MXPA00001152A - Adjuvants for use in vaccines - Google Patents

Abstract

The invention relates to adjuvants that contain a lecithin, oil and an amphiphilic surfactant and that are capable of forming a stable oil-in-water emulsion vaccine so as to minimize local reactions to the vaccine in the injected animal.

Description

ADJUVANTS FOR USE IN VACCINES FIELD OF THE INVENTION The invention relates to immunological adjuvants. In particular, the invention relates to adjuvants comprising an "oil in water" emulsion and a surfactant. The adjuvants of the invention are useful in a series of vaccine formulations, including vaccines comprising bacterial or viral components.

BACKGROUND OF THE INVENTION The generation of immunity to infectious organisms is a powerful tool in the control of diseases. Antigens that induce immunity to infection are known as immunogens. The protective antibodies that induce the antigens can collaborate with other natural defenses to inhibit the infectious process, or they can neutralize harmful products in the infected organism such as toxins. An effective means to improve the antibody response is the use of an adjuvant. Thus, an adjuvant in a vaccine is included as an additive or vehicle to improve the antigen response. An adjuvant can act by different mechanisms that include, (1) trapping the antigen in the body causing a slow release, (2) attracting the cells of the immune system to the injection site, (3) stimulating the cells of the immune system so that proliferate and activate; and (4) improve the dispersion of the antigen in the recipient's body. As adjuvants a number of agents with various chemical properties have been used, including "water in oil" and "oil in water" emulsions, mineral salts, polynucleotides and natural substances. An adjuvant, known by the trade name AMPHIGEN ™, is described in U.S. Patent No. 5,084,269. The AMPHIGEN ™ adjuvant is composed of a lecithin free of glycerides dissolved in an oil, normally clear liquid paraffin. In vaccine preparations, AMPHIGEN ™ is dispersed in an aqueous solution or suspension of the immunizing antigen in the form of an "oil in water" emulsion. When the AMPHIGEN ™ adjuvant was used according to U.S. Patent No. 5,084,269, problems were noted. For example, the lecithin in the AMPHIGEN ™ is not sufficient to produce a stable emulsion of the oil, thus giving rise to an accumulation or deposit of oil in the tissues in which it has been injected. The mineral oil can not be metabolized or eliminated by the animal. As a result, the oil becomes the source of severe chronic inflammation and the appearance of marks. The emulsification of AMPHIGEN ™ directly into the antigen preparation has the risk of damaging the antigen. Furthermore, if the desired emulsion is not formed, the valuable antigen must be discarded. It would be very desirable therefore a useful adjuvant in vaccines for animals, including humans, that is effective and that solves the above problems.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to an adjuvant useful for improving the immune response of an animal to an antigen. In particular, the invention relates to an adjuvant that can form an "oil in water" emulsion in a vaccine composition. The invention also relates to an adjuvant which, when used in a vaccine formulation, causes minimal inflammation and leaves minimal marks at the vaccination site. The invention further relates to a vaccine formulation containing an adjuvant of the invention. Finally, the invention relates to a method of using an adjuvant of the invention in a vaccination. In one embodiment, the adjuvant of the invention comprises a lecithin, an oil and an amphiphilic surfactant capable of emulsifying the adjuvant, for example, a Tween or Span surfactant. In another preferred aspect, the surfactant is Tween 80, Tween 85, Span 80 or Span 85. In another embodiment, the adjuvant of the invention comprises a lecithin, an oil and two amphiphilic surfactants capable of emulsifying the adjuvant or a vaccine composition that contain the adjuvant. In a preferred aspect, one of the two surfactants is found mainly in the aqueous phase, for example, Tween 80, and the other surfactant is found mainly in the oil phase, for example, Span 80. A lecithin is a phosphatide. Unpurified lecithin preparations may include triglycerides. For the purposes of the present invention, "lecithin" includes both purified and unpurified preparations. In a preferred aspect, lecithin is free of glycerides. Suitable oils include a mineral oil, for example DRAKEOL ™ light mineral oil. In a further embodiment, the adjuvant of the invention contains an aqueous carrier solution, for example, a physiologically acceptable buffer, water or a saline solution. In a preferred embodiment, the adjuvant of the invention contains a lecithin, a mineral oil, two amphiphilic surfactants and an aqueous vehicle solution (eg, saline). In another embodiment of the invention, a method is described for inactivating a culture of Bordetella bronchiseptica ("B. bronchiseptica") using formalin and glutaraldehyde. In another aspect, a ß culture is provided. bronchiseptica that is inactive using formalin and glutaraldehyde. In still another aspect, an antigen composition is provided from a B. bronchiseptica culture that is inactivated using formalin and glutaraldehyde. In still another aspect, a vaccine composition containing an antigen composition of a B. bronchiseptica culture that is inactivated using formalin and glutaraldehyde is provided.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents a graph representing the droplet size distribution of an emulsion prepared as described below. Lines (a) and (b) represent that approximately 94% of the droplets have a diameter of 1 μm or less.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to an adjuvant useful for improving the immune response to an antigen. In particular, the invention relates to an oil adjuvant that can emulsify a vaccine formulation. In addition, the invention relates to an adjuvant which, when used in a vaccine formulation, can substantially prevent inflammation or appearance of marks at the injection site, typical of vaccines containing mineral oil. The adjuvants of the invention comprise a lecithin, an oil and an amphiphilic surfactant capable of emulsifying the adjuvant or a vaccine composition containing the adjuvant. The invention is based in part on the discovery that by adding about 1.5% v / v (i.e., 1.5% volume concentration obtained, for example, by mixing 98.5 volumes of the vaccine comprising the adjuvant with 1.5 volumes of the amphiphilic surfactant) at 3.5% v / v of an amphiphilic surfactant to a vaccine containing an adjuvant such as that described in U.S. Patent No. 5,084,269 is effective for sufficiently emulsifying a vaccine composition formulated with said adjuvant and minimizing irritation at the injection site of the vaccinated animal. In one embodiment, the adjuvant of the invention contains a lecithin and an oil and an amphiphilic surfactant. In one embodiment, the adjuvant of the invention contains a lecithin and an oil and an amphiphilic surfactant capable of emulsifying a vaccine composition formulated with an adjuvant of the invention. In another preferred embodiment, two amphiphilic surfactants are used in an adjuvant of the invention, for example, a Tween surfactant and a Span surfactant. A preferred adjuvant, referred to herein as "Adjuvant No. 1", comprises about 2% v / v of lecithin, about 18% v / v of mineral oil and about 8% v / v of surfactant (e.g. , 6% v / v of Tween 80 and approximately 2.4% v / v of Span 80), the remaining volume being a saline solution. In a preferred aspect, a vaccine composition comprising an antigen in a concentration of about 75% v / v and an adjuvant, preferably, Adjuvant No. 1, in a concentration of about 25% v / v of the vaccine composition is formulated. . All concentrations expressed herein in percentage are indicated in volume by volume, unless the context indicates otherwise.

Surfactants Useful in the Adjuvant of the Invention The surfactants useful for the adjuvant of the invention are amphiphilic and acceptable for veterinary and medical use. Those skilled in the art can determine whether a particular surfactant is acceptable or not for medical or veterinary use. A surfactant is amphiphilic if one part of the surfactant molecule is hydrophobic and another part is hydrophilic. See U.S. Patent Nos. 5,690,942; 5,376,369; 4,933,179 and 4,606,918, which describe surfactants that can be used in the adjuvant of the invention. Examples of surfactants useful in the adjuvant of the invention include, but are not limited to, a Tween surfactant and a Span surfactant. The Tween and Span surfactants include, but are not limited to, monolaurate (Tween 20, Tween 21, Span 20), monopalmitate (Tween 40, Span 40), monostearate (Tween 60, Tween 61, Span 60), tristearate (Tween 65, Span 65), monooleate (Tween 80, Tween 81, Span 80) and trioleate (Tween 85, Span 85). In a preferred embodiment, Tween 80, Tween 85, Span 80 or Span 85 is used. It is preferred that a surfactant useful in the adjuvant of the invention be amphiphilic and have a hydrophilic-lipophilic balance ("HLB") value that is preferably at least half of the sum of the HLB values of the rest of the components of the adjuvant. More preferably, the surfactant has an HLB value that varies from about half to about twice the sum of the HLB values of the rest of the components of the adjuvant. More preferably, the surfactant has an HLB value that is approximately equal to the HLB value of the other components of the adjuvant. The HLB values for surfactants, lecithins, oils and vehicle solutions can be readily available or, if necessary, can be determined by routine experimentation. For example, see U.S. Patent Nos. 4,504,275 and 4,261,925 and references cited therein. Amphiphilic surfactants useful in the adjuvant of the invention have HLB values of from about 2 to about 20, preferably from about 3 to about 17. Methods for determining the HLB values of particular surfactants are known in the art. See, for example, U.S. Patent Nos. 5,603; 951; 4,933,179 and 4,606,918, which describe surfactants having particular values of HLB. The concentration of a surfactant in a vaccine composition formulated with the adjuvant of the invention ranges from about 1.5% to 3.5% v / v, more preferably from about 1.5% to about 3% v / v, more preferably from about 1.5% to about 2.5% v / v and, most preferably, about 2% v / v. When more than one surfactant is used, the sum of the concentrations of all the surfactants used in the vaccine composition formulated with the adjuvant of the invention will also vary from 1.5% to 3.5% v / v, more preferably from about 1.5% to about 3% v / v, more preferably from about 1.5% to about 2.5% v / v and, most preferably, about 2% v / v. The concentration of a surfactant in the adjuvant of the invention also depends on the concentration in which the adjuvant is used in a vaccine composition. For example, a vaccine composition can be formulated with the adjuvant of the invention so that approximately 25% of the volume of the vaccine composition is the adjuvant ("25% adjuvant") and the remainder, approximately 75%, it is constituted by the other components, for example, the antigen composition. In one aspect, the concentration of the surfactant in a 25% adjuvant ranges from about 6% to 14% v / v. More preferably, the concentration of the surfactant in a 25% adjuvant ranges from about 6% to 12% v / v, more preferably from about 6% to 10% v / v and, most preferably, about 8% v / v. The concentration of the surfactant in the adjuvant of the invention depends on several factors. For example, as the oil concentration in the adjuvant increases, more surfactant is needed to emulsify a vaccine composition formulated with the adjuvant of the invention. Another factor that is useful in determining the concentration of a surfactant is the concentration of lecithin. By increasing the concentration of lecithin in the adjuvant, the lower the amount of surfactant required for the emulsion. When an adjuvant of the invention is used in a vaccine composition in a concentration below 25% v / v, the concentration of the adjuvant components in the adjuvant should increase proportionally. The water vehicle is an exception since the vehicle always includes the volume that is left unoccupied by the other components; thus, if the concentration of all the components except the vehicle increases, the concentration of the vehicle in the adjuvant will decrease and vice versa. For example, when the adjuvant is used in a concentration of approximately 12.5% v / v in a vaccine composition, the concentration of the components in the adjuvant is approximately twice the concentration of the components in a 25% adjuvant. . Similarly, when the adjuvant of the invention is used in a vaccine composition in a concentration that is greater than 25% v / v, the concentration of the components in the adjuvant will have to decrease accordingly, for example, when the adjuvant used in a concentration of about 50% v / v in a vaccine composition, the concentration of the components in the adjuvant is about half the concentration of the components in a 25% adjuvant. In one embodiment, two amphiphilic surfactants may be used in the adjuvant of the invention. Preferably, the two surfactants will include a surfactant that would be more concentrated in an aqueous phase than in an oil phase of the adjuvant ("hydrophilic surfactant") and a surfactant that would be more concentrated in an oil phase of the adjuvant ("lipophilic surfactant"). For example, Tween 80 would concentrate more in an aqueous phase and Span 80 would concentrate more in an oil phase. A preferred hydrophilic surfactant has an HLB value of about 9 to about 20 and a preferred lipophilic surfactant has an HLB value of about 2 to about 9. See U.S. Patent Nos. 5,603,951; 4,933,179 and 4,606,918, which describe surfactants with HLB values in both ranges useful for the adjuvant of the invention. When two surfactants are used in the adjuvant of the invention, the total concentration of both surfactants combined in a vaccine composition formulated with the adjuvant of the invention ranges from about 1.5% to 3.5%, more preferably, about 1. , 5% to about 3%, more preferably, from about 1.5% to about 2.5% and, most preferably, about 2% v / v. The concentration of each of the two surfactants used in the adjuvant of the invention may differ from each other. For example, when a hydrophilic surfactant and a lipophilic surfactant are used, for example Tween 80 and Span 80, the concentration of Tween 80 may vary from about 1.2 to about 5 times the Span concentration, more preferably, about 1, 5 to about 4, more preferably from about 1, 8 to about 3, more preferably, from about 2 to about 2.5 and more preferably, about 2.3, preferably when used in an adjuvant with a lecithin and a concentration of oil as in Adjuvant No. 1. The concentration of the hydrophilic surfactant used in the adjuvant of the invention depends, in part, on the size of the aqueous phase, and the concentration of the lipophilic surfactant depends, in part, on the size of the phase oily In one embodiment, the adjuvant of the invention consisting of an 80% v / v aqueous phase and a 20% v / v oil phase, may contain a hydrophilic surfactant at a concentration of up to about four times (i.e., 80 / 20) the concentration of a lipophilic surfactant or, for example, up to about 2 times.

Non-Surfactant Components of the Adjuvant of the Invention In addition to the amphiphilic surfactant, the adjuvant of the invention contains a lecithin and an oil. In another aspect, the adjuvant of the invention contains an aqueous carrier solution. Any lecithin known in the art is useful for the adjuvant of the invention. The term "lecithin" refers to a mixture of phosphatides. When provided in the form of a crude extract, a lecithin may also contain triglycerides. The lecithins can be of vegetable or animal origin. In addition, lecithins can be obtained synthetically. In U.S. Patent Nos. 5,690,942; 5,597,602 and 5,084,269 examples of lecithins are described. In a preferred embodiment, the content of triglycerides in a lecithin used in the adjuvant of the invention is reduced when compared to its natural source, ie, the lecithin is free of glycerides. A number of ways to remove the glycerides from a lecithin are known in the art, for example, as described in U.S. Patent No. 5,597,602. The concentration of a lecithin in a vaccine composition formulated with the adjuvant of the invention ranges from about 0.25% to about 12.5% v / v, more preferably, from about 0.5% to about 10% v / v , more preferably from about 0.5% to about 7.5%, more preferably from about 0.5% to about 5%, more preferably from about 0.5% to about 2.5% and, most preferably, from about 0.5% to about 1.25% v / v. The concentration of a lecithin in a 25% adjuvant is at least about 1% v / v, preferably at least about 2% v / v. In another aspect, the concentration of lecithin in a 25% adjuvant ranges from about 1% to about 50% v / v, more preferably from about 2% to about 40% v / v, more preferably from about 2% to about 30. % v / v, more preferably from about 2% to about 20% v / v, more preferably from about 2% to about 10% v / v and, most preferably, from about 2% to about 5% v / v. The concentration of a lecithin in the adjuvant of the invention with a higher or lower concentration is determined as exemplified above. The adjuvant of the invention contains an oil, for example, an oil described in U.S. Patent Nos. 5,814,321 and 5,084,269. In a preferred aspect, the adjuvant of the invention contains a mineral oil, for example, DRAKEOLt. In another aspect, a mixture of oils is used.

The oil may be provided for the preparation of the adjuvant of the invention in the form of pure oil or as a mixture containing the oil and another component, for example, a lecithin. The concentration of oil in a vaccine composition formulated with the adjuvant of the invention ranges from about 1% to about 23% v / v, more preferably from about 1.5% to about 20% v / v, more preferably about 2%. , 5% to about 15%, more preferably from about 3.5% to about 10% v / v, more preferably from about 3.5% to about 7.5%, more preferably, from about 4% to about 6% and, most preferably, about 4.5%. The oil concentration in a 25% adjuvant is at least 5% v / v, preferably at least about 8% v / v and more preferably at least about 12% v / v. In another aspect, the oil concentration in a 25% adjuvant ranges from about 4% to about 92% v / v, more preferably, from about 6% to about 80% v / v, more preferably, about 10% a about 60% v / v, more preferably from about 14% to about 40% v / v, more preferably, from about 14% to about 30% v / v, more preferably, from about 16% to about 24%, and more preferable, of about 18%. The concentration of oil in the adjuvant of the invention with a higher or lower concentration is determined as exemplified above.

In another embodiment, an aqueous carrier, for example, saline (e.g., phosphate buffered saline), tris-HCl, citrate-phosphate buffer, Hepes buffers, other known pharmaceutically acceptable buffers, is used in the adjuvant of the invention. the technique or water. The pH of the vehicle is preferably physiologically acceptable, eg, from 6 to 8, more preferably, of about 7. The aqueous vehicle used in the adjuvant of the invention constitutes the volume that is not necessary for any of the other components. The adjuvant of the invention is preferably provided in a concentration ranging from about 2 to about 10 times the concentration after formulation of the adjuvant in a vaccine composition, more preferably, from about 2 to about 8, more preferably about 3 to about 6 and, most preferably, about 4.

Uses of the adjuvants of the invention The adjuvants of the invention can be used to improve the immune response to an antigen of a vaccine formulation. The adjuvants of the invention can be used with antigens derived from any bacterium or any virus, provided that the antigen is not destroyed or denatured. Non-limiting examples of antigens are antigens of Erysipelothrius rhusiopathiae, Bordetella bronchiseptica antigens, antigens of toxigenic strains of Pasteurella multocida, antigens of strains of Escherichia coli causing neonatal diarrhea, antigens of Actinobacillus pleuropneumoniae, antigens of Pasteurella haemolytica or any combination thereof. previous The adjuvants of the invention are also useful in vaccine compositions containing an antigen described in U.S. Patent Nos. 5,616,328 and 5,084,269. In a preferred embodiment, the adjuvant of the invention is used in a vaccine formulation containing an antigen obtained from the liquid phase of a culture of Erysipelothrix rhusiopathiae ("E. rhusiopathiae"). In a preferred aspect, a culture of E. rhusiopathiae is inactivated by adding formalin (approximately a final concentration of 0.5% v / v) and, after incubation for 24 hours at 37 ° C, the cells are separated, for example, by centrifugation or filtration. In a preferred embodiment, the culture supernatant is concentrated to approximately one tenth and aluminum hydroxide gel (preferably REHYDRAGEL ™) is added to the concentrated supernatant at a final concentration of about 30% v / v to stabilize the antigen. In another preferred embodiment, thimerosal preservatives (approximately 0.01% v / v final concentration), (Dimportex, Spain, imported by Flavine Inc., Klosters, New Jersey) with EDTA (approximately 0.07) are added as preservatives. % v / v final concentration). In another preferred embodiment, a vaccine composition comprising the antigen and the adjuvant of the invention is formulated (for example, Adjuvant No. 1) the adjuvant comprising, for example, about 25% v / v of the vaccine composition. This preferred E. rhusiopathiae antigen is described in U.S. patent application serial number, filed January 29, 1999, entitled "Erysipelothrix rhusiopathiae Antigens and Vaccine Compositions", which is incorporated herein by reference. In another preferred embodiment, the adjuvant of the invention is used in a vaccine composition containing antigens of a β culture. bronchiseptica that has been activated by the addition of formalin in the logarithmic phase, preferably in the late logarithmic phase, followed by the addition of glutaraldehyde. In addition to killing bacterial cells, the object of this novel and unique inactivation is to make non-toxic endotoxin and B. bronchiseptica exotoxin, while the antigens of B. bronchiseptica cells are still effective in eliciting the desired immune response. Formalin is added in the culture of B. bronchiseptica in a concentration of approximately 0, 2% v / v to about 1% v / v, more preferably from about 0.4% v / v to about 0.8% v / v and, most preferably, about 0.6% v / v. Glutaraldehyde is added from about 10 minutes to about 40 minutes after the addition of the formalin to the culture, more preferably from about 15 minutes to about 30 minutes and, most preferably, about 20 minutes. Glutaraldehyde is added in the ß culture. bronchiseptics in a concentration of about 0.2% v / v about 1% v / v, more preferably from about 0.4% v / v about 0.8% v / v and, most preferably, about 0.6% v / v. Before glutaraldehyde is added to the culture, it has a concentration of about 10% v / v about 50% v / v, more preferably about 15% v / v about 35% v / v and, most preferably, about 25% v / v. After adding formalin and glutaraldehyde to the ß culture. bronchiseptica, the resulting mixture is incubated by shaking from about 32 ° C to about 42 ° C, more preferably, from about 35 ° C to about 39 ° C and, most preferably at about 37 ° C. The mixture is incubated from about 12 hours to about 60 hours, more preferably from about 24 hours to about 48 hours. The rest of the processing steps in the preparation of the antigen composition of the invention from a β culture. bronchiseptics are described in Example 7, below, and in U.S. Patent Nos. 5,019,388 and 4,888,169.

Vaccine compositions comprising adjuvants of the invention and their administration The adjuvant of the invention can be used in a vaccine formulation to immunize an animal. In one embodiment, the vaccine formulation contains the adjuvant of the invention and an antigen. The optimal ratios of each component in the vaccine formulation can be determined by techniques well known to those skilled in the art. A vaccine formulation can be administered to a subject per se or in the form of a pharmaceutical or therapeutic composition. The pharmaceutical compositions comprising the adjuvant of the invention and an antigen can be manufactured by means of conventional methods for mixing, dissolving, granulating, entraining, dissolving, emulsifying, encapsulating, entrapping or lyophilizing. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable vehicles, diluents, excipients or auxiliary materials that facilitate the processing of the antigens of the invention into preparations that can be used pharmaceutically. The correct formulation depends on the chosen route of administration. For purposes of this application, "physiologically acceptable vehicle" encompasses vehicles that are acceptable for use by humans or animals without relatively detrimental side effects (with respect to the disorder being treated), as well as diluents, excipients or auxiliary materials that They are equally acceptable. Systemic formulations include those designed for administration by injection, for example, for subcutaneous, intradermal, intramuscular or intraperitoneal injection. For injection, the vaccine preparations can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as: Hank's solution, Ringer's solution, phosphate-buffered saline, or any other buffer based on physiological saline. The solution may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the proteins may be in powder form for reconstitution with a suitable vehicle before use, for example, with sterile, pyrogen-free water. It is within the knowledge of those skilled in the art to determine the effective amount of the vaccine formulation to be administered, especially in light of the detailed description set forth herein. An effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve the induction of an immune response using techniques that are well known. A person skilled in the art could easily optimize administration to all animal species based on the results described herein. The dosed quantity and the interval can be adjusted case by case. For example, when used as a vaccine, the vaccine formulations of the invention can be administered in about 1 to 3 doses for periods of 1 to 36 weeks. Preferably, 1 or 2 doses are administered, at intervals of about 3 weeks to about 4 months, and booster vaccinations may be administered periodically thereafter. Other protocols may be appropriate for animals. A suitable dose is an amount of the vaccine formulation which, when administered in the manner described above, can elicit an immune response in an immunized animal, sufficient to protect the animal from infection for at least 4 to 12 months. In general, the amount of antigen present in a dose ranges from about 1 pg to about 100 mg per kg of receptor, preferably from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 pg. The appropriate dose range will vary according to the route of injection and the weight of the patient, although it will typically vary from about 0.1 ml to about 5 ml. Having described the invention, the following examples are presented only in an illustrative and non-limiting sense.

EXAMPLE 1 Use of an adjuvant containing oil and lecithin The following example describes the use of an adjuvant containing glyceride-free lecithin dissolved in an oil ("oil-lecithin-based adjuvant"), usually mineral oil (clear liquid paraffin) in vaccines for veterinary use. See U.S. Patent No. 5,084,269, which discloses an oil-lecitin-based adjuvant. A vaccine preparation using an oil-lecithin-based adjuvant is an "oil in water" emulsion. Unless stated otherwise, all percentages herein are expressed in volume by volume. The percentage values, unless otherwise stated, of an oil-lecithin-based adjuvant, refer to the concentration of a mixture of lecithin (10% of the mixture) and a vehicle oil (DRAKEOL ™) ( 90% of the mixture) in an aqueous vehicle (continuous phase). For example, a 20% oil-lecithin adjuvant contains 2% v / v lecithin (Central Soya, Fort Wayne, Indiana), DRAKEOL ™ 5 at 18% v / v (Penreco, Kams City, Pennsylvania) and saline solution at 80% v / v (reducing the content of the saline solution if other components are added, for example, surfactants). Unless indicated otherwise, the percentage values of an oil-lecithin-based adjuvant in a vaccine composition, ie, after diluting the adjuvant solution with the antigen solution, refer to the concentration of a mixture of lecithin (10% of the mixture) and a carrier oil (DRAKEOL ™) (90% of the mixture) in the vaccine preparation comprising the adjuvant and a solution containing an antigen. Unless indicated otherwise, in all cases in which a surfactant is added to an adjuvant composition, the percentage values for the surfactant concentration refer to the total concentration of all the surfactants added in the adjuvant or the preparation. of vaccine. When an oil-lecithin-based adjuvant was used as an adjuvant in the vaccine formulations, it was found that it was not emulsified in aqueous preparations without the addition of additional surfactants since the lecithin in the oil-lecithin-based adjuvant is not sufficient for the emulsion. Thus, vaccines prepared using improperly dispersed oil-lecithin-based adjuvant formed an accumulation or deposit of most of the mineral oil in the tissues at the site of injection. This oil can not metabolize or eliminate the animal that has been injected and remains a source of chronic severe inflammation and appearance of marks. It was further determined that adding surfactants to the vaccine formulation, comprising an oil-lecitin-based adjuvant and an antigen, in order to emulsify the formulation was not a suitable solution. The problems encountered when adding oil and surfactants to the vaccine formulation before emulsifying were that the antigen could be damaged and, if a suitable emulsion was not achieved, the formulation had to be discarded, including the valuable antigen. Different adjuvant compositions comprising an oil-lecithin-based adjuvant in combination with surfactants were tested to emulsify the vaccine formulations.

EXAMPLE 2 Use of an adjuvant containing a surfactant in a low concentration The following example describes the use of an emulsion containing 40% acetyl-lecithin and 2% synthetic surfactants, ie, Tween 80 and Span 80 (Van Water &Rogers, Omaha, Nebraska) in phosphate buffered saline. This adjuvant was prepared aseptically and separated from the antigen. The emulsion was added to the antigen preparation without additional emulsion. The synthetic surfactants aided the oil-lecithin-based adjuvant to be dispersed in the form of a relatively stable emulsion with relatively large droplets. The adjuvant emulsion was added to the aqueous preparation of the antigen in a ratio of one to eight, decreasing the content of oil-lecithin-based adjuvant from 40% to 5% and the surfactants from 2% combined to 0.25%. The adjuvant was used in several vaccines. It was found that because the emulsion has coarse droplets and is not very stable, the oil droplets tend to coalesce and separate in the form of a permanent oil-irritant deposit in the injected tissues. Another problem observed with this adjuvant was that it was added with an Al gel. A series of vaccines contains Al gel for a number of purposes, for example, as an adjuvant or to stabilize an antigen or bind to an endotoxin. The oil-lecithin-based adjuvant has a negative charge that causes it to bind with the positively charged Al gel, forming large aggregates. These aggregates are unsightly, have difficulty passing through a hypodermic needle and are very irritating to the injected tissues.

EXAMPLE 3 Use of an adjuvant containing a surfactant at a high concentration An oil-lecitin-based adjuvant (5% v / v) was emulsified in the antigen preparation with the aid of Tween 80 and Span 80 surfactants, as before, albeit at a total surfactant concentration of 8% in the composition of vaccine. The emulsion was very fine and stable. The emulsion had almost the clarity of a solution and did not suffer cremation at rest. Under the microscope, with a maximum magnification (0.2 micrometer resolution), most of the droplets were too small to be visible. Therefore, it was a microemulsion. It was found that this adjuvant, when used in a vaccine formulation was practically free of reactivity at the injection site and, when Al gel was added, no aggregation of the oil and the gel was detected. As a result of its high surfactant content, this adjuvant is easy to emulsify, has an attractive appearance, is stable, does not react with Al gel and is practically free of irritant effects at the injection site. However, despite these advantages, this emulsion had a slightly lower adjuvant potency when compared to the thick droplet version made with surfactants at a lower concentration.

EXAMPLE 4 Use of an adjuvant containing a surfactant in an average concentration An attempt was made to find an adjuvant emulsion that was acceptably mild and with full potency as an adjuvant. A 20% oil-lecithin-based adjuvant was used in these experiments and it was found that a 20% oil-lecithin-based adjuvant emulsion is easier to prepare than an oil-lecithin-based adjuvant emulsion. 40% Its addition to vaccines in a ratio of one to four, to prepare a final concentration of oil of 5%, left 75% of the volume of the dose for the antigens. Previous experiments showed that a submicron soft emulsion could be prepared (most droplets had a diameter less than one micrometer, see Figure 1) with 20% oil and with 16% of the Tween 80 and Span 80 surfactants. Two emulsions were prepared for the tests. One contained 20% oil-lecithin adjuvant and 16% Tween 80 and Span 80 surfactants. Dilution one to four provided an emulsion comprising 5% lecithin-based adjuvant and 4% surfactants in the vaccine preparation. The other emulsion was prepared with an adjuvant based on 40% acetyl-lecithin and 2% of Tween 80 and Span 80 surfactants. The one to eight dilution provided an emulsion with 5% lecithin-based adjuvant and surfactants at 0.25%. Al gel was added (REHYDRAGEL ™, obtained from Reheis, Berkeley Heights, New Jersey) at a concentration of 10% to samples of each emulsion. In the emulsion with 0.25% surfactants, the oil and the Al gel were added and separated by forming a dense layer on the top of the column of liquid (cremated). In contrast, in the emulsion with 4% surfactants, no aggregation or creaming occurred. With 4% surfactants, the Al gel settled at the bottom of the tube, leaving the oil droplets dispersed in the supernatant liquid.

EXAMPLE 5 Inflammation of the sites of infection using an adjuvant containing a surfactant in an average concentration Vaccine preparations were tested in pigs to determine if inflammation of the injection site occurred when an adjuvant with a medium concentration of surfactant was used. Vaccine preparations containing a 5% oil-lecitin adjuvant and 0.25% or 4% surfactants did not cause inflammation in pigs at the injection site. When Al gel was added to the vaccine preparation in a concentration of 10%, the preparation with 0.25% of surfactants caused intense inflammation at the injection site, while the preparation with 4% surfactant almost no it produced inflammation. The experiments were carried out to determine the range of surfactant concentrations that was effective to prevent aggregation with the Al gel and inflammation of the injection site. When a surfactant concentration of 1 was used, 5% in the vaccine, a slight aggregation of the oil and the Al gel was observed. Aggregation was much more intense at lower concentrations of surfactant. At surfactant concentrations of 2% and 4%, no aggregation was observed. Inflammation induced in pigs by vaccine preparations containing 0.5% or less of surfactants was higher at 2 and 4 weeks after vaccination than that induced by preparations containing 1% or more of surfactants. At 6 months after vaccination, it was evident that the minimum necessary to avoid chronic inflammation was 1.5% of surfactants.

EXAMPLE 6 Adjuvants with useful properties in vivo and in vitro Assays were carried out to find an adjuvant that did not react with Al gel and that did not cause reactivity in the animal after vaccination. It was determined that a 20% oil-lecitin adjuvant containing 8% surfactants, and that originated a vaccine preparation with 5% adjuvant based on oil-lecithin and 2% surfactants, was sufficient to avoid both the in vitro reactivity with the Al gel and the irritation of the tissues at the injection site. The evidence for a relationship between the concentration of surfactant and the potency of the adjuvant was much less clear. There were occasional indications that 4% of surfactants in the vaccine was an excessive percentage, for example, in the induction of agglutinin to E. coli K99 and neutralization of antitoxin to the toxin of P. multocida Type D. Thus, it was determined that the optimum concentration of surfactants was 8% in a 20% oil-lecithin adjuvant, 2% of surfactants being obtained in the vaccine composition. This provided reasonably easy emulsification and good stability when stored cold. In vaccines with 5% lecithin-based adjuvant, 2% surfactants were ideal for the potency of the adjuvant and for the absence of irritation in the injected tissues. The size of the droplets in the submicrometric emulsion of a 20% oil-lecithin adjuvant with 8% surfactants was determined. The 8% surfactant was formed by 5.6% Tween 80 in the aqueous phase and 2.4% Span 80 in the oil phase. Approximately 94% of all droplets had a diameter of less than 1 micrometer, see Figure 1. A standard solution of 1000 ml of 20% acetyl-lecitin adjuvant with 8% surfactant was prepared from 200 ml of filter-sterilized lecithin-oil solution (10% lecithin in DRAKEOL ™ mineral oil), Tween 80 (56 ml) and Span 80 (24 ml) sterilized in autoclave, and phosphate buffered saline (720 ml) ( PBS by Dulbecco). The lecithin-oil and Span 80 solution were combined and mixed in a sterile tank for at least 1 hour at room temperature until the emulsion was complete. The saline solution and the Tween 80 were combined and mixed in a sterile tank for at least 1 hour at room temperature. The oil mixture was emulsified in the aqueous mixture using an emulsifying agent. The emulsion was continued by recirculation until all of the adjuvant was added to the saline. The emulsion was then passed twice through a homogenizer at room temperature. The adjuvant was stored at a temperature of 2 ° C to 8 ° C.

EXAMPLE 7 Vaccine for atrophic rhinitis using an adjuvant containing a surfactant in a medium concentration The adjuvant described in Example 4 was used with an average concentration of surfactants in a vaccine for atrophic rhinitis containing antigens of Bordetella bronchiseptica and toxigenic Pasteurella multocida. A Bordetella bronchiseptica-acte? Na-toxoid vaccine was prepared from Pasteurella multocida from B. bronchiseptica cells and P. multocida toxoid. The cells of ß. bronchiseptica, strain 2-9 NADL, were prepared as described in U.S. Patent Nos. 5,019,388 and 4,888,169, except that at the end of the growth cycle, cultures were mixed continuously and formalin solution was added until a final concentration of 0.6%. In a period of 20 minutes after the addition of formalin, a 25% glutaraldehyde solution was added to a final concentration of 0.6%. The culture was stirred for 24 to 48 hours at 37 ± 2 ° C to complete the inactivation and detoxification. (See Table 1). Then, the culture liquids were cooled to 15 ° C or less for processing. The unprocessed inactivated cultures were immediately stored at 2-8 ° C for a period of up to 14 days. After inactivation, the bacteria were separated from the culture liquid by centrifugation. The supernatant was discarded and the cells were resuspended in phosphate buffered saline to approximately one tenth of the original volume. The concentrated suspension was stored at 2-8 ° C. The treatment of B. bronchiseptica with two aldehydes inactivates both the endotoxin and the exotoxin, avoiding other safety treatments. The P. multocida toxoid was prepared in two different ways as described in U.S. Patent Nos. 5,536,496 and 5,695,769. In one of them, the toxin is converted into toxoid in the bacterial cells by the addition of formaldehyde to the culture; The toxoid remains inside the cells. In the alternative form, the live cells are mechanically lysed and the toxin is extracted. The toxin is converted to toxoid by exposure to high pH, as described in U.S. Patent 5,536,496. Both forms of toxoid are treated with Al gel to control free endotoxin by a patented process, as described in U.S. Patent 5,616,328 (see Table 1). The synergy between the two forms of pasteurella toxoid results in antitoxin responses that far exceed the sum of the responses of each when used separately as described in U.S. Patent 5,695,769.

TABLE 1. Cell treatment during the preparation of Bordetella bropc 7 / sepf / ca-Bacterin-toxoid vaccine from Pasteurella multocida The adjuvant described in Example 4 was added giving 5% concentrations of oil-lecitin-based adjuvant and 2% surfactants in the vaccine formulation. A clinical trial to determine the minimum immunizing dose of the vaccine for atrophic rhinitis demonstrated the properties as adjuvant of the adjuvant based on oil-lecithin with a medium concentration of surfactants. Pregnant sows were vaccinated with two doses of 2 ml in an interval of 4 weeks. These calved approximately 2 weeks after the second dose. At one month of age, their piglets underwent a severe infection, consisting of virulent cultures of B. bronchiseptica and P. multocida administered intranasally sequentially. Pigs born from 7 sows vaccinated with only a placebo developed severe atrophic rhinitis. The piglets of the 7 sows that were given a vaccine containing a full dose of the antigens were strongly protected by the maternal antibody still circulating. Sows given vaccines containing 1/2 or 1/8 doses of antigens did not provide satisfactory protection for their piglets.

EXAMPLE 8 Vaccine against erysipelas using an adjuvant containing a surfactant at an average concentration The adjuvant described in Example 4 was used with an average concentration of surfactants in an erysipelas vaccine containing E. rhusiopathiae antigens. Antigens for use in the vaccine were prepared from cultures of inactivated E. rhusiopathiae with 0.5% formalin for at least 24 hours. The inactivated cultures were clarified by centrifugation and concentrated approximately one-tenth by molecular filtration. The concentrates were stabilized by adding Al gel, ie, REHYDRAGEL ™, to a concentration of 30%. The previously adsorbed concentrates were included in the vaccine in such amount that each dose of 2 ml contained at least 3.2 units of opacity (UO), which were calculated from the optical density (OD) of the culture on inactivation . (The DO is multiplied by the final concentration factor giving a value in UO per ml). A vaccination against erysipelas was carried out to determine the efficacy of a vaccine containing the adjuvant based on oil-lectalin with an average concentration of surfactants. The adjuvant described in Example 4 was added to a final concentration of 25% v / v, giving a final lecithin-oil concentration of 5%. As a preservative, thimerosal (0.01% w / v) was added with EDTA (0.07% w / v). The vaccines prepared according to this formula were tested twice to determine their effectiveness in pigs. In each case, the pigs were vaccinated with two doses of 2 ml administered intramuscularly (IM), one dose at approximately 3 weeks (weaning) and the second dose 3 weeks later. The controls received phosphate buffered saline as a placebo. Immunity was tested for infection caused by intramuscular injection of virulent E. rusiopathiae at approximately 9 weeks of age in one study and at 6 months of age in the other. As shown in Table 2, the protection due to vaccination was 100% at 9 weeks and 75% at 6 months (ie, age of sacrifice). These results indicated that the vaccine provides satisfactory protection against erysipelas during the normal fattening period. The vaccine used in the group with infection caused at 9 weeks was already 12 months old. The results confirm that the protective antigen was adequately stabilized.

TABLE 2 Protection of swine against erysipelas Note: In the vaccinated group infected at 9 weeks, the pig number 20, a very unruly animal, was violently struggling when it was held, so that the resting temperature could not be determined. After the infection, this pig was totally healthy. The scope of the invention is not limited by the exemplary embodiments that will be construed as illustrations of individual aspects of the invention, and any adjuvant that is functionally equivalent is considered within the scope of the invention. In fact, from the foregoing description and the attached figures, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art. It is understood that such modifications are within the scope of the appended claims. All of the cited publications are incorporated herein by reference.

Claims (20)

NOVELTY OF THE INVENTION CLAIMS

1. - A vaccine composition comprising from about 0.25% to about 12.5% v / v of a lecithin, from about 1% to about 23% of an oil, of about 1.5% to 3.5% v / v of at least one amphiphilic surfactant and an antigen.

2. The vaccine composition according to claim 1, wherein said oil is a mineral oil and said lecithin is free of glycerides.

3. The vaccine composition according to claim 1, further comprising an aqueous vehicle.

4. The vaccine composition according to claim 1, wherein said antigen is selected from the group consisting of antigens of Erysipelothrix rhusiopathiae, antigens of Bordetella bronchiseptica, antigens of Pasteurella multocida, antigens of strains of Escherichia coli that cause neonatal diarrhea, antigens of Actinobacillus pleuropneumoniae, antigens of Pasteurella haemolytica and combinations thereof.

5. The vaccine composition according to claim 1, wherein two amphiphilic surfactants are used.

6. The vaccine composition according to claim 5, wherein a hydrophilic surfactant and a lipophilic surfactant are used.

7. - The vaccine composition according to claim 5, wherein said lecithin is in a concentration of about 0.5% v / v, said oil is in a concentration of about 4.5% v / v and said amphiphilic surfactants are found in a concentration of approximately 2% v / v.

8. The vaccine composition according to claim 5, further comprising an aqueous vehicle.

9. An adjuvant composition comprising from about 0.25% to about 12.5% v / v of a lecithin, from about 1% to about 23% of an oil, of about 1.5% to 3.5 % v / v of at least one amphiphilic surfactant, when said adjuvant is formulated in a vaccine composition.

10. A method for preparing a vaccine composition comprising adding an adjuvant composition according to claim 9 to an antigen composition.

11. The method according to claim 10, wherein said antigen is selected from the group consisting of antigens of Erysipelothrix rhusiopathiae, antigens of Bordetella bronchiseptica, antigens of Pasteurella multocida, antigens of strains of Escherichia coli that cause neonatal diarrhea, antigens of Actinobacillus pleuropneumoniae, antigens of Pasteurella haemolytica and combinations thereof.

12. An antigen composition comprising a culture of Bordetella bronchiseptica that has been inactivated by adding formalin, followed by the addition of glutaraldehyde.

13. A vaccine composition comprising the antigen composition according to claim 12 and an adjuvant.

14. The vaccine composition of claim 13, wherein said adjuvant is the adjuvant of claim 1.

15. A process for inactivating a culture of Bordetella bronchiseptica comprising adding formalin to said culture, followed by the addition of glutaraldehyde to said culture.

16. A composition of Bordetella bronchiseptica comprising a culture of Bordetella bronchiseptica, formalin and glutaraldehyde.

17. A vaccine to protect a mammal against an infection with Bordetella bronchiseptica comprising a number of cells of Bordetella bronchiseptica of an inactivated culture according to the method of claim 15, effective to protect said mammal against an infection by Bordetella bronchiseptica, and a physiologically acceptable carrier.

18. A vaccine according to claim 17, further comprising a quantity of toxigenic Pasteurella multocida antigens, effective to protect the mammal against an infection of toxigenic Pasteurella multocida.

19. The use of a quantity of Bordetella bronchiseptica cells from a formalin inactivated culture followed by glutaraldehyde in combination with a quantity of antigens of toxigenic Pasteurella multocida for the manufacture of a vaccine to protect a pig against an infection by Bordetella bronchiseptica and an infection with toxigenic Pasteurella multocida. 20.- The use of a quantity of Bordetella bronchiseptica cells from a culture inactivated by formalin followed by glutaraldehyde, in combination with antigens of toxigenic Pasteurella multocida for the manufacture of a vaccine to administer to a pregnant sow before calving, to produce colostrum produced by the sow to protect a piglet against atrophic rhinitis; said colostrum produced by the sow is administered to the piglet within approximately 24 hours after the birth of the piglet.