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

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for the stimulation of immune responses. Specifically, the present invention provides methods and compositions for the use of nanoemulsion compounds as mucosal adjuvants to induce immunity against environmental pathogens.

Accordingly, in some embodiments, the present invention provides a composition comprising a vaccine, the vaccine comprising an emulsion and an immunogen, the emulsion comprising an aqueous phase, an oil phase, and a solvent. In some embodiment, the immunogen comprises a pathogen (e.g., an inactivated pathogen). In other embodiments, the immunogen comprises a pathogen product (e.g., including, but not limited to, a protein, peptide, polypeptide, nucleic acid, polysaccharide, or a membrane component derived from the pathogen). In some embodiments, the immunogen and the emulsion are combined in a single vessel.

The present invention is not limited to a particular oil. A variety of oils are contemplated, including, but not limited to, soybean, avocado, squalene, olive, canola, corn, rapeseed, safflower, sunflower, fish, flavor, and water insoluble vitamins. The present invention is also not limited to a particular solvent. A variety of solvents are contemplated including, but not limited to, an alcohol (e.g., including, but not limited to, methanol, ethanol, propanol, and octanol), glycerol, polyethylene glycol, and an organic phosphate based solvent.

In some embodiments, the emulsion further comprises a surfactant. The present invention is not limited to a particular 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 particular cationic halogen containing compound. A variety of cationic halogen containing compounds are contemplated including, but not limited to, cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, and tetradecyltrimethylammonium halides. The present invention is also not limited to a particular halide. A variety of halides are contemplated including, but not limited to, halide selected from the group consisting of chloride, fluoride, bromide, and iodide.

In still further embodiments, the emulsion further comprises a quaternary ammonium containing compound. The present invention is not limited to a particular 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 ammonium chloride, n-Alkyl dimethyl benzyl ammonium chloride, n-Alkyl dimethyl ethylbenzyl ammonium chloride, Dialkyl dimethyl ammonium chloride, and n-Alkyl dimethyl benzyl ammonium chloride.

In certain embodiments, the immunogen is selected from the group consisting of virus, bacteria, fungus and pathogen products derived from the virus, bacteria, or fungus. The present invention is not limited to a particular virus. A variety of viral immunogens are contemplated including, but not limited to, influenza A, herpes simplex virus I, herpes simplex virus II, sendai, sindbis, vaccinia, parvo, human immunodeficiency virus, hepatitis B, virus hepatitis C virus, hepatitis A virus, cytomegalovirus, and human papilloma virus, picornavirus, hantavirus, junin virus, and ebola virus. The present invention is not limited to a particular bacteria. A variety of bacterial immunogens are contemplated including, but not limited to, Bacillus cereus, Bacillus circulans and Bacillus megaterium, Bacillus anthracis, Clostridium perfringens, Vibrio cholerae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumonia, Staphylococcus aureus, Neisseria gonorrhoeae, Haemophilus influenzae, Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, Proteus mirabilis, Pseudomonas aeruginosa, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The present invention is also not limited to a particular fungus. A variety of fungal immunogens are contemplated including, but not limited to, Candida and Aspergillus.

The present invention further provides a kit comprising a vaccine, the vaccine comprising an emulsion and an immunogen, the emulsion comprising an aqueous phase, an oil phase, and a solvent In some embodiments, the kit further comprises instructions for using the kit for vaccinating a subject against the immunogen.

In some embodiment, the immunogen comprises a pathogen (e.g., an inactivated pathogen). In other embodiments, the immunogen comprises a pathogen product (e.g., including, but not limited to, a protein, peptide, polypeptide, nucleic acid, polysaccharide, or membrane component derived from the pathogen). In some embodiments, the immunogen and the emulsion are combined in a single vessel.

The present invention is not limited to a particular oil. A variety of oils are contemplated, including, but not limited to, soybean, avocado, squalene, olive, canola, corn, rapeseed, safflower, sunflower, fish, flavor, and water insoluble vitamins. The present invention is also not limited to a particular solvent. A variety of solvents are contemplated including, but not limited to, an alcohol (e.g., including, but not limited to, methanol, ethanol, propanol, and octanol), glycerol, polyethylene glycol, and an organic phosphate based solvent.

In some embodiments, the emulsion further comprises a surfactant. The present invention is not limited to a particular 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 particular cationic halogen containing compound. A variety of cationic halogen containing compounds are contemplated including, but not limited to, cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, and tetradecyltrimethylammonium halides. The present invention is also not limited to a particular halide. A variety of halides are contemplated including, but not limited to, halide selected from the group consisting of chloride, fluoride, bromide, and iodide.

In still further embodiments, the emulsion further comprises a quaternary ammonium containing compound. The present invention is not limited to a particular 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 ammonium chloride, n-Alkyl dimethyl benzyl ammonium chloride, n-Alkyl dimethyl ethylbenzyl ammonium chloride, Dialkyl dimethyl ammonium chloride, and n-Alkyl dimethyl benzyl ammonium chloride.

In certain embodiments, the immunogen is selected from the group consisting of virus, bacteria, fungus and pathogen products derived from the virus, bacteria, or fungus. The present invention is not limited to a particular virus. A variety of viral immunogens are contemplated including, but not limited to, influenza A, herpes simplex virus I, herpes simplex virus II, sendai, sindbis, vaccinia, parvo, human immunodeficiency virus, hepatitis B, virus hepatitis C virus, hepatitis A virus, cytomegalovirus, and human papilloma virus, picornavirus, hantavirus, junin virus, and ebola virus. The present invention is not limited to a particular bacteria. A variety of bacterial immunogens are contemplated including, but not limited to, Bacillus cereus, Bacillus circulans and Bacillus megaterium, Bacillus anthracis, Clostridium perfringens, Vibrio cholerae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumonia, Staphylococcus aureus, Neisseria gonorrhoeae, Haemophilus influenzae, Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, Proteus mirabilis, Pseudomonas aeruginosa, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The present invention is also not limited to a particular fungus. A variety of fungal immunogens are contemplated including, but not limited to, Candida and Aspergillus.

In still further embodiments, the present invention provides a method of inducing immunity to an immunogen, comprising providing an emulsion comprising an aqueous phase, an oil phase, and a solvent; and an immunogen; combining the emulsion with the immunogen to generate a vaccine composition; and administering the vaccine composition to a 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 administration. In some preferred embodiments, the administering in under conditions such that the subject is immune to the immunogen.

In some embodiment, the immunogen comprises a pathogen (e.g., an inactivated pathogen). In other embodiments, the immunogen comprises a pathogen product (e.g., including, but not limited to, a protein, peptide, polypeptide, nucleic acid, polysaccharide, or membrane component derived from the pathogen). In some embodiments, the immunogen and the emulsion are combined in a single vessel.

The present invention is not limited to a particular oil. A variety of oils are contemplated, including, but not limited to, soybean, avocado, squalene, olive, canola, corn, rapeseed, safflower, sunflower, fish, flavor, and water insoluble vitamins. The present invention is also not limited to a particular solvent. A variety of solvents are contemplated including, but not limited to, an alcohol (e.g., including, but not limited to, methanol, ethanol, propanol, and octanol), glycerol, polyethylene glycol, and an organic phosphate based solvent.

In some embodiments, the emulsion further comprises a surfactant. The present invention is not limited to a particular 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 particular cationic halogen containing compound. A variety of cationic halogen containing compounds are contemplated including, but not limited to, cetylpyridinium halides, cetyltrimethylammonium halides, cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides, cetyltributylphosphonium halides, dodecyltrimethylammonium halides, and tetradecyltrimethylammonium halides. The present invention is also not limited to a particular halide. A variety of halides are contemplated including, but not limited to, halide selected from the group consisting of chloride, fluoride, bromide, and iodide.

In still further embodiments, the emulsion further comprises a quaternary ammonium containing compound. The present invention is not limited to a particular 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 ammonium chloride, n-Alkyl dimethyl benzyl ammonium chloride, n-Alkyl dimethyl ethylbenzyl ammonium chloride, Dialkyl dimethyl ammonium chloride, and n-Alkyl dimethyl benzyl ammonium chloride.

In certain embodiments, the immunogen is selected from the group consisting of virus, bacteria, fungus and pathogen products derived from the virus, bacteria, or fungus. The present invention is not limited to a particular virus. A variety of viral immunogens are contemplated including, but not limited to, influenza A, herpes simplex virus I, herpes simplex virus II, sendai, sindbis, vaccinia, parvo, human immunodeficiency virus, hepatitis B, virus hepatitis C virus, hepatitis A virus, cytomegalovirus, and human papilloma virus, picornavirus, hantavirus, junin virus, end ebola virus. The present invention is not limited to a particular bacteria. A variety of bacterial immunogens are contemplated including, but not limited to, Bacillus cereus, Bacillus circulans and Bacillus megaterium, Bacillus anthracis, Clostridium perfringens, Vibrio cholerae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumonia, Staphylococcus aureus. Neisseria gonorrhoeae, Haemophilus influenzae, Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, Proteus mirabilis, Pseudomonas aeruginosa, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The present invention is also not limited to a particular fungus. A variety of fungal immunogens are contemplated including, but not limited to, Candida and Aspergillus.

GENE DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for the stimulation of immune responses. Specifically, the present invention provides methods and compositions for the use of nanoemulsion compounds as mucosal adjuvants to induce immunity against environmental pathogens. Accordingly, in some embodiments, the present invention provides mucosal vaccines comprising a pathogen (e.g., an inactivated pathogen) and a nanoemulsion composition. In some embodiments, the pathogen is mixed with the nanoemulsion prior to administration for a time period sufficient to inactivate the pathogen. In others, purified protein components from an pathogen are mixed with the nanoemulsion.

The present invention is not limited to any mechanism of action. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that the nanoemulsion/pathogen compositions of the present invention stimulate a mucosal immune response against the pathogen component of the vaccine (See e.g., Richter and Kipp, Curr Top Microbiol Immunol 240:159-76 [1999]; Ruedl and Wolf, Int. Arch. Immunol., 108:334 [1995]; and Mor et al., Trends Micrbiol 6:449-53 [1998] for reviews of the mucosal immune system). Mucosal antigens stimulate the Peyer's Patches (PP) of the gastrointestinal tract. The M cells of the PP then transport antigens to the underlying lymph tissue where they encounter B cells and initiate B cell development. IgA is secreted by primed B cells that have been induced to produce IgA by Th2 helper T cells. Primed B cells are transported throughout the lymph system where they populate all secretory tissues. IgAs are then secreted in mucosal tissues where they serve as a first-line defense against many viral and bacterial pathogens.

An optimal prophylactic vaccine against influenza virus should include means to induce both Ab responses and cytotoxic T cell responses (McMichael, Curr. Top. Microbiol. Immunol. 189:75 [1994]). Experiments conducted during the coarse of development of the present invention (See e.g., Example 15) demonstrated that nanoemulsion vaccines of the present invention fulfill both requirements. Immunization with a single dose induced high titer of influenza specific IgG antibodies and titer of antibodies continued to increase after the lethal challenge. There was an early cytokine response (day 4) after single intranasal immunization with virus/nanoemulsion mixture with high levels of IL-12, IFN-.gamma., IL-2, TNF-.alpha. and IL-10 and absence of anti-inflammatory cytokine IL-4. Since IFN-.gamma. is the major cytokine produced in response to viral infection, kinetics of IFN-.gamma. production over the period of 20 days after immunization were measured. There was significant amount of IFN-.gamma. (200 pg of per milliliter of mouse serum) one day after immunization. Over 10 days, it gradually decreased to undetectable amounts. The immune response against virus was highly specific since mouse splenocytes harvested 20 days after immunization and stimulated with either congenic strain of virus (Ann Arbor) or heterogenic strain of virus (Puerto Rico) responded exclusively toward congenic strain of virus by production of IFN-.gamma. and proliferation. Moreover, mice immunized with Ann Arbor strain of virus and challenged with Puerto Rico strain did not survive the lethal challenge. However, the mice immunized with Ann Arbor strain and challenged with the same virus acquired the immunity against heterogenic strain of virus (Puerto Rico strain). The splenocytes from these animals were able to respond by profound production of IFN-.gamma. after in vitro stimulation with Puerto Rico virus. Furthermore, these animals were fully protected against lethal challenge with heterogenic virus, i.e Puerto Rico strain.

The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that this observation suggests an immunodominance effect (Sercarz et al., Anu Rev Immunol 11:729 [1993]; Perreault et al., Immunol Today 19:69 [1998]), which has been found to regulate cytotoxic T lymphocyte (CTL) responses to viruses (Silins et al., J Exp Med 184:1815 [1996]: Steven et al, J Exp Med 184:1801 [1996]). It appears that only a very small portion of epitopes, probably less than 10%, are dominant (Tremblay et al., Transplantation 58:59 [1994]; Brochu et al., J Immunol 155:5104 [1995]). During the process of vaccination, the presence of immunodominant epitopes prevented recognition of nondominant determinants and therefore animals responded exclusively toward congenic strain of virus. However, after both vaccination and the lethal challenge with congenic virus (Ann Arbor), animals expanded the epitope recognition and developed the response to nondominant determinants acquiring immune protection against heterogenic virus.

Experiments conducted during the course of the development of the present invention strongly support the notion that as little as a single intranasal instillation of virus/nanoemulsion mixture works as mucosal vaccine and is able to stimulate strong and specific immune response against influenza A virus. The vaccine was prepared by mixing the 5.times.10.sup.5 pfu of virus with equal volume of 4% nanoemulsion and incubated at RT for one hour prior to mucosal vaccination of animals. Although the reduction of virus was greater than three logs after one hour incubation of the virus with nanoemulsion, there was an incomplete viral inactivation with about 100 pfu of intact virus remaining, based on viral plaque assay. These finding led to an investigation of whether a small number of intact viral particles alone could be effective in immunization of mice. As shown in Table 28 (see Original Patent), up to 2.times.10.sup.3 pfu of virus per mouse administrated intranasally did not rest It in protected immunity since all animals challenged with lethal dose of virus succumbed to pneumonia and died. Low doses of virus were not effective and higher dose of intact virus caused sickness and death within the first 3 days after intranasal treatment. These data clearly demonstrated that, in addition to nanoemulsion and nanoemulsion-inactivated virus, a small dose of intact virus was useful for mucosal vaccination of experimental animals. This conclusion was also supported by the observation that formalin-inactivated virus mixed with nanoemulsion and administrated intranasally to animals did not protect them from lethal challenge with influenza A virus.

The nasally administered nanoemulsion vaccine compositions of the present invention have several advantages over parenterally administered vaccines. The vaccines can be easily administered when needed (e.g., immediately before or directly after exposure to the pathogen). When administered after exposure (e.g., after exposure of troops to a biological weapon), immune protection occurs specifically when needed. It is at this time that ongoing pathogen exposure might lead to infection. The administration methods of the present invention also avoid the need for expensive and problematic prophylactic vaccine programs. This approach provides the individual with specific immunity to the exact organisms exposed to, regardless of genetic or antigenic manipulation. The methods of the present invention are particularly valuable since they avoid the need for actual infection to induce immunity since even an attenuated infection can have undesired consequences. The present invention further provides methods of using nanoemulsions as adjuvants for parenteral administered vaccines. The present invention thus provides a rapid, killed vaccine for a range of naturally occurring and human administered pathological agents.
 

Claim 1 of 23 Claims

1. A method of inducing an immune response to an immunogen, comprising: a) providing: (i) a nanoemulsion, wherein said nanoemulsion comprises: 1. oil; 2. ethanol; 3. a surfactant; 4. a quaternary ammonium compound; and 5. distilled water; and (ii) an immunogen; b) combining said nanoemulsion with said immunogen; and c) administering said combined nanoemulsion and immunogen to a subject under conditions such that said subject produces an immune response to said immunogen, wherein said administering comprises contacting said combined nanoemulsion and immunogen with a mucosal surface of said subject.

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