified in incomplete freund's adjuvant on day 21 post RSV challenge. Mouse followerPost-received 1 intranasal challenge (15 μ g) 14 days later, and 2 intratracheal challenges (40 μ g) 5 and 7 days after intranasal challenge. Mice were evaluated for allergic disease 24 hours after the last intratracheal challenge.

One of the characteristics of allergic lung disease is hypersecretion of mucus. NE-RSV vaccinated mice showed a reduced allergen-induced mucus response compared to unvaccinated mice, as by Periodic Acid Schiff (PAS) staining via lung histological sections (see figure 8), and mucus genes in total lung RNAGob5Was evaluated (see fig. 7 and 8). Similar to the virus challenge alone, allergen challenged NE-RSV vaccinated animals had significantly higher IL-17 induction in the lungs as assessed via QPCR (see figure 9A). The Th2 cytokine is important for promoting allergic lung diseases. NE-RSV vaccinated mice showed attenuated Th2 cytokine production, including IL-4 (homogenized lung and BAL) and IL-5 (lung) (see fig. 9B). A trend towards reduced IL-13 mRNA was also noted. There was no enhancement of allergic disease in NE-RSV vaccinated animals. In addition, vaccinated animals had significantly lower expression of the alternatively activated macrophage marker Fizz-1. Alternatively activated macrophages are associated with Th2 responses and fibrotic diseases. Although an understanding of the mechanism is not necessary to practice the invention, and the invention is not limited to any particular mechanism of action, in some embodiments, the reduction in Fizz-1 reflects the result of reduced Th2 cytokine in vaccinated mice, and also provides a mechanism by which NE-RSV vaccination protects against subsequent development of allergic lung disease.

Example 6

RSV-nanoemulsion immunization induces RSV-specific antibody production

Intranasal vaccination of mice with NE/RSV resulted in the production of RSV-specific antibodies. Experiments were conducted during the development of embodiments of the present invention to determine NE-RSV vaccinationWhether an antibody response (e.g., associated with protection against viral infection) in a subject administered a NE-RSV composition will be promoted. The protocol of immunization by vaccinated mice receiving 2 intranasal doses of NE-RSV spaced 28 days apart was used. Mice were treated with a composition containing 10 on days 0 and 28⁵NE/RSV from the viral particle Line 19 was used for immunization. Total RSV-specific antibody levels in serum were determined at day 55 via ELISA using purified RSV protein extract. As shown in fig. 10, significant RSV-specific responses were generated systemically following vaccination with NE-RSV (see, e.g., fig. 10A). These included significant induction of total RSV-specific Ig without enhancement in RSV-specific IgE titers (see, e.g., fig. 10A).

Experiments were also performed during the development of embodiments of the present invention to determine whether vaccination could promote induction of RSV-specific antibodies locally in the lungs. In-use live RSV (10) ⁵) The presence of RSV-specific total Ig and IgA was assessed by ELISAs of bronchoalveolar (bronchalviolator) lavage samples (BAL) on day 2 post-intratracheal challenge. Specifically, total RSV-specific antibody levels in serum were determined via ELISA at day 55 using purified RSV protein extract. Total Ig measured from serum was evaluated for the 1:1600 diluted samples, and other samples were evaluated at 1: 50. (B)

As shown in figure 10B, vaccinated mice (NE-RSV) receiving 2 intranasal doses of NE-RSV spaced 28 days apart exhibited increased RSV-specific IgA and RSV-specific total Ig in bronchial lavage fluid at day 2 post challenge with live virus compared to control unvaccinated mice (naive controls) and mice receiving primary challenge with NE-RSV (primary RSV) (see, e.g., figure 10B). These data demonstrate that NE-RSV vaccination induces significant RSV-specific antibodies and enhances local induction of RSV-specific antibodies upon live virus challenge.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and alterations of the compositions and methods of this invention will be apparent to those skilled in the art without departing from the scope and spirit of this invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the invention.

Claims (13)

1. An immunogenic composition comprising nanoemulsion inactivated Respiratory Syncytial Virus (RSV), wherein the nanoemulsion for inactivating RSV comprises a surfactant selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate (TWEEN 20) and polyoxyethylene sorbitan monooleate (TWEEN 80), ethanol, cetylpyridinium chloride(CPC), oil and water。

2. The immunogenic composition of claim 1, wherein the nanoemulsion for inactivated RSV comprises about 5% by volume surfactant, about 8% by volume ethanol, about 1% by volume cetylpyridinium chloride(CPC), about 64% oil by volume, and about 22% water by volume.

3. The immunogenic composition of claim 1, wherein the composition comprises a 1-50% nanoemulsion solution.

4. The immunogenic composition of claim 1, wherein the composition comprises a 5-15% nanoemulsion solution.

5. The immunogenic composition of claim 1, wherein the composition comprises a 15% nanoemulsion solution.

6. The immunogenic composition of claim 1, wherein the composition comprises 10⁴PFU inactivated respiratory syncytial virus.

7. The immunogenic composition of claim 1, wherein the composition is thermotolerant.

8. The immunogenic composition of claim 1, further comprising a pharmaceutically acceptable carrier.

9. The immunogenic composition of claim 1, further comprising an adjuvant.

10. The immunogenic composition of claim 9, wherein the adjuvant biases a Th1 type immune response.

11. The immunogenic composition of any one of claims 1-10 for use in the treatment or prevention of respiratory syncytial virus infection.

12. Use of an immunogenic composition according to any one of claims 1 to 10 in the manufacture of a vaccine for the treatment or prevention of respiratory syncytial virus infection.

13. A vaccine comprising the immunogenic composition of any one of claims 1-8 and a pharmaceutically acceptable carrier.