This invention provides submicron oil-in-water emulsions useful as a vaccine adjuvant for enhancing the immunogenicity of antigens. The present invention also provides vaccine compositions containing an antigen combined with such emulsions intrinsically or extrinsically. Methods of preparing the emulsions and vaccines are also provided by the present invention.

SUMMARY OF THE INVENTION

It has been unexpectedly discovered by the present inventors that the adjuvant activity and the safety profile of non-metabolizable oil based oil-in-water emulsions can be improved through microfluidization. Antigens incorporated in microfluidized emulsions are stable even when the antigens are intrinsically incorporated into the emulsions prior to microfluidization.

Accordingly, in one embodiment, the present invention provides submicron oil-in-water emulsion formulations useful as a vaccine adjuvant. The submicron oil-in-water emulsions of the present invention are composed of a non-metabolizable oil, at least one surfactant, and an aqueous component, where the oil is dispersed in the aqueous component with an average oil droplet size in the submicron range. A preferred non-metabolizable oil is light mineral oil. Preferred surfactants include lecithin, TWEEN.RTM.-80 and SPAN.RTM.-80.

A preferred oil-in-water emulsion provided by the present invention is composed of an AMPHIGEN.RTM. formulation.

The oil-in-water emulsions of the present invention can include additional components that are appropriate and desirable, including preservatives, osmotic agents, bioadhesive molecules, and immunostimulatory molecules. Preferred immunostimulatory molecules include, e.g., Quil A, cholesterol, GPI-0100, dimethyldioctadecylammonium bromide (DDA).

In another embodiment, the present invention provides methods of preparing a submicron oil-in-water emulsion. According to the present invention, the various components of the emulsion, including oil, one or more surfactants, an aqueous component and any other component appropriate for use in the emulsion, are mixed together. The mixture is subjected to a primary emulsification process to form an oil-in-water emulsion, which is then passed through a microfluidizer to obtain an oil-in-water emulsion with droplets of less than 1 micron in diameter, preferably with a mean droplet size of less than 0.5 micron.

In still another embodiment, the present invention provides vaccine compositions which contain an antigen and a submicron oil-in-water emulsion described hereinabove. The antigen is incorporated into the emulsion either extrinsically or intrinsically, preferably, intrinsically.

The antigen which can be included in the vaccine compositions of the present invention can be a bacterial, fungal, or viral antigen, or a combination thereof. The antigen can take the form of an inactivated whole or partial cell or virus preparation, or the form of antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis.

In a further embodiment, the present invention provides methods of preparing vaccine compositions containing an antigen or antigens combined with a submicron oil-in-water emulsion.

In preparing the vaccine compositions of the present invention, the antigen(s) can be combined either intrinsically (e.g., prior to microfluidization) or extrinsically (e.g., after microfluidization) with the components of the oil-in-water emulsion. Preferably, the antigen is combined with the components of the oil-in-water emulsion intrinsically.

In still another embodiment, the present invention provides vaccine compositions which contain a microencapsulated antigen and a submicron oil-in-water emulsion described hereinabove, where the microencapsulated antigen is combined with the emulsion extrinsically.

It has also been surprisingly discovered that a saponin and a sterol, when combined in solution, associate with each other to form complexes in the form of helical micelles. According to the present invention, these helical micelle complexes have immunostimulating activities and are especially useful as adjuvants in vaccine compositions.

Accordingly, the present invention provides vaccine compositions containing a saponin and a sterol, wherein the saponin and the sterol form complexes in the form of helical micelles. The present invention also provides compositions containing a saponin, a sterol and an antigen, wherein the saponin and the sterol form complexes in the form of helical micelles, and wherein the antigen is admixed with but not incorporated within the helical micelles.

DETAILED DESCRIPTION OF THE INVENTION

It has been unexpectedly discovered by the present inventors that microfluidization of vaccine formulations adjuvanted with an oil-in-water emulsion comprised of a mixture of lecithin and mineral oil not only improves the physical appearance of the vaccine formulations, but also enhances the immunizing effects of the vaccine formulations. Microfluidized vaccine formulations are also characterized by an improved safety profile.

Based on these discoveries, the present invention provides submicron oil-in-water emulsions useful as an adjuvant in vaccine compositions. Methods of making these submicron oil-in-water emulsions by using a microfluidizer are also provided. Furthermore, the present invention provides submicron vaccine compositions in which an antigen is combined with a submicron oil-in-water emulsion. Methods for making such vaccine compositions are also provided. The present invention further provides vaccine compositions containing microencapsulated antigens combined with a submicron oil-in-water emulsion and methods for making such vaccines.

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections which describe or illustrate certain features, embodiments or applications of the invention.

Submicron Oil-in-Water Emulsions

In one embodiment, the present invention provides submicron oil-in-water emulsion formulations useful as a vaccine adjuvant. The submicron oil-in-water emulsions of the present invention enhance the immunogenicity of antigens in vaccine compositions, are safe for administration to animals and stable during storage.

The submicron oil-in-water emulsions of the present invention are composed of a non-metabolizable oil, at least one surfactant, and an aqueous component, where the oil is dispersed in the aqueous component with an average oil droplet size in the submicron range.

By "submicron" is meant that the droplets are of a size of less than 1 .mu.m (micron) and the average or mean oil droplet size is less than 1 .mu.m. Preferably, the mean droplet size of the emulsion is less than 0.8 .mu.m; more preferably, less than 0.5 .mu.m; and even more preferably, less than 0.4 .mu.m, or about 0.1 0.3 .mu.m.

The "mean droplet size" is defined as the Volume Mean Diameter (VMD) particle size within a volume distribution of particle sizes. The VMD is calculated by multiplying each particle diameter by the volume of all particles of that size and summing. This is then divided by the total volume of all particles.

The term "non-metabolizable oil" as used herein refers to oils that cannot be metabolized by the body of the animal subject to which the emulsion is administered.

The terms "animal" and "animal subject" as used herein refer to all non-human animals, including cattle, sheep, and pigs, for example.

Non-metabolizable oils suitable for use in the emulsions of the present invention include alkanes, alkenes, alkynes, and their corresponding acids and alcohols, the ethers and esters thereof, and mixtures thereof. Preferably, the individual compounds of the oil are light hydrocarbon compounds, i.e., such components have 6 to 30 carbon atoms. The oil can be synthetically prepared or purified from petroleum products. Preferred non-metabolizable oils for use in the emulsions of the present invention include mineral oil, paraffin oil, and cycloparaffins, for example.

The term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique. The term is synonymous with "liquefied paraffin", "liquid petrolatum" and "white mineral oil." The term is also intended to include "light mineral oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18.sup.th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, J.T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred mineral oil is light mineral oil commercially available under the name DRAKEOL.RTM..

Typically, the oil component of the submicron emulsions of the present invention is present in an amount from 1% to 50% by volume; preferably, in an amount of 10% to 45; more preferably, in an amount from 20% to 40%.

The oil-in-water emulsions of the present invention typically include at least one (i.e., one or more) surfactant. Surfactants and emulsifiers, which terms are used interchangeably herein, are agents which stabilize the surface of the oil droplets and maintain the oil droplets within the desired size.

Surfactants suitable for use in the present emulsions include natural biologically compatible surfactants and non-natural synthetic surfactants. Biologically compatible surfactants include phospholipid compounds or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by water-washing crude vegetable oils, and separating and drying the resulting hydrated gums. A refined product can be obtained by fractionating the mixture for acetone insoluble phospholipids and glycolipids remaining after removal of the triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylethanolamine. The phospholipids may be isolated from natural sources or conventionally synthesized.

Non-natural, synthetic surfactants suitable for use in the submicron emulsions of the present invention include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants (commercially available under the name SPAN.RTM. or ARLACEL.RTM.), fatty acid esters of polyethoxylated sorbitol (TWEEN.RTM.), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL M-53), polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL), polyoxyethylene fatty alcohol ethers (BRIJ.RTM.); polyoxyethylene nonphenyl ethers (TRITON.RTM. N), polyoxyethylene isooctylphenyl ethers (TRITON.RTM. X). Preferred synthetic surfactants are the surfactants available under the name SPAN.RTM. and TWEEN.RTM..

Preferred surfactants for use in the oil-in-water emulsions of the present invention include lecithin, Tween-80 and SPAN-80.

Generally speaking, the surfactant, or the combination of surfactants, if two or more surfactants are used, is present in the emulsion in an amount of 0.01% to 10% by volume, preferably, 0.1% to 6.0%, more preferably 0.2% to 5.0%.

The aqueous component constitutes the continuous phase of the emulsion and can be water, buffered-saline or any other suitable aqueous solution.

The oil-in-water emulsions of the present invention can include additional components that are appropriate and desirable, including preservatives, osmotic agents, bioadhesive molecules, and immunostimulatory molecules.

It is believed that bioadhesive molecules can enhance the delivery and attachment of antigens on or through the target mucous surface conferring mucosal immunity. Examples of suitable bioadhesive molecules include acidic non-naturally occurring polymers such as polyacrylic acid and polymethacrylic acid (e.g., CARBOPOL.RTM., CARBOMER); acidic synthetically modified natural polymers such as carboxymethylcellulose; neutral synthetically modified natural polymers such as (hydroxypropyl) methylcellulose; basic amine-bearing polymers such as chitosan; acidic polymers obtainable from natural sources such as alginic acid, hyaluronic acid, pectin, gum tragacanth, and karaya gum; and neutral non-naturally occurring polymers, such as polyvinylalcohol; or combinations thereof.

The phrase "immunostimulatory molecules", as used herein, refers to those molecules that enhance the protective immune response induced by an antigenic component in vaccine compositions. Suitable immunostimulatory materials include bacterial cell wall components, e.g., derivatives of N-acetyl muramyl-L-alanyl-D-isoglutamine such as murabutide, threonyl-MDP and muramyl tripeptide; saponin glycosides and derivatives thereof, e.g., Quil A, QS 21 and GPI-0100; cholesterol; and quaternary ammonium compounds, e.g., dimethyldioctadecylammonium bromide (DDA) and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine ("avridine").

Saponis are glycosidic compounds that are produced as secondary metabolites in a wide variety of plant species. The chemical structure of saponins imparts a wide range of pharmacological and biological activities, including some potent and efficacious immunological activity.

Structurally, saponins consist of any aglycone attached to one or more sugar chains. Saponins can be classified according to their aglycone composition: Triterpene glycosides, Steroid glycosides, and Steroid alkaloid glycosides.

Saponin can be isolated from the bark of Quillaja saponaria. Saponin has long been known as an immunostimulator. Dalsgaard, K., "Evaluation of its adjuvant activity with a special reference to the application in the vaccination of cattle against foot-and-mouth disease", Acta. Vet. Scand. 69: 1 40 1978. Crude extracts of plants containing saponin enhanced potency of foot and mouth disease vaccines. However, the crude extracts were associated with adverse side effects when used in vaccines. Subsequently, Dalsgaard partially purified the adjuvant active component from saponin by dialysis, ion exchange and gel filtration chromatography. Dalsgaard, K. et al., "Saponin adjuvants III. Isolation of a substance from Quillaja saponaria Morina with adjuvant activity in foot-and-mouth disease vaccines", Arch. Gesamte. Virusforsch. 44: 243 254 1974. An adjuvant active component purified in this way is known as "Quil A." On a weight basis Quil A showed increased potency and exhibited reduced local reactions when compared to crude saponin. Quil A is widely used in veterinary vaccines.

Further analysis of Quil A by high pressure liquid chromatography (HPLC) revealed a heterogenous mixture of closely related saponins and led to discovery of QS21 which was a potent adjuvant with reduced or minimal toxicity. Kensil C. R. et al., "Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex," J. Immunol. 146: 431 437, 1991. Unlike most other immunostimulators, QS 21 is water-soluble and can be used in vaccines with or without emulsion type formulations. QS21 has been shown to elicit a Th1 type response in mice stimulating the production of IgG2a and IgG2b antibodies and induced antigen-specific CD8+ CTL (MHC class 1) in response to subunit antigens. Clinical studies in humans have proved its adjuvanticity with an acceptable toxicological profile. Kensil, C. R. et al., "Structural and imunological charaterization of the vaccine adjuvant QS-21. In Vaccine Design: the subunit and Adjvuant Approach," Eds. Powell, M. F. and Newman, M.J. Plenum Publishing Corporation, New York. 1995, pp. 525 541.

U.S. Pat. No. 6,080,725 teaches the methods of making and using saponin-lilpophile conjugate. In this saponin-lipophile conjugate, a lipophile moiety such as lipid, fatty acid, polyethylene glycol or terpene is covalently attached to a non-acylated or desacylated triterpene saponin via a carboxy group present on the 3-O-glucuronic acid of the triterpene saponin. The attachment of a lipophilic moiety to the 3-O-glucuronic acid of a saponin such as Quillaja desacylsaponin, lucyoside P, or saponin from Gypsophila, saponaria and Acanthophyllum enhances their adjuvant effects on humoral and cell-mediated immunity. Additionally, the attachment of a lipophile moiety to the 3-O-glucuronic acid residue of non- or desacylsaponin yields a saponin analog that is easier to purify, less toxic, chemically more stable, and possesses equal or better adjuvant properties than the original saponin.

GPI-0100 is a saponin-lipophile conjugate described in the U.S. Pat. No. 6,080,725. GPI-0100 is produced by the addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid.

Quaternary ammonium compounds--A number of aliphatic nitrogenous bases have been proposed for use as immunological adjuvants, including amines, quaternary ammonium compounds, guanidines, benzamidines and thiouroniums. Specific such compounds include dimethyldioctadecylammonium bromide (DDA) and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine ("avridine").

U.S. Pat. No. 5,951,988 teaches adjuvant formulation containing quarternary ammonium salts such as DDA in conjunction with an oil component. This formulation is useful in conjunction with known immunological substances, e.g., viral or bacterial antigens in a vaccine composition, in order to enhance the immunogenic response. The composition is also useful without an incorporated antigen as nonspecific immunostimulatory formulation.

U.S. Pat. No. 4,310,550 describes the use of N,N-higher alkyl-N,N'-bis(2-hydroxyethyl)-propanediamine and N,N-higher alkyl-xylylenediamines formulated with fat or lipid emulsion as a vaccine adjuvant. A method of inducing or enhancing the immunogenic response of an antigen in man or an animal through parenteral administration of the adjuvant formulation is described in the U.S. Pat. No. 4,310,550.

In a preferred embodiment, the present invention provides a submicron oil-in-water emulsion useful as vaccine adjuvant, which is composed of an AMPHIGEN.RTM. formulation, with droplets of a size less than 1 .mu.m and a mean droplet size of about 0.25 .mu.m.

The term "AMPHIGEN.RTM. formulation" as used herein refers to a solution formed by mixing a DRAKEOL.RTM. lecithin oil solution (Hydronics, Lincoln, Nebr.) with saline solution in the presence of TWEEN.RTM. 80 and SPAN.RTM. 80. A typical AMPHIGEN.RTM. formulation contains 40% light mineral oil by volume (v/v), about 25% w/v lecithin, about 0.18% TWEEN 80 by volume (v/v) and about 0.08% Span 80 by volume (v/v).

Methods of Preparing Submicron Oil-in-Water Emulsions

In another embodiment, the present invention provides methods of preparing the suomicron oil-in-water emulsions described hereinabove.

According to the present invention, the various components of the emulsion, including oil, one or more surfactants, an aqueous component and any other component appropriate for use in the emulsion, are combined and mixed together.

The mixture formed is subjected to an emulsification process, typically by passage one or more times through one or more homogenizers or emulsifiers to form an oil-in-water emulsion which has a uniform appearance and an average droplet size of about 0.5 .mu.m. Any commercially available homogenizer or emulsifier can be used for this purpose, e.g., Ross emulsifier (Hauppauge, N.Y.), Gaulin homogenizer (Everett, Mass.).

The emulsion so formed is then subjected to microfluidization to bring the droplet size in the submicron range. Microfluidization can be achieved by use of a commercial mirofluidizer, such as model number 11OY available from Microfluidics, Newton, Mass.; Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.); and Rainnie Minilab Type 8.30H (Miro Atomizer Food and Dairy, Inc., Hudson, Wis.). These microfluidizers operate by forcing fluids through small apertures under high pressure, such that two fluid streams interact at high velocities in an interaction chamber to form emulsions with droplets of a submicron size.

Droplet size can be determined by a variety of methods known in the art, e.g., laser diffraction, by use of commercially available sizing instruments. The size may vary depending on the type of surfactant used, the ratio of surfactant to oil, operating pressure, temperature, and the like. The skilled artisan can determine the desired combination of these parameters to obtain emulsions with desired droplet size without undue experimentation. The droplets of the emulsions of the present invention are less than 1 .mu.m in diameter, preferably with a mean droplet size of less than 0.8 .mu.m, and more preferably with a mean droplet size less than 0.5 .mu.m, and even more preferably with a mean droplet size of less than 0.3 .mu.m.

In a preferred embodiment of the present invention, the DRAKEOL lecithin oil solution, which is commercially available from Hydronics (Lincoln, Nebr.) and contains 25% lecithin in light mineral oil, is combined and mixed with saline as well as surfactants TWEEN.RTM. 80 and SPAN.RTM. 80 to form an "AMPHGEN.RTM. solution" or "AMPHIGEN.RTM. formulation". The AMPHGEN.RTM. solution is then emulsified with a Ross.RTM. (Hauppauge, N.Y. 11788) emulsifier at approximately 3400 rpm to form an oil-in-water emulsion. Subsequently the emulsion is passed once through a Microfluidizer operating at about 4500.+-.500 psi. The microfluidized oil-in-water emulsion has droplets of a size less than 1 .mu.m, with a mean droplet size of about 0.25 .mu.m.

Vaccine Compositions Containing Antigens Incorporated in Submicron Oil-in-Water Emulsions

In another embodiment, the present invention provides vaccine compositions which contain an antigen(s) and a submicron oil-in-water emulsion described hereinabove. These vaccine compositions are characterized by having an enhanced immunogenic effect and an improved physical appearance (e.g., no phase separation is observed after an extended period of storage). In addition, the vaccine compositions of the present invention are safe for administration to animals.

According to the present invention, the antigen can be combined with the emulsion extrinsically, or preferably, intrinsically. The term "intrinsically" refers to the process wherein the antigen is combined with the emulsion components prior to the microfluidization step. The term "extrinsically" refers to the process where the antigen is added to the emulsion after the emulsion has been microfluidized. The extrinsically added antigen can be free antigen or it can be encapsulated in microparticles as further described herein below.

The term "antigen" as used herein refers to any molecule, compound or composition that is immunogenic in an animal and is included in the vaccine composition to elicit a protective immune response in the animal to which the vaccine composition is administered.

The term "immunogenic" as used in connection with an antigen refers to the capacity of the antigen to provoke an immune response in an animal against the antigen. The immune response can be a cellular immune response mediated primarily by cytotoxic T-cells, or a humoral immune response mediated primarily by helper T-cells, which in turn activates B-cells leading to antibody production.

A "protective immune response" is defined as any immune response, either antibody or cell mediated immune response, or both, occurring in the animal that either prevents or detectably reduces the occurrence, or eliminates or detectably reduces the severity, or detectably slows the rate of progression, of the disorder or disease caused by the antigen or a pathogen containing the antigen.

Antigens which can be included in the vaccine composition of the present invention include antigens prepared from pathogenic bacteria such as Mycoplasma hyopneumoniae, Haemophilus somnus, Haemophilus parasuis, Bordetella bronchiseptica, Actinobacillus pleuropneumonie, Pasteurella multocida, Manheimia hemolytica, Mycoplasma bovis, Mycoplasma galanacieum, Mycobacterium bovis, Mycobacterium paratuberculosis, Clostridial spp., Streptococcus uberis, Streptococcus suis, Staphylococcus aureus, Erysipelothrix rhusopathiae, Campylobacter spp., Fusobacterium necrophorum, Escherichia coli, Salmonella enterica serovars, Leptospiraspp.; pathogenic fungi such as Candida; protozoa such as Cryptosporidium parvum, Neospora canium, Toxoplasma gondii, Eimeria spp.; helminths such as Ostertagia, Cooperia, Haemonchus, Fasciola, either in the form of an inactivated whole or partial cell preparation, or in the form of antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis. Additional antigens include pathogenic viruses such as Bovine herpesviruses-1,3,6, Bovine viral diarrhea virus (BVDV) types 1 and 2, Bovine parainfluenza virus, Bovine respiratory syncytial virus, bovine leukosis virus, rinderpest virus, foot and mouth disease virus, rabies, swine fever virus, African swine fever virus, Porcine parvovirus, PRRS virus, Porcine circovirus, influenza virus, swine vesicular disease virus, Techen fever virus, Pseudorabies virus, either in the form of an inactivated whole or partial virus preparation, or in the form of antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis.

The amount of the antigen should be such that the antigen which, in combination with the oil-in-water emulsion, is effective to induce a protective immune response in an animal. The precise amount of an antigen to be effective depends on the nature, activity and purity of the antigen, and can be determined by one skilled in the art.

The amount of the oil-in-water emulsion present in the vaccine compositions should be sufficient for potentiating the immunogenicity of the antigen(s) in the vaccine compositions. When desirable and appropriate, additional amounts of surfactant(s) or additional surfactant(s) can be added in the vaccine composition in addition to the surfactant(s) provided by the oil-in-water emulsion. Generally speaking, the oil component is present in the final volume of a vaccine composition in an amount from 1.0% to 20% by volume; preferably, in an amount of 1.0% to 10%; more preferably, in an amount from 2.0% to 5.0%. The surfactant, or the combination of surfactants if two or more surfactants are used, is present in the final volume of a vaccine composition in an amount of 0.1% to 20% by volume, preferably, 0.15% to 10%, more preferably 0.2% to 6.0%.

In addition to the antigen(s) and the oil-in-water emulsion, the vaccine composition can include other components which are appropriate and desirable, such as preservatives, osmotic agents, bioadhesive molecules, and immunostimulatory molecules (e.g., Quil A, cholesterol, GPI-0100, dimethyldioctadecylammonium bromide (DDA)), as described hereinabove in connection with the oil-in-water emulsion.

The vaccine compositions of the present invention can also include a veterinarily-acceptable carrier. The term "a veterinarily-acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others.

In a preferred embodiment, the present invention provides a vaccine composition which includes at least one of a BVDV type I or BVDV type II antigen, incorporated intrinsically in an oil-in-water emulsion which has droplets of a size of less than 1 .mu.m, preferably with a mean droplet size of less than 0.8 .mu.m, more preferably less than 0.5 .mu.m, and even more preferably with a mean droplet size of about 0.5 .mu.m. The BVDV type I and/or II antigen is preferably in the form of an inactivated viral preparation. The submicron oil-in-water emulsion preferably is composed of an AMPHIGEN.RTM. formulation (i.e., a formulation which contains light mineral oil, lecithin, TWEEN.RTM. 80, and SPAN.RTM. 80). The vaccine composition preferably also includes Quil-A, cholesterol, and thimerosol.

In another preferred embodiment, the present invention provides a vaccine composition which includes a Leptospira antigen and at least one of a BVDV type I or BVDV type II antigen in an oil-in-water emulsion. The antigens, preferably in the form of inactivated cell or viral preparation, are incorporated intrinsically in the oil-in-water emulsion having droplets of a size of less than 1 .mu.m, preferably with a mean droplet size of less than 0.8 .mu.m, more preferably less than 0.5 .mu.m, and even more preferably with a mean droplet size of about 0.5 .mu.m. The submicron oil-in-water emulsion preferably is composed of an AMPHIGEN formulation (i.e., a formulation which contains light mineral oil, lecithin, TWEEN.RTM. 80, and SPAN.RTM. 80). The vaccine composition preferably also includes one or more immunostimulatory molecules selected from Quil-A, cholesterol, DDA, GPI-100 and aluminum hydroxide (AIOH).

In still another preferred embodiment, the present invention provides a vaccine composition which includes at least one bacterial antigen, e.g., the recombinant Streptococcus uberis PauA protein or a cell preparation of E. coli or a combination of both, in an oil-in-water emulsion. The antigen(s) is combined intrinsically with the oil-in-water emulsion which has droplets of a size of less than 1 .mu.m, preferably with a mean droplet size of less than 0.8 .mu.m, more preferably less than 0.5 .mu.m, and even more preferably with a mean droplet size of about 0.25 .mu.m. The submicron oil-in-water emulsion preferably is composed of an AMPHIGEN(.RTM. formulation (i.e., a formulation which contains light mineral oil, lecithin, TWEEN(.RTM. 80, and SPAN.RTM. 80). The vaccine composition preferably also includes one or more immunostimulatory molecules selected from Quil A, DDA and GPI-100.

The vaccine compositions of the present invention can be administered to an animal by known routes, including the oral, intranasal, mucosal, topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular) route. Administration can be achieved using a combination of routes, e.g., first administration using a parental route and subsequent administration using a mucosal route.

Methods of Preparing Vaccine Compositions

In a further embodiment, the present invention provides methods of preparing vaccine compositions containing an antigen or antigens and a submicron oil-in-water emulsion.

In preparing the vaccine compositions of the present invention, the antigen(s) can be combined either intrinsically or extrinsically with the components of the oil-in-water emulsion. Preferably, the antigen is combined with the components of the oil-in-water emulsion intrinsically.

The antigen can be combined with the various components of the emulsion, including oil, one or more surfactants, an aqueous component and any other appropriate component, to form a mixture. The mixture is subjected to a primary blending process, typically by passage one or more times through one or more homogenizers or emulsifiers, to form an oil-in-water emulsion containing the antigen. Any commercially available homogenizer or emulsifier can be used for this purpose, e.g., Ross emulsifier (Hauppauge, N.Y.), Gaulin homogenizer (Everett, Mass.), or Microfluidics (Newton, Mass.). Alternatively, the various components of the emulsion adjuvant, including oil, one or more surfactants, and an aqueous component can be combined first to form an oil-in-water emulsion by using a homogenizer or emulsifier; and the antigen is then added to this emulsion. The mean droplet size of the oil-in-water emulsion after the primary blending is approximately 1.0 1.2 micron.

The emulsion containing the antigen is then subjected to microfluidization to bring the droplet size in the submicron range. Microfluidization can be achieved by use of a commercial mirofluidizer, such as model number 11OY available from Microfluidics, Newton, Mass.; Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.); and Rainnie Minilab Type 8.30H (Miro Atomizer Food and Dairy, Inc., Hudson, Wis.).

Droplet size can be determined by a variety of methods known in the art, e.g., laser diffraction, by use of commercially available sizing instruments. The size may vary depending on the type of surfactant used, the ratio of surfactant to oil, operating pressure, temperature, and the like. One can determine a desired combination of these parameters to obtain emulsions with a desired droplet size. The oil droplets of the emulsions of the present invention are less than 1 .mu.m in diameter. Preferably the mean droplet size is less than 0.8 .mu.m. More preferably, the mean droplet size is less than 0.5 .mu.m. Even more preferably, the mean droplet size is about 0.1 to 0.3 .mu.m.

In a preferred embodiment of the present invention, the DRAKEOL.RTM. lecithin oil solution, which contains 25% lecithin in light mineral oil, is combined and mixed with surfactants TWEEN.RTM. 80 and SPAN.RTM. 80 and saline solution to form a mixture that contains 40% light mineral oil, lecithin, 0.18% TWEEN.RTM. 80, and 0.08% SPAN.RTM. 80. The mixture is then emulsified with a Ross.RTM. (Hauppauge, N.Y. 11788) emulsifier at approximately 3400 rpm to form an emulsion product, which is also referred to as an "AMPHIGEN.RTM. formulation" or "AMPHIGEN.RTM. solution". Subsequently, the desired antigen(s) are combined with the AMPHIGEN.RTM. solution and any other appropriate components (e.g., immunostimulatory molecules) with the aid of an emulsifier, e.g., a Ross homogenizer, to form an oil-in-water emulsion containing the antigen(s). Such emulsion is passed once through a Microfluidizer operating at about 10000.+-.500 psi. The microfluidized oil-in-water emulsion has droplets of a size of less than 1 .mu.m, with the mean droplet size of about 0.25 .mu.m.

In another preferred embodiment, prior to combining an oil-in-water emulsion (e.g., an AMPHIGEN.RTM. formulation) with a desired antigen(s), the antigen(s) is combined with a saponin glycoside, e.g., Quil A, to form a mixture. This antigen(s)-saponin mixture is subjected to homogenization, e.g., in a homogenization vessel. A sterol, e.g., cholesterol, is then added to the homogenized antigen(s)-saponin mixture. The mixture containing the antigen(s), saponin and sterol is then subjected to further homogenization. The homogenized antigen(s)-saponin-sterol mixture is then combined with an oil-in-water emulsion (e.g., an AMPHIGEN.RTM. formulation) with the aid of a homogenizer, for example. The homogenized oil-in-water emulsion containing the antigen(s), saponin and sterol is then subjected to high pressure homogenization, such as microfluidization.

Vaccine Compositions Containing Microencapsulated Antigens in a Submicron Oil-in-Water Emulsion and Methods of Preparation

In still another embodiment, the present invention provides vaccine compositions which contain an antigen encapsulated in microparticles (or "microencapsulated antigen"), where the microencapsulated antigen is extrinsically incorporated into a submicron oil-in-water emulsion described hereinabove.

Methods for absorbing or entrapping antigens in particulate carriers are known in the art. See, e.g., Pharmaceutical Particulate Carriers: Therapeutic Applications (Justin Hanes, Masatoshi Chiba and Robert Langer. Polymer microspheres for vaccine delivery. In: Vaccine design. The subunit and adjuvant approach. Eds. Michael F. Powell and Mark J. Newman, 1995 Plenum Press, New York and London ). Particulate carriers can present multiple copies of a selected antigen to the immune system in an animal subject and promote trapping and retention of antigens in local lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release. Particulate carriers have also been described in the art and include, e.g., those derived from polymethyl methacrylate polymers, as well as those derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. Polymethyl methacrylate polymers are non-biodegradable while PLG particles can be biodegrade by random non-enzymatic hydrolysis of ester bonds to lactic and glycolic acids which are excreted along normal metabolic pathways.

Biodegradable microspheres have also used to achieve controlled release of vaccines. For example, a continuous release of antigen over a prolonged period can be achieved. Depending upon the molecular weight of the polymer and the ratio of lactic to glycolic acid in the polymer, a PLGA polymer can have a hydrolysis rate from a few days or weeks to several months or a year. A slow, controlled release may result in the formation of high levels of antibodies similar to those observed after multiple injections. Alternatively, a pulsatile release of vaccine antigens can be achieved by selecting polymers with different rates of hydrolysis. The rate of hydrolysis of a polymer typically depends upon the molecular weight of the polymer and the ratio of lactic to glycolic acid in the polymer. Microparticles made from two or more different polymers with varying rates of antigen release provide pulsatile releases of antigens and mimics multiple-dose regimes of vaccination.

According to the present invention, an antigen, including any of those described hereinabove, can be absorbed to a particulate polymer carrier, preferably a PLG polymer, by using any procedure known in the art (such as one exemplified in Example 17), to form a microencapsulated antigen preparation. The microencapsulated antigen preparation is then mixed with and dispersed in a submicron oil-in-water emulsion, which emulsion has been described hereinabove, to form the vaccine composition.

In a preferred embodiment, the present invention provides a vaccine composition which contains an antigen encapsulated in a PLG polymer, wherein the microencapsulated antigen is dispersed extrinsically in a microfluidized oil-in-water emulsion which is composed of light mineral oil, lecithin, TWEEN80, SPAN80 and saline, and has a mean droplet size of less than 1.0 .mu.m.

Complexes Formed by a Saponin and Sterol

In one embodiment, the present invention provides compositions containing a saponin and a sterol, wherein the saponin and the sterol form complexes in the form of helical micelles. According to the present invention, these complexes have immunostimulating activities.

By "immunostimulating" is meant that the complexes can enhance the immune response induced by an antigenic component, or that the complexes can induce an immune response independent of a separate antigenic component.

In accordance with the present invention, a preferred saponin for use in a composition of the present invention is Quil A.

Preferred sterols for use in the adjuvant compositions of the present invention include beta-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 11th Edn., page 341, as a naturally occurring sterol found in animal fat. Most preferably the sterol is cholesterol.

The ratio of saponin:sterol in the composition is typically on the order of 1:100 to 5:1 weight to weight. Preferably, the ratio is 1:1.

In another embodiment, the present invention provides vaccine compositions containing a saponin, a sterol and an antigen, wherein the saponin and the sterol form complexes in the form of helical micelles, and wherein the antigen is admixed with but not incorporated within the helical micelles.
 

Claim 1 of 8 Claims

1. A vaccine comprising a saponin glycoside, a sterol and an antigen, wherein said saponin glycoside and said sterol associate with each other to form complexes in the form of helical micelles, and wherein said antigen is in admixture with, but not integrated within, said helical micelles.

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