Patent 6,814,971

The invention relates to adjuvants that contain a lecithin, an 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 of the Invention

FIELD OF THE INVENTION

The invention relates to immunological adjuvants. In particular, the invention relates to adjuvants which comprise an oil-in-water emulsion and a surfactant. Adjuvants of the invention are useful in a variety 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 disease control. Those antigens that induce immunity to infection are known as immunogens. The protective antibody they induce may collaborate with other natural defenses to inhibit the infective process, or they may neutralize harmful products of the infective organism such as toxins.

An effective means of enhancing the antibody response is the use of an adjuvant. Thus, an adjuvant is included in a vaccine as an additive or vehicle to enhance the response to the antigen. An adjuvant may function by different mechanisms, including (1) trapping the antigen in the body to cause a slow release, (2) attracting cells of the immune system to the injection site, (3) stimulating cells of the immune system to proliferate and to become activated, and (4) improving antigen dispersion in the recipient's body.

A number of agents with diverse chemical properties have been used as adjuvants, including water-in-oil and oil-in-water emulsions, mineral salts, polynucleotides and natural substances. One adjuvant, known under the trademark AMPHIGEN.TM., is described in U.S. Pat. No. 5,084,269. AMPHIGEN.TM. adjuvant consists of de-oiled lecithin dissolved in an oil, usually light liquid paraffin. In vaccine preparations AMPHIGEN.TM. is dispersed in an aqueous solution or suspension of the immunizing antigen as an oil-in-water emulsion.

Problems were observed when using an AMPHIGEN.TM. adjuvant according to U.S. Pat. No. 5,084,269, above. For example, the lecithin in the AMPHIGEN.TM. does not suffice to produce a stable emulsion of the oil, thus leading to a pool or depot of oil in the injected tissues. Mineral oil can not be metabolized or removed by the animal. As a result, the oil becomes a source of severe chronic inflammation and scarring. Emulsifying the AMPHIGEN.TM. directly in the antigenic preparation carries the risk of damaging the antigen. Also, if the desired emulsion fails to form, the valuable antigen must be discarded.

An adjuvant useful in vaccines for animals, including humans, that is effective and solves the above problems would therefore be highly desirable.

SUMMARY OF THE INVENTION

The invention relates to an adjuvant useful for the enhancement of the immune response of an animal to an antigen. In particular, the invention relates to an adjuvant that is capable of forming an oil-in-water emulsion in a vaccine composition. The invention also relates to an adjuvant that, when used in a vaccine formulation, causes minimal inflammation and scarring at the vaccination site. The invention further relates to a vaccine formulation that contains 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 a 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 contains the adjuvant. In a preferred aspect, one of the two surfactants is predominantly found in the aqueous phase, for example, Tween 80, and the other surfactant is predominantly found in the oil phase, for example, Span 80.

A lecithin is a phosphatide. Crude preparations of lecithin may include triglycerides. For purposes of the present invention, "lecithin" encompasses both purified and crude preparations. In a preferred aspect, the lecithin is de-oiled.

Suitable oils include a mineral oil, for example, DRAKEOL.TM. 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 carrier solution (e.g., saline).

In another embodiment of the invention, a method to inactivate a culture of Bordetella bronchiseptica ("B. bronchiseptica") using formalin and glutaraldehyde is described. In another aspect, a culture of B. bronchiseptica is provided that was inactivated using formalin and glutaraldehyde. In yet another aspect, an antigen composition from a B. bronchiseptica culture is provided that was inactivated using formalin and glutaraldehyde. In yet another aspect, a vaccine composition is provided that contains an antigen composition from a B. bronchiseptica culture that was inactivated using formalin and glutaraldehyde.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an adjuvant useful for the enhancement of the immune response to an antigen. In particular, the invention relates to an oily adjuvant that is capable of emulsifying a vaccine formulation. Further, the invention relates to an adjuvant that, when used in a vaccine formulation, is capable of substantially avoiding the inflammation or scarring at the injection site, typical of vaccines containing mineral oil. Adjuvants of the invention comprise a lecithin, an oil and an amphiphilic surfactant capable of emulsifying the adjuvant or a vaccine composition that contains the adjuvant.

The invention is based, in part, on the discovery that adding from about 1.5% v/v (i.e., 1.5% volume per volume concentration obtained by, e.g., mixing 98.5 volumes of the vaccine comprising the adjuvant with 1.5 volumes of the amphiphilic surfactant) to 3.5% v/v of an amphiphilic surfactant to a vaccine containing an adjuvant as described in U.S. Pat. No. 5,084,269 is effective to sufficiently emulsify a vaccine composition formulated with such an adjuvant and to minimize irritation in 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 and a Span surfactant.

A preferred adjuvant, herein referred to as "No.1 Adjuvant", comprises about 2% v/v lecithin, about 18% v/v mineral oil, and about 8% v/v surfactant (e.g., about 5.6% v/v Tween 80 and about 2.4% v/v Span 80), with the remaining volume being a saline solution. In a preferred aspect, a vaccine composition is formulated comprising an antigen at a concentration of about 75% v/v and an adjuvant, preferably No. 1 Adjuvant, at a concentration of about 25% v/v of the vaccine composition. All concentrations provided herein in percentage are indicated in volume per volume unless the context indicates otherwise.

Surfactants Useful in the Adjuvant of the Invention

Surfactants useful for the adjuvant of the invention are amphiphilic and acceptable for veterinary or medical use. Whether or not a particular surfactant is acceptable for medical or veterinary use can be determined by those of ordinary skill in the art. A surfactant is amphiphilic if a part of the surfactant molecule is hydrophobic and a part is hydrophilic. See U.S. Pat. Nos. 5,690,942; 5,376,369; 4,933,179 and 4,606,918, which describe surfactants than 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. Tween and Span surfactants include, but are not limited to, monolaureate (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 is amphiphilic and has a hydrophilic-lipophilic balance ("HLB") value that is preferably at least about half the sum of the HLB values of all other components of the adjuvant. More preferably, the surfactant has an HLB value that is from about half to about twice the sum of the HLB values of all other components of the adjuvant. More preferably, the surfactant has an HLB value that is about the same as the HLB value of all other components of the adjuvant. HLB values are readily available for surfactants, lecithins, oils and carrier solutions or, if necessary, can be determined through routine experimentation. For example, see U.S. Pat. Nos. 4,504,275 and 4,261,925 and references provided therein.

Amphiphilic surfactants useful in the adjuvant of the invention have HLB values from about 2 to about 20, preferably from about 3 to about 17. Methods for determining the HLB value of particular surfactants are known in the art. See for example U.S. Pat. Nos. 5,603,951; 4,933,179 and 4,606,918, which describe surfactants having particular HLB values.

The concentration of a surfactant in a vaccine composition formulated with the adjuvant of the invention is 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%, and most preferably about 2% v/v. When more than one surfactant is used, the sum of the concentrations of all surfactants used in a vaccine composition formulated with the adjuvant of the invention is also from about 1.5% to 3.5%, more preferably from 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 a surfactant in the adjuvant of the invention also depends on the concentration at which the adjuvant is used in a vaccine composition. For example, a vaccine composition may be formulated with the adjuvant of the invention so that about 25% of the volume of the vaccine composition is the adjuvant ("25% adjuvant") and the remaining about 75% is made up of other components, for example the antigen composition. In one aspect, the concentration of the surfactant in a 25% adjuvant is from about 6% to 14% v/v. More preferably, the surfactant concentration in a 25% adjuvant is from about 6% to about 12%, more preferably from about 6% to about 10%, and most preferably about 8% v/v.

The concentration of the surfactant in the adjuvant of the invention is dependent on different factors. For example, the higher the concentration of oil in the adjuvant the more surfactant is required to emulsify a vaccine composition formulated with the adjuvant of the invention. Another factor that is useful to determine the concentration of a surfactant is the concentration of a lecithin. The higher the concentration of a lecithin in the adjuvant, the less surfactant may be required for emulsification.

When the adjuvant of the invention is used in a vaccine composition at a concentration of less than 25% v/v, the concentration of the adjuvant components in the adjuvant has to be increased accordingly. The aqueous carrier is an exception as the carrier always comprises the volume that remains unoccupied by all other components; thus if the concentration of all components except the carrier increases, the concentration of the carrier in the adjuvant will decrease and vice versa. For example, when the adjuvant is used at a concentration of about 12.5% v/v in a vaccine composition, the concentration of the components in the adjuvant is about twice the concentration of the components in a 25% adjuvant. Similarly, when the adjuvant of the invention is used in a vaccine composition at a concentration that is above 25% v/v, the concentration of the components in the adjuvant has to be decreased accordingly, for example when the adjuvant is used at 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 would include one surfactant that would be more concentrated in an aqueous phase than in an oil phase of the adjuvant ("hydrophilic surfactant") and one 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 from about 9 to about 20 and a preferred lipophilic surfactant has an HLB value from about 2 to about 9. See U.S. Pat. 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 is from about 1.5% to 3.5%, more preferably from 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 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 be from about 1.2x to about 5x, more preferably from about 1.5x to about 4x, more preferably from about 1.8x to about 3x, more preferably from about 2x to about 2.5x and more preferably about 2.3x as high as the concentration of Span 80, preferably when used in an adjuvant with a lecithin and an oil concentration as in No. 1 Adjuvant.

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 oil phase. In one embodiment, the adjuvant of the invention that consists of an aqueous phase at 80% v/v and of an oil phase at 20% v/v, may contain a hydrophilic surfactant at a concentration of up to about 4 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 an 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. Lecithin refers to a mixture of phosphatides. When provided as a crude extract, a lecithin may also contain triglycerides. Lecithins may be of plant or animal origin. In addition, lecithins may be synthetically derived. Examples of lecithins are described in U.S. Pat. Nos. 5,690,942; 5,597,602 and 5,084,269. In a preferred embodiment, the contents of triglycerides in a lecithin used in the adjuvant of the invention is lowered compared to its natural source, i.e., the lecithin is de-oiled. A number of ways are known in the art to de-oil a lecithin, for example as described in U.S. Pat. No. 5,597,602.

The concentration of a lecithin in a vaccine composition formulated with the adjuvant of the invention is 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 lecithin concentration in a 25% adjuvant is 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. Pat. Nos. 5,814,321; 5,084,269. In a preferred aspect, the adjuvant of the invention contains a mineral oil, for example DRAKEOL.TM.. In another aspect, a mixture of oils is used. The oil may be provided for preparation of the adjuvant of the invention as pure oil or as a mixture that contains the oil and another component, for example the lecithin.

The concentration of an oil in a vaccine composition formulated with the adjuvant of the invention is from about 1% to about 23% v/v, more preferably from about 1.5% to about 20% v/v, more preferably from about 2.5% to about 15%, more preferably from about 3.5% to about 10%, more preferably from about 3.5% to about 7.5%, more preferably from about 4% to about 6% v/v, and most preferably about 4.5%.

The concentration of an oil in a 25% adjuvant is at least about 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 is from about 4% to about 92% v/v, more preferably from about 6% to about 80% v/v, more preferably from about 10% to 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 most preferably about 18%. The concentration of an oil in the adjuvant of the invention with a higher or lower concentration is determined as exemplified above.

In another embodiment, an aqueous carrier is used in the adjuvant of the invention, for example saline (e.g., phosphate-buffered saline), tris-HCl, citrate-phosphate buffer, Hepes buffers, other pharmaceutically acceptable buffers known in the art or water. The pH of the carrier preferably is physiologically acceptable, for example between 6 and 8, most preferably around 7. The aqueous carrier used in the adjuvant of the invention preferably takes up the volume that is not needed for any of the other components.

The adjuvant of the invention is preferably provided at a concentration that is from about 2x to about 10x the concentration after formulation of the adjuvant in a vaccine composition, more preferably from about 2x to about 8x, more preferably from about 3x to about 6x and most preferably about 4x.

Uses of Adjuvants of the Invention

Adjuvants of the invention may be used to enhance the immune response to an antigen of a vaccine formulation. Adjuvants of the invention can be used with antigens derived from any bacteria or from any virus, provided the antigen does not get destroyed or denatured. Examples of antigens, and not by way of limitation, are Erysipelothrix rhusiopathiae antigens, Bordetella bronchiseptica antigens, antigens of toxigenic strains of Pasteurella multocida, antigens of Eschericia coli strains that cause neonatal diarrhea, Actinobacillus pleuropneumoniae antigens, Pasteurella haemolytica antigens, or any combination of the above. Adjuvants of the invention are also useful in vaccine compositions that contain an antigen described in U.S. Pat. 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 an Erysipelothrix rhusiopathiae ("E. rhusiopathiae") culture. In a preferred aspect, a culture of E. rhusiopathiae is inactivated by adding formalin (about 0.5% v/v final concentration) and, after incubation for 24 hours at 37^oC., the cells were removed, for example by centrifugation or filtration. The culture supernatant, in a preferred embodiment, is concentrated about 10 fold and aluminum hydroxide gel (preferably REHYDRAGEL.TM.) is added to the concentrated supernatant at a final concentration of about 30% v/v to stabilize the antigen. In another preferred embodiment, thimerosal (about 0.01% v/v final concentration) (Dimportex, Spain, imported through Flavine Inc., Klosters, N.J.) with EDTA (about 0.07% v/v final concentration) are added to the antigens as preservatives. In another preferred embodiment, a vaccine composition is formulated comprising the antigen and the adjuvant of the invention (e.g. No. 1 Adjuvant) with 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 No. 60/117,704, filed Jan. 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 from a B. bronchiseptica culture that has been inactivated by adding formalin thereto in log phase, preferably late log phase, followed by the addition of glutaraldehyde. In addition to killing the bacterial cells, the purpose of this novel and unique inactivation is to make nontoxic the endotoxin and exotoxin B. bronchiseptica, while leaving the antigens of B. bronchiseptica cells effective in eliciting the desired immune response. Formalin is added to a concentration in the B. bronchiseptica culture of from about 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 following the addition of formalin to the culture, more preferably from about 15 minutes to about 30 minutes and most preferably about 20 minutes. Glutaraldehyde is added to a concentration in the B. bronchiseptica culture of from about 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. Prior to adding the glutaraldehyde to the culture, it has a concentration of from about 10% v/v to about 50% v/v, more preferably from about 15% v/v to about 35% v/v and most preferably about 25% v/v. Following the addition of formalin and glutaraldehyde to the B. bronchiseptica culture, the resulting mix is incubated under stirring at from about 32^oC. to about 42^oC., more preferably at from about 35^oC. to about 39^oC. and most preferably at about 37^oC. The mix is incubated from about 12 hours to about 60 hours, more preferably from about 24 hours to about 48 hours. All other processing steps in preparing an antigen composition of the invention from B. bronchiseptica culture are described in Example 7, infra, and in U.S. Pat. Nos. 5,019,388 and 4,888,169.

Vaccine Compositions Comprising Adjuvants of the Invention and their Administration

The adjuvant of the invention may 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 may be determined by techniques well known to those skilled in the art.

A vaccine formulation may be administered to a subject per se or in the form of a pharmaceutical or therapeutic composition. Pharmaceutical compositions comprising the adjuvant of the invention and an antigen may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the antigens of the invention into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For purposes of this application, "physiologically acceptable carrier" encompasses carriers that are acceptable for human or animal use without relatively harmful side effects (relative to the condition being treated), as well as diluents, excipients or auxiliaries that are likewise acceptable.

Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intradermal, intramuscular or intraperitoneal injection.

For injection, the vaccine preparations may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, phosphate buffered saline, or any other physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the proteins may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Determination of an effective amount of the vaccine formulation for administration is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

An effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve an induction of an immune response using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to all animal species based on results described herein. Dosage amount and interval may be adjusted individually. For example, when used as a vaccine, the vaccine formulations of the invention may be administered in about 1 to 3 doses for a 1-36 week period. Preferably, 1 or 2 doses are administered, at intervals of about 3 weeks to about 4 months, and booster vaccinations may be given periodically thereafter. Alternative protocols may be appropriate for individual animals. A suitable dose is an amount of the vaccine formulation that, when administered as described above, is capable of raising an immune response in an immunized animal sufficient to protect the animal from an infection for at least 4 to 12 months. In general, the amount of the antigen present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 .mu.g. Suitable dose range will vary with the route of injection and the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

Claim 1 of 3 Claims

What is claimed is:

1. A vaccine composition comprising a Bordetella bronchiseptica culture that has been inactivated by adding formalin followed by adding glutaraldehyde to the culture, and an adjuvant, wherein the adjuvant includes 23% v/v of an oil, from 1.5% to 3.5% v/v of at least one amphiphilic surfactant and from 0.25% to 12.5% v/v of a lecithin.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]