MXPA06011569A - Microfluidized oil-in-water emulsions and vaccine compositions - Google Patents

Abstract

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.

Description

empower the immune response. Many adjuvants modify the network of cytokines associated with immune response. These immunomodulatory adjuvants can exert their effect even when they are not combined with antigens. In general, immunomodulatory adjuvants cause a regulation by general increase of certain cytokines and a concomilanie regulation by decreasing of hearing causing a cellular response Th1 and / or a humoral Th2. Some adjuvants have the ability to preserve the integrity of the configuration of an antigen so that the antigens can be efficiently presented to the appropriate immune cells. As a result of this preservation of the nutrient configuration mediated by the adjuvant formulation, the vaccine would have a longer shelf life than that demonstrated for the immunosuppressant complexes (ISCOM). Ozel M. et al .; Qualernary Structure of the Immunoestimmulafing Complex (Iscom), J. of Ultrastruc. and Molec. Struc. Res. 102, 240-248 (1989). Some adjuvants have the property of retaining the antigen in deposit form in the area of the injection. As a result of this depot effusion, the amphigen is not rapidly lost by hepatic clearance. The aluminum salts and the water emulsions in oil accrue to the effect of this deposition effect with a shorter duration. For example, a long-term depot can be achieved by using Freund's complete adjuvant (FCA) which is a water-in-oil emulsion. The FCA typically remains in the injection zone until biodegradation allows the antigen to be removed by the antigen-presenting cells. Based on their physical nature, adjuvants can be grouped into two very broad categories, specifically particulate adjuvants and non-particulate adjuvants. The particulate adjuvants exist in the form of microparticles. The immunogen is capable of incorporating or associating with the microparticles. Aluminum salts, water emulsions in oil, emulsions of oil in water, immunosimulatory complexes, liposomes and nanoparticles and microparticles are examples of particulate adjuvants. Non-particulate adjuvants are generally immunomodulators and are generally used in June with particulate adjuvants. Muramil dipeptide (a component of the active adjuvant of a pepifidoglycan extracted from Mycobacillus), non-ionic block copolymers, saponins (a complex mixture of extruded fyleerpenoids from the bark of the Quillaja saponaria tree), Lipid A (a glucosamine disaccharide with two phosphate groups and five or six chains of fatty acids generally of C12 to C16 in length), cytokines, carbohydrate polymers, derivatized polysaccharides, and bacterial toxins such as cholera toxin and labile ioxin of E. coli (LT) are examples of non-particulate adjuvants . Some of the best-known adjuvants are combinations of non-particulate immunomodulators and particulate materials that could confer a depot effect to the adjuvant formulation. For example, FCA combines the immunomodulatory properties of components of Mycobacterium tuberculosis with the short-term deposition effect of oil emulsions. Oil emulsions have been used as vaccine adjuvants for a long time. Le Moignic and Pinoy found in 1916 that a suspension of inactivated Salmonella typhimurium in paraffin oil increased the immune response. Subsequently, in 1935, Ramón described starch as one of the substances that increased the antimicrobial response to the diphtheria dioxoid. However, oil emulsions did not become popular until 1937 when Freund achieved his formulation of adjuvant, which is now known as Freund's Adjuvant Complement (FCA). The FCA is an emulsion of water in oil composed of mineral oil (paraffin) mixed with inactivated Mycobacteria and Arlacel A. Arlacel A is mainly monooleate of mannide and is used as an emulsionanfe agent. Although FCA is excellent for inducing an antibody response, causes severe pain, abscess formation, fever and granulomatous inflammation. To avoid these undesirable side reactions, the Incomplete Freund's Adjuvant (IFA) was developed. The IFA is similar to the FCA in its composition except for the absence of mycobacterial components. The IFA acíúa medlanfe formulation in depósiío in the area of the injection and liberation lenía of the aníígeno simulating the producing cells of aníibodies. Another strategy to improve FCA was based on the notion that replacing paraffin oil with a biocompatible oil would help eliminate FCA-associated reactions in the injection area. It was also believed that the emulsion should be one of oil in water instead of water in oil because that produces a long-term deposit in the area of the injection. Hilleman et al, described an Adjuvant 65 oil based adjuvant, which consisted of 86% peanut kernel, 10% Arlacel A as an emulsifier and 4% aluminum monoesiearafoxide as a destabilizer. Hilleman, 1966, Prog. Med. Viral. 8: 131-182; Hilleman and Beale, 1983, in New Approaches I Vaccine Developmení (Editors Bell, R. and Torrigiani, G.), Schwabe, Basel. In humans, the use of Adjuvaní 65 was safe and powerful, but it had less adjuvant capacity than the IFA. However, the use of Adjuvant 65 was suspended due to the reactogenic capacity in man with certain batches of vaccines and the reduction of adjuvant capacity when purified or synthetic emulsifier was used in place of Arlacel A. US Pat. 5,718,904 and 5,690,942 teach that the paraffin oil in the water-in-water emulsion can be suspended by meabolizable oil in order to improve the safety profile. In addition to adjuvant capacity and safety, the physical appearance of an emulsion is also an important commercial consideration. The physical aspect depends on the emulsion's stability. Phase separation, sedimentation and coalescence are indicators of the instability of the emulsion. Phase separation occurs when the oleaginous and aqueous phases of the emulsion have a different specific gravity. Phase separation also occurs when the droplet size of the emulsion is large and the emulsion does not have Brownian movement. When the size of the drop is large, there is a tendency for the interface to break and the drops merge into large particles. The stability of the emulsion is determined by a number of natural factors such as the nature and amount of emulsifier used, the size of the gouge in the emulsion and the difference in density between the oil and water phase. The emulsifiers promote the stabilization of dispersed goites reducing the free energy of the inferic and creating physical or electrophysical barriers to the coalescence of the goias. Nonionic as well as ionic detergents have been used as emulsifiers. The nonionic emulsifiers are oriented in the infervescence and produce relatively voluminous esírucfuras, which entails the spherical evolution of the dispersed goies. The anionic or cationic emulsifiers induce the formation of a double electric layer by reducing the conjugated ions; the repellent forces of the double layer cause the drops to repel one another as they approach. In addition to using emulsifiers, the stability of the emulsion can also be achieved by reducing the size of the emulsion gouge by mechanical means. Typically, propellant mixers, turbine rotors, colloid mills, homogenizers, and ultrasonic devices have been used to make emulsions. Microfluidization is another way to increase the homogeneity of the size of the gouge in the emulsion. Microfluidization can produce an elegant, physically flammable emulsion with a particle size consistent in the submicrometric range. In addition to increasing emulsion stability, the microfluidization process allows for final filtration which is a preferred way of ensuring the sterility of the final product. In addition, the submicromergic oil particles can pass from the injection sites to the lymphatics and then to the lymph nodes of the drainage, blood and spleen chain. This reduces the likelihood of isolating an oil deposit in the area of the injection that can produce local inflammation and a significant reaction in the area of the injection. Microfluidizers are now commercially available.

The formation of emulsions is produced in a microfluidizer by introducing two fluidized jets at high velocities in an interaction chamber. The microfluidizer is driven by air or nilrogen and can operate with internal pressures above 137,900 kPa. U.S. Patent 4,908,154 teaches the use of a microfluidizer to obtain emulsions essentially free of any emulsifying agents. A number of submicromerial water adjuvant formulations in water have been described in the literature. U.S. Patent 5,376,369 describes a submicron emulsion adjuvant of oil in water which is known as Syntax Adjuvanf Formulafion (SAF). SAF contains squalene or squalane as an oleaginous component, a polymericity of Pluronic block polymer L121 (polyoxypropylene and polyoxyethylene) which forms emulsions and a nimmunopoinizing canine of muramyl dipépfide.

Squalene is a precursor of linear hydrocarbon colesferol that is found in many tissues, nobly in the liver of fish and other sharks. Squalane is prepared by hydrogenation of squalene and is completely purified. Tancula squalene such as squalane can be metabolized and have a good background in toxicological studies. Squalene and squalane emulsions have been used in vaccines against cancer in humans with mild side effects and with a desirable efficacy. See, for example, Anthony C. Allison, 1999, Squalene and Squalane emulsions as adjuvants, Methods 19: 87-93. US Pat. No. 6,299,884 and International Patent Publication WO 90/14837 teach that polyoxypropylene and polyoxy-ethylene block copolymers are not essential for the formation of submicron emulsions of oil in water. In addition, these references teach the use of non-toxic melabolizable oil and expressly exclude the use of paraffin oil and toxic petroleum oil oils in their emulsion formulations. U.S. Patent 5,961,970 describes another submicron emulsion of oil in water for use as adjuvant in vaccines. In the emulsion described in this patent, the hydrophobic component is selected from the group consisting of a medium-chain glyceride oil, a vegetable oil and a mixture thereof. The surfactant that is included in this emulsion may be a biologically comparative, non-ionic agent such as a phospholipid (for example lecltin) or a pharmaceutically acceptable non-naive medical agent such as TWEEN-80. This patent teaches also the incorporation of the antigen to the emulsion at the moment in which the emulsion is formed, contrasted with the mixture of the anígen with the emulsion after the emulsion has been formed independently and exirinseca. U.S. Patent 5,084,269 teaches that an adjuvant formulation containing leciin combined with paraffin oil causes a decrease in irritation in the host animal and simultaneously induces an increased seismic immunity. The formulation of the adjuvant resulting from U.S. Patent 5,084,269 is commercially used in veterinary vaccines under the trade name AMPHIGEN®. The formulation of AMPHIGEN® is formed by micelles - oil gofas surrounded by lecithin. These micelles allow more complete cellular anigenes to bind than the adjuvant ones with oleaginous base. In addition, formulations of AMPHIGEN®-based vaccines contain a low oil content of 2.5 to 5% paraffin wax, compared to other formulations of vaccines containing oleaginous adjuvants, which typically contain 10% to 20% oil Its low oil content makes this adjuvant-based vaccine less irritating to the tissues in the injection site, resulting in fewer injuries and less waste when sacrificed. In addition, the lecithin coating surrounding the oil droplets further reduces reactions in the injection site resulting in a vaccine that is safe as well as effective. The formulation of AMPHIGEN® is used as adjuvant in a number of veterinary vaccines and there is a need to maintain the physical appearance of the vaccine during short and long storage periods as well as at the time of reconstitution. further, a lyophilized antigen is mixed with the adjuvant formulation prepared previously just before the injection. This practice does not always ensure that there is a uniform distribution of the antigen in the emulsion of oil in water and the appearance of the emulsion may not be desirable. In addition, when standing, the homogenized emulsion may show phase separation. Therefore, there is a need for a stable adjuvant formulation that does not reveal phase separation over a long shelf life. One way to prevent phase separation is to reduce the size of the glaze and increase the homogeneity of the emulsion particles. Although the process of microfluidization of meta bolite oil-based emulsion formulations has been documented, microfluidization of acetyl emulsions in water has not yet been carried out as the AMPHIGEN® formulation. In the present invention, microfluidization has been used to bring the size of the paraffin oil goiters surrounded by lecithin to submicron size. Unexpectedly, the present inventors have discovered that microfluidization of vaccine formulations with adjuvants of an oil-in-water emulsion formed by a mixture of leciin and oil not only improves the physical appearance of the formulations, but also enhances the effects immunizers of the formulations. The microfluidized formulations are also characterized by an improved safety profile.

BRIEF DESCRIPTION OF THE INVENTION The present inventors have unexpectedly discovered that the adjuvant activity and the safety profile of non-metabolizable oil-based oil emulsions in water can be improved by microfluidization. The antigens that are incorporated into the microfluidized emulsions are stable even when the antigens are intrinsically incorporated into the emulsions before microfluidization. Accordingly, in one embodiment, the present invention provides formulations of oil-in-water submicron emulsions useful as vaccine adjuvants. The oil-in-water submicron emulsions of the present invention are compounded by a non-meabolizable oil, at least one surfactant and an aqueous component, in which the oil is dispersed in the aqueous component with an average size of the oil golas in the submicrometric interval. A preferred non-meyabolizable oil is fluid paraffin oil. Preferred surfactants include lecithin, Tween-80 and SPAN-80. A preferred water-in-oil emulsion provided by the present invention is compounded by an AMPHIGEN® formulation.

The oil-in-water emulsions of the present invention may include additional components that are appropriate and desirable, including preservatives, osmotic agents, bioadhesive molecules and immune-limiting molecules. Preferred immunosuppressant molecules include, for example, Quil A, cholesterol, GPI-0100, dimethyl diacid bromide (DDA). In another embodiment, the present invention provides methods for preparing a submicroméral oil-in-water emulsion. In accordance with the present invention, the various components of the emulsion are mixed, including the oil, one or more lensioacids, an aqueous component and any other component suitable for use in the emulsion. 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 of goats with a diameter of less than 1 micrometer, preferably with an average size of drops below 0.5 micrometers. In yet another embodiment, the present invention provides compositions of vaccines that contain an antigen and a submicromerric emulsion of oil in water described above. The anigen is incorporated into the emulsion exírinseca or inírínseca, preferably in an intrinsic manner. The antigen that can be included in the compositions of the vaccines 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 preparation of whole or partial cells or viruses, or have 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 for preparing compositions of vaccines containing an antigen or combined antigens (s) with a submicron emulsion of oil in water. In order to prepare the compositions of the vaccines of the present invention, the antigen (s) can be combined in an intrinsic manner (for example before microfluidization) or extrinsically (for example after microfluidization) with the components of the oil in water emulsion. Preferably, the antigen is combined with the components of the oil emulsion in water in an inhysninic manner. In still another embodiment, the present invention provides compositions of vaccines conferring a microencapsulated anion and an oil-in-water submicron emulsion described above, in which the microencapsulated anion is combined with the emulsion in an extrinsic manner. It has also surprisingly been found that a saponin and a sterol, when combined in solution, they are associated with yes to form complexes in the form of helical micelles. In accordance with the present invention, these helical micelle complexes have immunostimulatory properties and are especially useful as adjuvants in vaccine compositions. Accordingly, the present invention provides compositions of vaccines containing a saponin and a eseryol, in which saponin and eserol form complexes in the form of helical micelles. The present invention also provides compositions comprising a saponin, a seriol and an anigen, wherein the saponin and the eserol form complexes in the form of helical micelles and in which the antigen is mixed but not incorporated into the helical micelles.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the process for batch preparation of non-microfluidized vaccine compositions. In this procedure the various components of the vaccine are added to the addition vessel on the left and finally pumped into the mixing vessel where the components are mixed by simple mechanical means. Figure 2 represents the process for the preparation of compositions of the microfluidized vaccines containing the antigen incorporated in an inverse way. The various components of the vaccine are added to the addition vessel and transferred to the pre-emulsion mixing unit for mixing by simple mechanical means. Subsequently, the emulsion is passed through a microfluidizer and collected in the post-microfluidization chamber. Figure 3 represents the distribution of the size of the vaccines of the non-microfluidized AMPHIGEN® formulation-based vaccine, the microfluidized AMPHIGEN® formulation-based vaccine and the preparation of the vaccine mixed in a bank. Figure 4 shows the absence of phase separation in the preparation of microfluidized vaccine. Figure 5 represents a comparison of the stability of the antigens that are incorporated in a non-inverse manner to the preparation of the vaccine based on the formulation of AMPHIGEN® microfluidized (A907505) and fresh preparations of the vaccines based on formulation of AMPHIGEN® non-microfluidized conírol (A904369, A904370 and A904371). The preparations of the vaccines were stored at 4 ° C for two years. At different times during storage (0, 6, 12 or 24 months), the formulations were used to vaccinate cows of months of age.

Vaccination was carried out on Day 0 and 21 with 2 ml of a vaccine dose and the sera were collected two weeks after the second vaccination. The assessment of neutralizing antibodies for the BVD virus was determined Type II in each of the serum samples. Damages are presented in terms of the geometric mean of 5 animals. Figure 6 shows the mean of least squares of the rectal temperature of cattle before and after the administration of microfluidized and non-microfluidized vaccines. T01: Placebo group - single dose; Placebo Group - Double Dose; T03: Non-microfluidized formulation - Single dose; T04: Non-microfluidized formulation - Double dose; T05: Microfluidized formulation - single dose; T06: Microfluidized formulation - double dose. Figure 7 represents the average of the least squares of injection reaction volumes observed in cattle after administration of microfluidized and non-microfluidized vaccines. T03: Non-microfluidized formulation - Single dose; T04: Non-microfluidized formulation - Double dose; T05: Microfluidized formulation - single dose; T06: Microfluidized formulation - double dose. Figure 8 represents the geometric mean of the IgG titers for PauA recombinant aniigen of Streptococcus uberis iras vaccination with the various formulations of vaccines containing PauA recombinant anigen as an antigen of E. coli cells. Figure 9 represents the geometrical average of the IgG titers for E. coli cell antigens of Streptococcus uberis iras vaccination with the various formulations of the vaccines containing recombinant PauA antigen tannin as an E. coli whole cell antigen. Figures 10A and 10B depict the particle size distribution of a microfluidized Amphigen formulation at the start of production (Figure 10A) and 22 months after production (Figure 10B). Figure 11 is a photograph of electron microscopy showing helical micelles formed with Quil A micelles and cholesterol crystals. Figure 12 is a photograph of electron microscopy showing helical immunogenic complexes formed by Quil A and cholesterol on the surface of the BVD Type I antigen.

DETAILED DESCRIPTION OF THE INVENTION The present inventors have unexpectedly discovered that microfluidization of formulations of vaccines with oil-in-water emulsion adjuvants composed of a mixture of lecithin and paraffin oil not only improves the physical appearance of vaccine formulations, but also it also enhances the immunizing effects of the vaccine formulations. Microfluidized bottle formulations are also characterized by an improved safety profile. Based on these findings, the present invention provides oil-in-water submicron emulsions useful as adjuvants in vaccine compositions. Methods are also provided to prepare these submicromergic emulsions of aceifee in water using a microfluidizer. In addition, the present invention provides compositions of submicromergic vaccines in which an anigen is combined with a submicron emulsion of oil in water. Methods for preparing such vaccine compositions are also provided. The present invention further provides compositions of vaccines containing microencapsulated antigens combined with a submicron emulsion of oil in water and methods for preparing vaccines. To make the description clearer, and not by way of limitation, the detailed description of the invention is divided into the following subsections which describe or illustrate certain characteristics, embodiments or applications of the invention.

Sub-micron oil-in-water emulsions In one embodiment, the present invention provides oil-in-water submicron emulsions useful as vaccine adjuvants. The oil-in-water submicron emulsions of the present invention enhance the immunogenic capacity of the antigens of the vaccine compositions, are safe to be administered to animals and remain stable during storage. The oil-in-water submicron emulsions of the present invention are composed of a non-metabolizable oil, at least one surfactant and an aqueous component, in which the oil is dispersed in the aqueous component with an average size of the oil droplets in the oil. submicrometric interval. By "submicroméírico" it is meant that the goias are of a size less than 1 μm (micrometer) and the average size of the oil drops is less than 1 μm. Preferably, the average droplet size of the emulsion is less than 0.8 μm; more preferably less than 0.5 μm; and even more preferably less than 0.4 μm or approximately 0.1-0.3 μm. The "average droplet size" is defined as the size of the particles of the mean volume diameter (DMV) in a volume distribution of the particle sizes. The DMV is calculated by multiplying each particle diameter by the volume of all particles of that size and adding. This is then divided by the total volume of all the particles. The term "non-metabolizable oil" as used herein refers to oils that can not 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. The non-metabolizable oils suitable for use in the emulsions of the present invention include alkenes, alkenes, alkynes and their corresponding acids and alcohols, their ethers and esters, and mixtures thereof. Preferably, the individual compounds of the oil are light hydrocarbon compounds, ie, the components of that type have from 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 "paraffin oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum by a distillation technique. The term is synonymous with "liquid paraffin", "liquid petrolatum" and "white paraffin oil". The term is intended to also include "fluid paraffin oil", that is, oil that is similarly made by petrolatum distillation but that has a specific gravity slightly less than petrolatum. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Easinio, Pa.; Mack Publishing Company, 1990, pages 788 and 1323). The paraffin oil can be obtained in various commercial sources, for example J.T. Baker (Phillipsburg, PA), USB Corporation (Cleveland, OH). The preferred paraffin oil is the fluid paraffin oil commercially available under the name DRAKEOL®. Typically, the oleaginous component of the submicron emulsions of the present invention is present in an amount of 1% or 50%) by volume; preferably, in an amount of 10% to 45; more preferably in an amount of 20% to 40%. The oil-in-water emulsions of the present invention typically include at least one (i.e., one or more) surfactant. The surfactants and emulsifiers, terms that are used interchangeably herein, are agents that stabilize the surface of the oil droplets and keep the oil droplets to the desired size. Suitable surfactants 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 phosphatidyl cholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained in the form of a mixture of phosphatides and triglycerides by washing crude vegetable oils with water, and separating and drying the resulting hydrated gums. A refined product can be obtained by fractionating the mixture to obtain the phospholipids and acetone insoluble glycolipids that remain after removing the triglycerides and the vegetable oil by washing with acetone. Alternatively, lecithin can be obtained in various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin and phosphatidylethanolamine. Phospholipids can be isolated from natural sources or conventionally synthesized. Non-natural synthetic surfactants suitable for use in the submicron emulsions of the present invention include non-ionic surfactants with sorbitan base, for example sorbitan surfactants substituted with fatty acids (commercially available under the name SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR); polyethoxylated fatty acid (for example stearic acid available under the name SIMULSOL M-53), polyethoxylated isooctylphenol polymer and formaldehyde (TYLOXAPOL), ethers of polyoxyethylene fatty alcohols (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethylene isocycylphenyl ethers (TRITON® X). Preferred synthetic tensides are the surfactants available under the name SPAN® and TWEEN®. Preferred tensio-acids for use in the oil-in-water emulsions of the present invention include lecithin, Tween-80 and SPAN-80. In general, the surfactant, or 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 from 0.1% to 6.0%, more preferably 0.2% by volume. 5.0% The aqueous component consti- tutes the coninuous phase of the emulsion and may be water, pH regulated saline or any other suitable aqueous solution. The oil-in-water emulsions of the present invention may include additional components that are appropriate and desirable, including preservatives, osmotic agents, bioadhesive molecules, and immunostimulatory molecules. It is believed that bioadhesive molecules can potentiate the administration and binding of antigens on or through the target mucosal surface conferring mucosal immunity. Examples of suitable bioadhesive molecules include non-natural acidic polymers such as polyacrylic acid and polymethacrylic acid (e.g. CARBOPOL®, CARBOMER); synthetic modified acidic polymers such as carboxymethylcellulose; syntectically neutral modified natural polymers such as (hydroxypropyl) methylcellulose; polymers that carry basic amines such as chitosan; acidic polymers obtainable from natural sources such as alginic acid, hyaluronic acid, pectin, gum tragacanth and karaya gum; and natural non-natural neutral polymers, such as polyvinyl alcohol; or their combinations. The phrase "immunostimulatory molecules", as used herein, refers to molecules that enhance the immune protective response induced by an antigen component in the vaccine compositions. Suitable immunosorbent materials include bacterial cell wall compounds, for example N-acetylmuramyl-L-alanyl-D-isoglutamine derivatives such as murabutide, threonyl-MDP and tripiplymuramuram; saponin glycosides and their derivatives, for example Quil A, QS 21 and GPI-0100; cholesterols and quaternary ammonium compounds, for example dimethyldiocylammonium bromide (DDA) and N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamna ("avridine"). Saponins are glycosidic compounds that are produced as secondary metabolites in a wide variety of plant species. The chemical structure of saponins confers a wide range of pharmacological and biological activities, including some potent and effective immunological activity.

Structurally, saponins are constituted by any aglucone linked to one or more chains of sugars. Saponins can be classified according to their agglutin composition: triterpenic glycosides, steroidal glycosides and steroidal alkaloid glycosides. Saponin can be isolated from the bark of Quillaja saponaria.

Saponin has long been known as an immunostimulator. Dalsgaard, K., "Evaluation of adjutant activity with special reference to the application in the vaccination of cattle against foot-and-mouth disease", Act. See. Scand. 69: 1-40 1978. The crude extracts of plants containing saponins improved the potency of foot-and-mouth disease vaccines. However, crude extracts were associated with adverse side effects when used in vaccines. Subsequently, Dalsgaard partially purified the active component of the saponin adjuvant by dialysis, ion exchange and gel filtration chromatography. Dalsgaard, K. and cois. "Saponin Adjuvants III Isolation of a substance from Quillaja saponaria Morina with adjuvant activity in foot-and-mouth disease vaccines", Aren. Gesamte. Virusforsch. 44: 243-254 1974. An aqueous adjuvant component purified in this way is known as "Quil A". Based on weight, Quil A demonstrated increased potency and showed reduced local reactions when compared to crude saponin. Quil A is widely used in veterinary vaccines. Further analysis of Quil A by high performance liquid chromatography (HPLC) revealed a heterogeneous mixture of very similar saponins and led to the discovery of QS21 which was a potent adjuvant with reduced or minimal toxicity. Kensil C.R. et al., "Separation and characíerizafion of saponins with adjuvant activity from Quillaja saponaria Molina cortex", J. Immunol. 146: 431-437, 1991. In contrast to most other immunostimulators, QS21 is soluble in water and can be used in vaccines with or without emulsion-type formulations. It has been shown that QS21 elicits a Th1-like response in mice by stimulating the production of IgG2 and IgG2b antibodies and induced antigen-specific CD8 + CTL (MHC class I) in response to subunit antigens. Clinical studies in humans have shown their capacity as an adjuvant with an acceptable toxicological profile. Kensil, C.R. et al., "Structural and immunological characterization of vaccine adjuvant QS-21, In Vaccine Design: the subunit and Adjuvaní Approach," Editors Powell, M.F. and Newman, M.J. Plenum Publishing Corporation, New York 1995, pages 525-541. U.S. Patent 6,080,725 teaches methods for preparing and using saponin and lipophilic conjugate. In this saponin and lipophilic conjugate, a lipophilic moiety such as a lipid, fatty acid, polyethylene glycol or irepene is covalently linked to a triterpenic saponin not adhered or deacylated by a carboxy group present in the 3-O-glucuronic acid of the triterpene saponin . The binding of a lipophilic moiety to 3-O-glucuronic acid of a saponin such as Quillaja's desacylsaponin, P-lucioside or Gypsophila saponin, saponaria and Acanthophyllum enhances its adjuvant effects on humoral and cell-mediated immunity.

In addition, the binding of a lipophilic moiety to the 3-O-glucuronic acid moiety of a non-adiated or deacylated saponin provides a saponin analog that is easier to purify, less toxic, chemically more stable and possesses properties as an adjuvant or better than the original saponin. GPI-0100 is a saponin and lipophilic conjugate that is described in United States Patent 6,080,725. GPI-0100 is produced by the addition of an 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 tiouronios. Specific compounds of that type include dimethyldioctadecylammonium bromide (DDA) and N, N-dioctadecyl-N, N-bis (2-hydroxy-yl) -propanediamine ("avridine"). U.S. Patent 5,951,988 teaches an adjuvant formulation containing quaternary ammonium salts such as DDA together with an oleaginous component. This formulation is useful together with known immunological substances, for example viral or bacterial antigens in a vaccine composition, to enhance the immunogenic response. The composition is also useful without an antigen incorporated as a nonspecific nonsostimulatory formulation. U.S. Patent 4,310,550 discloses the use of N, N-higher alkyl-N, N'-bis (2-hydroxy-yl) propanediamna, and N, N-higher alkyl-xylylenediamines formulated with fat or lipid emulsion as adjuvant of vaccines. A method for inducing or enhancing the immunogenic response of an antigen in man or an animal by parenteral administration of the adjuvant formulation is described in U.S. Patent 4,310,550. In a preferred embodiment, the present invention provides a submicron oil-in-water emulsion useful as a vaccine adjuvant, which is composed of an AMPHIGEN® formulation, with droplets of a size less than 1 μm and an average droplet size of approximately 0.25 μm. The term "AMPHIGEN® formulation" as used herein refers to a solution formed by mixing a solution of DRAKEOL® leciin oil (Hydronics, Lincoln, NE) with saline in the presence of TWEEN® 80 and SPAN® 80. A typical AMPHIGEN® formulation contains 40% fluid paraffin oil >; in volume (v / v), approximately 25% w / v of lecithin, approximately 0.18% by volume (v / v) of TWEEN 80 and approximately 0.08% > in volume (v / v) of Span 80.

Means for preparing submicron emulsions of oil in water In another embodiment, the present invention provides methods for preparing the submicron emulsions of oil in water described above.

In accordance with the present invention, the various components of the emulsion, including the oil, one or more surfactants, an aqueous component and any other suitable component for use in the emulsion are combined and mixed together. The mixture formed is subjected to an emulsification process, typically by passing one or more times through one or more homogenizers or emulsifiers to form an oil-in-water emulsion which has a uniform appearance and a mean size of the gums of approximately 0.5 μm. . For this purpose any commercially available homogenizer or emulsifier can be used, for example Ross emulsifier (Hauppauge, NY), Gaulin homogenizer (Everetí, MA). The emulsion thus formed is then subjected to microfluidization to bring the size of the goies to the submicron range. Microfluidization can be achieved using a commercial microfluidizer such as model number 110Y available from Microfluidics, Newton, Mass; the model 30CD of Gaulin (Gaulin, Inc., Evereíí, Mass.); and Rainnie Minilab at 8.30 am (Miro Alomizer Food and Dairy Inc., Hudson, Wis.). These microfluidizers operate by forcing the fluids through small openings at elevated pressure, in such a way that the two fluid jets move at high velocities in an interaction chamber to form emulsions with beams of a submicron size. The size of the drop can be determined by a variety of methods known in the art, for example, laser diffraction, using commercially available measuring instruments. The size may vary depending on the type of lens used, the ratio of surfactant to oil, the pressure at which it is worked, the temperature and the like. The expert in the art can determine the desired combination of these parameters to obtain emulsions with the desired droplet size without undue experimentation. The gofas of the emulsions of the present invention have a diameter of less than 1 μm, preferably a mean size of the gouges of less than 0.8 μm, and more preferably an average size of the drops of less than 0.5 μm, and even more preferably of an average size of the drops lower than 0.3 μm. In a preferred embodiment of the present invention, the DRAKEOL lecithin oil solution, which is commercially available from Hydronics (Lincoln, NE) and contains 25% leciin in fluid paraffin oil, is combined and mixed with saline as well as with the TWEEN® 80 and SPAN® 80 agents to form an "AMPHIGEN® solution" or "AMPHIGEN® formulation". The AMPHIGEN® solution is then emulsified with a Ross® emulsifier (Hauppauge, NY 11788) at approximately 3400 rpm to form a water-in-oil emulsion. Subsequently the emulsion is passed once through a Microfluidizer working at 31.027.5 kPa ± 3.447.5 kPa. The oil-in-water emulsion contains beads less than 1 μm in size, with an average droplet size of approximately 0.25 μm.

Compositions of Vaccines Containing Antigens Incorporated in Submicroméeric Oil-in-Water Emulsions In another embodiment, the present invention provides compositions of vaccines that contain an antigen (s) and a submicromer emulsion of oil in water described above. These compositions of the vaccines are characterized in that they have an enhanced immunogenic effect and an improved physical appearance (for example, phase separation is not observed after a long period of storage). In addition, the compositions of the vaccines of the present invention are safe for administration to animals. According to the present invention, the enzyme can be combined with the emulsion in an extrinsic or, preferably, in an irresistible way. The term "nirinase" refers to the process in which the antigen is combined with the components of the emulsion prior to the microfluidization step. The term "ex-insulin" refers to the process in which the antigen is added to the emulsion after the emulsion has been microfluidized. The antigen added extrinsically may be free antigen or may be encapsulated in microparticles as described in more detail below. The term "antigen" as used in the present specification refers to any molecule, compound or composition that is immunogenic in an animal and is included in the vaccine composition to elicit a pro-cyclic immune response in the animal to which it is administered. the vaccine composition. The term "immunogenic" as used in connection with an animal refers to the ability of the animal to elicit an immune response in an animal conferred by the animal. The immune response may be a primordially mediated cellular immune response by cytoxic T lymphocytes, or a humoral immune response primarily mediated by cooperating T-lymphocytes, which in turn acylate B-lymphocytes that cause the production of antibodies. An "immune pro-cyclic response" is defined as any immune response, either antibody-mediated or cell-mediated immune response, or both, that occurs in the animal that prevents or reduces detectable appearance or eliminates or reduces detectable severity, or slows down in a detectable way the speed of progression, the disorder or disease caused by the antigen or a pathogen that contains the antigen. The antigens which may be included in the vaccine composition of the present invention include antigens prepared from pathogenic bacteria such as Mycoplasma hyponeumoniae, Haemophilus somnus, Haemophilus parasuis, Bordetella bronchiseptica, Actinobacillus pleuropneumonie, Pasteurella multocida, Manheimia hemolitica, 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, Leptospira spp.; 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 inacíivated 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 viral pathogenic viruses such as bovine herpesvirus 1, 3, 6, bovine viral diarrhea virus (BVDV), lipos 1 and 2, bovine pseudoinfluenza virus, bovine respiratory syncytial virus, bovine leukosis virus, rinderpest virus, foot-and-mouth disease virus, rabies, swine virus, African swine fever virus, porcine parvovirus, PRRS virus, porcine circovirus, influenza virus, porcine vesicular disease virus, Techen fever virus, pseudorbism virus, either in the form of a preparation of whole inactivated or partial viruses, or in the form of antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis. The amount of the antigen must be such that the antigen, combined with the oil in water emulsion, is effective to induce a protective immune response in an animal. The precise amount for an effective antigen depends on the nature, activity and purity of the antigen and can be determined by those skilled in the art. The amount of oil in water emulsion present in the vaccine compositions should be sufficient to enhance the immunogenic capacity of the antigen (s) in the vaccine compositions. When desirable and appropriate, additional amounts of surfactant (s) or additional surfactant (s) may be added to the vaccine composition in addition to the surfactant (s) provided by the emulsion of aceifeide in water. Generally, the oleaginous component is present in the final volume of a vaccine composition in an amount of 1.0% to 20% or by volume; preferably at a rate of 1.0% to 10% >; more preferable in a rate of 2.0% to 5.0%. The density, or the combination of nutrients if two or more substances are used, is present in the final volume of a vaccine composition in a quantity of 0.1%) to 20% by volume, preferably 0.15%) to 10%, more preferably 1.2% to 6.0%. In addition to the enzyme (s) and the emulsion of oil in water, the vaccine composition may include other components that are appropriate and desirable, such as preservatives, osmoic agents, bioadhesive molecules, and immunostimulatory molecules (eg, Quil A). , cholesterol, GPI-0100, dimethyldiocylammonium bromide (DDA)), as described above in relation to the oil in water emulsion. The compositions of the vaccines of the present invention can also include a veterinarily acceptable vehicle. The term "an acceptable carrier vehicle" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, silicone agents, adsorption retarding agents, and the like. The diluyenles may include water, saline, dextrose, ethanol, glycerol and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbifol and lactose in other. Stabilizers include albumin in others. In a preferred embodiment, the present invention provides a vaccine composition that includes at least one of a BVDV type I or BVDV type II antigen, which is intrinsically incorporated into an oil-in-water emulsion which is sized less than 1 μm, preferably of an average size of the gouges of less than 1.8 μm, more preferably less than 1.5 μm, and even more preferably of an average droplet size of about 1.5 μm. The BVDV antigen I I and / or II is preferably in the form of a viral activated preparation. The submicron emulsion of oil in water is preferably composed of an AMPHIGEN® formulation (ie, a formulation containing fluid paraffin oil, lecithin, TWEEN® 80 and SPAN® 80). The vaccine composition preferably also includes Quil A, cholesterol and thimerosol. In another preferred embodiment, the present invention provides a vaccine composition that includes a Lepfospira anígen and at least one of a BVDV ipo I or BVDV type II anigen in an oil-in-water emulsion. The antigens, preferably in the form of an inactivated cellular or viral preparation, are incorporated intrinsically into the submicron emulsion of oil in water having droplets of less than 1 μm in size.; preferably of a mean gage size of less than 1.8 μm, more preferably less than 1.5 μm, and even more preferably of an average gum size of about 1.5 μm. The submicroméric emulsion of oil in water is preferably composed of an AMPHIGEN® formulation (ie, a formulation containing fluid paraffin oil, lecithin, TWEEN® 80 and SPAN® 80). The vaccine composition preferably also includes one or more immunosphimulatory molecules which are selected from Quil-A, cholesterol, DDA, GPI-100 and aluminum hydroxide (AIOH). In yet another preferred embodiment, the present invention provides a vaccine composition that includes at least one bacterial agent, for example the PauA protein of recombinant Streptococcus uberis or an E. coli cell preparation or a combination of both in an emulsion of oil in water. The antigen (s) is intrinsically combined with the submicron emulsion of oil in water having droplets of size less than 1 μm, preferably of an average size of the goias less than 1.8 μm, more preferably lower at 1.5 μm, and even more preferably from an average droplet size of approximately 1.25 μm. The submicron oil-in-water emulsion is preferably composed of an AMPHIGEN® formulation (ie, a formulation containing fluid paraffin oil, lecillin, TWEEN® 80 and SPAN® 80). The vaccine composition preferably also includes one or more immunostimulatory molecules that are selected from Quil-A, DDA, GPI-100. The compositions of the vaccines of the present invention can be administered to an animal by known routes, including oral, inanal, mucosal, topical, transdermal and parenteral (for example intravenous, iniraperitoneal, nfradermal, subcutaneous or intramuscular). Administration can be achieved using a combination of routes, for example the first administration using a parenteral route and subsequent administration using a mucosal route.

Means for preparing compositions of vaccines In a further embodiment, the present invention provides methods for preparing compositions of vaccines conferring an antigen or antigens and a submicromerric emulsion of oil in water. To prepare the compositions of the vaccines of the present invention, the antigen (s) can be combined 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 in an intrinsic manner. The antigen can be combined with the various components of the emulsion, including oil, one or more compounds, an aqueous component and any other suitable component to form a mixture. The mixture is subjected to a mixing method typically by passing it one or more times through one or more homogenizers or emulsifiers to form an emulsion of oil in water which the antigen confers. Any commercially available homogenizer or emulsifier can be used, for example Ross emulsifier (Hauppauge, NY), Gaulin homogenizer (Everett, MA) or Microfluidics (Newton, Ma). Alternatively, the various components of the adjuvant emulsion, including the oil, one or more surfactants and an aqueous component may be first combined to form a submicron emulsion of oil in water using a homogenizer or emulsifier; and the antigen is then added to this emulsion. The average particle size of the emulsion of oil in water after primary mixing is about 1.0-1.2 micrometers. The emulsion containing the animal is then subjected to microfluidization to bring the size of the drops to the submicron range. Microfluidization can be achieved using a commercial microfluidizer such as model number 110Y available from Microfluidics, Newton, Mass; the 30CD model of Gaulin (Gaulin, Inc., Everetf, Mass.); and Rainnie Minilab at 8.30 am (Miro Aomizer Food and Dairy Inc., Hudson, Wis.). The size of the goulash can be determined by a variety of methods known in the art, for example, laser diffraction, using commercially available measuring instruments. The size may vary depending on the type of surfactant used, the relationship between the surfactant and oil, the pressure at which it is worked, the temperature and the like. A desired combination of these parameters can be determined to obtain emulsions with a desired gage size. The oil content of the emulsions of the present invention is less than 1 μm in diameter. Preferably the average size of the goles is less than 1.8 μm. More preferably, the average droplet size is less than 1.5 μm. Even more preferably, the average droplet size is between 0.1 μm and 1.3 μm. In a preferred embodiment of the present invention, the DRAKEOL® lecithin oil solution, containing 25% lecithin in fluid paraffin oil, is combined and mixed with the surfactants TWEEN® 80 and SPAN® 80 and saline to form a mixture which contains 40% fluid paraffin oil, TWEEN® 80 at 0.18%) and SPAN® 80 at 0.08%. The mixture is then emulsified with a Ross® emulsifier (Hauppauge, NY 11788) at approximately 3400 rpm to form an emulsified product which is also referred to as "AMPHIGEN® formulation" or "AMPHIGEN® solution". Subsequently, the desired antigen (s) are combined with the AMPHIGEN® solution and any other suitable compounds (for example immunosfigurative molecules) with the aid of an emulsifier, for example, Ross homogenizer, to form a water emulsion in oil that contains the antigen (s). Such an emulsion is passed once through a Microfluidizer that works at 6895 ± 3447.5 pKa. The emulsion of oil in water has sizes less than 1 μm, with an average droplet size of approximately 1.25 μm. In another preferred modality, before combining an oil-in-water emulsion (for example an AMPHIGEN® formulation) with a desired antigen (s), the antigen (s) is combined with a saponin glycoside , for example Quil A, forming a mixture. This mixture of the enzyme (s) and saponin is subjected to homogenization, for example, in a homogenization vessel. Then a stellar, for example, cholesterol, is added to the homogenized mixture of antigen (s) and saponin. Then, the mixture that contains the anigen (s), saponin and stellar is again subjected to homogenization. Then, the mixture of antigen (s), saponin and homogenized spherical is combined with an emulsion of aceifene in water (for example an AMPHIGEN® formulation) with the aid of a homogenizer, for example. Then, the oil-in-water emulsion containing the amphiphile (s), saponin and stellar is subjected to homogenization at elevated pressure, as well as microfluidization. .

Compositions of Vaccines Containing Microencapsulated Anigenes in a Submicromerric Emulsion of Oil in Water and Preparation Methods Still in another embodiment, the present invention provides compositions of vaccines that contain a microparticle encapsulated anigen (or "microencapsulated agent"), those that the microencapsulated anigen is incorporated exlrinsically to a submicromeriric oil-in-water emulsion described above. Methods for absorbing or trapping antigens in particulate vehicles are well known in the art. See for example Pharmaceutical Particulate Carriers: Therapeutic Applications (Justin Hanes, Masaioshi Chiba and Robert Langer, Polymer microspheres for vaccine delivery, In: Vaccine design, The subunit and adjuvaní approach, Editors Michael F. Powell and Mark J. Newman, 1995 Plenum Press , New York and London). The particulate carriers can present multiple copies of a selected antigen to the immune system in an animal subject and promote the capylation and retention of amphigens in the local lymph nodes. The particles can be phagocytosed by macrophages and can potentiate the presentation of antigens through the release of cytokines. Particulate carriers have also been disclosed in the art and include, for example, those derived from polymethylacrylate polymers of methyl, as well as those derived from poly (lactides) and poly (lacyda-co-glycolides), which are known as PLG. Polymethyl methacrylate polymers are not biodegradable while PLG particles can be biodegradable by non-enzymatic random hydrolysis of ester linkages to lactic and glycolic acids that are excreted by normal metabolic pathways. Biodegradable microspheres have also been used to achieve controlled release of vaccines. For example, a sustained release of antigen can be achieved over a prolonged period. Depending on the molecular weight of the polymer and the ratio of lactic and 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 and controlled release can cause the formation of high levels of antibodies similar to those observed after multiple injections. Alternatively, a pulsatile release of antigens from the vaccines can be achieved by selecting polymers with different rates of hydrolysis. The rate of hydrolysis of a polymer typically depends on the molecular weight of the polymer and the ratio of lactic acid to glycolic acid in the polymer. Microparticles that are formed by two or more different polymers with variable rates of release of anligens provide pulsatile releases of antigens and mimic multiple-dose vaccination schedules. In accordance with the present invention, an antigen, including any of those described above, can be absorbed onto a particulate polymeric carrier, preferably a PLG polymer, using any method known in the art (such as that exemplified in the example). 17) to form a microencapsulated animal preparation. The microencapsulated antigen preparation is then mixed and dispersed in a submicron emulsion of oil in water, emulsion which has been described above, to form the vaccine composition. In a preferred embodiment, the present invention provides a vaccine composition containing an antigen encapsulated in a PLG polymer, in which the microencapsulated antigen is extrinsically dispersed in an oil emulsion in microfluidized water that is composed of paraffin oil. fluid, lecifin, TWEEN80, SPAN80 and saline solution and has an average droplet size of less than 1.0 μm.

Complexes formed by a saponin and a stellate In one embodiment, the present invention provides compositions containing a saponin and a esoteric, in which the saponin and the steral form complexes in the form of helical micelles. According to the present invention, these complexes have immunosimulatory activities. By "immunostimulatory" it is meant that the complexes can potentiate the immune response induced by an antigen component or that the complexes can induce an immune response independent of a separate antigen component. According to the present invention, a preferred saponin for use in a composition of the present invention is Quil A. Preferred spherals for use in the adjuvant compositions of the present invention include beía-siioslerol, esigmasterol, ergosterol, ergocalciferol and cholesterol. These esterales are notorious in the art, for example, cholesterol is described in Merck Index, 11th Edition, page 341, as a natural esteral found in animal fat. More preferably, the esteral is cholesterol. The ratio of saponin to stellar in the composition is typically from about 1: 100 to 5: 1 by weight. Preferably, the ratio is 1: 1.

In another embodiment, the present invention provides vaccine compositions containing a saponin, a sterol and an antigen, in which saponin and eserol form complexes in the form of helical micelles and in which the anigen is mixed but not incorporated into the helical micelles. The following are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

EXAMPLE 1 Preparation of an AMPHIGEN® formulation A formulation of AMPHIGEN® was prepared by a two-stage method. In the first stage 80 liters of Drakeol lecifin oil solution, 116 liters of Tinane Toxoid saline, 1.2 liters of SPAN 80, and 2.8 liters of Tween 80 were mixed and emulsified using a Ross emulsifier. The resulting solution in Drakeol lecithin oil contained 25% soy lecillin and 75% paraffin wax. The emulsified product was redrilled by the Ross emulsifier for a minimum of 5 volumes or a minimum of 10 minutes. The emulsified product was stored at 2-7 ° C for a maximum of 24 hours for further processing. The emulsion of the Ross emulsifier tank was transferred to a Gaulin homogenizer and homogenized for 20 minutes at a pressure of 3,102.75 kPa. The 40% drakeol lecithin oil solution (hereinafter "AMPHIGEN® formulation" or "AMPHIGEN® solution") was then dispensed in sterile polypropylene carboxy containers. The distribution was made within a class 100 distribution hood located in a controlled class 10,000 environment. The containers were stored at 2-7 ° C. This formulation of AMPHIGEN® was used in the experiments described below unless otherwise indicated.

EXAMPLE 2 Primary mixing by homogenization by ultrafast mixing of BVD vaccine The apparatus used for this homogenization method is shown in Figure 1. Using aseptic technique or cross-vapor valves, a vial containing a BVD Type I antigen (an inactivated BVD Type I viral preparation) was attached to the port of the lower part of the mixing vessel. After completing the transfer of the required volume of the BVD Type I antigen, the BVD Type I bottle was replaced by the container containing a BVD Type II viral preparation (an inactivated BVD Type II viral preparation). After completing the transfer of the required density of a Type II BVD anigen, the Ross homogenizer was attached to the portable container and recirculation started at the maximum RPM (3300 rpm).

The agitation of the vessel was maintained at medium speed. Using aseptic technique or cross-vapor valves, a bottle containing Quil-A with a concentration of 50 mg / ml was attached to the in-line port of the homogenizer of the mixing vessel. A required amount of the Quil-A solution was passed to the vessel through in-line suction. After completing the transfer of Quil-A solution, the bottle was removed. In the same way, a required amount of cholesterol in eilanol solution (18 mg / ml) was transferred to the mixing vessel. Subsequently, a required amount of the AMPHIGEN® formulation, 10% thimerosol solution and dilution solutions of Eagle's Modified Basic ("BME") medium were added to the mixing vessel. Once the additions were completed, mixing continued for another 15 minutes. The resulting formulation was divided into 2 ml aliquots and represented a BVD vaccine with AMPHIGEN® formulation base not microfluidized. Each dose of the vaccine contained 500 μg of Quil-A, 500 μg of cholesterol, 2.5% AMPHIGEN® formulation and 0.009% thimerosol. The antigen concentration for the two different BVD strains was determined in terms of the ELISA assay for gp53.

EXAMPLE 3 Secondary mixing by microfluidization Figure 2 illustrates the method used for secondary mixing mediating microfluidization. The microfluidizer was steam sterilized. First the camera of the auxiliary processing module was installed in the unit and the camera of the target was installed in the second position of the camera. The container conferring the BVD vaccine with all the adjuvants prepared as described in Example 2 was connected to the microfluidizer by attaching a transfer line from the drain valve of the supply container to the intake of the microfluidizer. Nitrogen gas was connected to the air intake of the supply vessel and the vessel pressure was adjusted to 137.9 +/- 34,475 kPa. The drain valve of the collecting vessel was connected to the transfer line of the microfluidizer outlet. After making all the necessary connections, the valves were opened and microfluidization was started with an opening pressure of 6895 +/- 3447.5 kPa. The entire contents of the vaccine were passed through the microfluidizer once and collected in the post-microfluidization chamber. This preparation was divided into 2 ml aliquots and represents the BVD vaccine with a microfluidized AMPHIGEN® formulation base.

EXAMPLE 4 Preparation of a vaccine composition by bank mixing The AMPHIGEN® formulation prepared as described in Example 1 was diluted to 2.5% by adding BVD antigens and the diluent. The resulting solution was mixed on the bench using a stir bar instead of a homogenizer. The final preparation contained the following composition: BVD Type 1 and Type 2 antigens, 2.5% AMPHIGEN® formulation (which contains oil, lecithin, SPAN® and TWEEN®, as described in Example 1) and saline solution. . TWEEN 80 and SPAN 80 are present in the final vaccine preparation at 0.18% and 0.08% or in volume respectively.

EXAMPLE 5 Comparison of the droplet size distribution between preparations of the non-microfluidized and microfluidized AMPHIGEN® formulation based vaccines The non-microfluidized AMPHIGEN® formulation-based vaccine prepared as described in example 2, the microfluidized AMPHIGEN® formulation-based vaccine prepared as described in Example 3 and the preparation prepared by bench-mix as described in Example 3. described in Example 4 were used to compare the size of the drops of the vaccine preparations. 2 ml of the sample from each of the preparations was added to a Malvern 2000 laser diffractometer and the size distribution of the lozenges was determined. As shown in Figure 3, the results indicate that the microfluidifi- cated AMPHIGEN® formulation-based vaccine preparation had the maximum particle volume around 0.1 micrometers while the non-microfluidifi- cated AMPHIGEN® formulation-based vaccine preparation had the maximum volume of particle distribution around 1 micron.

EXAMPLE 6 Reduction of phase separation of vaccines.

Fresh preparations of the different vaccines were compared: the non-microfluidized AMPHIGEN® formulation-based vaccine prepared as described in example 2, the microfluidized AMPHIGEN®-based formulation vaccine prepared as described in example 3 and the vaccine prepared by bank mixing as described in example 4, to determine its phase separation properties after prolonged storage. All these preparations were allowed to stand at 4 ° C for about a month and the phase separation was carried out considering the appearance of a creamy layer in the upper part of the vaccine preparations. As shown in FIG. 4, no phase separation occurred in the microfluidized AMPHIGEN® formulation-based preparation when compared to the other two preparations.

EXAMPLE 7 Preparation of vaccine for microfluidized and non-microfluidized bovine cattle against bovine viral diarrhea virus Viral antigen of bovine diarrhea virus was incorporated into the AMPHIGEN® formulation by microfluidization. The term "intrinsically incorporated" refers to the method by which the antigen was added to the AMPHIGEN® formulation prior to microfluidization. The antigen was subjected to the physical forces of the microfluidization method together with the components of the adjuvant formulation. In the non-microfluidized control group, the antigen preparation was dispersed in the AMPHIGEN® formulation by mixing. The final composition of the control and microfluidized preparations was as follows: BVD type I with an ELISA titration after inactivation of 2535 UR / dose for gp53, BVD Type II with an ELISA titration at inactivation of 3290 UR / dose for gp53 , Quil-A with a concentration of 1.25 mg / dose, cholesterol with a concentration of 1.25 mg / dose, the formulation of AMPHIGEN® with a final concentration of 2.5% and thimerosol with a final concentration of 0.009%). The dose of the vaccine was 5 ml.

EXAMPLE 8 Long-Term Stability of BVD Viral Antigens Incorporated in an Intrinsic Way to the Preparation of Microfluidifiated AMPHIGEN® Formulation-Based Vaccine This experiment was performed to determine the stability of the antigen incorporated intrinsically during long-term storage. The BVD Type II viral antigen was incorporated in an inírinseca form Activated to the formulation of AMPHIGEN® during the microfluidization method to obtain a microfluidized vaccine preparation (A907505). Three other preparations of the vaccines containing the same antigen in a non-microfluidized AMPHIGEN® formulation (A904369, A904370 and A904371) served as conirols. In the non-microfluidized preparations, the antigen was mixed with the AMPHIGEN® formulation and mixed by beating with a Ross homogenizer. The four preparations of the vaccines were stored at 4 ° C for two years. At different times during storage (0, 6, 12 or 24 months), the formulations were used to vaccinate months-old cows. On days 0 and 21 three month old cows were vaccinated subcutaneously with a 2 ml vaccine formulation. The serum of the vaccinated animals was extracted on day 35 and the serological response to the vaccine was measured by titrating the antibodies by means of a BVDV-E2 ELISA. As shown in Figure 5, the microfluidized vaccine preparation showed a higher antibody titration at all analyzed times (0, 6, 12 and 24 months), which suggests that the stability of the antigen preparation was not loses during the intrinsic incorporation of the antigen during the microfluidization method. In addition, it was also surprisingly found that the preparation of microfluidized vaccine induced an enhanced immune response at all times.

EXAMPLE 9 Reduction in rectal temperature increase induced by the vaccine after microfluidization The preparations of the microfluidized and non-microfluidized vaccines were used as described in example 7 to vaccinate the cattle on day zero and the rectal temperature was monitored during the period from one day before the vaccination until four days after the vaccination. The dose of the vaccine was 2 ml. The groups were vaccinated with a single or double dose of the vaccine. Rectal temperatures were measured and recorded daily from Day 1 to Day 4 inclusive. Rectal temperatures were measured on day 0 before administration of the experimental article.

As shown in Figure 6, the results indicate that there was a sharp increase in rectal temperature approximately 24 hours after vaccination in animals vaccinated with a single or double dose of the non-microfluidized vaccine formulation. However, in the animals vaccinated with microfluidized forms of the vaccine, the increase in the temperature after vaccination was minimal and significantly lower than in the animals vaccinated with the non-microfluidized formulation (Figure 6).

EXAMPLE 10 The reaction volume in the injection site resolved faster when it was vaccinated with formulations of the microfluidized vaccines preparations of the vaccines used microfluidifizadas not microfluídifizadas prepared as described in Example 7 to immunize cattle on day zero. The animals included in this study were hybrid beef cattle. There were three animals in each of the placebo treatment groups (T01 and T02). There were six animals in each of groups T03 to T06. The vaccine dose was 2 ml and the groups were vaccinated with one or two doses of the vaccine on day zero. On day 0, the test article was administered on the right side of the neck. The animals that received the double dose (4 ml) of the experimental article (T02, T03 and T06) received the full double dose in the form of a single injection on one side. The observation of the injection zones, including the estimation of the reaction zone in the area of the injection was made on the right side of the neck from Day 0 to Day 4 inclusive, and Days 6, 9 and 14. The Day 0 the injection zones were observed before the administration of the experimental articles. Groups vaccinated with one or two doses of placebo showed no significant increase in reaction volume in the injection site and therefore these data are not shown in Figure 7. In the case of non-microfluidized vaccine formulation, produced a proportional increase in the volume of reaction in the area of the injection between vaccination with one dose and with two doses. On the other hand, in the case of the microfluidized vaccine formulation, although the single dose induced a larger reaction volume in the area of the injection, the injection with the second dose did not cause an additional increase. However, in the case of the animals injected with the microfluidized vaccine formulation, the reaction volume in the injection site was resolved with a higher velocity compared to that of the animals injected with a vaccine. formulation of non-microfluidized vaccine. These results are shown in figure 7.

EXAMPLE 11 Preparation of a Microfluidifi- cated AMPHIGEN® Formulation-Based Vaccine. Preparations with intrinsically incorporated BVD and Leptospira viral antigens and immunostimulatory molecules such as Quil A and DDA Leptospira hardjo-bovis of the CSL strain inactivated in formalin was formulated with the appropriate adjuvant with direct counts of approximately 1.4 x 109 organisms / 5 ml dose. The Lepiospira Pomona T262 strain born in formalin was formulated at approximately 2400 nephromeric units / 5 ml dose. Nephalomeric units were calculated based on the nephrometric measurement of previously processed fermentation fluid. Type 1 BVD virus was formulated with an E2 Elisa titre of approximately 3000 relative units / 5 ml dose. Type 2 BVD virus was formulated with an E2 ELISA titration of approximately 3500 relafive units / 5 ml dose. The relative unit was calculated based on the evaluation in E2 ELISA of bulk fluid after inactivation before assembly. Quil-A tannery was used as colesterol with a concentration of 1.5 mg per dose. Thimerosol and the AMPHIGEN® formulation were used with final concentrations of 0.009% and 2.5% respectively. Aluminum hydroxide (Rehydragel LV) with a final concentration of 2.0% was used. When DDA was used as an immunomodulator, DDA was included in the AMPHIGEN® formulation. The AMPHIGEN® formulation (ie, the 40% Drakeol-lecithin stock solution) contained 1.6 mg / ml DDA and, when properly diluted, the final vaccine preparation contained 2.5% AMPHIGEN® formulation and 0.1 mg / ml of DDA. Fractions of BVD, Leptos, Quil-A, cholesterol, thimerosol were added in the preparation of different formulations of the vaccines., the formulation of AMPHIGEN® and saline as diluent to a Silverson homogenizer and mixed for 15 minutes at 10,000 + 500 rpm. The components were then microfluidised with a 200 micron sieve at 68,950 kPa. When the vaccine formulation contained aluminum hydroxide, the microfluidization was carried out without aluminum hydroxide. When the microfluidization was completed, aluminum hydroxide was added and mixed with a stir bar until the next morning at 4 ° C.

EXAMPLE 12 Preparation of BVD virus vaccine for exposure studies The vaccine preparation used in this experiment congested BVD Type 1 virus amphe- lenes and BVD Type 2 virus. BVD1-5960 antigen was used with an ELISA titration of 2535 UR / dose for gp53. The BVD2-890 antigen was used with a post-inactivation ELISA titre of 3290 UR / dose for gp53. Quil-A and cholesferol were used at a concentration of 1.5 mg / ml. Iimersol and the AMPHIGEN® formulation were used with a final concentration of 0.009% and 2.5% respectively. When DDA was used as an immunomodulator, DDA was included in the AMPHIGEN® formulation. The stock solution of AMPHIGEN® (40% Drakeol-lecifin solution >) contained varying amounts of DDA and, when properly diluted, the final vaccine preparation contained 2.5% AMPHIGEN® formulation and a DDA concentration which varied in the range of 1.5 mg / dose to 2.0 mg / dose. Aluminum gel (Rehydragel-LV) with a final concentration of 2% was used. GPI-0100 was used in the range of 2, 3 and 5 mg / dose. All components were added to a homogenizer Silverson and were mixed for 15 minutes at 10,500 rpm and then microfiluted by passing them through a 200 micrometer chamber at 68,950 kPa. When the vaccine formulation contained aluminum hydroxide, the microfluidization was carried out without aluminum hydroxide. When the microfluidization was completed, aluminum hydroxide was added and mixed with a stir bar until the next morning at 4 ° C.

EXAMPLE 13 Protection against exposure to Leptospira after vaccination with a microfluidized formulation of Amphigen vaccine with Leptospira antigens TABLE 1 Treatment groups Table 1 shows the composition of the adjuvant formulations of the vaccine preparations that are experienced in this study. The preparations of the vaccines were prepared as described in example 11. There were six animals in each group. In this study, hybrid beef steers of approximately seven months of age were used. Vaccination was carried out on Day 0 and Day 21 subcutaneously with a volume of 5 ml vaccine. The exhibition was carried out with L. hardjo-bovis strain 203 of the NADC (National Agricultural Disease Center). The exposure was carried out during days 57-59 with an inoculum of 1 ml. The exposure was administered via the ocular conjunctiva and vaginally. The exposure material contained 5.0 x 106 leprospiros / ml. The urine was collected weekly to perform a culture of leptospira, FA and PCR. The kidneys were removed during Days 112 and 113.

TABLE 2 Results of the Leptospira exposure study Table 2 shows the data from the exposure study Leptospira. To determine the percentage of Leptospira infection in the exposed animal, the following criteria were used. If the kidney culture was positive in only one sample, the animal is considered positive for Leptospira. If an animal is positive only in a sample for FA or PCR, the animal is considered negative. If the sample is positive for FA and PCR in only one sample, it is considered positive for Leptospira. The results shown in table 2 indicate that the duration of presence in urine in all vaccinated groups was significantly shorter based on the fres trials. In regard to urinary and renal colonization, the efficiencies of the formulations containing QAC and DDA without AIOH were comparable. The AIOH did not improve or even reduce the efficacy of the vaccines containing QAC or DDA in this exposure study.

TABLE 3 Evaluation interval of microscopic agglutination on the day of assessment with maximum geometric mean before exposure (Day 35) Serological responses were detected against both Leptospira antigens of the vaccine formulation in the vaccinated animals and the maximum response was observed on Day 35. There was no correlation between the serological response and the protection against exposure. The presence of aluminum gel in the vaccine formulation reduced the level of protection although the serological response was enhanced by the presence of aluminum gel in the vaccine.

EXAMPLE 14 Provocation of immune response to BVD viral antigen and Protection against BVD Type 2 virus exposure after immunization with a microfluidized vaccine preparation containing AMPHIGEN® and DDA formulation In this experiment, seronegative calves from four to seven months of age were used. There were six different groups and each group had ten animals (table 4). On Day 0 and Day 21 each animal received a subcutaneous dose of 2 ml of the vaccine or placebo on the lateral neck approximately midway between the scapula and the head.

TABLE 4 Treatment groups of the virus (approximately 2.5 ml per tose) intranasally on day 44 of the study. In this study BVD type 2 virus was used, isolated strain n ° 24515 (Ellis strain), batch no. 46325-70 non-cytopathic as the exposure strain. The retained samples of exposure material (two repetitions per assessment) were assessed at the time of the start of the exposure and immediately after completion. The average live virus titration per 5 ml dose was 5.3 log-io FAID50 / 5 ml before exposure and 5.4 log10 IDAD5o / 5 ml at exposure (FAID is equivalent to TCID50). Animals were convalesced every day from Day 3 to day 58. Clinical disease scores of 0, 1, 2, or 3 were assigned based on clinical signs attributable to BVD 2 infection for each animal on Days 42 to 58. Day 44 scores were recorded before the exhibition. Blood samples (two 13 ml serum separation tubes, SST) from each animal were taken on Days 0, 21, 35, 44 and 58 to determine serum titers of BVD Type 1 and BVD virus neutralization antibodies. Item 2. Blood samples were taken from each animal from Day 42 to Day 58 inclusive, and the presence of BVD virus in the buffy coat was determined. On Day 44, samples were taken before the exhibition. Blood samples (a 4 ml EDTA tube) were taken to determine leukocytometry from Day 42 to Day 58 inclusive. On Day 44 samples were obtained before exposure. Leukopenia was defined as a 40% or greater decrease in leukocilomelry compared to the initial level (leukocytometry prior to the mean exposure of two days before and on the day of exposure). Clinical disease punctuations were used to define the disease state as follows; if the puniuation is < 1, then disease = no; if the puniuation is > 2, then disease = yes. As shown in Tables 5 and 6, the groups that were vaccinated with vaccines containing BVD June viral antigens with the formulation of AMPHIGEN®, Quil A or DDA and microfluidized, were seroconverted with significant serum virus neuralization titers. for BVD Type 1 and BVD Type 2 viruses. In those groups there was also a significant reduction in the percentage of animals showing viraemia after exposure, while in the control group 100% of the animals showed viremia (Table 7) . In addition, in the vaccinated groups the frequency of the disease was also significantly reduced (Table 8). Similarly, the percentage of animals showing leucopenia was also reduced in the vaccinated groups and the reduction in leukopenia was more significant in the group containing DDA than in the group containing Quil A (Table 9). In the control group there was a significant decrease in the weight gain compared with the vaccinated groups (Table 10).

Serology Prior to vaccination on Day 0, all the animals in the study were seronegative (SVN <1: 2) for antibodies to BVD Virus Types 1 and 2 (data not shown). Fourteen days after the second vaccination (Day 35) all the animals given placebo (T01) were still seronegative for antibodies against the BVD virus Types 1 and 2; and all the animals vaccinated with the ITA (Anígen in research experimentation) (T02, T03, T04, T05 and T06) were seropositive (SVN >; 1: 8) for antibodies against the BVD virus, Types 1 and 2. An animal that was administered the adjuvanted vaccine of the AMPHIGEN® formulation at 2 mg / dose of DDA had an SVN rating of 3 for antibodies against BVD Type 2 virus on Day 35 (Table 11 and 12). Prior to exposure on Day 44, all controls (T01) except one, were seronegative (SVN < 1.2) for antibodies against BVD viruses Types 1 and 2 (data not shown). One of the controls (n ° 2497) was seropositive (SVN = 10) for antibodies against the BVD Type 1 virus and seronegative for the BVD Type 2 virus. Fourteen days after exposure, all the animals in the study were seropositive for antibodies against the viruses. BVD virus Types 1 and 2.

TABLE 5 Geometric mean of serum virus neutralization titers for BVD Type 1 virus TABLE 6 Geometric mean of serum virus neutralization titers for BVD Type 2 virus TABLE 7 Isolation of BVD virus after exposure TABLE 8 Clinical signs of BVD disease after exposure TABLE 9 Leukopenia after exposure TABLE 10 Virus Isolation As shown in the data in Table 13, during the exposure period (Days 44 to 58), the ten animals of the control group (T01) suffered viremia (the BVD virus was isolated one or more days). In the groups to which the ITAs had been administered, the frequency of animals with viremia was one, zero, fres, two and two in each group of ten (T02, T03, T04, T05 and T06, respectively). The difference between the groups of conírol and the groups to which the ITAs had been administered was statistically significant (P <0.05). The mean of the least squares of the number of days of viremia was also significantly higher (11.4 days) for the control compared with the groups to which the ITAs had been administered (0.0 to 1.5 days).

Clinical Disease It was considered that animals with clinical signal scores of 2 or 3 showed signs of BVD disease. As shown in Table 14, the frequency of animals with clinical signs of BVD virus disease was nine out of ten in the control (T01) and one, two, zero and zero out of ten in each of the groups at that they were administered the ITA (T02, T03, T04, T05 and T06 respectively). The difference between the control and the groups to which the ITAs were administered was statistically significant (P <0.05).

Leukopenia As shown in Table 15, during the exposure period (Days 44 to 58), the ten control animals (T01) suffered from leukemia (a reduction of 40% or in leukocytry compared with the initial before exposure). , Days 42-44). The frequency of animals with leukemia was six, two, four, fres and two of the ten animals of each of the groups to which the ITAs were administered (T02, T03, T04, T05 and T06 respectively). The difference between the confrol and the groups to which the vaccine containing the formulation of AMPHIGEN® was administered as an adjuvant at 1.5 mg / dose and aluminum hydroxide (T03) was statistically significant (P <0.05). The mean of the least squares of the number of days of leukemia was significantly higher (7.8 days) for the control compared with the groups to which the ITAs were administered (1.2 to 1.2 days).

EXAMPLE 15 Provocation of immune response to BVD viral antigen and protection against BVD type 2 virus challenge after immunization with a microfluidized vaccine preparation containing GPI-0100 A group of experimental conditions such as the one described in Example 14 was followed and a direct comparison was made between Quil A and GPI-0100. As shown in Tables 11 and 12, animals vaccinated with BVD antigens in the microfluidifi- cated AMPHIGEN® formulation preparation that contained Quil-A or GPI-0100 had a significant antibody titre for BVD Type 1 and BVD viruses. Type 2. The antibody titre for the BVD Type 1 virus was much higher than that of the BVD Type 2 virus. However, the subsequent exposure to BVD Type 2 virus demonstrated a potent protection and significantly reduced the incidence of the disease in the BVD Type 2 viruses. calves vaccinated with the vaccine preparation based on AMPHIGEN® microfluidized formulation containing GPI-0100.

TABLE 11 Geometric mean of serum virus neutralization titers for BVD virus type 1 TABLE 12 Geometric mean of serum virus neutralization titers for BVD Type 2 virus TABLE 13 Isolation of BVD virus after exposure TABLE 14 Clinical signs of BVD disease after exposure Treatment Frequency Frequency (%) observations with Total (%) with a disease score Obs. Clinical disease of 0 1 2 T01 Saline solution 5/10 (50.0) 103 (60.6) 55 (32.4) 12 (7.1) 170 T02 Amphigen. 5/10 (50.0) 115 (67.6) 48 (28.2) 7 (4.1) 170 Quil A T03 Amphigen. 0/10 (0.0) 128 (75.3) 42 (24.7) 0 (0) 170 2 mg of GPI-0100. AIOH T04 Amphigen. 0/10 (0.0) 124 (72.9) 46 (27.1) 0 (0) 170 2 mg of GPI-0100 T05 Amphigen. 0/10 (0.0) 104 (61.2) 66 (38.8) 0 (0) 170 3 mg of GPI-0100 T06 Amphigen. 0/10 (0.0) 128 (75.3) 42 (24.7) 0 (0) 170 5 mg of GPI-0100 TABLE 15 Leukopenia after exposure In conclusion, the safety of each vaccine was demonstrated by the absence of adverse death reactions in vaccinated animals.

The potency of each vaccine was demonstrated by seroconversion (titers of SVN antibodies for BVD-1 and BVD-2> 1: 8) in 100% of the vaccinated animals. The vaccine with 2 mg of GPI-0100 only as adjuvant showed satisfactory resistance to exposure.

EXAMPLE 16 Preparation of vaccine containing microencapsulated antigen in water-in-water microfluidized emulsion Three grams of Trehalose (Fluka) were added to water to obtain a stock solution of 333 mg / ml Trehalose solution. Recombinant PauA antigen solubilized in 1.8% SDS solution was added (SDS / rPauA) to the Trehalose solution to obtain a final concentration of 494 μg rPauA / ml. In the next step, 10 grams of polylactidaglucolic acid (PLG-Resomer RE 503H, Boeringher Ingelheim) was dissolved in 200 ml of methylene chloride (MeCl2). The resulting solution of PLG / MeCI2 was combined with the SDS-rPauA / trehalose solution prepared in the first step. The combined solution was subjected to microfluidization using Microfluidizer (from Microfluidics Model M110EH) and microfluidized preparation was spray dried using Temco Spray Dryer (Model SD-5). The spray dried material was collected using a 500 micron sieve. The concentration of rPauA in this spray-dried material was quantified using a Western blot analysis. 1.04 mg of spray-dried material was dissolved in 50 μl of acetone and centrifuged at 13,200 rpm at ambient temperature for 10 minutes. The supernatant was eliminated. The supernatant and sediment fractions were dried in a biological safety hood for 2.5 hours. The pellet was resuspended in 47.43 μl of sample solution (25 μl of sample pH regulator + 10 μl of reducing agent + 65 μl of water). The dried supernatant fraction was resuspended with 20 μl of sample solution. In the Western analysis, purified PauA was used as a payroll to quantify the rPauA content of the spray dried material. A stock solution of 20% manifold or dissolving was prepared 100 grams of maniol (Sigma) in 500 ml of water for injection (WFI). The solution was heated to 40 ° C with heating plate / agilator and cooled to 30 ° C. The solution was sterilized by filtration with a sterile 1.22 micromembrane filter (Millipore). A 2.5% carboxymethylcellulose solution was prepared) by dissolving 12.5 grams of carboxymethylcellulose (Sigma) in 500 ml of WFI and mixed overnight at 4 ° C. The solution was sterilized in an autoclave at 121 ° C. The powder resulting from the spray drying was reconsiluted in a solution containing 5% mannol or, 1.3% carboxymethyl cellulose) and 1: 5000 imimerosol. The final solution was grown in vials in 3 ml aliquots and lyophilized using a Lyophilizer (USIFROID). The lyophilized powder represents the microencapsulated rPauA. The microencapsulated protein subunit antigen was resuspended in 2 ml of microfluidized water-in-water emulsion containing an AMPHIGEN® formulation (as the microfluidized emulsion described in Example 20) and used as a vaccine.

EXAMPLE 17 Preparation of a microfluidized vaccine formulation containing both whole cell bacterial antigen and recombinant protein antigen in oil in water emulsion Two preparations of the PauA recombinant Streptococcus uberis-containing vaccines were prepared as bacterial cells of Escherichia coli, intrinsically added to oil-in-water emulsions as described in examples 2 and 3. The recombinant PauA anigen was at a concentration of 100 μg per dose and the E. coli cells gave a final count of 4 x 109 per dose. The compositions of the emulsion adjuvants of the two formulations of the vaccines are shown in Table 16.

TABLE 16 Formulations of vaccines containing both recombinant protein and whole cells of E. coli EXAMPLE 18 Immune response to the microfluidized vaccine containing the oil in water emulsion with rPauA and whole cell bacterial agents In this experiment, mature dairy cows were used. The animals were at the end of their first or second lactation at the time of admission. Two ml of each vaccine formulation were administered subcutaneously one fold times, one at the time of drying (D-0), 28 days later (D = 28) and again 4 to 10 days after delivery (C + 4 - C + 10). The first and third doses were administered on the left side of the neck and the second dose was administered on the right side of the neck. Blood was drawn before each vaccination and approximately 14 days and 32 days after the first vaccination.

Antibody titers against E. coli and the rPauA antigen were determined by ELISA. As shown in Figure 8, the results indicate that the assessment of antibodies to rPauA was higher in the group vaccinated with the vaccine formulation containing GPI-0100 as immunostimulant and was maximum on day 70 after the initial vaccination. The titration of antibodies to the antigen against E. coli is shown in Figure 9. The titration of antibodies against E. coli antigen was comparable in both formulations of the vaccines, although the presence of GPI-0100 as immunoscleragen induced an assessment of Relatively greater antibodies compared to the formulation with DDA as immunosimulant.

EXAMPLE 19 Analysis of virucidal activity of the preparations of the vaccines based on the formulation of AMPHIGEN® microfluidized To determine if microfluidization inactivates the virus, the viricidal activity of fresh preparations of the microfluidifi- cated AMPHIGEN® formulation-based vaccines was determined. The preparations involved different infectious bovine virus, specifically bovine herpesvirus (BHV), parainfluenza virus 3 (PI3) and bovine respiratory syncytial virus (BRSV). The detection of the virucidal activity of the three preparations of the vaccines was carried out in accordance with the requirements of the USDA regulation 9CFR-113.35. The results shown in Table 17 indicate that the microfluidization of AMPHIGEN® formulation-based vaccine preparations does not cause significant degradation of the vaccine preparation.

TABLE 16 Analysis of virucidal activities of microfluidized vaccines A = cholesterol added at 650 ml / minute B = cholesterol added at 28 ml / minute C = cholesterol added at 5 ml / minute M200 = microfluidized with 200 micron sieve M75 = microfluidized with 75 micron sieve M75 at 37C = heated fluids at 37 ° C before microfluidization A value above 1.7 indicates a virucidal effect.

EXAMPLE 20 Preparation of a microfluidized AMPHIGEN® formulation An AMPHIGEN® formulation was prepared by combining lecithin oil solution DRAKEOL (fluid paraffin oil with 25% lecithin) and TWEEN 80 (with a final concentration of 0.18%) and Span 80 (with a final concentration of 0.08% > ) mixing for 8-22 hours at 36 + 1 ° C. The oil mixture was then added to saline with the aid of a Ross® emulsifier (Hauppage, NY 11788) at approximately 3400 rpm. Subsequently the mixture was passed once through a microfluidizer with an interaction chamber at 31,027.5 kPa ± 3.447.5 kPa. Figures 10A and 10B show the stability of the microfluidized AMPHIGEN® formulation. The size distribution of the particles, measured by laser diffraction, at the start (FIG. 10A) was almost identical to the particle size distribution after 22 months of storage at 4 ° C (FIG. 10b).

EXAMPLE 21 Analysis by electron microscopy of the immunogenic complex of Quil A and cholesterol To determine the nature of the immunogenic complex formed by Quil A and cholesterol, a mixture of these two components was prepared either in the presence or in the absence of an antigen. In a flask containing 50 ml of HR-Hals pH regulator, BVD Type I antigen was added while stirring the solution with a magnetic bar. Then, a concentrated stock solution of Quil A (50 mg / ml) was added drop by drop while stirring the solution to a final concentration of 50 μg / ml. The addition of Quil A was followed with the addition of a stock solution of cholesterol in ethanol (18 mg / ml) to a final concentration of 50 μg / ml. In a second flask, Quil A and cholesterol were added in the same way to the 50 ml pH regulator without BVD Type I ampigen. For electron transmission microscopy, 10 μl of each sample was adsorbed onto 400 mesh copper gratings with a formvar and carbon support platform (Elecíron Microscopy Sciences, Inc., Fort Washington, PA). The samples were negatively stained using 10 μl of filtered 2% phosphotungsic acid, at pH 5.2 as a conirase agent. The samples were examined using a JEOL 1230 electronic transmission microscope (JEOL Inc., Tokyo, Japan) with an acceleration voltage of 80 kV.

Digital imaging was performed with a Gafan BioScan 792 camera. Film microscopy was performed on 4489 EM film and printed on Kodabrome II RC F3 paper (Eastman Kodak Company, Rochester, NY.). In the solution containing only cholesterol and Quil A without BVD Type I antigen, helical micelles were detected together with Quil A micelles and cholesterol crystals (Figure 11). The helical micelles were cross-linked and had a mesh appearance. In the sample containing the BVD Type I antigen, it was observed that helical micelles randomly surrounded certain dense areas (Figure 12). The dense areas represent the BVD Type 1 antigen and the helical immunogenic complex that is produced by the association of Quil A and cholesterol is adsorbed on the surface of the BVD Type I antigen. Having described the invention as above, it is declared as property content in the following:

Claims (8)

NOVELTY OF THE INVENTION CLAIMS

1. - A vaccine comprising a saponin glycoside, a stellar and an animal, wherein said saponin glycoside and said esoteric are associated with each other to form complexes in the form of helical micelles and in which said antigen is mixed, but not innate , in said helical micelles.

2. The vaccine composition according to claim 1, further characterized in that it also comprises a veterinarily acceptable vehicle.

3. The vaccine composition according to claim 2, further characterized in that said verieterly acceptable vehicle is an oil in water emulsion.

4. The vaccine composition according to claim 1, further characterized in that said saponin glycoside is an iodidenoid.

5. The vaccine composition according to claim 4, further characterized in that said triterpenoid is Quil A.

6. The vaccine composition according to claim 1, further characterized in that said steral is cholesterol.

7. The vaccine composition according to claim 1, further characterized in that said antigen comprises a viral agent, a bacterial agent or a combination thereof.

8. The vaccine composition according to claim 7, further characterized in that said antigen comprises a type 1 or type 2 BVDV antigen.