Oil-based adjuvants

Abstract

Novel vaccine compositions and adjuvant formulations useful in vaccines are provided. [Solution] Various formulations containing combinations of immunostimulatory oligonucleotides, polycationic carriers, sterols, saponins, quaternary amines, TLR-3 agonists, glycolipids, and MPL-A or analogs thereof in oil emulsions, their use in the preparation of immunogenic compositions and vaccines, and their use in the treatment of animals are provided.

Description

[Technical field]

[0001]

[0001] The present invention relates generally to novel adjuvant formulations that enhance immune responses to antigens for use in immunogenic and vaccine compositions. The present invention also relates to methods of preparation and use of the adjuvants, immunogenic and vaccine compositions. [Background technology]

[0002] Bacterial, viral and parasitic infections are widespread in humans and animals. Diseases caused by these infectious agents often show resistance to antimicrobial pharmaceutical therapy, leaving no effective means of treatment. As a result, vaccinological approaches are increasingly being utilized to control infectious diseases. Whole infectious agents can be adapted for use in vaccine formulations after chemical inactivation or appropriate genetic engineering. Alternatively, protein subunits of the agents can be expressed in recombinant expression systems and purified for use in vaccine formulations. Vaccines can be made more effective by including suitable adjuvants in the composition.

[0003]

[0003] The term "adjuvant" generally refers to any material that enhances the humoral or cellular immune response to an antigen. Adjuvants are used to serve two purposes: to delay the release of antigens from the injection site and to enhance the stimulation of the immune system. Traditional vaccines are generally composed of crude preparations of inactivated or killed or denatured live pathogenic microorganisms. Impurities associated with these cultures of pathogenic microorganisms can act as adjuvants to enhance the immune response. However, the immunity elicited by vaccines that use homogenous preparations of pathogenic microorganisms or purified protein subunits as antigens is often insufficient. Therefore, the addition of certain foreign substances such as adjuvants is necessary. Furthermore, in some instances, synthetic and subunit vaccines can be expensive to produce. Also, in some instances, the pathogen cannot be grown on a commercial scale, making synthetic / subunit vaccines the only viable option. The addition of an adjuvant allows the use of smaller amounts of antigens that stimulate a similar immune response, thereby reducing the cost of producing the vaccine. Thus, the effectiveness of some injectable pharmaceuticals can be significantly increased when they are used together with an adjuvant.

[0004]

[0004] Many factors must be considered in the selection of an adjuvant. An adjuvant must cause antigen release and absorption at a relatively slow rate in an efficient manner with minimal toxicity, allergenicity, irritation, and other undesirable effects on the host. To be desirable, an adjuvant must be non-killing virally active, biodegradable, capable of consistently high levels of immune generation, capable of stimulating cross-protection, compatible with multiple antigens, effective in multiple species, non-toxic, and safe for the host (e.g., no injection site reaction). Other desirable properties of an adjuvant are that it can be microdosed, is dose-sparing, has good storage stability, can be adapted to drying, can be oil-free, can exist as either a solid or liquid, isotonic, easily manufactured, and inexpensive to manufacture. Finally, it is necessary and desirable that the adjuvant can be configured to induce either a humoral or cellular immune response, or both, depending on the requirements of the vaccination scenario. However, the number of adjuvants that can meet the above requirements is limited.

[0005] The choice of adjuvant depends on whether it is to increase the magnitude or function of the antibody response, to increase the cell-mediated immune response, to induce mucosal immunity, or to reduce the antigen dose. The adjuvant used depends on the requirements of the vaccine. Several adjuvants have been proposed, but no adjuvant has been shown to be ideally suited for all vaccines. The first adjuvant reported in the literature was Freund's complete adjuvant (FCA), which contains water-in-oil emulsions and extracts of Mycobacteria. Unfortunately, FCA is poorly tolerated and can cause uncontrolled inflammation. Since the discovery of FCA more than 80 years ago, efforts have been made to reduce the inappropriate side effects of adjuvants.

[0006]

[0006] Some other materials that have been used as adjuvants include metal oxides (e.g., aluminum hydroxide), alum, inorganic chelating agents of salts, gelatin, various paraffin-type oils, synthetic resins, alginates, mucoid and polysaccharide compounds, caseinates, blood-derived substances such as fibrin clots, etc. Although these materials are generally effective in stimulating the immune system, none have been found to be entirely sufficient due to adverse effects in the host (e.g., formation of sterile abscesses, organ damage, carcinogenic, or allergenic responses) or undesirable pharmaceutical properties (e.g., rapid or poorly controlled dispersion from the injection site, or swelling of the material). Summary of the Invention [Problem to be solved by the invention]

[0007]

[0007] The present invention provides novel vaccine compositions and adjuvant formulations useful in vaccines. [Means for solving the problem]

[0008]

[0008] In a first aspect, the present invention provides an adjuvant formulation comprising an oil phase and an aqueous phase, wherein the oil phase accounts for at least 50 v / v% of the formulation, and wherein the formulation comprises monophosphoryl lipid A (MPL-A) or an analogue thereof and at least one of an immunostimulatory oligonucleotide, with the proviso that a) if the immunostimulatory oligonucleotide is not present, the formulation comprises poly I:C and a glycolipid and optionally a quaternary amine; or a polycationic carrier, and b) if the monophosphoryl lipid A (MPL-A) or an analogue thereof is not present, the formulation comprises a source of aluminium and optionally a polycationic carrier.

[0009]

[0009] In different embodiments, the oil phase may comprise an oil and, optionally, a fat-soluble emulsifier.

[0010]

[0010] In some embodiments, both the monophosphoryl lipid A (MPL-A) or analog thereof are present in the adjuvant formulation. In these embodiments, the formulation further comprises a sterol (e.g., cholesterol), poly I:C, or a combination thereof.

[0011]

[0011] In a particular set of embodiments, the adjuvant formulation comprises, in addition to the oil and the optional emulsifier(s), a combination of monophosphoryl lipid A (MPL-A) or an analog thereof, a sterol and an immunostimulatory oligonucleotide ("TCMO"). The adjuvant formulation may also optionally include poly I:C ("TCMYO") and / or a saponin ("QTCMO" or "QTCMYO", respectively).

[0012]

[0012] In yet another embodiment, the adjuvant formulation also includes, in addition to the oil and the optional emulsifier(s), a combination of a quaternary amine, a glycolipid, MPL-A or an analog thereof, and poly I:C ("ODYRM").

[0013] In a further set of embodiments, the adjuvant formulation also comprises the oil and and in addition to the optional emulsifier(s), a combination of a saponin, a sterol, a quaternary amine, and a polycationic carrier, with the proviso that the polycationic carrier is dextran DEAE, and the antigen is E. coli J-5 bacterin ("QC DXO").

[0014]

[0014] In a further embodiment, the adjuvant may include, in addition to the oil and the optional emulsifier(s), an immunostimulatory oligonucleotide, an aluminum source, and optionally a polycationic carrier ("TOA" and "TXO-A", respectively).

[0015] In a second aspect, an adjuvant formulation according to any of the above cited embodiments may include an antigen component to form a vaccine composition, provided that when the adjuvant formulation consists (or consists essentially) of DEAE dextran, Quil A, cholesterol, and DDA, or when the adjuvant formulation consists (or consists essentially) of DEAE dextran and an immunostimulatory oligonucleotide, the antigen is not E. coli J-5 protein. In certain embodiments, the vaccine of this aspect includes antigen(s) derived from a pathogen affecting bovine, ovine, equine, or porcine animals. In other embodiments, the vaccine of this aspect includes antigen(s) derived from a pathogen affecting poultry or felines.

[0016] In further aspects of the invention, different combinations of said antigenic compounds and said adjuvant formulations are provided.

[0017] More specifically, in a third aspect, the present invention relates to a method for the preparation of a vaccine comprising administering to a patient a vaccine comprising administering to said patient an Eimeria maxima and / or Clostridium perfringens antigen and and an adjuvant formulation. In a different embodiment of this third aspect, the adjuvant formulation may comprise an oil phase present in an amount of at least 50% v / v of the composition, a polycationic carrier, and optionally an immunostimulatory oligonucleotide. In another embodiment of this aspect of the invention, the invention provides a vaccine composition comprising an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, a sterol, and an adjuvant component comprising monophosphoryl lipid A (MPL-A) or an analogue thereof.

[0018]

[0018] In a fourth aspect, the invention provides a vaccine composition comprising a Neospora antigen and an adjuvant formulation. In a different embodiment of the invention according to this aspect, the adjuvant formulation comprises an oil phase present in an amount of at least 50% v / v of the composition, and monophosphoryl lipid A (MPL-A) or an analogue thereof. In another embodiment, the adjuvant formulation comprises an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, and a polycationic carrier.

[0019] In a fifth aspect, the present invention provides a vaccine composition comprising a Chlamydophila abortis antigen and an adjuvant formulation, the adjuvant formulation being The vaccine composition comprises an oil phase present in an amount of at least 50% v / v of the composition, a sterol, an immunostimulatory oligonucleotide, monophosphoryl lipid A (MPL-A) or an analog thereof, and poly I:C.

[0020] In a sixth aspect, the present invention provides a vaccine composition comprising a Streptococcus uberis (S. uberis) antigen and an adjuvant formulation. In accordance with this sixth aspect of the invention, there is provided a vaccine composition, wherein the adjuvant formulation comprises an oil phase present in an amount of at least 50% v / v of the composition, and a polycationic carrier. In various different embodiments, the adjuvant formulation also includes an immunostimulatory oligonucleotide.Alternatively, or in addition, the adjuvant formulation may include a saponin, a sterol, and a quaternary amine.

[0021]

[0021] In a seventh aspect, the present invention provides a vaccine composition comprising myostatin as an antigenic component and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, and either a polycationic carrier or MPL-A or an analogue thereof. In one set of embodiments according to this aspect of the invention, the adjuvant formulation comprises MPL-A or an analogue thereof. In some embodiments of this set, the adjuvant formulation comprises less than 0.5 μg sterol per 50 μl of the vaccine composition, and preferably is cholesterol-free. The choice of myostatin depends on the species of the subject. In one selected embodiment, the selected animal species is chicken and the source of myostatin is chicken myostatin.

[0022]

[0022] In an eighth aspect, the present invention provides a vaccine composition comprising an A. pyogenes (formerly known as Arcanobacterium pyogenes, Actinomyces pyogenes or Corynebacterium pyogenes, now known as Trueperella pyogenes) antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, and a polycationic carrier.

[0023]

[0023] In a ninth aspect, the present invention provides a vaccine composition comprising an E. coli antigen, a BRV antigen or a BCV antigen, and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, and at least one of a polycationic carrier and an aluminum source.

[0024] In a tenth aspect, the present invention provides a vaccine composition comprising a Rhipicephalus microplus antigen and an adjuvant, the adjuvant comprising: a) an aqueous adjuvant comprising an immunostimulatory oligonucleotide, a saponin, a sterol, a quaternary amine, a polyacrylic polymer, and a glycolipid; and b) an oil-based adjuvant comprising an oil phase present in an amount of at least 50% v / v of the vaccine composition, the oil phase comprising an immunostimulatory oligonucleotide and a polycationic carrier.

[0025] In an eleventh aspect, the present invention provides a vaccine composition comprising a foot and mouth disease virus (FMDV) antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the vaccine composition, an immunostimulatory oligonucleotide, and a polycationic carrier. In different embodiments, the foot and mouth disease virus antigen can be either a wild-type FMDV, a genetically modified and / or attenuated FMDV strain, or a virus-like particle (VLP) of recombinantly expressed FMDV structural proteins, such as serotypes A, C, O, Asia 1, SAT1, SAT2, or SAT3.

[0026]

[0026] In a twelfth aspect, the present invention provides a method for producing a diagnostic or therapeutic antibody, comprising immunizing a source animal with an adjuvant formulation according to any of the embodiments according to the first aspect of the invention and an antigen, then extracting a source of the antibody from the source animal and, if necessary, purifying the antibody.

[0027] In certain embodiments, the source animal is a rat, a mouse, a guinea pig, a hamster, a bovine animal, a goat, a rabbit, a horse, a porcine animal, or a sheep. In some other embodiments, the source animal is a cat or a dog.

[0028] In some embodiments particularly suitable for polyclonal antibodies, the source of the antibody is serum or milk. In embodiments suitable for monoclonal antibodies, a suitable source of the antibody is spleen cells.

[0029]

[0029] In certain embodiments, the adjuvant formulation comprises an immunostimulatory oligonucleotide and a polycationic carrier. The adjuvant may optionally contain an aluminum source, including an aluminum source, and the aluminum source may be aluminum hydroxide gel. In certain embodiments, the immunostimulatory oligonucleotide is CpG and the polycationic carrier is DEAE dextran.

[0030] In certain embodiments, the antigen is FeLV gp70, bovine parainfluenza-3 BPI-3 (HN protein), Histophilus somni p31, Bordetella FHA, Parapox, BVDV1 gp53 , BVDV2 gp53, Clostridia toxins, Canine circovirus, Brachyspira hyodysenteriae (porcine species) antigens; inactivated and Pepsi The recombinant protein may be selected from digested, inactivated whole cells.

[0031]

[0031] The present invention also provides methods of using the vaccines according to the third to twelfth aspects of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032]

[0032] "About" or "approximately" when used in connection with a number of measurable variables refers to the indicated value of the variable and all values ​​of the variable that fall within experimental error of the indicated value (e.g., within a 95% confidence interval of the mean) or within 10% of the indicated value, whichever is greater, except when about is used in reference to weekly time intervals, where "about 3 weeks" is 17-25 days and about 2 to about 4 weeks is 10-40 days.

[0033]

[0033] "Adjuvant" means any substance that enhances the humoral or cellular immune response to an antigen. Adjuvants are generally used to serve two purposes: to control the release of antigens from the injection site and to stimulate the immune system.

[0034]

[0034] "Adjuvant formulation" refers to a formulation that has immunostimulatory properties.

[0035]

[0035] "Alkyl" refers to both straight-chain and branched saturated hydrocarbon moieties.

[0036]

[0036] "Amine" refers to a compound containing nitrogen. Amines are a group of compounds derived from ammonia by replacing the hydrogen atom with a hydrocarbon group. "Quaternary amine" refers to a compound of ammonium base having four hydrocarbon groups.

[0037]

[0037] "Antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen as a result of an immune response to that antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains having "constant" and "variable" regions, and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of their constant regions.

[0038]

[0038] "Antigen" or "immunogen" refers to any substance that is recognized by an animal's immune system and produces an immune response. The term includes killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term "antigen" also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, individually or in any combination. The term "antigen" also includes antibodies, such as anti-idiotypic antibodies or fragments thereof, as well as synthetic peptide mimotopes that can mimic antigens or antigenic determinants (epitopes).

[0039]

[0039] "Bacterin" means a suspension of one or more killed bacteria that can be used as a component of a vaccine or immunogenic composition.

[0040]

[0040] "Buffer" means a chemical system that prevents changes in the concentration of another chemical, for example, a proton donating and accepting system acts as a buffer to prevent significant changes in hydrogen ion concentration (pH). A further example of a buffer is a solution containing a mixture of a weak acid and its salt (conjugate base) or a weak base and its salt (cojugate acid).

[0041]

[0041] A "cellular immune response" or "cell-mediated immune response" is an immune response mediated by T lymphocytes or other white blood cells, or both, and includes cytokines, chemokines and similar molecules produced by activated T cells, white blood cells, or both; or the response of T lymphocytes or other immune cells that kill infected cells.

[0042]

[0042] "Companion animals" refers to dogs, cats and horses.

[0043]

[0043] "Consisting essentially of" as applied to an adjuvant formulation refers to a formulation that does not contain an additional unlisted immunostimulatory or immunomodulatory agent in an amount that exerts a measurable immunostimulatory or immunomodulatory effect.

[0044] "Delayed-type hypersensitivity (DTH)" refers to an inflammatory reaction that develops 24 to 72 hours after exposure to an antigen that the immune system recognizes as foreign. This type of immune response involves primarily T cells rather than antibodies (produced by B cells).

[0045]

[0045] "Dose" refers to a vaccine or immunogenic composition given to a subject. "First dose" or "prime vaccine" refers to the dose of such a composition given on day 0. "Second dose" or "third dose" or "annual dose" refers to the amount of such a composition given subsequent to the first dose, which may or may not be the same vaccine or immunogenic composition as the first dose.

[0046]

[0046] The term "emulsifier" is used broadly in this disclosure and includes any material generally accepted as an emulsifier, such as those manufactured by TWEEN® or SPAN®. There are different products in the range (fatty acid esters of polyethoxylated sorbitol, and fatty acid substituted sorbitan surfactants, respectively) and solubility enhancers with different solubilities, such as PEG-40 castor oil or other PEGylated hydrogenated oils.

[0047]

[0047] "Humoral immune response" refers to an immune response mediated by antibodies.

[0048]

[0048] An "immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or both a humoral and a cellular immune response to an antigen. An immune response is generally defined as a response to an antigen, as defined by the art. The antibody titer can be determined using standard immunoassays and neutralization assays known in the art.

[0049] An "immunologically protective amount" or "immunologically effective amount" or "amount effective to generate an immune response" of an antigen is an amount effective to induce an immunogenic response in a recipient. The immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of a disease, including adverse health effects or complications caused by infection with a disease pathogen. Either humoral or cellular immunity, or both, may be induced. The immunogenic response of an animal to an immunogenic composition may be assessed indirectly, for example, through measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms after challenge with a wild strain, while protective immunity conferred by a vaccine may be assessed by measuring reduction in clinical signs, such as mortality, morbidity, body temperature, general physical condition, and general health and behavior of the subject. The immune response may include, but is not limited to, induction of cellular and / or humoral immunity.

[0050]

[0050] "Immunogenic" means eliciting an immune or antigenic response. Thus, an immunogenic composition is any composition that induces an immune response.

[0051]

[0051] "Immunostimulatory molecule" refers to a molecule that stimulates a non-antigen-specific immune response.

[0052]

[0052] "Lipid" refers to any of a group of organic compounds, including fats, oils, waxes, sterols, and triglycerides, that are insoluble in water, soluble in nonpolar organic solvents, oily in texture, and which, together with carbohydrates and proteins, constitute the major structural materials of living cells.

[0053]

[0053] "Pharmaceutically acceptable" refers to materials that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit-risk ratio, and are effective for their intended use.

[0054] The term "poly I:C" refers, for example, to a polypeptide having a stable backbone and preferably TLR-3. Polyinosinic acid: Refers to naturally occurring polymers of polycytidylic acid and their synthetic forms, which have agonistic activity.

[0055] "Reaction-eliciting" refers to an adverse reaction induced in a subject in response to administration of an adjuvant, immunogenic or vaccine composition. It may occur at the site of administration and is usually evaluated in terms of the development of a number of symptoms. These symptoms may include inflammation, redness, and abscesses. It is also evaluated in terms of incidence, duration, and severity. A "low" reaction is of short duration, including, for example, swelling that can only be detected by palpitations and not by eye. A more severe reaction is of short duration, for example, visible to the eye or of long duration.

[0056]

[0056] "Room temperature" means a temperature between 18 and 25°C.

[0057]

[0057] "Saponin" refers to a group of surface active glycosides of plant origin composed of a hydrophilic region (usually multiple sugar chains) associated with a hydrophobic region of either a steroidal or triterpenoid structure.

[0058]

[0058] "Steroids" are compounds that are readily soluble in organic solvents and slightly soluble in water. Refers to any of a group of organic compounds that belong to the biochemical classification of lipids. Steroids contain a four-fused ring system of three fused cyclohexane (six carbon) rings plus a fourth cyclopentane (five carbon) ring.

[0059] "Sterol" refers to compounds in animals that are biologically produced from terpenoid precursors. They contain a steroid ring structure with a hydroxyl (OH) group, usually attached to the 3-carbon. The hydrocarbon chain of the fatty acid substituent usually varies in length from 16 to 20 carbon atoms and can be saturated or unsaturated. Sterols generally contain one or more double bonds in the ring structure and a variety of substituents attached to the ring. Sterols and their fatty acid esters are essentially insoluble in water.

[0060]

[0060] "Subject" refers to any animal to which administration of the adjuvant composition is desired. It includes mammals and non-mammals such as primates, livestock, companion animals, laboratory animals, captive wild animals, birds (including eggs), reptiles, and fish. Thus, this term includes, but is not limited to, monkeys, humans, pigs, cows, sheep, goats, horses, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, chickens, turkeys, ducks, other poultry, frogs, and lizards.

[0061]

[0061] "TCID 50 " refers to the "tissue culture infectious dose" and is defined as the virus dilution required for 50% infection of a given batch of inoculated cell cultures. Various methods have been used to determine the viral infection dose, including the Spearman-Karber method utilized throughout this specification. TCID 50 can be calculated. See BW Mahy & HO Kangro, Virology Methods Manual, p. 25-46 (1996).

[0062]

[0062] "Therapeutically effective amount" refers to an amount of an antigen or vaccine that induces an immune response in a subject receiving the antigen or vaccine that is sufficient to prevent or reduce the signs or symptoms of a disease, including adverse health effects or complications thereof, caused by infection with a pathogen such as a virus or bacteria. Humoral or cellular immunity, or both humoral and cellular immunity, can be induced. The immunogenic response of an animal to a vaccine can be evaluated indirectly, for example, through measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms after challenge with a wild strain. Protective immunity conferred by a vaccine can be evaluated by measuring the reduction of clinical signs, such as, for example, mortality, morbidity, body temperature, general physical condition, and general health and behavior of the subject. The amount of vaccine that is therapeutically effective may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, and can be determined by one of skill in the art.

[0063]

[0063] "Treating" refers to preventing the disorder, disease or condition to which such term applies, or preventing or reducing one or more symptoms of such disorder, disease or condition.

[0064] "Treatment" refers to the act of "treating" as defined above.

[0065]

[0065] "Triterpenoids" refers to a large and diverse class of naturally occurring organic molecules derived from six five-carbon isoprene (2-methyl-1,3-butadiene) units that can be constructed and modified in thousands of ways. Most are polycyclic structures that differ from one another in functional groups and basic carbon skeletons. These molecules can be found in all classes of living organisms.

[0066]

[0066] "Vaccine" refers to a composition comprising an antigen as defined herein. Administration of a vaccine to a subject generally results in an immune response against one or more specific diseases. The amount of vaccine that is therapeutically effective will vary depending on the particular antigen used or the condition of the subject. The amount of the ionic liquid may vary and can be determined by one of ordinary skill in the art.

[0067] Adjuvant formulations and methods of manufacture The present application discloses several adjuvant formulations suitable for the present invention. A common feature of these adjuvants is the presence of an oil and one or more emulsifiers, with the oil phase making up more than 50% of the vaccine composition comprising the adjuvant formulations disclosed herein.

[0068]

[0068] A number of oils and their combinations are suitable for use in the present invention. These oils include, but are not limited to, animal oils, vegetable oils, and non-metabolizable oils. Non-limiting examples of vegetable oils suitable for the present invention are corn oil, peanut oil, soybean oil, coconut oil, and olive oil. Non-limiting examples of animal oils are squalene. Suitable non-limiting examples of non-metabolizable oils include light mineral oils, linear or branched saturated oils, etc.

[0069] In one set of embodiments, the oil used in the adjuvant formulations of the invention is a light mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petroleum through distillation techniques. The term is synonymous with "liquid paraffin," "liquid petroleum," and "white mineral oil." The term is also intended to encompass "light mineral oil," i.e., oils similarly obtained by distillation of petroleum, but having a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, pages 788 and 1323). Mineral Oils were obtained from a variety of commercial sources, e.g., J.T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). A preferred mineral oil is available under the trade name DRAKEOL®. It is a light mineral oil sold commercially under the trade name .

[0070] Typically the oil phase is present in an amount of 50-95% by volume of the vaccine composition, preferably from greater than 50% to 85%, more preferably from greater than 50% to 60%, more preferably from greater than 50% to 52%. If an optional emulsifier (e.g., SPAN® 80, TWEEN® 80, etc.) is present, the oil phase may be present in the form of oil and and such emulsifier. The volume of the oil phase is calculated as the sum of the volume of oil and the volume of emulsifier(s). Thus, for example, if the volume of oil is 40% of the composition and the volume of emulsifier(s) is 12% of the composition, the oil phase will be present at 52% v / v of the composition. Similarly, if the oil is present at about 45% of the composition and the emulsifier(s) is present at about 6% of the composition, the oil phase will be present at about 51% v / v of the composition.

[0071]

[0071] It should also be understood that since the adjuvants of the present invention form only a part of the vaccine of the present invention, the oil phase is present in an amount of 50 to 95% by volume of each of the adjuvants of the present invention, preferably in an amount of 50 to 85%, more preferably in an amount of 50 to 60%, more preferably in an amount of 50 to 52% v / v.

[0072] In a subset of embodiments applicable to all adjuvants / vaccines of the present invention, the volume percentage of oil and lipophilic emulsifier together is at least 50% of the vaccine composition, such as 50-95% by volume, preferably 50-85% by volume, more preferably 50-60% by volume, more preferably 50-52% v / v. Thus, by way of example and not limitation, the oil may be present in an amount of 45% and the lipophilic emulsifier is present in an amount of more than 5% v / v. Thus, the volume percentage of oil and lipophilic emulsifier together is at least 50%.

[0073] In yet another subset applicable to all vaccines of the invention, the volume percentage of the oil is greater than 40% of the vaccine composition, for example 40-90% by volume, 40-85%. , 43~60%, and 44~50% v / v.

[0074]

[0074] Emulsifiers suitable for use in the emulsions of the present invention include natural biocompatible emulsifiers and non-natural synthetic surfactants. Biocompatible emulsifiers include phospholipid compounds or mixtures of phospholipids. A preferred phospholipid is phosphatidylcholine (lecithin), such as soybean or egg lecithin. Lecithin can be obtained as a mixture of phosphatides and triglycerides by washing crude vegetable oil with water and separating and drying the resulting hydrated gum. A purified product can be obtained by fractionating the mixture for acetone-insoluble phospholipids and glycolipids remaining after removing the triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from a variety of commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylethanolamine. The phospholipids can be isolated from natural sources or conventionally synthesized.

[0075]

[0075] In additional embodiments, the emulsifier used herein is free of lecithin or uses a non-immunologically effective amount of lecithin.

[0076]

[0076] Non-natural synthetic emulsifiers suitable for use in the adjuvant formulations of the invention include sorbitan-based nonionic surfactants, such as fatty acid substituted sorbitan surfactants (commercially available under the trade names SPAN® or ARLACEL®), poly Fatty acid esters of ethoxylated sorbitol (TWEEN®), castor oil, etc. Polyethylene glycol esters of fatty acids from natural sources (EMULFOR®), ethoxylated fatty acids (e.g., stearic acid available under the trade name SIMULSOL® M-53), polyethoxylated isooctylphenol / formaldehyde polymers (TYLOXAPOL®), polyoxyethylene fatty acid alcohol ethers (BRIJ®), ), polyoxyethylene nonylphenyl ethers (TRITON® N), polyoxyethylene isooctylphenyl ether (TRITON Preferred synthetic surfactants include those sold under the trade names SPAN® and and TWEEN®, such as TWEEN®-80 (polyoxyethylene (20) sorbitan monooleate) and SPAN®-80 (sorbitan monooleate). It is a surfactant available.

[0077]

[0077] Generally speaking, the emulsifying agent(s) may be present in the vaccine composition in an amount of from 0.01 to 40% by volume, preferably from 0.1 to 15% by volume, more preferably from 2 to 10%.

[0078]

[0078] Additional raw materials present in the adjuvant formulations of the invention include cationic carriers, immunostimulatory oligonucleotides, monophospholipid A and its analogs (MPL-A), polyinosinic:polycytidylic acid (poly I:C), saponins, quaternary amines, sterols, glycolipids, aluminum sources (e.g., REHYDRAGEL® or VAC 20® wet gels) and combinations thereof.

[0079]

[0079] Suitable cationic carriers include, but are not limited to, dextran, dextran DEAE (and its derivatives), PEG, guar gum, chitosan derivatives, polycellulose derivatives such as hydroxyethyl cellulose (HEC) polyethyleneimine, polyaminos such as polylysine, and the like.

[0080] Suitable immunostimulatory oligonucleotides include ODN (DNA-based), ORN (RNA-based) oligonucleotides, or chimeric ODN-Os. Included are RN structures, which may have modified backbones, including but not limited to phosphorothioate modifications, halogenations, alkylations (e.g., ethyl or methyl modifications), and phosphodiester modifications. In some embodiments, polyinosinic-cytidylic acid or derivatives thereof (poly I:C) may be used.

[0081] CpG oligonucleotides are a recently described class of pharmacotherapeutic agents characterized by the presence of unmethylated CG dinucleotides in specific base sequence contexts (CpG motifs) (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229-232, incorporated herein by reference). These CpG motifs are not found in eukaryotic DNA, where CG dinucleotides are suppressed and, if present, are usually methylated, but are present in bacterial DNA and confer immune stimulating properties.

[0082] In selected embodiments, the adjuvants of the invention utilize so-called P-class immunostimulatory oligonucleotides, more preferably modified P-class immunostimulatory oligonucleotides, more preferably E-modified P-class oligonucleotides. P-class immunostimulatory oligonucleotides are characterized by the presence of a palindrome and are generally CpG oligonucleotides of 6-20 nucleotides in length. P-class oligonucleotides have the ability to spontaneously self-assemble into concatamers either in vitro and / or in vivo. These oligonucleotides are strictly single-stranded, but the presence of a palindrome allows for the formation of concatamers, or possibly stem-loop structures. The total length of the P-class immunostimulatory oligonucleotides is 19-100 nucleotides, e.g., 19-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, 90-100 nucleotides.

[0083]

[0083] In one embodiment of the invention, the immunostimulatory oligonucleotide comprises a 5' TLR activation domain and at least two palindrome regions, one of which is a 5' palindrome region at least 6 nucleotides in length, linked directly or via a spacer to a 3' palindrome region at least 8 nucleotides in length.

[0084]

[0084] P class immunostimulatory oligonucleotides may be modified by techniques known in the art. For example, J modification refers to an iodo-modified nucleotide. E modification refers to an ethyl-modified nucleotide(s). Thus, an E-modified P class immunostimulatory oligonucleotide is a P class immunostimulatory oligonucleotide in which at least one nucleotide, preferably the 5' nucleotide, is ethylated. Additional modifications include 6-nitrobenzimidazole, O-methylation, modification with propynyl-dU, inosine modification, 2-bromovinyl linkage (preferably to uridine).

[0085]

[0085] P class immunostimulatory oligonucleotides may also contain modified internucleotide linkages, including, but not limited to, phosphodiester and phosphorothioate linkages. The oligonucleotides of the invention may be synthesized or obtained from commercial sources.

[0086]

[0086] P class oligonucleotides and modified P class oligonucleotides are further disclosed in published PCT Application No. WO2008 / 068638, filed Jun. 12, 2008. Suitable non-limiting examples of modified P class immunostimulatory oligonucleotides are shown below (in SEQ ID NOs 1-10, " * " refers to a phosphorothioate bond and "_" refers to a phosphodiester bond). In O11-14, all of the bonds are phosphodiester bonds. SEQ ID NO: 1 5' T * C_G * T * C_G * A*C_G * A * T * C_G * G * C * G * C_G * C * G * C * C * G3' SEQ ID NO: 25' T * C_G * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G* C * C * G 3' SEQ ID NO: 3 5' T * C * G * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G * T 3' SEQ ID NO: 4 5' JU * C_G * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G 3' SEQ ID NO: 5 5' JU * C_G * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C* G * C * C * G * T 3' SEQ ID NO: 6 5' JU * C * G * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G * T 3' SEQ ID NO: 7 5' EU * C_G * A * C * G * T * C * G * A * T * C * G * G * C * G * C * G * C * G * C * C * G 3' SEQ ID NO: 8 5' JU * C_G * T * C * G * A * C * G * A * T * C * G * G * C * G * G* C * C * G * C * C * G * T 3' SEQ ID NO: 9 5' JU * C * G * T * C * G * A * C * G * A * T * C * G * G * C * G * G * C * C * G * C * C * G * T 3' SEQ ID NO: 10 5' T * C_G * T * C_G * A * C_G * A * T * C_G * G * C * G * C_G * C * G * C * C * G 3' SEQ ID NO: 11 5'-GUGGUGUGUGUG-3' SEQ ID NO: 12 5'-UUAUUAUUAUUAUUAUU-3' SEQ ID NO: 13 5′-AAACGCUCAGCCAAAGCAG-3′ SEQ ID NO: 14 5'-dTdCdGdTdCdGdTdTdTrGrUrUrGrUrGrUdTdTdTdT-3'

[0087] The amount of P class immunostimulatory oligonucleotide used in the adjuvant composition will depend on the nature of the P class immunostimulatory oligonucleotide employed and the intended species.

[0087]

[0088] Suitable analogs of MPL-A include, but are not limited to, structurally modified, unmodified, or synthetic natural LPS of bacterial origin, glucopyranosyl lipid adjuvant (GLA), pertactin, various substitutions at the 3-O-position of reducing sugars, and synthetic forms of lipid A analogs with reduced endotoxicity.

[0088]

[0089] Sterols share a common chemical core, which is a steroid ring structure(s) with a hydroxyl (OH) group, usually attached to the 3-carbon. The hydrocarbon chains of the fatty acid substituents vary in length, usually from 16 to 20 carbon atoms, and may be saturated or unsaturated. Sterols have in common one or more double bonds in the ring structure, and a variety of substituents attached to the ring. Sterols and fatty acid esters are essentially insoluble in water. Thus, in view of their chemical similarity, sterols that share this chemical core are likely to have similar properties when used in the vaccine compositions of the present invention. Sterols are well known in the art and can be purchased commercially. Cholesterol, for example, is disclosed in the Merck Index, 12th Ed., p. 369. Suitable sterols Examples of such fatty acids include, but are not limited to, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

[0089]

[0090] Suitable saponins include triterpenoid saponins. These triterpenoids are a group of surface-active glycosides of plant origin, which have a common chemical core composed of a hydrophilic region (usually multiple sugar chains) associated with a hydrophobic region of either steroid or triterpenoid structure. Because of these similarities, saponins that share these chemical cores may have similar immunostimulatory properties. Triterpenoids suitable for use in the adjuvant composition can be obtained from many sources, either plant-derived or synthetic equivalents, including but not limited to Quillaja saponaria, tomatine, ginseng extract, mushrooms, and alkaloid glycosides that are structurally similar to steroid saponins.

[0090]

[0091] When a saponin is used, the adjuvant composition generally comprises an immunologically active saponin fraction derived from the bark of Quillaja saponaria. A or another purified or partially purified saponin preparation, which may be commercially obtained. Thus, the saponin extract may be QS-7, QS-17, QS-18, and QS Quil A can be used as a mixture such as Quil A-21, or as purified individual components. In one embodiment, Quil A is at least 85% pure. In another embodiment, Quil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.

[0091]

[0092] A quaternary amine compound is an ammonium-based compound having four hydrocarbon groups. In practice, the hydrocarbon groups are generally limited to alkyl or aryl groups. In one set of embodiments, the quaternary amine compound is composed of four alkyl chains, two of which are C10-C20 alkyl and the remaining two are C1-C4 alkyl. In one set of embodiments, the quaternary amine is dimethyldioctadecylammonium bromide or chloride, or a pharma- ceutically acceptable counterion (DDA).

[0092]

[0093] Suitable glycolipids are generally those that activate a Th2 response, including, but not limited to, those provided by Formula I and generally described in U.S. Patent Application Publication No. 20070196384 (Ramasamy et al.).

[0093] [ka]

[0094] In the structure of formula I, R 1 and R 2 are independently hydrogen or a saturated alkyl radical having up to 20 carbon atoms; X is -CH2-, -O-, or -NH-; R 2 is hydrogen or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R 3 , R 4 , and R 5 are independently hydrogen, -SO4 2- , -PO4 2- , -COC 1-10 R is alkyl; 6 is L-alanyl, L-α-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl, or their D-isomers.

[0095] In one set of embodiments, a suitable glycolipid is N-(2-deoxy-2-L-leucylamino-bD-glucopyranosyl)-N-octadecyldodecanoylamide or an acetate salt thereof.

[0096]

[0096] Aluminum is a known adjuvant or component of adjuvant formulations and is commercially available in forms such as Brentag alhydrogel REHYDRAGEL® or VAC 20® wet gel from Reheis, Inc. REHYDRAGEL® is a mineralogic It is a crystalline aluminum oxyhydroxide known in the art as boehmite. It is effective in vaccines where there is a need to bind negatively charged proteins. The amount of Al2O3 ranges from 2% to 10% depending on the grade, and its viscosity is 1000 to 1300 cP. It may generally be described as an adsorbent aluminum hydroxide. VAC 20® Wet Gel is a white or nearly white, translucent, viscous colloidal gel. In certain embodiments, the Al2O3 is about 2% w / v.

[0097]

[0097] In other embodiments, the aluminum source can also be prepared by the process of precipitation of aluminum hydroxide.

[0098]

[0098] In a particular set of embodiments, the adjuvant formulation also includes (or consists essentially of, or consists of) a combination of monophosphoryl lipid A (MPL-A) or an analog thereof, a sterol, and an immunostimulatory oligonucleotide, in addition to the oil and the optional one or more emulsifiers. Adjuvants that include these raw materials are referred to as "TCMO". TCMO adjuvant formulations may optionally include poly I:C ("TCMYO") and / or saponin. Thus, adjuvant formulations that include, or consist essentially of, or consist of a combination of monophosphoryl lipid A (MPL-A) or an analog thereof, a sterol, an immunostimulatory oligonucleotide, and a saponin are referred to as "QTCMO". In addition, the adjuvant formulation may also include poly I:C. Such adjuvants are referred to as "QTCMYO".

[0099] In one set of embodiments, the TCMO adjuvant comprises a light mineral oil in an amount of 40-50% v / v of the total volume of the vaccine composition. The emulsifiers comprise TWEEN-80 and SPAN-80 in a total amount of 0.1-40% v / v of the total volume of the vaccine composition, with the proviso that provided that the lubitan monooleate and oil together constitute about 50.5-52% v / v of the composition. The immunostimulatory oligonucleotide is an ODN, preferably a palindrome containing ODN, optionally with a modified backbone.

[0100]

[0100] In a specific embodiment, one dose of TCMO comprises about 1 μg to about 400 μg of an immunostimulatory oligonucleotide, about 1 μg to about 1000 μg of a sterol, and about 0.1 to 500 μg of MPL-A or an analog thereof.

[0101]

[0101] The other compounds per dose are selected based on the subject species.

[0102]

[0102] In some embodiments, suitable for example for cattle, sheep or adult pigs, one dose of TCMO comprises about 50 to 400 μg (e.g., 50 to 300 or 100 to 250 μg, or about 50 to about 100 μg for adult pigs and about 100 to about 250 μg for cattle) of immunostimulatory oligonucleotide, about 100 to about 1000 μg (e.g., 200 to 1000, 250 to 700 μg, or about 400 to 500 μg) of a sterol, such as cholesterol, and about 5 to about 500 μg (e.g., 5 to 100 μg, or 5 to 50 μg, or 10 to 25 μg) of MPL-A or an analogue thereof.

[0103]

[0103] In some embodiments suitable for companion animals or piglets, one dose of TCMO comprises about 5 to 100 μg (e.g., 10 to 80 or 20 to 50 μg) of immunostimulatory oligonucleotide, about 5 to about 100 μg (e.g., 10 to 80, or 20 to 50 μg) of a sterol, such as cholesterol, and about 0.5 to about 200 μg (e.g., 1 to 100 μg, or 5 to 50 μg, or 5 to 20 μg) of MPL-A or an analogue thereof.

[0104] In some embodiments suitable for poultry, a dose of TCMO adjuvant comprises about 0.1 to about 5 μg (e.g., 0.5 to 3 μg, or 0.9 to 1.1 μg) of an immunostimulatory oligonucleotide, about 0.5 to about 50 μg (e.g., 1 to 20 μg, or about 1 to 10 μg) of a sterol, such as cholesterol, and about 0.1 to about 10 μg (e.g., 0.5 to 5 μg, or 1 to 5 μg) of MPLA or an analog thereof. MPL-A is present in an amount of 0.1 μg / dose to 2,000 μg / dose.

[0105] In certain embodiments, the TCMO adjuvant is prepared as follows: a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in light mineral oil. The resulting oily solution is sterilized by filtration; b) dissolving the immunostimulatory oligonucleotide and polyoxyethylene (20) sorbitan monooleate in an aqueous phase, thereby forming an aqueous solution; c) The aqueous solution is added to the oily solution with continuous homogenization to form the adjuvant formulation TCMO.

[0106] In the TCMYO adjuvant, cholesterol, oil, optional emulsifier, MPL-A, and immunostimulatory oligonucleotides are present as in the TCMO adjuvant formulation for each species. Poly I:C may be present in an amount of about 1 μg to about 100 μg per dose.

[0107] More specifically, poly I:C may be present in an amount of 5-100 μg / dose (e.g., 5-50 μg or 10-30 μg) in certain embodiments suitable for cattle, live pigs, or sheep. In certain embodiments suitable for companion animals or piglets, a dose of TCMO contains about 1-50 μg (e.g., 5-50 μg, or 10-20 μg) of poly I:C. In certain embodiments suitable for poultry vaccines, a dose of TCMO contains about 1-10 μg (e.g., 1-5 μg, or 3-5 μg) of poly I:C.

[0108]

[0108] In certain embodiments, a TCMYO adjuvant is prepared similarly to a TCMO adjuvant, with poly I:C being added to the aqueous solution.

[0109] In one set of embodiments, in the QTCMO adjuvant, cholesterol, oil, optional emulsifier, MPL-A, and immunostimulatory oligonucleotides are present as in the TCMO adjuvant formulation for each species. The saponin is preferably Quil A or a purified fraction thereof and may be present in an amount of about 0.1 to about 1000 μg / dose.

[0110] More specifically, in certain embodiments suitable for poultry vaccines, the saponin may be present in an amount of 0.1-5 μg / 50 μl of vaccine composition (e.g., 0.5-30 μg / 50 μl of the composition, or more preferably 1-10 μg) per dose. In certain embodiments suitable for companion animal and piglet applications, the saponin, e.g., Quil A or a purified fraction thereof, is present in an amount of about 10 to about 100 μg / dose (e.g., 10-50 μg or 20-50 μg / dose). In certain embodiments suitable for cattle, adult pigs or sheep, the saponin, e.g., Quil A or a purified fraction thereof, is present in an amount of about 100 to about 1000 μg / dose (e.g., 200-800 μg or 250-500 μg / dose).

[0111]

[0111] In certain embodiments, the QTCMO adjuvant is prepared similarly to the TCMO adjuvant, with saponin being added to the aqueous solution.

[0112]

[0112] In one set of embodiments, the saponin is present in the QTCMYO adjuvant as in the QTCMO adjuvant for each species, and the remaining ingredients are present as in TCMYO.

[0113] In certain embodiments, the QTCMYO adjuvant is a TCMYO adjuvant. It is prepared similarly to an adjuvant, in that saponin is added to the aqueous solution.

[0114]

[0114] In another embodiment, the adjuvant formulation also comprises (or consists essentially of, or consists of) monophosphoryl lipid A (MPL-A) or an analog thereof in combination with a polycationic carrier, in addition to the oil and optional emulsifier(s). These adjuvants are referred to as "XOM".

[0115]

[0115] In one set of embodiments, in a XOM adjuvant for companion animals or piglets, the polycationic carrier is present in an amount of 1 to 50 mg / dose (e.g., 1 to 25 μg / dose, or 10 to 25 mg / dose) and the MPL-A or analogue thereof is present in an amount of about 1 to 50 μg / dose (e.g., 1 to 25 μg / dose, or 10 to 25 μg / dose).

[0116]

[0116] In specific embodiments suitable for cattle, sheep and adult pigs, the polycationic carrier is present in an amount of about 5 to about 500 mg per dose (e.g., 10 to 500 mg, or 10 to 300 mg, or 50 to 200 mg per dose) and the MPL-A or analogue thereof is present in an amount of about 1 to about 100 μg per dose (e.g., 5 to 100 μg, or 5 to 50 μg, or 10 to 30 μg).

[0117]

[0117] In certain embodiments suitable for companion animals or piglets, the polycationic carrier is present in an amount of about 1 to about 50 mg / dose (e.g., 1 to 25 mg / dose, or 10 to 25 mg / dose) and the MPL-A or analogue thereof is present in an amount of about 0.5 to about 200 μg / dose (e.g., 1 to 100 μg, or 5 to 50 μg, or 5 to 20 μg).

[0118]

[0118] In certain embodiments suitable for poultry vaccines, the polycationic carrier is present in an amount of 0.5 to 25 mg per dose (e.g., 1 to 20 mg, 1 to 10 mg, or 5 to 10 mg) and the MPL-A or analogue thereof is present in an amount of about 0.5 to 10 μg per dose (e.g., 1 to 10 μg, or 1 to 5 μg, or 2 to 5 μg).

[0119]

[0119] In certain embodiments, the XOM adjuvant is prepared as follows: a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in light mineral oil. The resulting oily solution is sterilized by filtration; b) dissolving dextran DEAE and polyoxyethylene (20) sorbitan monooleate in the aqueous phase, thereby forming an aqueous solution; c) The aqueous solution is added to the oily solution with continuous homogenization to form the adjuvant formulation XOM.

[0120] In additional alternative embodiments, the adjuvant formulation also comprises (or consists essentially of, or consists of) a combination of immunostimulatory oligonucleotides and a polycationic carrier in addition to the oil and optional emulsifier(s), with the proviso that when the polycationic carrier is dextran DEAE, the antigen is not E. coli J-5 bacterin. These adjuvants are referred to as "TXO". In certain embodiments, the TXO-adjuvanted vaccine comprises an antigen(s) comprising a pathogen affecting cattle, sheep, horses, or pigs. In other embodiments, the antigen is derived from said pathogen. In other embodiments, the TXO-adjuvanted vaccine comprises an antigen(s) comprising a pathogen affecting poultry or cats, or the antigen may be derived from such a pathogen. In one set of embodiments, the TXO adjuvant may also comprise an aluminum source, such as Al(OH)3 gel. Al The TXO adjuvant containing minium is referred to as "TXO-A."

[0121] In one set of embodiments, the immunostimulatory oligonucleotide, preferably ODN, preferably containing a palindromic sequence and optionally having a modified backbone, may be present in an amount of 0.5-400 μg / dose, and the polycationic carrier may be present in an amount of 0.5-400 mg / dose in the TXO adjuvant, with attention paid to the species of the subject.

[0122]

[0122] In certain embodiments suitable for example for cattle, sheep or adult pigs, a dose of TXO comprises about 50 to 400 μg (e.g., 50 to 300 or 100 to 250 μg, or about 50 to about 100 μg for live pigs, or about 100 to about 250 μg for cattle) of immunostimulatory oligonucleotide, and the polycationic carrier may be present in an amount of about 5 to about 500 mg / dose (e.g., 10 to 500 mg, or 10 to 300 mg, or 50 to 200 mg / dose).

[0123]

[0123] In certain embodiments suitable for companion animals or piglets, a dose of TXO contains about 5 to 100 μg (e.g., 10 to 80 μg, or 20 to 50 μg) of immunostimulatory oligonucleotide, and the polycationic carrier may be present in an amount of 1 to 50 mg / dose (e.g., 1 to 25 mg dose, or 10 to 25 mg / dose).

[0124]

[0124] In certain embodiments suitable for poultry, a dose of TXO is about 0.1 to 5 μg (e.g., 0.5 to 3 μg, or 0.9 to 1.1 μg) of immunostimulatory oligonucleotide and the polycationic carrier may be present in an amount of 0.5 to 25 mg per dose (e.g., 1 to 20 mg, or 1 to 10 mg, or 5 to 10 mg).

[0125] In certain embodiments, the TXO adjuvant is prepared as follows: a) Sorbitan monooleate is dissolved in light mineral oil. The resulting oily solution is sterilized by filtration; b) dissolving the immunostimulatory oligonucleotide, dextran DEAE and polyoxyethylene (20) sorbitan monooleate in the aqueous phase, thereby forming an aqueous solution; c) The aqueous solution is added to the oily solution with continuous homogenization to form the adjuvant formulation TXO.

[0126] In one set of embodiments, the immunostimulatory oligonucleotides in a TXO-A adjuvant are present as in a TXO adjuvant and the aluminum source is present in an amount of up to 40% v / v (e.g., 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%). In one set of embodiments, the aluminum source is present in an amount of 2 to 20% v / v, more preferably about 5 to about 17% v / v, of the vaccine composition.

[0127]

[0127] In certain embodiments, a TXO-A adjuvant is prepared similarly to a TXO adjuvant, and an aluminum source is added to the aqueous solution.

[0128] In a further embodiment, the adjuvant of the invention comprises an oil, an optional emulsifier(s), an immunostimulatory oligonucleotide, and an aluminum source. These compounds are present within the ranges disclosed for the TXO-A adjuvant, except that the polycationic carrier is not present in TOA. The TOA adjuvant is prepared similarly to the TXO adjuvant, except that the aqueous phase comprises an aluminum source rather than DEAE dextran.

[0129] In certain embodiments, the adjuvant formulation also comprises (or consists essentially of, or consists of) a combination of a polycationic carrier and an aluminum source in addition to the oil and optional emulsifier(s). This adjuvant is called AXO. These compounds can be present in amounts similar to those present in the adjuvant TXO-A for each species, and the adjuvant AXO can be prepared similarly to TXO-A, but without the addition of immunostimulatory oligonucleotide.

[0130] In certain other embodiments, the adjuvant formulation also comprises (or consists essentially of, or consists of) a combination of saponin and sterol in addition to the oil and optional emulsifier(s). This adjuvant is called QCO. The nature and amount of the raw materials of QCO are similar to the amounts of saponin, sterol, oil and emulsifier(s) in adjuvant QTCMO. QCO can be prepared by adding, with continuous homogenization, an aqueous solution containing saponin, sterol and preferably water-soluble emulsifier to an oil phase containing oil and preferably fat-soluble emulsifier.

[0131] In yet another embodiment, the adjuvant formulation also comprises (or consists essentially of, or consists of) in addition to the oil and optional emulsifier(s) a combination of a quaternary amine, a glycolipid, MPL-A or an analog thereof, and poly I:C. These adjuvants are referred to as "ODYRM".

[0132]

[0132] In ODYRM adjuvants, the oil is generally a mixture of phospholipids such as phosphatidylcholine. AMPHIGEN® is a suitable example of such an oil, present in amounts similar to those of the oils described above.

[0133]

[0133] In one set of embodiments, the quaternary amine in the ODYRM adjuvant, such as DDA, is present in an amount of about 1 μg to about 200 μg / dose, Poly I:C is present in an amount of about 0.5 μg to about 100 μg / dose, the glycolipid is present in an amount of about 0.5 μg to about 2000 μg / dose, and MPL-A or an analogue thereof is present in an amount of about 0.5 μg to about 100 μg / dose.

[0134]

[0134] More specifically, in certain embodiments suitable for cattle, adult pigs or sheep, the quaternary amine may be present in an amount of about 50 to about 200 μg / dose (e.g., 50 to 150 μg or about 100 μg), the poly I:C may be present in an amount of about 1 to about 100 μg / dose (e.g., 1 to 50 μg or about 5 to 50 μg), the glycolipid may be present in an amount of about 500 to about 2000 μg / dose (e.g., 500 to 100 μg or about 1000 μg), and the MPLA or analogue thereof may be present in an amount of about 5 to about 100 μg / dose (e.g., 5 to 50 μg or about 10 to 50 μg).

[0135]

[0135] In certain embodiments suitable for companion animals and piglets, the quaternary amine may be present in an amount of about 5 to about 500 μg / dose (e.g., 10 to 100 μg / dose or 20 to 50 μg / dose), the Poly I:C may be present in an amount of about 5 μg to about 25 μg / dose (e.g., 50 to 20 μg or about 10 μg), the glycolipid may be present in an amount of about 10 to about 100 μg / dose (e.g., 20 to 100 μg or 25 to 50 μg), and the MPL-A or analogue thereof may be present in an amount of about 5 to about 50 μg / dose (e.g., 5 to 20 or about 10 to 20 μg).

[0136]

[0136] In certain embodiments suitable for poultry vaccines, one dose comprises about 1 µg to about 10 µg of a quaternary amine compound (e.g., 5-10 µg or about 5 µg), about 0.5 to about 10 µg of poly I:C (e.g., 1-10 µg or 1-5 µg), about 0.5 to 10 µg of glycolipid (e.g., 1-10 µg or 5-10 µg or 1-5 µg), and about 0.5 to 10 µg of glycerol (e.g., 1-10 µg or 5-10 µg or 1-5 µg). 0.5 μg to about 5 μg of MPL-A or an analogue thereof (e.g., 0.5-5 μg or 1-5 μg).

[0137]

[0137] In certain embodiments, the ODYRM adjuvant is prepared as follows: a) Sorbitan monooleate, MPL-A, is dissolved in light mineral oil. The resulting oily solution is filter sterilized and dispersed in water containing some surfactant, ethanol, and acetic acid; b) dissolving polyoxyethylene (20) sorbitan monooleate, a quaternary amine, such as DDA, and poly I:C in the aqueous phase, thereby forming an aqueous solution; c) The aqueous solution is added to the oily solution with continuous homogenization to form the adjuvant formulation ODYRM.

[0138] In a further set of embodiments, the adjuvant formulation also comprises (or consists essentially of, or consists of) in addition to the oil and optional emulsifier(s) a combination of a saponin, a sterol, a quaternary amine, and a polycationic carrier, with the proviso that when the polycationic carrier is dextran DEAE, the antigen is not E. coli J-5 bacterin. These adjuvants are referred to as "QCDXO".

[0139]

[0139] In a particular embodiment of the QCDXO adjuvant, the saponin, e.g., Quil A, is present in an amount of about 0.1 μg to about 1000 μg / dose, the sterol, e.g., cholesterol, is present in an amount of about 1 μg to about 1000 μg / dose, the quaternary amine, e.g., DDA, is present in an amount of about 1 μg to about 200 μg / dose, and the polycationic carrier may be present in an amount of 0.5 to 400 mg / dose. The dosage is cautious depending on the species of the subject.

[0140]

[0140] In certain embodiments suitable for cattle, sheep and adult pigs, the saponin may be present in an amount of about 100 to about 1000 μg / dose (e.g., 200 to 800 μg, or 250 to 500 μg / dose), the sterol may be present in an amount of about 100 to about 1000 μg (e.g., 200 to 1000, 250 to 700 μg, or about 400 to 500 μg), the quaternary amine may be present in an amount of about 50 μg to about 200 μg / dose (e.g., 50 to 150 μg, or about 100 μg), and the polycationic carrier may be present in an amount of about 5 to about 500 mg / dose (e.g., 10 to 500 mg, or 10 to 300 mg, or 50 to 200 mg / dose).

[0141]

[0141] In certain embodiments suitable for companion animal and piglet applications, the saponin, such as Quil A or a purified fraction thereof, may be present in an amount of about 10 to about 100 μg / dose (e.g., 10 to 50 μg, or 20 to 50 μg / dose), the sterol may be present in an amount of about 5 to 100 μg (e.g., 10 to 80 or 20 to 50 μg), the quaternary amine may be present in an amount of about 5 to about 500 μg / dose (e.g., 10 to 100 μg / dose, or 20 to 50 μg / dose), and the polycationic carrier may be present in an amount of 1 to 50 mg / dose (e.g., 1 to 25 mg / dose, or 10 to 25 mg / dose).

[0142] In some embodiments suitable for poultry vaccines, the saponin may be present in an amount of 0.1-5 μg / 50 μl of vaccine composition (e.g., 0.5-30 μg / 50 μl of the composition, or more preferably 1-10 μg), the sterol may be present in an amount of about 0.5 to about 50 μg (e.g., 1-20 μg, or 1-10 μg), the quaternary amine may be present in an amount of about 5 to about 500 μg / dose (e.g., 10-100 μg / dose, or about 20-50 μg / dose), and the polycationic carrier may be present in an amount of 0.5-25 mg / dose (e.g., 1-20 mg, or 1-10 mg, or 5-10 mg). .

[0143]

[0143] In certain embodiments, the QCDXO adjuvant is prepared as follows: a) Sorbitan monooleate is dissolved in light mineral oil. The resulting oily solution is sterilized by filtration; b) dissolving polyoxyethylene (20) sorbitan monooleate, a quaternary amine, such as DDA, a polycationic carrier, a sterol and a saponin in the aqueous phase, thereby forming an aqueous solution; c) The aqueous solution is added to the oily solution with continuous homogenization to form the adjuvant formulation QCDXO.

[0144]

[0144] In some cases, especially in large-scale commercial applications, it may not be possible or feasible to concentrate the antigen and low-concentration antigen solutions must be used. Thus, in some embodiments, the vaccine composition of the present invention comprises the adjuvant formulations described above, in which the contents of the oil phase in these adjuvant formulations are diluted and the vaccine composition is a water-in-oil emulsion.

[0145]

[0145] In fact, it is possible to produce water-in-oil emulsions in which the oil phase is less than 50% v / v.

[0146]

[0146] Briefly, first, an adjuvant formulation of the present invention is prepared as described below. In said adjuvant formulation, the oil phase accounts for more than 50% v / v of the adjuvant formulation. The amounts of ingredients other than the oil and the emulsifier(s) can be increased based on the final target concentration and the desired dilution, respectively. For example, if it is aimed to prepare a vaccine composition in which the adjuvant formulation accounts for 80% v / v, the amounts of ingredients other than the oil are increased by a factor of 1.25 (1 / 0.8). The amount of emulsifier, if present (e.g., TWEEN® 80 and / or SPAN® 100) is increased by 1.25 (1 / 0.8). ) 80), preferably, but not necessarily, increased, the volume ratio of the oil to the emulsifier(s) is kept the same in the adjuvant formulation as in the final vaccine.

[0147]

[0147] The antigen solution is then added to the adjuvant formulation.

[0148]

[0148] The integrity of a water-in-oil emulsion can be maintained as long as the dispersed spherical water droplets are not present in a form more concentrated than the maximum packing ratio for random packing of monodisperse droplets, i.e., 0.64. Tadros, Emulsion Formation, Stability and Rheology, 1 st ed. 2013, Wiley-VCH GmbH & Co KGaA. The total volume fraction occupied by aqueous droplets is 0.64 , i.e. unless it exceeds 64% v / v. Conversely, this suggests that the oil phase should not be dripped at less than 36% v / v.

[0149]

[0149] Thus, in different embodiments of this aspect of the invention, there is provided a vaccine formulation comprising an antigenic compound and a diluted adjuvant formulation according to the previous embodiments, wherein the oil phase comprises more than 36% v / v of the vaccine composition, and the vaccine composition is a water-in-oil emulsion. Non-limiting adjuvant formulations suitable for this aspect of the invention include TCMO, TCMYO, QTCMO, QTCMYO, XOM, TXO, TXO-A, TAO, AXO, QCO, ODYRM, QCDXO. The volume of the oil phase is, in different embodiments, 37% v / v, 38% v / v, 39% v / v, 40% v / v, 41% v / v, 42% v / v, 43% v / v, 44% v / v, 45% v / v, 46% v / v, 47% v / v, 48% v / v, 49% v / v, or 50% v / v of the vaccine composition.

[0150]

[0150] The concentration of the oil phase must be high enough to create a depo effect and protect the antigen and immunomodulatory substance(s) from rapid degradation by the host's immune system, preferably greater than 20% v / v of the vaccine composition.

[0151]

[0151] Thus, in another embodiment, in the vaccine composition of the invention, the amount of oil phase of the adjuvant formulation is diluted so that the vaccine formulation is an oil-in-water emulsion or a water-in-oil-in-water emulsion, with the oil phase making up 20% v / v or more of the vaccine composition. The amounts of ingredients other than the oil and the emulsifier are increased, respectively, based on the final target concentration and the desired dilution. For example, to prepare a vaccine composition in which the adjuvant formulation makes up 33.3% v / v, the amounts of ingredients other than the oil and the emulsifier(s) are increased by a factor of 3 (1 / 0.333). The amount of emulsifier, if present (e.g., TWEEN® 80 and / or SPAN® 80), does not need to be increased, but is preferably increased. Preferably the volume ratio of the oil to the emulsifier(s) is kept the same in the adjuvant formulation and in the final vaccine composition.

[0152]

[0152] In different embodiments, the vaccine composition is an oil-in-water emulsion or a water-in-oil-in-water emulsion, wherein the oil phase comprises 21% v / v, 22% v / v, 23% v / v, 24% v / v, 25% v / v, 26% v / v, 27% v / v, 28% v / v, 29% v / v, 30% v / v, 31% v / v, 32% v / v, 33% v / v, 34% v / v, 35% v / v, 36% v / v, 37% v / v, 38% v / v, 39% v / v, 40% v / v, 41% v / v, 42% v / v, 43% v / v, 44% v / v, 45% v / v, 46% v / v, 47% v / v, 48% v / v, 49% v / v, or 50% v / v of the vaccine composition.

[0153]

[0153] Adjuvant formulations suitable for this aspect of the invention include TCMO, TCMYO, QTCMO, QTCMYO, XOM, TXO, TXO-A, TAO, AXO, QCO, ODYRM, QCDXO, provided that the oil phase of the adjuvant formulation may represent less than 50% v / v and more than 20% v / v of the final vaccine composition.

[0154] Antigens and diseases The composition may contain one or more antigens. The antigens may be any of a wide variety of substances, individually or in any combination, capable of generating a desired immune response in a subject, including but not limited to one or more viruses (inactivated, attenuated, and modified live viruses), bacteria, parasites, nucleotides (including but not limited to nucleic acid-based antigens, e.g., DNA vaccines), polynucleotides, peptides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, carbohydrates, fatty acids, teichoic acids, peptidoglycans, lipids, or glycolipids.

[0155]

[0155] Antigens for use with the adjuvants of the present invention also include immunogenic fragments of nucleotides, polynucleotides, peptides, polypeptides that may be isolated from the organisms referenced herein.

[0156]

[0156] Live, modified, and attenuated virus strains that do not induce disease in subjects have been isolated in a non-virulent form or have been attenuated using methods well known in the art, such as subculture in appropriate cell lines or exposure to ultraviolet light or chemical mutagens. Inactivated or killed virus strains have been inactivated by methods known to those skilled in the art, including treatment with formalin, beta-propriolactone (BPL), binary ethyleneimine (BEI), radiation sterilization, heating, or other such methods.

[0157]

[0157] Two or more antigens can be combined to produce a multivalent composition that can protect subjects against a wide variety of diseases caused by pathogens. Currently, commercial manufacturers and end users of vaccines prefer multivalent vaccine products. Conventional adjuvants are often limited to the variety of antigens that can be effectively used (either monovalent or polyvalent), but the adjuvants described herein can be effectively used with a wide range of antigens, both monovalent and polyvalent. Thus, the antigens described herein can be mixed into one composition that includes the adjuvants described herein.

[0158]

[0158] Some examples of bacteria used as antigens together with the adjuvant composition include Aceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacillus pleuropneumoniae, Aeromonas hydrophila, Alicyclobacillus acidocaldarius, Arhaeglobus fulgidus, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bordetella bronchiseptica, Burkholderia cepacia, Burkholderia glumae, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter hyointestinalis, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila genus, Chromobacterium viscosum, Erysipelothrix rhusiopathieae, Listeria monocytogenes, Ehrlichia canis, Escherichia coli, Haemophilus influenzae, Haemophilus somnus, Helicobacter suis, Lawsonia intracellularis, Legionella pneumophilia, Moraxellsa genus, Mycobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasma mycoides subspecies, mycoides LC, Clostridium perfringens, Odoribacter denticanis, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida, Photorhabdus luminescens, Porphyromonas gulae, Porphyromonasgingivalis, Porphyromonas salivosa, Propionibacterium acnes, Proteus vulgaris, Pseudomonas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas fragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp. B11-1, Alcaliges eutrophus, Psychrobacter immobilis, Rickettsia prowazekii, Rickettsia rickettsia, Salmonella enterica all serotypes (e.g., Salmonella enterica Typhimurium, Salmonella enterica Bongori, Salmonella enterica Dublin, Salmonella enterica Choleraseuis, and Salmonella enterica Newport), Serratia marcescens, Spirlina platensis, Staphlyoccocus aureus, Staphyloccoccus epidermidis, Staphylococcus hyicus, Streptomyces albus, Streptomyces cinnamoneus, Streptococcus uberis, Streptococcus suis, Streptomyces exfoliates, Streptomyces scabies, Sulfolobus acidocaldarius, Syechocystis genus, Vibrio cholerae, Borrelia burgdorferi, Treponema denticola, Treponema minutum, Treponema phagedenis, Treponema refringens, Treponema vincentii, Treponema palladium, Trueperella pyogenes, and Leptospira genus, such as known In particular, the pathogens Leptospira canicola, Leptospira grippotyposa, Leptospira hardjo, Leptospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii hardjo-prajitno, Leptospira interrogans, Leptospira icterohaemorrhagiae, Leptospira pomona, and Leptospira bratislava, and combinations thereof, may be included.

[0159] Both inactivated and live attenuated viruses can be used in the adjuvant composition.Some examples of viruses that can be used as antigens include avian herpesvirus, bovine herpesvirus, canine herpesvirus, equine herpesvirus, feline viral rhinotracheitis virus, Marek's disease virus, ovine herpesvirus, porcine epidemic diarrhea virus (PEDv), pseudorabies virus, avian paramyxovirus, bovine respiratory syncytial virus, canine distemper virus, canine parainfluenza virus, canine adenovirus, canine parvovirus, bovine parainfluenza virus 3, ovine parainfluenza virus 3, rinderpest virus, and border disease virus. , bovine viral diarrhea virus (BVDV), BVDV type I, BVDV type II, classical hog fever virus, avian leukemia virus, bovine immunodeficiency virus, bovine leukemia virus, bovine tuberculosis virus, equine infectious anemia virus, feline immunodeficiency virus, feline leukemia virus (FeLV), Newcastle disease virus, ovine progressive pneumonia virus, ovine pulmonary adenocarcinoma virus, canine coronavirus (CCV), pantropic CCV, canine respiratory coronavirus, bovine coronavirus, feline calicivirus, feline enteric coronavirus, feline infectious peritonitis, virus, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine parvovirus, porcine circovirus (PCV) type I, PCV These include, but are not limited to, type II, porcine reproductive and respiratory syndrome (PRRS) virus, transmissible gastroenteritis virus, turkey coronavirus, bovine ephemeral fever virus, rabies, rotovirus, vesicular stomatitis virus, lentivirus, avian influenza, rhinovirus, equine influenza virus, swine influenza virus, canine influenza virus, feline influenza virus, human influenza virus, Eastern Equine Encephalitis Virus (EEE), Venezuelan Equine Encephalitis Virus, West Nile Virus, Western Equine Encephalitis Virus, human immunodeficiency virus, human papilloma virus, varicella zoster virus, hepatitis B virus, rhinovirus, and measles virus, and combinations thereof.

[0160]

[0160] Examples of peptide antigens include Bordetella bronchiseptica p68, GnRH, IgE peptides, Fel d1, and cancer antigens, as well as derivatives thereof. Examples of other antigens include nucleotides, carbohydrates, lipids, glycolipids, peptides, fatty acids, lipoteichoic acid, teichoic acid and peptidoglycan, and combinations thereof.

[0161] Some examples of parasites that may be used as antigens in conjunction with the adjuvant compositions include, but are not limited to, Anaplasma, Fasciola hepatica (liver fluke), Coccidia, Eimeria spp., Neospora caninum, Toxoplasma gondii, Giardia, Dirofilaria (heartworms), Ancylostoma (hookworms), Cooperia, Haemonchus contortus (barberpole worms), Ostertagia ostertagi (stomach worms), Dictyocaulus viviparous (lung worms), Trypanosoma spp., Leishmania spp., Trichomonas spp., Cryptosporidium parvum, Babesia, Schistosoma, Taenia, Strongyloides, Ascaris, Trichinella, Sarcocystis, Hammondia, and Isopsora, and combinations thereof. Also contemplated are ectoparasites such as mites, including, but not limited to, Ixodes, Rhipicephalus, Dermacentor, Amblyomma, Boophilus, Hyalomma, and Haemaphysalis genera, and combinations thereof. It is a live insect.

[0162] The amount of antigen used to induce an immune response varies considerably depending on the antigen used, the subject, and the level of response desired, and can be determined by one of skill in the art. For vaccines containing modified live or attenuated viruses, a therapeutically effective amount of antigen is generally about 10 2 Tissue Culture Infectious Dose (TCID) 50 ~about 10 10 TCID 50 For many such viruses, a therapeutically effective dose is generally within the range of about 10 2 TCID 50 ~about 10 8 TCID 50 (all values ​​inclusive). In some embodiments, the therapeutically effective dose range is about 103 TCID 50 ~about 10 6 TCID 50 (inclusive). In some other embodiments, the therapeutically effective dose range is about 10 4 TCID 50 ~about 10 5 TCID 50 (both numbers are inclusive).

[0163] For vaccines containing inactivated viruses, a therapeutically effective amount of antigen will generally be at least about 100 relative units / dose, and often will be within the range of about 1,000 to about 4,500 relative units / dose (all values ​​inclusive). In other embodiments, a therapeutically effective amount of antigen will be from about 250 to about 4,000 relative units / dose (all values ​​inclusive), from about 500 to about 3,000 relative units / dose (all values ​​inclusive), from about 750 to about 2,000 relative units / dose (all values ​​inclusive), or from about 800 to about 1000 relative units / dose (all values ​​inclusive). or in the range of about 1,000 to about 1,500 relative units / dose (inclusive).

[0164]

[0164] The therapeutically effective amount of antigen in a vaccine containing an inactivated virus can also be measured in terms of relative potency (RP) per mL. The therapeutically effective amount is often within the range of about 0.1 to about 50 RP / mL (inclusive). In other embodiments, the therapeutically effective amount of antigen is within the range of about 0.5 to about 30 RP / mL (inclusive), about 1 to about 25 RP / mL (inclusive), about 2 to about 20 RP / mL (inclusive), about 3 to about 15 RP / mL (inclusive), or about 5 to about 10 RP / mL (inclusive).

[0165] The cell number for the bacterial antigen administered in the vaccine is approximately 1×10 6 ~Approx. 5×10 10 In another embodiment, the cell number is about 1×10 7 ~Approx. 5×10 10CFU / dose (inclusive), or approximately 1 x 10 8 ~Approx. 5×10 10 In yet another embodiment, the cell count is within the range of about 1×10 CFU / dose, inclusive. 2 ~Approx. 5×10 10 CFU / dose (inclusive), or approximately 1 x 10 4 ~Approx. 5×10 9 CFU / dose (inclusive), or approximately 1 x 10 5 ~Approx. 5×10 9 CFU / dose (inclusive), or approximately 1 x 10 6 ~Approx. 5×10 9 CFU / dose (inclusive), or approximately 1 x 10 6 ~Approx. 5×10 8 CFU / dose (inclusive), or approximately 1 x 10 7 ~Approx. 5×10 9 CFU / dose (inclusive).

[0166] The cell number for the parasite antigen administered in the vaccine is approximately 1×10 2 ~Approx. 1×10 10 In another embodiment, the cell number is about 1×10 3 ~Approx. 1×10 9 / dose (inclusive), or approximately 1 × 10 4 ~Approx. 1×10 8 / dose (inclusive), or approximately 1 × 10 5 ~Approx. 1×10 7 / dose (inclusive), or approximately 1 × 10 6 ~Approx. 1×10 8 / Dose (inclusive) range.

[0167] It is well known in the art that in conventional adjuvants, substantially more inactivated virus is required to stimulate the same level of serological response than modified live virus or attenuated virus. However, surprisingly, in the adjuvant composition described herein, it has been found that approximately the same amount of inactivated virus and modified live virus stimulates similar levels of serological response. In addition, the adjuvants described herein require less modified live virus, attenuated virus, and inactivated virus than conventional adjuvants to achieve the same level of serological response. These unexpected findings demonstrate the conservation of supply resources and cost reduction in preparing immunogenic and vaccine compositions. Vaccines with widespread applications require the production of millions of doses per year, so these savings can be substantial.

[0168] Administration of the Composition The dose size of the composition is typically in the range of about 1 mL to about 5 mL (both inclusive), depending on the subject and antigen. For example, for dogs or cats, a dose of about 1 mL is typically used, while for cattle, a dose of about 2 to 5 mL is typically used. However, these adjuvants can also be formulated in microdoses, in which case a dose of about 100 μL can be used.

[0169]

[0169] The administration route of the adjuvant composition may be parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transdermal, intradermal, intraperitoneal, intraocular, intravenous, or into eggs. The composition can be administered by any suitable device, such as a syringe, a dropper, a needleless injector, a patch, etc. The route and device selected for use depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to those skilled in the art.

[0170] Use of the composition One of the requirements of any vaccine adjuvant preparation for commercial use is to ensure the stability of the adjuvant solution during long-term storage. Provided herein is an adjuvant formulation that is easy to manufacture and stable for at least 18 months. In one embodiment, the formulation is stable for about 18 months. In another embodiment, the formulation is stable for about 18-24 months. In another embodiment, the formulation is stable for about 24 months. Accelerated testing procedures have also shown that the formulations described herein are stable.

[0171]

[0171] An advantageous feature of the adjuvant compositions of the present invention is that they can be safely and effectively administered to a wide range of subjects. It is predicted in the art that adjuvant combinations will be more reactogenic than the individual components. However, the compositions described herein show lower reactogenicity compared to compositions in which any one or two components are used, while maintaining the effectiveness of the adjuvant. Also surprisingly, it has been found that the adjuvant compositions described herein show improved safety compared to other adjuvant compositions.

[0172]

[0172] The adjuvant compositions described herein are useful for inducing a desired immune response in a subject. They are effective in multiple species. Suitable species are any animal to which administration of the adjuvant composition is desired. It includes mammals and non-mammals, including primates, livestock, companion animals, laboratory animals, captive wild animals, birds (including eggs), reptiles, and fish. Thus, the term includes, but is not limited to, monkeys, humans, pigs, cows, sheep, goats, horses, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, chickens, turkeys, ducks, other poultry, frogs, and lizards.

[0173] The adjuvants described herein can be used to show serological discrimination between infected and vaccinated animals.Therefore, they can be used in marker vaccines, where the antigens in the vaccine induce antigen patterns in vaccinated animals that are different from wild-type viruses.Marker vaccines are generally used in conjunction with companion diagnostic tests, where the difference in antibody patterns is measured to demonstrate which animals have been vaccinated and which have been infected with wild-type viruses.Such techniques are useful for controlling and eradicating viruses from target populations.

[0174] The present invention also provides a method for the detection of Nipah virus infection using antigens provided by the Hendra virus G protein (and fragments, dimers, multimers, and modified forms thereof). The present invention provides novel vaccine compositions useful for preventing infection and disease caused by the Hendra and Nipah viruses, all of which are adjuvanted as described herein. In certain embodiments, the adjuvant is selected from the group consisting of TXO, TAO, and TXO-A. Such vaccines are useful for preventing infection and disease in, for example, horses, dogs, pigs and humans. In a most preferred embodiment, both pigs and dogs are protected against both Hendra and Nipah viruses. will be done.

[0175]

[0175] In recent years, the re-emergence of NiV causing significant human deaths has become a problem, see, e.g., Butler, Nature, vol. 429, page 7 (2000); and Gurley et al., Emerging Infectious Diseases, vol. 13(7), pp. 1031-1037 (2007). Case Study Studies have linked disease in humans to zoonotic transmission from pigs, see Parashar et al., J. Infect. Dis. vol 181, pp. 1755-1759 (2000). Hendra virus has also been positively linked to human deaths via infection from horses. Currently, there is one licensed vaccine for the prevention of infection or disease from Hendra virus (Equivac® HeV; Zoetis) approved for use in horses, but Nipah virus There is no licensed vaccine to prevent infection. There remains a need for a Nipah or Hendra virus vaccine that could be clinically effective.

[0176] Paramyxoviruses, such as Hendra virus and Nipah virus, The virus particle envelope contains two major membrane-bound glycoproteins. One glycoprotein is required for virion binding to the receptor on the host cell and the other is the hemagglutinin-neutralizing protein. They are designated either laminidase protein (HN) or hemagglutinin protein (H), the other being a glycoprotein (G) with neither hemagglutination nor neuraminidase activity. These fusion glycoproteins are type II membrane proteins with the amino (N) terminus of the molecule pointing towards the cytoplasm and the carboxy (C) terminus of the protein being extracellular. The other major glycoprotein is the fusion (F) glycoprotein, which is a trimeric type I fusogenic envelope glycoprotein containing two heptad repeat (HR) regions and a hydrophobic fusion peptide. After binding to the receptor, Hendra and Nipah viruses assemble their Through the concerted action of the bound G and F glycoproteins, it infects recipient host cells via a pH-independent membrane fusion process.

[0177] Hendra virus G glycoprotein inhibits Nipah virus infection and disease The potential cross-protection of HIV-1 and HIV-2 has been reported by K. Bossart et al., Journal of Virology, vol. 79, pp. 6690-6702 (2005) and B. Mungall et al., Journal of Virology, vol. 80, pp. 12293-12302 (2006). However, previous studies have not shown that any mammal For this species, no vaccine composition has been provided that is actually clinically effective in this regard. Thus, the present invention provides a method for the preparation of a vaccine composition that induces clinically effective protection against Hendra and / or Nipah viruses. In one embodiment, the present invention provides an immunogenic composition comprising a Hendra virus G protein, an adjuvant as described in accordance with the practice of the present invention, and one or more excipients, in an amount effective to inhibit the production of the Hendra virus G protein.

[0178]

[0178] With regard to Hendra virus G glycoprotein polypeptides useful in the practice of the present invention, and recombinant expression thereof, reference is made to the entire disclosures of published International Patent Applications WO2012 / 158643 and WO2006 / 085979, in which such information is clearly set forth. Preferred examples of specific Hendra virus G protein polypeptides useful herein are disclosed in WO2012 / 158643, including, for example, the full-length G protein (SEQ ID NO:2 thereof); a soluble fragment thereof (encoding amino acids 73-604 of SEQ ID NO:2 of WO2012 / 58643); and an Ig(κ) leader sequence (SEQ ID NO:3 of WO2012 / 158643). 16). In general, a soluble form of the Hendra virus G glycoprotein is produced by deleting all or part of the transmembrane domain and all or part of the cytoplasmic tail of the G glycoprotein, including all or part of the ectodomain. Preferably, the encoding gene sequence is codon-optimized for expression.

[0179] In some embodiments, the HendraG glycoprotein may be in dimeric and / or tetrameric form. Such dimers depend on the formation of disulfide bonds formed between cysteine ​​residues in the G glycoprotein. Such disulfide bonds may correspond to those formed in the native G glycoprotein, or different disulfide bonds may be formed resulting in different G glycoprotein dimeric and / or tetrameric forms that enhance antigenicity. In addition, the G glycoprotein may also be in a number of Given that they present conformation-dependent epitopes (i.e., arising from tertiary three-dimensional structures) and that conservation of many such natural epitopes is highly favorable for conferring neutralizing antibody responses, non-dimeric and tetrameric forms will also be useful in the practice of the invention.

[0180] Generally speaking, the construction of an expression vector for HendraG glycoprotein is as described in Example 1 of WO2012 / 158643, and the expression of the resulting protein from CHO cells may be as described in Example 2 thereof or may utilize a vaccine system (see Example 3 thereof) or 293 cells (see Example 4 thereof). In a specific preferred embodiment, the soluble G protein is provided as amino acids 73-604 of the native Hendra virus G glycoprotein (see SEQ ID NO:2 of WO2012 / 158643). The dimerization occurs spontaneously upon expression from CHO cells, and the resulting G protein is approximately 50% dimer and 50% tetramer, with very little monomer remaining. Expression in 293F cells results in approximately 70% dimer. The resulting protein fraction is mixed with an adjuvant as described throughout this application. As described in WO2012 / 158643, preferred antigen doses for large animals are in the range of 50-200 micrograms / dose, with 100 micrograms being the most preferred dose. As will be appreciated by those skilled in the art, smaller doses, such as 25-50 micrograms, may be required for small animals, such as dogs.

[0181] In addition, the adjuvant according to any of the above-described embodiments can be used to produce diagnostic or therapeutic antibodies. In this aspect of the invention, a donor animal is immunized with a formulation comprising the adjuvant composition of the invention and an antigen. The choice of antigen is determined by the person who needs to obtain said therapeutic or diagnostic antibody, and includes, but is not limited to, viruses, viruses, bacteria, virus particles, extracts, recombinant antigens, cell wall structures, etc. Antigens can also include venom for producing snake bite medicines.

[0182] Antigens suitable for this aspect of the invention may be of feline, canine, equine, porcine, bovine, ovine or avian origin. In particular embodiments, the antigen is FeLVgp70, bovine parainfluenza-3 BPI-3 (HN protein), Histophilus somni p31, Bordetella FHA, parapox, BVDV1 gp53, BVDV2 gp53, Clostridia toxin, canine circovirus, Brachyspira hyodysenteriae (porcine species) The antigen may be selected from an inactivated whole cell, and an inactivated pepsin digest.

[0183] At a certain time after immunization, an antibody source is extracted from the source animal (e.g., mouse, rat, hamster, pig, guinea pig, rabbit, goat, sheep, poultry, cow, horse). In certain other embodiments, the source animal is a cat or dog. The source of the antibody depends on whether a monoclonal or polyclonal antibody is ultimately required. For polyclonal antibodies, the use of serum or milk can be considered. For monoclonal antibodies, spleen cells are a suitable source. Such antibodies can be used in a variety of applications, including but not limited to antivenoms, medicines for transplant rejection, serum neutralization assays, ELISA, ELISPOT, Western blots, cell assays, potency assays, and immunohistochemistry. The present invention can be used for diagnostic, research, or therapeutic purposes, including, but not limited to, antivenoms, medicines for transplant rejection, serum neutralization assays, ELISA, ELISPOT, Western blots, cell assays, potency assays, and immunohistochemical assays. For the purposes of this invention, monoclonal and polyclonal antibodies extracted from source animals are provided for use in diagnostic and therapeutic applications.

[0184] In certain embodiments, immunization with a composition of the invention involves administering to at least one animal (preferably at least two animals, or at least three animals, or to a mammalian animal, such as a mammalian animal, a mammalian animal, or ... Induce a sufficiently high (greater than 1000, or more preferably greater than 5000, or more preferably greater than 10000, or more preferably greater than 50000, or more preferably greater than 100000, or more preferably greater than 250000, or more preferably greater than 500000, or more preferably greater than 1000000) serum titer against the desired antigen in 50% of the animals, or at least 75% of the treated animals, or most preferably in each treated animal) to thereby obtain sufficient antibody quantities for diagnostic or research applications.

[0185]

[0185] Typically antibodies of the immunoglobulin G (IgG) isotype are used for these applications, although antibodies of other isotypes, such as immunoglobulin M (IgM), are also used. The source of the antibody will ultimately depend on whether a polyclonal or monoclonal antibody is desired. For polyclonal antibodies, serum or milk can be used as the antibody source. For monoclonal antibodies, spleen cells are a suitable source. Further purification of the antibody, if required, or preparation of monoclonal antibodies is well described in the literature, and the skilled artisan will have no undue difficulty in carrying out these procedures. Furthermore, the antibody may be adapted to the target species, if necessary (e.g., canonized or The techniques for performing the same are well known in the art and need not be described in this application.

[0186]

[0186] Application of antibodies Antibodies are used in serum neutralization assays, ELISA, ELISPOT, Western blots, and cell assays. The present invention is suitable as a reagent for immunohistochemical assays, potency assays and immunohistochemical measurements, which techniques are known in the art.

[0187]

[0187] The antibodies of the invention may also be used as therapeutic agents, e.g., in transplant rejection, e.g., for the production of antithymocyte globulin (ATG) agents. Currently, two such agents are on the market: Atgam® and Thymoglobulin®. Methods for producing antithymocyte globulin in general are described in U.S. Patent Application Publication No. 20040023340.

[0188]

[0188] It may also be used to prepare antivenom medicines. In these embodiments, snake venom components are used as antigens. The venom and its components are also well known in the art.

[0189]

[0189] Many species of animals can be used as feeder animals, including but not limited to poultry, mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, goats, pigs, cattle, and horses. The choice of feeder animal depends on the task at hand and the judgment of the skilled artisan.

[0190]

[0190] Specific non-limiting embodiments are as follows:

[0191] In a first embodiment, the invention provides an adjuvant formulation comprising an oil phase and an aqueous phase, wherein the oil phase comprises at least 50% v / v of the formulation, and wherein the formulation comprises at least one of monophosphoryl lipid A (MPL-A) or an analogue thereof and an immunostimulatory oligonucleotide, with the proviso that a) if the immunostimulatory oligonucleotide is not present, the formulation i. Poly I:C, a glycolipid, and optionally a quaternary amine, or ii. Polycationic Carriers Including, b) if the monophosphoryl lipid A (MPL-A) or analogue thereof is not present, the formulation comprises a source of aluminium; The present invention provides an adjuvant formulation,

[0191]

[0192] In a second embodiment, the invention provides the adjuvant formulation of the first embodiment, wherein the immunostimulatory oligonucleotide, if present, is CpG or an oligoribonucleotide; the polycationic carrier, if present, is selected from the group consisting of dextran, dextran DEAE (and its derivatives), PEG, guar gum, chitosan derivatives, polycellulose derivatives such as hydroxyethylcellulose (HEC) polyethyleneimine, polyamino; and the quaternary amine, if present, is selected from the group consisting of DDA and avridine.

[0192]

[0193] In a third embodiment, the invention provides an adjuvant formulation according to the first or second embodiment, wherein the immunostimulatory oligonucleotide, if present, is CpG; the polycationic carrier, if present, is dextran DEAE; and the quaternary amine, if present, is DDA.

[0193]

[0194] In a fourth embodiment, the present invention relates to a glycolipid, when present, having formula I:

[0194] [ka]

[0195] (In the formula, R 1 and R 2 are independently hydrogen or a saturated alkyl radical having up to 20 carbon atoms; X is -CH2-, -O-, or -NH-; R 2 is hydrogen or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R 3 , R4 , and R 5 are independently hydrogen, -SO4 2- , -PO4 2- , -COC 1-10 R is alkyl; 6 provides an adjuvant formulation according to any one of the first to third embodiments, comprising a compound represented by the formula: L-alanyl, L-α-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl, or a D-isomer thereof.

[0196]

[0195] In a fifth embodiment, the present invention provides the adjuvant formulation of the fourth embodiment, wherein the glycolipid is N-(2-deoxy-2-L-leucylamino-bD-glycopyranosyl)-N-octadecyldodecanoylamide or a salt thereof.

[0197]

[0196] In a sixth embodiment, the present invention provides the adjuvant formulation of the fifth embodiment, wherein the salt is an acetate salt.

[0198]

[0197] In a seventh embodiment, the present invention comprises both the monophosphoryl lipid A (MPL-A) or an analog thereof, and at least one of a sterol and poly I:C. Any one of the fourth embodiments, which fists thought further includes either The present invention provides two adjuvant formulations.

[0199]

[0198] In an eighth embodiment, the present invention provides an adjuvant formulation according to the seventh embodiment, comprising a sterol and further comprising a saponin.

[0200]

[0199] In a ninth embodiment, the present invention provides an adjuvant formulation of either the seventh or eighth embodiment, wherein the saponin, if present, is a triterpenoid saponin and the sterol, if present, is selected from the group consisting of ergosterol, lanosterol, and cholesterol.

[0201]

[0200] In a tenth embodiment, the present invention provides an adjuvant formulation according to the ninth embodiment, wherein the saponin, if present, is Quil A and the sterol, if present, is cholesterol.

[0202]

[0201] In an eleventh embodiment, the present invention provides an adjuvant formulation according to the seventh embodiment, comprising poly I:C and further comprising at least one of a quaternary amine and a glycolipid.

[0203]

[0202] In a twelfth embodiment, the present invention provides the adjuvant formulation of any one of the first to eleventh embodiments, comprising the MPL-A or an analogue thereof in an amount of 0.5-100 μg / dose.

[0204]

[0203] In a thirteenth embodiment, the present invention provides an adjuvant formulation according to the twelfth embodiment, wherein the MPL-A or an analogue thereof is present in an amount of 5 to 50 μg / dose, or 5 to 20 μg / dose, or 1 to 5 μg / dose.

[0205]

[0204] In a fourteenth embodiment, the invention provides the adjuvant formulation of any one of the first to thirteenth embodiments, comprising the immunostimulatory oligonucleotide in an amount of 0.5 to 400 μg / dose.

[0206]

[0205] In a fifteenth embodiment, the present invention provides the adjuvant formulation of the fourteenth embodiment, wherein the immunostimulatory oligonucleotide is present in an amount of about 100 to about 250 μg / dose, or about 20 to about 50 μg / dose, or about 1 μg / dose.

[0207]

[0206] In a sixteenth embodiment, the present invention provides the adjuvant formulation of any one of the first to fifteenth embodiments, comprising the polycationic carrier in an amount of about 0.5 to about 400 mg / dose.

[0208]

[0207] In a seventeenth embodiment, the present invention provides the adjuvant formulation of the sixteenth embodiment, wherein the polycationic carrier is present in an amount of 50-300 mg / dose, or 1-25 mg / dose, or 1-10 mg / dose.

[0209]

[0208] In an eighteenth embodiment, the present invention provides the adjuvant formulation of any one of the first to seventeenth embodiments, comprising the glycolipid in an amount of about 0.5 to about 2000 µg / dose.

[0210]

[0209] In a nineteenth embodiment, the present invention provides the adjuvant formulation of the eighteenth embodiment, wherein the glycolipid is present in an amount of about 1000 μg / dose, or 25-50 μg / dose, or 1-10 μg / dose.

[0211]

[0210] In a twentieth embodiment, the present invention provides the adjuvant formulation of any one of the first to nineteenth embodiments, comprising the sterol in an amount of about 0.1 to about 1000 μg / dose.

[0212]

[0211] In a twenty-first embodiment, the present invention provides an adjuvant formulation according to the twentieth embodiment, wherein the sterol is present in an amount of 250-500 μg / dose, or 20-50 μg / dose, or 1-10 μg / dose.

[0213]

[0212] In a twenty-second embodiment, the present invention provides the adjuvant formulation of any one of the first to twenty-first embodiments, comprising the saponin in an amount of 0.1-1000 μg / dose.

[0214]

[0213] In a twenty-third embodiment, the present invention provides the adjuvant formulation of the twenty-second embodiment, wherein the saponin is present in an amount of 250-500 μg / dose, or 20-50 μg / dose, or 1-10 μg / dose.

[0215]

[0214] In a twenty-fourth embodiment, the present invention provides the adjuvant formulation of any one of the first to twenty-third embodiments, comprising the poly I:C in an amount of about 0.5 to about 100 µg / dose.

[0216]

[0215] In a twenty-fifth embodiment, the present invention provides the adjuvant formulation of the twenty-fourth embodiment, wherein the Poly I:C is present in an amount of 5 to 50 μg / dose, or 5 to 20 μg / dose, or 1 to 5 μg / dose.

[0217]

[0216] In a twenty-sixth embodiment, the present invention provides the adjuvant formulation of any one of the first to twenty-fifth embodiments, comprising an aluminium source which is an aluminium hydroxide gel.

[0218]

[0217] In a twenty-seventh embodiment, the invention provides the adjuvant formulation of the twenty-sixth embodiment, wherein said aluminium source is present in an amount of 5-20% v / v of the formulation.

[0219]

[0218] In a twenty-eighth embodiment, the invention provides the adjuvant formulation of the twenty-seventh embodiment, wherein said aluminium source is present in an amount of 10% v / v of the formulation.

[0220]

[0219] In a twenty-ninth embodiment, the present invention provides the adjuvant formulation of any one of the first to twenty-eighth embodiments, wherein the oil phase comprises an oil and a fat-soluble emulsifier.

[0221]

[0220] In a thirtieth embodiment, the invention provides the adjuvant formulation of any one of the first to twenty-ninth embodiments, wherein the oil phase is present in an amount of up to 85% v / v.

[0222]

[0221] In a thirty-first embodiment, the present invention provides an adjuvant formulation according to the thirty-first embodiment, wherein the oil phase is present in an amount of 51%.

[0223]

[0222] In a thirty-second embodiment, the present invention provides an adjuvant formulation of any one of the twenty-ninth to thirty-first embodiments, wherein the oil comprises 40-84% v / v of the formulation and the fat-soluble emulsifier comprises 1-11% v / v of the formulation.

[0224]

[0223] In a thirty-third embodiment, the present invention provides the adjuvant formulation of the thirty-second embodiment, wherein the oil comprises 45% v / v of the formulation and the fat-soluble emulsifier comprises 6% v / v of the formulation.

[0225]

[0224] In a thirty-fourth embodiment, the present invention provides an adjuvant formulation according to any one of the first to thirty-third embodiments, wherein the oil is selected from the group consisting of squalane, vegetable oils, triglycerides, non-metabolizable linear alkane oils, and combinations thereof.

[0226]

[0225] In a thirty-fifth embodiment, the present invention provides an adjuvant formulation according to the thirty-fourth embodiment, wherein the oil is a light mineral oil.

[0227]

[0226] In a thirty-sixth embodiment, the present invention provides a vaccine composition comprising an effective amount of an antigen and an adjuvant formulation according to any one of the first to thirty-fifth embodiments, wherein the oil phase of the composition is at least 50% v / v.

[0228] In a thirty-seventh embodiment, the present invention provides a vaccine composition comprising an effective amount of an antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase and an aqueous phase, the oil phase comprising at least 50% v / v of the formulation, a polycationic carrier, and a. a combination of a saponin, a sterol, and optionally a quaternary amine; provided that when the adjuvant formulation consists essentially of DEAE dextran, Quil A, cholesterol, and DDA, the antigen is not E. coli J-5 bacterin; or b. immunostimulatory oligonucleotides; provided that when the adjuvant formulation consists essentially of DEAE dextran and immunostimulatory oligonucleotides, the antigen comprises or is derived from a pathogen affecting cattle, sheep, horses, or pigs and is not E. coli J-5 bacterin; The present invention provides a vaccine composition comprising:

[0229]

[0228] In a thirty-eighth embodiment, the present invention provides a vaccine composition according to the thirty-seventh embodiment, wherein the saponin, if present, is a triterpenoid saponin; the sterol, if present, is selected from the group consisting of ergosterol, lanosterol, and cholesterol; the polycationic carrier, if present, is selected from the group consisting of dextran, dextran DEAE (and derivatives), PEG, guar gum, chitosan derivatives, polycellulose derivatives such as hydroxyethylcellulose (HEC) polyethyleneimine, polyamino; and the quaternary amine, if present, is selected from the group consisting of DDA and avridine.

[0230]

[0229] In a thirty-ninth embodiment, the present invention provides a vaccine composition according to the thirty-eighth embodiment, wherein the saponin is Quil A, the sterol is cholesterol, the polycationic carrier is dextran DEAE; and the quaternary amine is DDA.

[0231]

[0230] In a fortieth embodiment, the invention provides the vaccine composition of any one of the thirty-seventh to thirty-ninth embodiments, wherein the immunostimulatory oligonucleotide is CpG.

[0232]

[0231] In a forty-first embodiment, the present invention provides the vaccine composition of any one of the thirty-seventh to fortieth embodiments, wherein the polycationic carrier is present in an amount of about 0.5 to about 400 mg / dose.

[0233]

[0232] In a forty-second embodiment, the present invention provides a vaccine composition of the forty-first embodiment, wherein the polycationic carrier is present in an amount of 50 to 300 mg / dose, or 1 to 25 mg / dose, or 1 to 10 mg / dose.

[0234]

[0233] In a forty-third embodiment, the present invention provides a vaccine composition of any one of the thirty-seventh to forty-second embodiments, comprising the saponin in an amount of about 0.1 to about 1000 μg / dose.

[0235]

[0234] In a forty-fourth embodiment, the present invention provides a vaccine composition of the forty-third embodiment, wherein the saponin is present in an amount of 250-500 μg / dose, or 20-50 μg / dose, or 1-10 μg / dose.

[0236]

[0235] In a forty-fifth embodiment, the present invention provides a vaccine composition of any one of the thirty-seventh to forty-fourth embodiments, comprising the sterol in an amount of about 0.1 to about 1000 μg / dose.

[0237]

[0236] In a forty-sixth embodiment, the present invention provides a vaccine composition according to the forty-fifth embodiment, wherein the sterol is present in an amount of 250-500 μg / dose, or 20-50 μg / dose, or 1-10 μg / dose.

[0238]

[0237] In a forty-seventh embodiment, the present invention provides a vaccine composition of any one of the thirty-seventh to forty-sixth embodiments, comprising the quaternary amine in an amount of about 1 to about 200 μg / dose.

[0239]

[0238] In a forty-eighth embodiment, the present invention provides a vaccine composition according to the forty-seventh embodiment, wherein the quaternary amine is present in an amount of about 10 μg / dose, or about 10 to about 100 μg / dose, or about 5 μg / dose.

[0240]

[0239] In a forty-ninth embodiment, the present invention provides a vaccine composition of any one of the thirty-seventh to forty-eighth embodiments, comprising the immunostimulatory oligonucleotide in an amount of about 0.5 to about 400 μg / dose.

[0241]

[0240] In a fiftieth embodiment, the present invention provides a vaccine composition of the forty-ninth embodiment, wherein the immunostimulatory oligonucleotide is present in an amount of 100 to 250 μg / dose, or 20 to 50 μg / dose, or about 1 μg / dose.

[0242]

[0241] In a fifty-first embodiment, the present invention provides a vaccine composition of any one of the thirty-seventh to fifty-first embodiments, wherein the oil phase comprises an oil and a fat-soluble emulsifier.

[0243]

[0242] In a fifty-second embodiment, the present invention provides a vaccine composition of any one of the thirty-seventh to fifty-first embodiments, wherein the oil phase is present in an amount of up to 85% v / v.

[0244]

[0243] In a fifty-third embodiment, the present invention provides a vaccine composition according to the fifty-second embodiment, wherein the oil phase is present in an amount of 51% v / v.

[0245]

[0244] In a fifty-fourth embodiment, the present invention provides a vaccine composition of any one of the fifty-first to fifty-third embodiments, wherein the oil constitutes 40 to 84% v / v of the vaccine composition and the fat-soluble emulsifier constitutes 1 to 11% v / v of the vaccine composition.

[0246]

[0245] In a fifty-fifth embodiment, the present invention provides a vaccine composition of the fifty-third embodiment, wherein the oil constitutes 45% v / v of the formulation and the fat-soluble emulsifier constitutes 6% v / v of the formulation.

[0247] In a fifty-sixth embodiment, the present invention provides a vaccine composition comprising an Eimeria maxima or Clostridium perfringens antigen and an adjuvant formulation, The hubant formulation is a) an oil phase, a polycationic carrier, and optionally an immunostimulatory oligonucleotide, present in an amount of at least 50% v / v of the composition; or b) an oil phase, an immunostimulatory oligonucleotide, a sterol, and monophosphoryl lipid A (MPL-A) or an analog thereof, present in an amount of at least 50% v / v of the composition; The present invention provides a vaccine composition comprising:

[0248] In a fifty-seventh embodiment, the present invention provides a vaccine composition of the fifty-sixth embodiment, comprising antigens against Eimeria maxima and Clostridium perfringens. do.

[0249]

[0248] In a fifty-eighth embodiment, the present invention provides a vaccine composition of the fifty-sixth embodiment or the fifty-seventh embodiment, wherein the polycationic carrier is DEAE dextran.

[0250] In a fifty-ninth embodiment, the present invention relates to a method for the treatment or prevention of infections caused by Eimeria maxima or Clostridium perfringens in poultry, Use of a vaccine composition according to an embodiment of claims 56 to 58 is provided.

[0251] In a sixtieth embodiment, the present invention provides a vaccine composition comprising a Neospora antigen and an adjuvant formulation, the adjuvant formulation being present in an amount of at least 50% v / v of the composition; and a) monophosphoryl lipid A (MPL-A) or an analog thereof; or b) combination of immunostimulatory oligonucleotides with polycationic carriers; The present invention provides a vaccine composition comprising:

[0252]

[0251] In a sixty-first embodiment, the present invention provides a vaccine composition of the sixtieth embodiment, comprising a combination of an immunostimulatory oligonucleotide and dextran DEAE.

[0253]

[0252] In a sixty-second embodiment, the present invention provides a vaccine composition of the sixtieth embodiment, comprising monophosphoryl lipid A (MPL-A) or an analog thereof and further comprising an immunostimulatory oligonucleotide.

[0254]

[0253] In a sixty-third embodiment, the present invention provides a vaccine of the sixty-second embodiment, further comprising a sterol.

[0255]

[0254] In a sixty-fourth embodiment, the present invention provides a vaccine of the sixty-third embodiment, wherein the sterol is cholesterol.

[0256] In a sixty-fifth embodiment, the present invention relates to a method for the treatment of a pulmonary edema, comprising administering to a subject a patient infected with the pulmonary edema aerobic pulmonary edema. The vaccine according to any one of the sixtieth to sixty-fourth embodiments is a caninum antigen. .

[0257]

[0256] In a sixty-sixth embodiment, the present invention provides a vaccine of any one of the sixtieth to sixty-fifth embodiments for the treatment or prevention of an infection caused by Neospora.

[0258] In a sixty-seventh embodiment, the present invention relates to a Chlamydophila abortis antigen. and an adjuvant formulation comprising: an oil phase present in an amount of at least 50% v / v of the composition; a sterol; an immunostimulatory oligonucleotide; monophosphoryl lipid A (MPL-A) or an analogue thereof; and poly I:C.

[0259]

[0258] In a sixty-eighth embodiment, the present invention provides the use of a vaccine according to the sixty-seventh embodiment for the treatment or prevention of abortion caused by C. abortis in ewes.

[0260] In a sixty-ninth embodiment, the present invention provides a vaccine composition comprising myostatin and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, and a) a polycationic carrier; or b) MPL-A or an analogue thereof; The present invention provides a vaccine composition comprising any one of the following:

[0261]

[0260] In a seventieth embodiment, the present invention provides a vaccine composition of the sixty-ninth embodiment comprising MPL-A or an analogue thereof, wherein the formulation contains less than 0.5 μg of sterol per 50 μl of the composition.

[0262]

[0261] In a seventy-first embodiment, the present invention provides a vaccine composition of the seventieth embodiment, which does not contain a sterol.

[0263]

[0262] In a seventy-second embodiment, the present invention provides the vaccine composition of the seventieth embodiment, wherein the sterol is cholesterol.

[0264]

[0263] In a seventy-third embodiment, the present invention provides the use of a vaccine according to any one of embodiments 69 to 72 for reducing the amount of myostatin in an animal.

[0265]

[0264] In a seventy-fourth embodiment, the present invention provides a use according to the seventy-third embodiment, wherein the animal is a poultry animal.

[0266]

[0265] In a seventy-fifth embodiment, the present invention provides a vaccine composition comprising a Trueperella pyogenes antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, and a polycationic carrier.

[0267]

[0266] In a seventy-sixth embodiment, the present invention provides a vaccine composition of the seventy-fifth embodiment, wherein the Trueperella pyogenes antigen is pyolysin.

[0268] In a seventy-seventh embodiment, the present invention relates to a vaccine of the seventy-fourth or seventy-fifth embodiment for the treatment or prevention of an infection caused by Trueperella pyogenes. The present invention provides uses of the chin composition.

[0269]

[0268] In a seventy-eighth aspect, the present invention provides a vaccine composition comprising an E. coli antigen, a BRV antigen or a BCV antigen, and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the vaccine composition, an immunostimulatory oligonucleotide, and at least one of a polycationic carrier and an aluminium source.

[0270]

[0269] In a seventy-ninth aspect, the present invention provides a vaccine composition of the seventy-eighth embodiment, comprising an E. coli antigen, a BRV antigen and a BCV antigen.

[0271]

[0270] In an eightieth embodiment, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a E. coli antigen, if present, is selected from the group consisting of E. coli K99, E. coli F41, and combinations thereof; b. The BRV antigen, if present, is selected from the group consisting of BRV G6, BRV G10, and combinations thereof; The vaccine composition of the seventy-eighth or seventy-ninth embodiment is provided.

[0272]

[0271] In an eighty-first embodiment, the present invention provides a vaccine composition according to any one of the seventy-eighth to eighty-first embodiments, wherein the polycationic carrier, if present, is dextran DEAE; and the immunostimulatory oligonucleotide is CpG.

[0273]

[0272] In an eighty-second embodiment, the present invention provides a vaccine composition according to any one of the seventy-eighth to eighty-first embodiments, comprising an aluminum source which is an aluminum hydroxide gel.

[0274]

[0273] In an eighty-third embodiment, the present invention provides the vaccine composition of the eighty-second embodiment, wherein the aluminum source is present in an amount of 5-20% v / v.

[0275]

[0274] In an eighty-fourth embodiment, the present invention provides the vaccine composition of the eighty-third embodiment, wherein the aluminum source is present in an amount of 10-17% v / v.

[0276]

[0275] In an eighty-fifth embodiment, the present invention provides the use of a vaccine composition according to any one of the seventy-eight to eighty-fourth embodiments for the treatment or prevention of enteritis caused by E. coli, BCV or BRV in a bovine animal.

[0277]

[0276] In an eighty-sixth embodiment, the present invention provides a use according to the ninety-first embodiment, wherein the vaccine induces immunity to the antigen(s) for at least six months.

[0278] In an eighty-seventh embodiment, the present invention relates to a Rhipicephalus microplus antibacterial agent. A vaccine composition comprising an antigen and an adjuvant, the adjuvant comprising: a) an aqueous adjuvant comprising an immunostimulatory oligonucleotide, a saponin, a sterol, a quaternary amine, a polyacrylic polymer, and a glycolipid; and b) an oil-based adjuvant comprising an oil phase present in an amount of at least 50% v / v of said vaccine composition, said oil-based adjuvant comprising an immunostimulatory oligonucleotide and a polycationic carrier; The present invention provides a vaccine composition selected from the group consisting of:

[0279]

[0278] In an eighty-eighth embodiment, the present invention relates to a method for the preparation of a glycerol-based glycerol extract comprising the steps of: The vaccine composition of an eighty-seventh embodiment is provided, wherein is N-(2-deoxy-2-L-leucylamino-bD-glucopyranosyl)-N-octadecyldodecanoylamide or a salt thereof, and the immunostimulatory oligonucleotide is CpG.

[0280]

[0279] In an eighty-ninth embodiment, the present invention provides the vaccine composition of the eighty-seventh embodiment, wherein the polycationic carrier is dextran DEAE and the immunostimulatory oligonucleotide is CpG.

[0281]

[0280] In a ninetieth embodiment, the present invention relates to the Rhipicephalus microplus antibacterial agent. The vaccine composition of any one of the eighty-seventh to eighty-ninth embodiments is provided, wherein the origin is a Bm86 protein.

[0282] In a ninety-first embodiment, the present invention relates to a method for producing a medaka strain of Rhipicephalus microplus. The present invention provides a use of a vaccine composition according to any one of the eighty-seventh to ninetyth embodiments for the treatment or prevention of an infection caused by B. cerevisiae.

[0283] In a ninety-second embodiment, the present invention provides a vaccine composition comprising a foot and mouth disease (FMD) antigen and an adjuvant formulation, wherein the adjuvant formulation comprises an oil phase present in an amount of at least 36% v / v of the vaccine composition, an immunostimulatory oligonucleotide, and a polycationic carrier, and wherein the vaccine composition is a water-in-oil emulsion. In different embodiments, the foot and mouth disease virus antigen can be a wild-type FMDV, a genetically modified and / or attenuated FMDV strain, or a recombinantly expressed FMDV structural protein, such as a virus-like particle (VLP) of serotype A, C, O, Asia1, SAT1, SAT2, or SAT3.

[0284]

[0283] In a ninety-third embodiment, the present invention provides a ninety-second vaccine composition, wherein the immunostimulatory oligonucleotide is CpG and the polycationic carrier is DEAE dextran.

[0285] In a ninety-fourth embodiment, the present invention provides the vaccine composition of the ninety-second or ninety-third embodiment, in which the antigen is derived from a genetically modified FMD-LL3B3D platform virus, specifically FMD-LL3B3D-A24 Cruzeiro, which is attenuated in bovine and porcine. The composition is provided.

[0286]

[0285] In a ninety-fifth embodiment, the present invention provides the use of a vaccine composition of any one of the ninety-second or ninety-fourth embodiments for the treatment or prevention of FMD in cattle.

[0287] In a ninety-sixth embodiment, the present invention provides a vaccine composition comprising a Streptococcus uberis (S. uberis) antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the composition, a polycationic carrier, and a) immunostimulatory oligonucleotides; b) a combination comprising a saponin, a sterol, and a quaternary amine; or c) any combination thereof; The present invention provides a vaccine composition comprising:

[0288]

[0287] In a ninety-seventh embodiment, the present invention provides the vaccine composition of the ninety-sixth embodiment, wherein the antigen is a Suberis adhesion molecule or an immunogenic fragment thereof.

[0289]

[0288] In a ninety-eighth embodiment, the present invention provides the use of a vaccine according to any one of the ninety-sixth or ninety-seventh embodiments for the treatment or prevention of infection caused by Suberis.

[0290]

[0289] The following examples are presented as illustrative embodiments and should not be construed as limiting the scope of the present invention. Many changes, modifications, variations, and other uses and applications of the present invention will be apparent to those skilled in the art. EXAMPLES

[0291] Example 1. Development of a recombinant vaccine strategy to enhance immunity to phlegmonous enteritis The objective of this study was to evaluate the efficacy of adjuvanted recombinant Clostridium vaccinia against live challenge with Eimeria maxima and Clostridium perfringens in a disease model of phlegmonous enteritis. The objective of this study was to evaluate the effect of in vivo vaccination with Cutin.

[0292] Materials and Methods Recombinant Proteins: The full-length coding sequences of the genes encoding C. perfringens (ATCC 13124, American Type Culture Collection, Manassas, VA) NetB and EF-Tu were cloned by PCR into the pET32a(+) vector with an NH2-terminal polyhistidine epitope tag. The cloned genes were transformed into the antagonist Escherichia coli, the bacteria were grown at 37°C for 16 hours, and induced with 1.0 mM isopropyl β-D-thiogalactopyranoside (Amresco, Cleveland, OH) for 5 hours at 37°C. Bacteria were harvested by centrifugation at 10,000 rpm for 10 min at 4°C, resuspended in PBS, disrupted by sonication, and centrifuged at 10,000 rpm for 15 min. The supernatant was incubated with Ni-NTA agarose (Qiagen, Valencia, CA) for 1 h at 22°C. The resin was washed with PBS and the purified clostridial proteins were eluted with 250 mM imidazole in PBS pH 9.2. Protein purity was confirmed on a Coomassie Blue stained SDS-acrylamide gel. Protein concentration was determined using a commercial kit from Sigma. I decided to use it.

[0293]

[0292] Animals: 1-day-old mice hatched at Longeneckers Hatchery (Elizabethtown, PA). Young broiler birds (Ross / Ross) were transported to BARC-East, Building 1082, and the chickens were raised in a Petersime small animal incubator in accordance with established guidelines of the BARC Small Animal Care Committee. Birds were raised in Eimeria-free facility incubators and in large hanging incubators in a separate location. They were transferred to cages where they were infected and kept until the end of the experimental period for live challenge. All procedures for transportation, weight measurement, infection, and blood and spleen collection were approved by the BARC Small Animal Care Committee (with SOPs). The ARS BARC Small Animal Care Committee establishes the guidelines for animal experiments at BARC and conducts regular inspections of all animal facilities.

[0294] Immunization: Primary immunization was performed by subcutaneous injection of 100 μl of vaccine (100 μg Ag / dose) into 1-day-old broiler chickens. Secondary immunization was performed by subcutaneous injection of 100 μl of vaccine (100 μg Ag / dose) into 7-week-old broiler chickens.

[0295] Eimeria challenge: BARC strains of the genus Eimeria grown at the Animal Parasitic Diseases Laboratory and propagated according to established procedures. E. maxima (41A) was incubated with 5% hypochlorous acid. They were clarified by floating in sodium chlorate, washed three times with PBS, and viable counts were made using a hemocytometer with trypan blue. Six days after the booster immunization, chickens were inoculated intraesophagically with 10,000 E. maxima using a needle.

[0296] C. perfringens challenge: Four days after Eimeria infection, Using 1×10 9 CFU of Clostridium perfringens was inoculated into the esophagus of NE birds. Ta.

[0297]

[0296] Analysis: Birds were weighed on the day of arrival, immediately prior to challenge with EM, prior to challenge with C. perfringens, 2 days after CP challenge, and 10 days after CP challenge to measure weight gain.

[0298] To score intestinal lesions, birds (5 / group) were sacrificed 2 days after CP infection. Approximately 20 cm intestinal segments extending 10 cm anterior and posterior to the diverticulum were obtained and cut longitudinally. Lesion scores were assessed by two independent observers on a scale of 0 to 4 in order of increasing lesion severity.

[0299]

[0298] The two major C. perfringens virulence factors in chickens are alpha toxin and NetB (necrotic enteritis B-like) toxin, both of which are involved in the pathogenesis of NE. Additional C. elegans proteins that may be involved in bacterial pathogenesis and host defense immunity include pyruvate:ferredoxin oxidoreductase (PFO) and elongation factor G (EF-G). perfringens proteins have previously been reported to induce protective immunity against experimental challenge infection with C. perfringens. Therefore, antibody titers against these agents were determined as described below.

[0300] Five birds per group were randomly selected for blood collection by cardiac puncture immediately after euthanasia. Serum was obtained by low speed centrifugation and used in enzyme-linked immunosorbent assays (ELISA) to measure α-toxin-, NetB-, EF-, and PFO-specific antibody levels. Briefly, 96-well microtiter plates were coated overnight with 1.0 μg / well of purified recombinant α-toxin-, NetB-, EF-, and PFO-proteins. Plates were washed with PBS containing 0.05% Tween (PBS-T) and incubated overnight at 4°C for 1 h. The plate was blocked with PBS containing 1% BSA. Serum (100 μl / well) was added with gentle stirring. The plates were washed with PBS-T and incubated with peroxidase-conjugated rabbit anti-chicken IgG (Sigma, St. Louis, MO) for 2 hours. Bound antibodies were detected with a peroxidase-specific substrate. The optical density (OD) at 450 nm was measured with an automated microplate reader (Bio-Rad, Richmond, Calif.).

[0301] Statistical Analysis: All values ​​are expressed as mean ± SEM. Weight gain and lesion scores Mean values ​​are compared between groups by analysis of variance with Turkey's test using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL). Differences in means are considered significant at p<0.05.

[0302]

[0301] The experimental design is shown in Table 1.

[0303] [Table 1]

[0304]

[0302] The composition of the adjuvant was as follows (per 50 μl):

[0303] TXO:SEQ ID NO:8 was present in an amount of 1 μg, Dextran DEAE was present in an amount of 5 μg, and light mineral oil was present in an amount of 51% v / v of the composition.

[0305]

[0304] TCMO:SEQ ID NO:8 was present in an amount of 1 μg, cholesterol was present in an amount of 1 μg, MPL-A was present in an amount of 1 μg / 50 μl dose, and light mineral oil was present in an amount of 51% v / v of the composition.

[0306]

[0305] XO: Dextran DEAE was present in an amount of 5 μg and light mineral oil was present in an amount of 51% v / v of the composition.

[0307]

[0306] XOM: Dextran DEAE was present in an amount of 5 μg, light mineral oil was present in an amount of 51% v / v of the composition, and MPL-A was present in an amount of 1 μg.

[0308] 5% AMPHIGEN® + Poly I:C: Poly I:C was present in an amount of 1 μg.

[0309] 5% AMPHIGEN® + CpG: SEQ ID NO:8, 1 μl It was present in amounts of g.

[0310]

[0309] 5% AMPHIGEN® + DEAE Dextran: DEAE Dextran was present in an amount of 25 μg.

[0311]

[0310] 5% AMPHIGEN® + DDA: DDA was present in an amount of 1 μg.

[0312] The weight gain rate was significantly decreased by EM and CP infection in the NE control group (p<0.05). However, the weight gain rate was generally increased by 4-21% in the groups immunized with recombinant CP protein (NetB+EF). A significant difference from the NE control was found in the Prot TCMO group immunized with CP protein conjugated with TCMO adjuvant.

[0313] [Table 2]

[0314] [Table 3]

[0315] Serum antibody responses to alpha toxin, Net-B, EF, and PFO were evaluated 6 days after EM infection and 2 days after CP infection. The results are shown in Table 4. In brief, CP proteins generally increased Ab titers against CP antigens in birds immunized with CP proteins. Ab responses to Net B, EF, and PFO antigens were significantly higher than responses to alpha toxin.

[0316] [Table 4]

[0317] Example 2: Female Chicken Anti-Myostatin Vaccine Myostatin is a secreted growth and differentiation factor that is a member of the TGFβ protein family that inhibits muscle differentiation and growth. Myostatin is produced primarily in skeletal muscle cells, circulates in the blood, and acts in muscle tissue by binding to a cell-bound receptor called the activin type II receptor. Thus, inhibition of myostatin results in animals with greater amounts of meat / muscle. One approach to reducing the amount of myostatin in an animal is to generate an anti-myostatin immune response, which can be conventionally measured by titers of anti-myostatin antibodies. In this example, a female avian model was used.

[0318]

[0314] Cobb 500 Parent Stock and Ross 308 female birds (12-10 each) Mice (age 14-28 weeks) were primed with a vaccine containing myostatin conjugated peptide and an adjuvant formulation. The adjuvant formulations used in the study are shown in Table 5.

[0319] [Table 5]

[0320]

[0315] The components of the adjuvant are set out in Table 6.

[0321] Cobb 500 Parent Stock and Ross 308 females were cultured at 12 and 10 weeks of age. Mice were primed in the eye and boosted at week 18. Serum titers of anti-myostatin antibodies were measured by ELISA prevaccination and every 2 weeks postprime until the age of 22 and 20 weeks, respectively.

[0322] Groups T06, T07, T09 and T10 produced the best responses (antibody geometric mean titers of 50,000-15,000 at week 22). Of these four groups, Cobb 500 birds in groups T06 and T07 showed geometric mean titers of over 100,000.

[0323] [Table 6]

[0324] Example 3. Vaccine against T. pyogenes Truepurella pyogenes (formerly Arcanobacterium pyogenes, and formerly Actinomyces pyogenes, and Corynebacterium pyogenes) causes a severe clinical form of metritis in cattle, often characterized by a cloudy, purulent discharge. The putrid odor that sometimes accompanies the disease is probably caused by anaerobic bacteria that are present but not detected by routine culture methods. The disease is most common in dry cows or heifers before or at calving, and in lactating animals it may occur as a result of nipple or udder injury. Economically important diseases caused by this organism include metritis, abortion in dairy cows, and liver abscesses in feedlot cattle. Pyolysin (PLO), a cholesterol-dependent cytolysin expressed by Truepurella pyogenes, is an important host defense antigen.

[0325] Angus crossbred cattle, approximately 14 months of age, were used in this study. At the time of enrollment, the animals were in good overall health and free of any complications. Animals had free access to food and water.

[0326]

[0320] Formulation: Total bacteria (E. coli and T. pyogenes) at 1 x 10 9 Pyolysin was administered to animals in groups T02 to T07 at 150 micrograms / dose. Group T01 was used as the control group.

[0327]

[0321] The adjuvant formulations tested in this study were as follows: ISC / PolyIC-ISC 1000μg / PolyI:C 50μg in 2mL dose ISC / CpG-ISC 1000μg / CpG(SEQ ID NO:8)100μg in 2mL dose TXO-CpG (SEQ ID NO:8) 100 μg / DEAE dextran / mineral oil 5LT NF in 2 mL dose QCDCRT- Quil A 150μg / Cholesterol 150μg / DDA in 2mL dose 100μg / CARBOPOL® (polyacrylic polymer) 0.0375% / R1005 1000μg / CpG (SEQ ID NO:8) 100μg QAC-Quil A 500μg / cCholesterol 500μg / AMPHIGEN(registered trademark) in a 2mL dose (Lecithin oil emulsion) 2.5%

[0322] Pyolysin antibodies were measured using an indirect ELISA, measuring antigen on a plate followed by serum samples (primary antibody) followed by anti-bovine IgG conjugate on days 0, 28, and 56.

[0328] All samples and controls were diluted 1:2000 and responses were determined by calculating the ratio of the OD of the sample to the OD of the positive control (the positive control was a serum pool of convalescent animals). Antibodies were detected with HRP-conjugated sheep anti-bovine IgG.

[0329] [Table 7]

[0330]

[0324] The results are shown in Table 8.

[0331] [Table 8]

[0332]

[0325] Groups T04 and T06 (adjuvants TXO and QAC) performed significantly better than the control (P<0.05). In addition, several trends (selected as differences of p<0.1) were found between the different treatment groups. These trends are summarized in Table 9.

[0333] [Table 9]

[0334] Example 4. Evaluation of pyolisin vaccine formulations in lactating dairy cows against metritis challenge The objective of this study was to evaluate the efficacy of TXO-adjuvanted native and recombinant pyolysin vaccine formulations in non-pregnant, lactating Holstein or Holstein-cross dairy cows using an artificial metritis challenge model.

[0335] Animals were in overall good health, free of any comorbidities, and had not received any chemotherapy, systemic antibiotic or other anti-inflammatory medications in the 7 days prior to and after vaccination and challenge. Animals were primiparous to 3rd parous, had no history of metritis, and had not been culture positive for T. pyogenes pre-challenge (days -1 or 0). Animals that developed clinically significant complications during the study were withdrawn.

[0336]

[0328] Animals should be kept in a cool, dark place for at least a minimum of each 24-hour period, except when they are being milked. All animals were allowed free access to food for 20 hours. A formulated basal mixed diet representative of the lactation industry was used. The animals were allowed to acclimate for at least 7 days before the start of the study. The combined vaccine administered to the cows (n=20 / group) contained the following components: T01 - saline; T02 - TXO + native pyolysin (nPLO); T03 - TXO recombinant pyolysin (rPLO). Recombinant pyolysin was obtained by cloning, expression, and purification of the antigen from Corynebacterium glutamicum. The purified protein was then inactivated by formalin treatment. Native pyolysin expressed and purified from Trueperella pyogenes was also inactivated by formalin treatment. The TXO adjuvant contains CpG oligonucleotides in a mixture of DEAE dextran, mineral oil, and the surfactants Span 80 and Tween 80. was born.

[0337] On the vaccination day, the appropriate IVP (Table 10) was administered by subcutaneous route. Vaccine was administered on day 0 in the neck and on the contralateral side of the neck on day 28. Vaccine administration sites were evaluated for injection site reactions on study days 0, 1, 2, 3, 7, 28, 29, 30, 31, 35, 49, and 77. On the vaccination day, administration sites were evaluated to ensure the absence of swelling prior to vaccine administration. Both sides of the neck were observed on study days 28, 49, and 77. Injection site evaluations were recorded. Rectal temperatures were also measured and recorded on study days 0 (prior to first vaccination), 1, 2, 3, 7, 28 (prior to second vaccination), 29, 30, 31, and 35 of the vaccination period. Rectal temperatures were also measured and recorded on challenge days 0-28.

[0338] Post-vaccination clinical observations were recorded on study days 0, 1, 2, 3, 7, 28, 29, 30, 31, and 35 (vaccination period). In addition, clinical observations were observed and recorded during the challenge period starting on day 49 through day 77.

[0339] Antibody responses to pyolysin were measured by ELISA on study days 0, 28, 49, and the final day of the study (day 77). A hemolysis inhibition assay was also performed on each serum sample. This assay measures anti-pyolysin antibody responses and correlates with biological activity (protection).

[0340] [Table 10]

[0341]

[0332] All cows were synchronized in ovarian cycle before challenge. Progesterone was administered before challenge and daily during the 28-day challenge period. A sterile cannula similar to the breeding cannula was used to infuse 10 mL of Escherichia coli challenge strain and 10 mL of Trueperella pyogenes challenge strain, each collected in a separate syringe, into the uterus of all cows on challenge day 0. The cannula was flushed with 10 mL of sterile medium to ensure complete delivery of the challenge material.

[0342] If at least 60% of the animals in treatment group T01 (control group) developed metritis, The challenge was considered successful if the fetus was >25 years old. The presence of metritis was indicated by the presence of mucopurulent uterine / vaginal discharge with a score of 2 or greater. (This scoring system was adapted from the method described in Sheldon et al., Theriogenology, 65:1516-1530, 2006, where scores of 0 and 1 were considered normal.) The primary variable was the presence of mucopurulent uterine / vaginal discharge with a score of 2 or greater, indicating the presence of metritis. Uterine / vaginal discharge was assessed using a sterile Simcro MetriCheck™ device. Collected using a bacterial cup, the samples were scored starting on challenge day 0 through day 28 (study days 49–77).

[0343] Treatment was considered effective if only T01 cows developed clinical metritis or if the duration and / or day rate of mucopurulent vaginal discharge (score ≥2) was significantly shorter than controls (p=<0.1). If there were no significant differences between groups with respect to duration and day rate of metritis, the occurrence of T. pyogenes isolates from uterine bacterial swabs was compared with the vaccine. The safety of each vaccine was assessed based on injection site evaluation, rectal temperature, and any adverse effects on lactation.

[0344]

[0336] The metritis data collected (with or without the presence of vaginal / uterine discharge; vaginal / uterine discharge score) were summarized for each animal at each time point and used to determine the frequency distribution of each category for each treatment at each time point. The frequency distribution of whether animals were normal / abnormal (normal is a score of 0 or 1, abnormal is a score of 2 or more) for each metritis sign (e.g., vaginal / uterine discharge score) was summarized by treatment and time point. Uterine discharge scores were summarized by treatment using a generalized linear mixed model (Proc Glimmix) with a binomial error distribution and logit link function, whether the animal was previously abnormal (score 2 or more). The statistical model included a fixed effect of treatment and a random effect of batch. Treatment groups were contrasted. This was performed repeatedly for each metritis variable described in this paragraph. If Proc Glimmix did not converge for a metritis variable, Fisher's exact test was used instead of treatment group comparison.

[0345] The duration of abnormal scores (for each metritis variable) was determined for each animal and calculated as "(last abnormal time point-first abnormal time point)+1". The duration of abnormal scores was set to 0 for animals that did not have a time point at which the metritis variable was an abnormal score. For animals that left the study before the last planned data collection time point for the metritis variable, the duration of abnormal scores was calculated as "(last planned data collection time point-first abnormal time point)+1". The duration of abnormal scores (for each metritis variable) was log-transformed and then analyzed by a generalized linear mixed model with fixed effect: treatment, and random effect: remaining. Linear combinations of parameter estimates were used for a priori contrasts after testing for significant (p≦0.10) treatment effects. Comparisons were made between treatments. Back-transformed least squares means, their standard errors, and 90% confidence intervals were calculated for each treatment group from the least squares parameter estimates obtained from the analysis.

[0346] The proportion of days with abnormal scores (for each metritis variable) and the proportion of days with both normal E. coli and T. pyogenes absent from the excrement (absence considered a value <=1+) were determined for each animal. Each was then transformed using an arcsin square root transformation prior to analysis. These transformed percentage of days variables were then analyzed with a generalized linear mixed model with fixed effect: treatment, and random effect: remaining, respectively. Linear combinations of parameter estimates were used for a priori contrasts after testing for significant (p≦0.10) treatment effects. Comparisons were made between treatments. Back-transformed least squares means, their standard errors, and 90% confidence intervals were calculated for each treatment group from the least squares parameter estimates obtained from the analysis. Animals were assessed for the presence of E. coli (presence determined as a value >1+), T. pyogenes (presence determined as a value >1+), and both E. coli and T. pyogenes. The frequency distribution of whether both were present (presence was determined as a value >1+) by treatment at each time point. Summarised.

[0347] Results. Antibody responses to pyolysin were assessed by ELISA to measure serum IgG levels. Results (Table 11), expressed as least squares mean (LSM) titers, show that the titers were significantly higher in T02 and T03 cows compared to T01 on study days 28, 49, and 77. They also suggest that there was no statistically significant difference in titers between the T02 and T03 groups. With regard to intrauterine antibody titers, also assessed by ELISA, results (Table 12) demonstrate that there were significantly higher titers in T02 and T03 cows on days 49 and 77 compared to T01 on those days. With regard to hemolysis inhibitory antibodies, results in Table 13 show that T02 animals had significantly higher titers than T01 and T03 animals on study days 49 and 77.

[0348] [Table 11]

[0349] [Table 12]

[0350] [Table 13]

[0351]

[0340] With regard to the primary variable evaluated, the level of mucopurulent uterine / vaginal discharge (Vaginal Discharge Score or VDS), the duration of metritis was measured and was significantly shorter in group T02 compared to groups T01 and T03 measured on days 7 and 10 after bacterial challenge (Tables 14, 15).

[0352] [Table 14]

[0353] [Table 15]

[0354] Metritis was evident within 10 days after challenge (i.e., VDS > Regarding the percentage of days (Tables 16 and 17), it is clear that the T02 group had fewer abnormal days compared to the T01 and T03 groups. It was demonstrated that the most abundant isolates were from T02 (data not shown). Therefore, the vaccine effect was most prominent in group T02 (native pyolysin + TXO).

[0355] [Table 16]

[0356] [Table 17]

[0357] An additional study was conducted to evaluate the efficacy of an experimental metritis vaccine in a novel adjuvant formulation in pregnant dairy cows. In this study, pregnant cows were vaccinated during the dry period. Efficacy was measured during the first 10 days after parturition (calving).

[0358]

[0343] Pregnant Holstein or Holstein cross cows in lactation 1-3 were selected for the study. All cows selected were in good overall health, had no history of metritis, and had no known All animals had a known due date for parturition. The animals were also free of any comorbidities and had not received any chemotherapy, systemic antibiotic medications or other anti-inflammatory medications prior to or for 7 days after vaccination. Animals that developed clinically significant complications at any time during the study were withdrawn. Animals had free access to food for at least 20 hours of each 24-hour period, except during the lactation period only, during the course of the study. Animals also had free access to water during the study.

[0359]

[0344] The vaccines administered to the groups (n=15 / group) were as follows: animals in T01 received 2 ml of vaccine consisting of saline; animals in T02 received 2 ml of vaccine consisting of ISCOMS / Poly I:C+nPLO; animals in T03 received 2 ml of vaccine consisting of TXO+nPLO; animals in T04 received 2 ml of vaccine consisting of TXO+Escherichia coli+Trueperella pyogenes+nPLO (all vaccine antigens were formalin inactivated).

[0360]

[0345] Upon arrival, animals were allowed to acclimate for 7 days. Approximately 2 months prior to calving (study day 0), animals received their first vaccination subcutaneously on the left side of the neck (Table 18), except for animals in group T02, which received the vaccine intranasally. 28 days later, all animals received a second vaccination subcutaneously on the right side of the neck (Table 18). All cows were dry off since their first vaccination.

[0361] [Table 18]

[0362] Beginning on the day of delivery and continuing for 21 days thereafter, the presence of uterine / vaginal discharge was assessed and, if present, a score was collected and assigned, with a score of 2 or greater indicating the presence of metritis. Approximately 30 mL of blood was collected (on study days 0, 28, and 49) for measurement of antibody responses to E. coli, T. pyogenes, and pyolysin by ELISA. Any adverse reactions other than those obtained as part of procedural data collection were recorded.

[0363] The primary variable was the presence of mucopurulent uterine / vaginal discharge, with a score of 2 or greater indicating the presence of metritis. Treatment was considered effective if only T01 cows developed clinical metritis or if the duration of mucopurulent vaginal discharge (score ≥ 2) was significantly shorter than controls (p = < 0.1). Mucopurulent discharge, if present, was collected after calving.

[0364] At each time point, comparisons between treatments were performed. Least squares means (for serological data) The mean mean (w / w, back transformed for each group), its standard error, and 90% confidence intervals were calculated from least squares parameter estimates obtained from the analysis. The range and number of animals according to the data were calculated in each treatment group at each time point.

[0365] The collected metritis data (presence or absence of vaginal / uterine discharge; vaginal / uterine discharge score; clinical signs) were summarized for each animal at each time point and used to determine the frequency distribution of each category for each treatment at each time point. The frequency distribution of animals as normal / abnormal (normal is a score of 0 or 1, abnormal is a score of 2 or more) for each metritis sign (e.g., vaginal / uterine discharge score) was summarized by treatment and time point. Uterine discharge scores were summarized whether the animal was previously abnormal (score 2 or more) or not and analyzed using a generalized linear mixed model (Proc Glimmix) with a binomial error distribution and a logit link function. The statistical model included a fixed effect of treatment and a random effect of batch and blocks within batch. Treatment groups were contrasted (this was done repeatedly for each metritis variable described in this paragraph). If Proc Glimmix did not converge for a metritis variable, Fisher's exact test was used instead of treatment group comparison.

[0366] The duration of abnormal scores (for each metritis variable) was determined for each animal and calculated as "(last abnormal time point-first abnormal time point)+1". The duration of abnormal scores was set to 0 for animals that did not have a time point at which the metritis variable had an abnormal score. For animals that left the study before the last planned data collection time point for the metritis variable, the duration of abnormal scores was calculated as "(last planned data collection time point-first abnormal time point)+1". The duration of abnormal scores was analyzed with a generalized linear mixed model with fixed effects: treatment, and random effects batch, block within batch, and remaining. Linear combinations of parameter estimates were used for a priori contrasts after testing for significant (p≦0.10) treatment effects. Comparisons were made between treatments. Least squares means, their standard errors, and 90% confidence intervals were calculated for each treatment group from the least squares parameter estimates obtained from the analysis.

[0367] Results. All cows that delivered twins were withdrawn from the study because they may have a predisposition to metritis and skew the data. Among the withdrawn cows were 6 cows in the control group T01, 2 cows each in groups T02 and T03, and 1 cow in group T04. The metritis incidence and estimated days of metritis were calculated for the remaining cows in each group. As seen in Table 19, the metritis incidence in groups T03 and T04 was numerically lower compared to the other groups. The data also showed that groups T03 and T04 had a shorter metritis period than animals in groups T01 and T02 during the first 10 days after calving. Therefore, it can be concluded that native pyolysin, whether alone or in combination with E. coli and T. pyogenes, is effective in reducing the natural metritis incidence in cows when adjuvanted with TXO.

[0368] [Table 19]

[0369] Example 5. Metritis vaccine in cattle E. coli bacterin J-5 is a known antigen for treating metritis. In this study, different adjuvants in combination with J-5 bacterin were evaluated for their anti-metritis effects. .

[0370]

[0353] The study design is summarized in Table 20. Calving occurred approximately on day 49. Blood and milk samples were collected on days 0, 7, 28, 35, 49, 63, 70 and 84. Cows were challenged on day 70.

[0371] [Table 20]

[0372] The duration of infection caused by E. coli in groups T01-T06 was as follows: T01 252.1 hours, T02 213 hours, T03 191.6 hours, T04 190.2 hours, T05 198.7 hours. Treatment with VACCIMAX® resulted in the shortest duration of infection. VACCIMAX® is an oil-in-water emulsion containing multilamellar liposomes in which the antigen is encapsulated between the bilayers of the liposome.

[0373] The protective effect of the treatment was also estimated using the stratified mitigated fraction. The higher the stratified reduction rate, the greater the protective effect. The formulation with VACCIMAX® also had the greatest effect (13.9% of the control). 5-17.19-fold), and treatment with TXO was also effective (6.24-fold over the control).

[0374] Total antibody response of serum J-5 specific IgG in whole cells was measured using an indirect capture ELISA. The results are summarized in Tables 21 and 22.

[0375] [Table 21]

[0376] [Table 22]

[0377] Example 6: Neospora Caninum Vaccine Neospora Caninum is a coccidian parasite that was identified as a species in 1988. It is an important cause of spontaneous abortion in infected livestock. In addition to being an important cause of births, it is also a significant disease of dogs worldwide. If the disease is identified early, dogs can be successfully treated with clindamycin and other antiprotozoal drugs. However, the disease is often fatal to young dogs. A preventative vaccine has been tested in cattle. An inactivated vaccine has been commercially available with mixed results. A live vaccine using attenuated N. caninum tachyzoite has been more successful but is expensive to produce. In this study, we determined the effect of different adjuvants on the properties of a vaccine against N. caninum using N. caninum cyclophilin (NcCYP) and profilin (NcPro) as antigens.

[0378]

[0358] 8-10 week old female BALB / c mice were used in this study. All animals were Mice were immunized twice with rNcCyP and rNcProf, three weeks apart, in the presence of the indicated adjuvants. Three weeks after the second immunization, all animals were euthanized and spleens and blood were harvested. NcCyP / NcProf-specific splenocyte proliferation responses were measured by proliferation assay (days 3-4). NcCyP / NcProf-specific splenocyte cytokine responses were measured by stimulating splenocytes with Neospora antigen for 48 hours and supernatant cytokine levels were measured by cytokine-specific ELISA. Serum antibody levels were measured by ELSIA. Animals were treated as summarized in Table 23.

[0379] [Table 23]

[0380]

[0359] Characteristics of the above treatment groups are summarized in Table 24.

[0381] [Table 24]

[0382]

[0360] Taken together, these data demonstrate the superior results obtained with TXO and TCMO.

[0383] Example 7. Effect of Chlamydophila abortus on the immune response to genital infection The effect of different adjuvants C. abortus is an intracellular bacterium that causes abortion in sheep and goats. Infection generally occurs when unprimed ewes are exposed to abortive material (e.g., placenta, body fluids, fetus). The bacteria remain latent in the infected ewe's bay until parturition and during mid- or late gestation, are present in the placenta, and are expressed in the placenta when an antibody response is mounted. After abortion, ewes typically avoid reinfection.

[0384] It is believed that vaccination may be advantageous pre-exposure to prevent primary infection and to prevent homing of the bacteria to the placenta. IFNg associated antibody responses in post-abortion immunity are important correlates of protection. IFNg may also be associated with persistence observed in non-pregnant ewes.

[0385] Ewes were vaccinated on days 0 and 28 and challenged on day 49. Animals were sacrificed on day 63 and necropsies were performed. Vaginal and whole blood samples were taken for qPCR on day 0. Blood was sampled weekly for serological results and on days 0, 7, 28 and 35 for cytokine and Elispot measurements.

[0386]

[0364] Treatment groups are shown in Table 25.

[0387] [Table 25]

[0388] The antigen was prepared from aborted fetal sheep kidney and expanded in McCoy cells. Elementary bodies were purified by centrifugation and sonication. Antigens were fixed at 100 μg / dose with 0.1% formaldehyde in 0.9% sodium chloride for vaccination.

[0389] [Table 26]

[0390] Serological results were obtained using the Chek-it ELISA kit and are listed in Table 26 above. It summarises.

[0391]

[0367] IFNg, IL-2 and IL-4 expression levels were measured in ovine PMBC stimulated with Chlamydia AG. The results are shown in Table 27.

[0392] [Table 27]

[0393] The response of sheep PBMC to Chlamydia abortus antigens is summarized in Table 28. .

[0394] [Table 28]

[0395]

[0369] In addition, the amount of white blood cells was analyzed (data not shown). Two-way ANOVA showed that group F had significantly higher WBC amount than groups A and B, and group E had significantly higher WBC amount than group B.

[0396]

[0370] Nodules upon injection were also analyzed. As expected, groups A-C had larger nodules than groups C-D. Of the three adjuvants used (groups A-C), group C had the largest nodule size, followed by group B, then group A.

[0397] Nodule volumes were measured. Groups A-C also had larger nodule volumes than groups D-F. Of groups A-C, group A had the smallest volume. Groups A and B nodules had more hemorrhagic and / or necrotic tissue. Group C nodules had more fibrosis. Cellular characteristics were similar in all three groups, although group C may have more lymphocytic components.

[0398] Example 8. Addition of aluminum to TXO results in improved stability The current TXO blend formulation contains 50 mg / ml DEAE dextran. DEAE-dextran may induce reactions at the injection site in animals if present in high concentrations during subcutaneous injection. Therefore, it is proposed to test DEAE-dextran at various concentrations to check whether it can provide safety and good therapeutic value without compromising the stability of the vaccine formulation.

[0399] Characterization and stability testing is important as it informs whether the vaccine can be formulated consistently and with good manufacturing shelf life. Viscosity tests are performed over a range of shear rates to explore the flow properties of non-Newtonian fluids: shear thinning (as shear rate increases, apparent viscosity decreases) or shear thickening (as shear rate increases, apparent viscosity increases). Syringe force tests are performed to ensure the vaccine is easily drawn and can be easily dispensed at the farm in several doses. Ensure that the drug is administered at the appropriate dose.

[0400]

[0374] Immunostimulatory oligonucleotides were not added to the adjuvant mixture in this example because they are not expected to change the stability of the formulation. AXO (aluminum + dextran + oil) blends with various REHYDRAGEL® (5%-16%) and DEAE dextran (50mg / ml-10mg / ml) concentrations are formulated and tested for viscosity, syringe force and sedimentation, using XO (dextran + oil) blends as controls. The compositions tested were as follows:

[0401] Approximately 10 ml of sample was poured into each of five 15 ml Corning centrifuge tubes. The samples were filled and left for a week to observe the accelerated settling of the emulsion due to the narrow dimensions and conical bottom of the centrifuge tubes. Samples were also tested for syringeability and viscosity. The results are shown below.

[0402] [Table 29]

[0403]

[0376] These data indicate that the blend with 16% REHYDRAGEL® is the most stable when subjected to accelerated precipitation in centrifuge tubes. Furthermore, from previous work by the inventors, it is known that higher DEAE Dextran concentrations are associated with higher viscosity and possible shear thickening. The results of these experiments indicate that the addition of more REHYDRAGEL® compensates for the predicted loss of shear thickening (pseudoplasticity) imparted by DEAE Dextran. 16% REHYDRAGEL® It was also observed that even though the formulation had a higher syringe force, injection was not significantly more difficult (syringe force of 3 N for water).

[0404] [Table 30]

[0405]

[0377] From the overall data it is apparent that a blend of 16% REHYDRAGEL® and 10 mg / ml DEAE dextran is optimal for use in vaccine formulations, particularly those requiring endotoxin-free binding and / or where a longer emulsion shelf life may be desirable.

[0406] Example 9. BRV, BCV and E. coli antigens In this example, we consider the use of the adjuvant of the present invention in a vaccine against enteritis. Enteritis is caused by bacterial, viral, and / or parasitic infections. Cattle, especially newborn dairy and beef cattle, are susceptible to calf diarrhea because they are under a lot of stress during the first few hours of life when their immune systems are not fully developed. Fluid loss from calf diarrhea leads to dehydration and often death. Animals that survive calf diarrhea are often still vulnerable and perform poorly throughout their lives. Pathogens associated with diarrhea include bacteria, especially E coli K99 and F41, and viruses, such as bovine coronavirus. Examples of such viruses include bovine rotavirus (BCV) and bovine rotavirus (BRV).

[0407] Ten month old Holstein steers were used in this study. The animals were seronegative for E coli (K99 and F41), BRV (B223 and Lincoln), and BCV. or were low tittered.

[0408]

[0380] The treatment groups were as follows:

[0409] [Table 31]

[0410] Blood samples were collected every 21 days for 6 months for serological testing. Injection site reactions were measured on days 0 (pre-vaccination), 1, 2, 3, 7, 14, 21, and every 21 days thereafter. Responses to E. coli K96, E. coli F41, BRV Lincoln, BRV B223, and BCV were measured by quantitating antibody titers on selected days. The results are summarized below (different letters indicate differences at α=0.1):

[0411] [Table 32]

[0412] Treatment with T05 and T06 resulted in the highest antibody titers from day 21 through the end of the study (day 189). Of note, the commercial vaccine (ROTAVEC®) outperformed T05 and T06 in inducing antibodies against the viral component of enteritis. did not work to the same extent.

[0413] [Table 33]

[0414]

[0383] Treatments T02 and T05-T06 induce responses to K99 Treatment with T02 induced the best response against E. coli F41. Treatment with T05 was second most effective in eliciting responses to that antigen.

[0415] Taken together, these data indicate that T05 and T06 appear to be the most promising formulations. Both provided targeted IgG responses with multiple fractions through day 189. Both T05 and T06 were superior to ROTAVEC® (T0 2, IM). T03 and T04 maintained high serum titers for a shorter period of time than T05 and T06. T03, T04, T05 and T06 were able to immunize BRV G6, BRV 1, and BRV 2 with a single dose of vaccination. G10 and BCV. T04, T05 and T06 provided serum titers above target levels against E. coli K99 with a single dose of vaccination. T04, T05 and T06 maintained anti-viral serum titers above target levels for 6 months. T05 and T06 maintained anti-E. coli K99 serum titers above target levels for 6 months. All formulations evaluated provided sufficient Safety has been proven.

[0416]

[0385] Rectal temperatures were measured on days 0, 1, 2, and 3. There was a statistically significant difference between group T01 (control) and groups T02-T06, but the temperature differences (LSM) were not large (within 1°F).

[0417] [Table 34]

[0418]

[0386] Preliminary testing of the formulation in pregnant dairy cows has demonstrated safety. Groups T01, T03 and T05 were tested with 5 cows per group. Thirteen of the 15 cows gave birth, 12 were normal and one was stillborn.

[0419]

[0387] Example 10. Anti-tick vaccine Experimental design Two vaccine formulations based on the Bm86 antigen were tested: one formulation contained an aqueous adjuvant (QCDCRT) and the other contained an oil-based adjuvant (TXO), as summarized below.

[0420] [Table 35]

[0421]

[0388]

[0422] Twenty-four calves were randomly assigned to one of three treatment groups of eight calves each. Calves in each treatment group were individually vaccinated with 2 cc of one of the two Bm86+adjuvant formulations or saline (control group). Vaccinations were performed on days 0 and 28, and calves were placed in stanchions and vaccinated with R. annulatus larvae 25 ml on day 42. The animals were infected with 0.0 mg of mites. The ticks used in this study were originally collected on a farm in Val Verde County, Texas. All adult female ticks that had bitten were removed and collected from each calf daily on days 63-84. On day 85, the calves were removed from the stanchions. Collected ticks were counted and up to 10 ticks were weighed from each calf on each of the 13 collection days and placed in an environmental chamber. 14 days after collection, weakened females were discarded and eggs laid were weighed. 14 days after first hatching, the number of hatched intact eggs was recorded and a measure of % hatchability was calculated. Before each vaccine injection and for 3 days after each subsequent injection, each calf was monitored for swelling at the injection site and rectal temperatures were measured. Serum was collected from each calf on days -7, 0, 14, 28, 42 and 85 for measurement of Bm86 antibody titers throughout the study period as measured by ELISA.

[0423]

[0390] Results Preliminary results show 98.6 and 99.6% control from T02 and T03 formulations respectively, significantly higher than T01. These % control calculations only take into account the reduction in weight of bitten females and eggs. The reduction in % hatching will be measured at a later date and added to the final results. One of 24 calves in the study develops a small swelling after each injection (formulation with oily adjuvant). The swelling is less than 10 cm in length and less than 3 cm deep. The swelling is soft and not painful to the animal. There is no increase in rectal temperature of treated animals throughout the study.

[0424] Serological results demonstrate statistically significant differences between treatments at each time point, except for the 14-day time point where there was no statistical significance between treatments with QCDCRT and TXO (p=0.114). Both effectively increased BM86 antibody titers at each time point tested. TXO was superior (p<0.05) to QCDCRT at each time point tested except day 14 (p=0.114).

[0425] [Table 36]

[0426] Example 11. Foot and mouth disease (guinea pigs) The objective of this study was to compare humoral immune responses in guinea pigs vaccinated with trivalent FMD vaccines adjuvanted with different adjuvant formulations. Guinea pigs were vaccinated on days 0 and 28 as summarized in Table 37.

[0427] For each dose of T03-T07, the antigens were a combination of FMVD O (9 μg), A (5 μg) and Asia 1 (5 μg / ml). The antigen composition for T02 was proprietary manufacturer information and therefore was not available.

[0428]

[0413] Blood samples were taken for serological testing on days -3, 25 and 53. Serum titers of antibodies to serotypes O, A and Asia1 are summarized below.

[0429] Responses to serotypes O and A were also low in the positive control group (T02), but responses to Asia1 were higher in T07 (TXO adjuvant) than in the positive control group and greater than in any other treatment. The low responses to serotypes O and A may be abolished by the presence of low levels of O and A antigens in the formulation.

[0430]

[0413] Of note, the liposome-based VACCIMAX® groups (T03-T05) did not show significant responses to any of the antigens (O, A, Asia1).

[0431] [Table 37]

[0432]

[0397]

[0433] [Table 38]

[0434] Example 12. Foot and mouth disease (cattle) In this study, the effect of different adjuvants used in a vaccine against FMD in a challenge model was measured. Three adjuvants were tested. The vaccine was the ARS experimental vaccine against FMD in a challenge model developed by PIADC. FMD-LL3B3D-A24 Cruzeiro used both as the antigen component of the vaccine (10 μg). and wild-type FMDV A-24 Cruzeiro was used as the challenge virus. Antigens have been previously described, for example in U.S. Patent Application Publication No. 20120315295 (Rieder et al., filed June 9, 2011, published December 13, 2012). Briefly, FMD-LL3B3D-A24 Cruzeiro comprises a genetically modified FMDV (foot and mouth disease virus). The FMDV is a genetic It is genetically modified, i.e., the leader (L pro It is a leaderless virus that contains a deletion of the coding region of the 3B protein, such that the FMD virus lacking this protein is attenuated in cattle and pigs. It also contains mutations (negative markers) introduced into two nonstructural viral proteins, thus eliminating two antigenic epitopes recognized by specific antibodies, one located in protein 3B and the other in protein 3D (which are replaced in these proteins by the corresponding sequences of bovine rhinovirus acting as negative antigenic epitopes), thus providing two potential targets for the DIVA (Differentiation of Naturally Infected and Vaccinated Animals) serological test.

[0435]

[0399] In each group, 4-7 cattle were used. The total volume of injection was 2 ml. The animals were vaccinated with wild type FMDV by IM injection (2.0 ml / dose) on day 0 and challenged by intradermal route on day 21. Clinical scores were assessed on days 0, 3, 7, and 10 according to the following scale: no clinical signs: 0, vesicular foot lesions: 1 point for each foot involvement. The maximum score is 4. The experimental results are as follows:

[0436] [Table 39]

[0437]

[0400] The differences between T01 and T02 and between T01 and T04 were statistically significant.

[0438] From the above table, it can be concluded that the adjuvants TXO and MONTANIDE® ISA 206 VG are approximately equally effective, at least based on the clinical scores. However, serological analysis measuring serum neutralizing activity against FMDV-A24 showed that T04 (adjuvant TXO) had a higher potency than T02 (MONTANIDE® ISA 206 VG). It is demonstrated that the invention has value.

[0439] [Table 40]

[0440]

[0402] These results demonstrate that the TXO adjuvant was able to provide 100% protection in cattle against challenge with the FMD-causing pathogen and conferred higher antibody titers than the saline control and the other two adjuvants tested.

[0441]

[0403] FMDV RNA (copies / mL) was measured in nasal swabs and serum The data are shown in Tables 41-44. In brief, these data demonstrate that the T02 and T04 groups produced lower amounts of FMDV in the nasal swabs. Of the T02 and T04 groups, it is notable that the animals in the T04 group showed a faster decrease (or lack of increase) in FMDV RNA load, thus demonstrating the superior properties of the adjuvant TXO.

[0442] [Table 41]

[0443] [Table 42]

[0444] [Table 43]

[0445] [Table 44]

[0446] All animals in group T01 developed fever after challenge, whereas none of the animals in group T04 had fever. The response in groups T02 and T03 was inconsistent (some animals developed fever, others did not). This observation confirms the conclusion that TXO is generally superior to other adjuvants used in this study.

[0447] Example 13: TXO activates cellular immunity The effect of the adjuvant on cellular immunity was analyzed using FMD as an exemplary antigen in the animal model described in the previous examples. Peripheral blood mononuclear cells (PBMCs) were purified from bovine whole blood collected on days 4, 7, 14 and 21 after vaccination. FMDV-specific T cell proliferation responses were evaluated using carboxyfluorescein diacetate succinimidyl ester (CFSE) staining.

[0448]

[0406] The results are shown in Table 45.

[0449] [Table 45]

[0450] These data demonstrate the beneficial effect of TXO on cell-mediated immunity at both days 14 and 21. These data also suggest that the adjuvant TXO may potentially be a factor in the progression of inflammatory bowel disease, as cell-mediated immunity is responsible for duration off immunity. It has shown the potential to provide longer-lasting immunity than ISA 206 VG.

[0451] Example 14. Generation of antibodies for diagnostic use The adjuvant TXO of the present invention was used to generate antibodies for diagnostic use. Briefly, feeder animals were immunized every 2-4 weeks with a formulation containing selected recombinant antigens supplemented with TXO, with the following composition: SEQ ID NO:8 125 μg; DEAE dextran 125 mg; mineral oil 46.56% v / v of the formulation; TWEEN® 80 1.5% v / v of the formulation; SPAN® 80 6.518% v / v of the formulation.

[0452]

[0409] The final volume was 2 ml.

[0453]

[0410] A small visible injection site reaction was observed following injection, but was within the expected reaction size. The reaction observed above the ribs was not believed to be painful to the goat based on routine observations.

[0454]

[0411] Blood samples were taken 2-3 weeks after each immunization and various assays were performed to assess antibody titers. A serological ELISA titer of over 1000 was deemed sufficient to initiate antibody collection.

[0455]

[0412] Animals were bled weekly (7.5% of blood volume based on body weight). At the end of the study, goats were euthanized by terminal exsanguinations and blood was also collected for antibody collection. Animals were reused if necessary or for further testing.

[0456] [Table 46]

[0457] Serum antibodies to FeLV gp70 were successfully purified using either Protein A or Protein G columns in-house for small scale purification, or using Protein G chromatography for large scale purification at Maine Biotechnology Services (MBS), Portland, ME. Polyclonal antibodies were concentrated to a final concentration of approximately 1 mg / ml using Millipore 30K Ultra Filter Units. Antibodies to other antigens disclosed in Table 46 were not purified.

[0458]

[0414] Antibodies were isolated from milk obtained from naturally lactating goats immunized with FeLVgp70 by a method comprising the following steps: a) The pH of the milk was titrated to 4.6 with 2M HCl and incubated at room temperature for 30 minutes for casein precipitation. Stirred; b) the milk was centrifuged at 17,000 × g for 30 minutes and the supernatant was collected; c) equilibration buffer was added to the supernatant to yield 3.3 M NaCl, 0.3 M glycine and 0.2 M Tris base; d) the supernatant was clarified by centrifugation at 3000 × g for 15 min; e) the clarified milk supernatant was applied to the MabSelect column equilibrated with buffer in step "c"; f) The column was washed with equilibration buffer and eluted with 0.15 M glycine pH 3.0; g) The eluted fraction was neutralized with 0.2 M sodium phosphate.

[0459]

[0415] As a non-limiting example, methods for making anti-PI-3 and anti-FeLV gp70 are provided below.

[0460] One of the objectives was to generate goat polyclonal antisera to purified bovine parainfluenza-3 (BPI-3) HN protein for use in an in vitro assay. The study was set up to vaccinate goats with purified bovine parainfluenza-3 (BPI-3) HN protein formulated with TXO-adjuvanted BPI-3 HN protein used as the antigen.

[0461] Approximately seven weeks after the first injection, all four goats had serum antibody concentrations high enough (SN>1000) to initiate production bleeds for serum. ) were determined to have a BPI-3 HN-based antibody titer of 1. Production bleeds began one week after the fourth immunization. Blood was collected for serum once a week at three week intervals. Serum from each goat was pooled with its individual production bleed. A total of 3,187.50 mL of serum was collected approximately three weeks into the production bleed. Serum was processed and stored at -80°C for evaluation in the BPI-3 HN-based assay.

[0462] All serum collected from three goats (#30, 31 and 35) was thawed to room temperature. Serum from goat #34 was not used due to low antibody response in screening ELISA. See Table 47 (PI-3 HN Polyclonal Antibody Production: SN Response and Antigen Potency ELISA).

[0463] Goat 35, harvested on Nov. 20, 2013, had a minimum volume of 117 mL of serum available. Therefore, the 117 mL from each goat per harvest was pooled into sterile 1 L Nalgene PETG bottles. Approximately 1053 mL of serum (9 x 117 mL) was distributed into twenty 50 mL aliquots and fifty 1.0 mL aliquots in 60 mL Nalgene PETG bottles.

[0464] [Table 47]

[0465]

[0420] As a result, upon completion of this study, a total of 3,187.5 mL of whole blood was successfully generated from four goats repeatedly immunized with purified BPI-3 protein formulated with TXO over a three week period of production bleeds. Good polyclonal antibody titers were generated in the serum. Sufficient amounts of purified reagent were obtained for use in in vitro assay applications.

[0466] In 2010, the USDA banned LEUKOCELL and VERSIFEL. The industry was notified that the FeLV gp85 / 70 capture reagent used in the FeLV gp70 (FeLV Gp70) assay would no longer be supplied. Therefore, the purpose of this study was to generate goat polyclonal antisera to recombinant FeLV gp70 protein for use in an in vitro assay.

[0467] Previous attempts to generate antibodies after vaccination with Freund's adjuvant have been unsuccessful. This study used recombinant E. coli-expressed FeLV in an adjuvant. A 444 amino acid fragment of gp70 protein was designed to inoculate goats. Starting with the fourth injection, the injection dose was reduced to 100 μg FeLV gp70 protein (instead of the original dose of 282 μg FeLV gp70 protein). The dose was modified because the initial dose of 282 μg / ml frequently caused injection site reactions. The dose was initially reduced to 100 μg / ml, but then increased to 150 μg / mL for the seventh immunization and maintained at that level until the end of the study (a total of 15 immunizations). The difference was adjusted using PBS buffer to maintain the administration volume at 1 mL.

[0468]

[0423] Blood was collected from the goats and once sufficiently high antibody titers were measured by direct and sandwich ELISA, serum was collected and polyclonal antibodies were purified.

[0469] Six healthy female goats of the LaMancha and Alpine breeds, 1-3 years old and weighing over 100 pounds, were obtained for use in this study. During the study, the goats consumed hay and grain and had water ad libitum. General health observations were performed once daily. A 1 mL dose of the experimental vaccine was administered subcutaneously to each goat at 21-day intervals, for a total of 15 immunizations in each of the five goats that completed the study. Immunizations were initially administered to the neck or hind leg, with subsequent immunizations alternating sides and sites. Small visible injection site reactions were observed following immunization. Immunizations administered to the loose skin on the cranial side of the right hind leg of the goats were reported to produce small swelling, tenderness, and moderate lameness the next day in all goats. Subsequent injections were administered to the other side of the neck or the area above the ribs and were generally well tolerated. However, the area above the ribs was ultimately found to be the location best tolerated by the goats.

[0470] Approximately 8 weeks after the first injection, 4 of 6 goats (21, 22, 24, 25) were determined to have serum antibody concentrations high enough to initiate production bleeds for serum. Production bleeds from the remaining 2 goats (23, 26) began 5 weeks later. Blood was collected for serum at weekly intervals. Goat #25 was removed from the study 6 weeks after production bleed. The goat was lame on arrival and continued to exhibit lameness despite Banamine treatment. Euthanasia was indicated for the final bleed and treated at sites to ensure maximum collection. Serum from each goat was pooled with its individual production bleed. A total of 26.7 L of serum was collected over the approximately 7 months of production bleeds.

[0471] Unexpectedly, goat #24 developed a pseudopregnancy during the study. Milk was collected from this goat for over 3 months, making a total of 300 L of milk available for antibody collection. A protocol was developed for high-level purification of FeLV gp70 polyclonal antibodies from milk.

[0472] On two separate days, antibodies were isolated from 500 mL of pooled serum from goat 24 by Protein G affinity chromatography at Maine Biotechnology Services. A total of 6388 mg (321 mL of 19.9 mg / mL) and 7343 mg (348 mL of 21.1 mg / mL) of purified goat anti-FeLV gp70 antibody were prepared for evaluation in FeLV-based assays.

[0473]

[0428] 14 days after each vaccination, blood samples (approximately 25 mL / sample) were collected into 12.5 mL serum separator tubes (SST) to measure antibody concentrations to FeLV gp70. The SST was labeled with the goat ID and collection date. Once the animals had acceptable levels of FeLV gp70 antibody titers based on ELISA signal intensity as determined by the assay, production collections were initiated from the animals. The volume of blood to be extracted from each goat was determined based on the goat's weight to obtain a maximum blood volume. IACUC guidelines allow collection of up to 7.5% of the blood volume once a week.

[0474]

[0429] Blood was collected at 12.5 mL SST for production collection. At the end of the study, goats were euthanized by terminal bleeding and blood was also collected for antibody collection. All test tubes were labeled with the goat ID and collection date.

[0475]

[0430] Blood was left at room temperature to clot. After centrifugation, serum was collected and transferred to polypropylene vials. Serum from the same goat at different SSTs on the same collection day was pooled. Serum was kept on ice until transported for purification. A production summary is shown in Table 48.

[0476] [Table 48]

[0477]

[0431] Goat number 24 produced serum with the highest antibody concentration of all the goats.

[0478] Purified serum antibodies from goat 24 showed a similar dose response compared to USDA 94-06 as a capture reagent using FeLV gp70 C11D8 detection mAb in a sandwich ELISA assay. Purified serum from goat 24 was compared to USDA 94-06 by Western blot for its ability to detect FeLV gp85 / 70 protein. Similar Western blot results were obtained with the current capture 94-06 and the new capture 24 goat, except that an additional approximately 15 kD band was observed in the capture 24 goat. Data with the new capture antibody showed that the current reference had a different dose-response curve shape than the current reagent when used to capture the reference and a range of analytes tested.

[0479] Both anrendati-FeLV gp70 purified from serum and milk were found to be It performed well as a capture reagent in an ELISA assay.

[0480]

[0434] Further trials are ongoing as shown in Table 49.

[0481]

[0435] Each of the formulations (antigens with TXO as disclosed in Table 49) is expected to induce a serum titer sufficiently high (greater than 1000, or more preferably greater than 5000, or more preferably greater than 10000, or more preferably greater than 50000, or more preferably greater than 100000, or more preferably greater than 250000, or more preferably greater than 500000, or more preferably greater than 1000000) in at least one animal (preferably at least two, or more preferably at least three, or most preferably each animal treated) and thus provide sufficient amounts of antibodies for diagnostic or research applications.

[0482] [Table 49]

[0483]

[0436] All publications cited in this specification, both patent and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. These publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0484]

[0437] Although the invention herein has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. It should therefore be understood that numerous modifications can be made to the exemplary embodiments, and that other arrangements can be devised, without departing from the spirit and scope of the invention as defined in the following claims. Without being limited thereto, the present invention includes the following aspects. [Aspect 1] 1. An adjuvant formulation comprising an oil phase and an aqueous phase, said oil phase comprising at least 50% v / v of said formulation, said formulation comprising at least one of monophosphoryl lipid A (MPL-A) or an analogue thereof and an immunostimulatory oligonucleotide, provided that a) when the immunostimulatory oligonucleotide is not present, the formulation i. Poly I:C, a glycolipid, and optionally a quaternary amine, or ii. Polycationic Carriers Including, b) if the monophosphoryl lipid A (MPL-A) or analogue thereof is not present, the formulation comprises a source of aluminum; 2. An adjuvant formulation, [Aspect 2] the immunostimulatory oligonucleotide, when present, is a CpG or an oligoribonucleotide; said polycationic carrier, when present, is selected from the group consisting of dextran, dextran DEAE (and its derivatives), PEG, guar gum, chitosan derivatives, polycellulose derivatives such as hydroxyethyl cellulose (HEC) polyethyleneimine, polyamino; 2. The adjuvant formulation of embodiment 1, wherein the quaternary amine, if present, is selected from the group consisting of DDA and avridine. [Aspect 3] The glycolipid, when present, has Formula I: [ka] (In the formula, R 1 and R 2 are independently hydrogen or a saturated alkyl radical having up to 20 carbon atoms; X is -CH2-, -O-, or -NH-; R 2 is hydrogen or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R 3 , R 4 , and R 5 are independently hydrogen, -SO4 2- , -PO4 2- , -COC 1-10 R is alkyl; 6 is L-alanyl, L-α-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl, or a D-isomer thereof. [Aspect 4] The adjuvant formulation according to aspect 3, wherein the glycolipid is N-(2-deoxy-2-L-leucylamino-β-D-glycopyranosyl)-N-octadecyldodecanoylamide or a salt thereof. [Aspect 5] 5. The adjuvant formulation according to any one of aspects 1 to 4, comprising both the monophosphoryl lipid A (MPL-A) or an analog thereof, and further comprising at least one of a sterol and poly I:C. [Aspect 6] 6. The adjuvant formulation of aspect 5, comprising the sterol and further comprising a saponin. [Aspect 7] The adjuvant formulation according to any one of aspects 1 to 6, comprising the poly I:C and further comprising at least one of the quaternary amine and the glycolipid. [Aspect 8] 7. The adjuvant formulation according to any one of aspects 1 to 6, comprising an aluminium source which is an aluminium hydroxide gel. [Aspect 9] 9. A vaccine composition comprising an effective amount of an antigen and the adjuvant formulation according to any one of aspects 1 to 8, wherein the oil phase of said composition is at least 50%. [Aspect 10] 1. A vaccine composition comprising an effective amount of an antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase and an aqueous phase, the oil phase comprising at least 50% v / v of the formulation, a polycationic carrier, and a. a combination of a saponin, a sterol, and optionally a quaternary amine; provided that when the adjuvant formulation consists essentially of DEAE dextran, Quil A, cholesterol, and DDA, the antigen is not E. coli J-5 bacterin; or b. immunostimulatory oligonucleotides; provided that when the adjuvant formulation consists essentially of DEAE dextran and immunostimulatory oligonucleotides, the antigen comprises or is derived from a pathogen(s) affecting bovine, ovine, or porcine and is not E. coli J-5 bacterin; 23. A vaccine composition comprising: [Aspect 11] 11. The vaccine composition of aspect 10, wherein the saponin is Quil A, the sterol is cholesterol, the polycationic carrier is dextran DEAE, the quaternary amine is DDA, and the immunostimulatory oligonucleotide is CpG. [Aspect 12] 1. A vaccine composition comprising at least one of an Eimeria maxima or Clostridium perfringen antigen and an adjuvant formulation, the adjuvant formulation comprising: a) an oil phase, a polycationic carrier, and optionally an immunostimulatory oligonucleotide, present in an amount of at least 50% v / v of said composition; or b) an oil phase, an immunostimulatory oligonucleotide, a sterol, and monophosphoryl lipid A (MPL-A) or an analog thereof, present in an amount of at least 50% v / v of said composition; 23. A vaccine composition comprising: [Aspect 13] 13. The vaccine composition of embodiment 12, wherein the polycationic carrier is DEAE dextran. [Aspect 14] 14. Use of a vaccine composition according to embodiment 12 or 13 for the treatment or prevention of infections caused by Eimeria maxima or Clostridium perfringens in poultry. [Aspect 15] 1. A vaccine composition comprising a Neospora antigen and an adjuvant formulation, the adjuvant formulation being present in an amount of at least 50% v / v of the composition; and a) monophosphoryl lipid A (MPL-A) or an analog thereof; or b) combination of immunostimulatory oligonucleotides with polycationic carriers; 23. A vaccine composition comprising: [Aspect 16] 16. The vaccine composition according to embodiment 15, comprising a combination of said immunostimulatory oligonucleotide and dextran DEAE. [Aspect 17] 16. The vaccine composition of embodiment 15, comprising monophosphoryl lipid A (MPL-A) or an analog thereof, and further comprising said immunostimulatory oligonucleotide. [Aspect 18] 18. The vaccine of embodiment 17, further comprising a sterol. [Aspect 19] Use of a vaccine according to any one of aspects 15 to 18 for the treatment or prevention of an infection caused by Neospora. [Aspect 20] A vaccine composition comprising a Chlamydophila abortis antigen and an adjuvant formulation, the adjuvant formulation comprising: an oil phase present in an amount of at least 50% v / v of the composition; a sterol; an immunostimulatory oligonucleotide; monophosphoryl lipid A (MPL-A) or an analogue thereof; and poly I:C. [Aspect 21] 21. Use of a vaccine according to embodiment 20 for the treatment or prevention of abortion caused by chlamydiosis (C. abortis) in ewes. [Aspect 22] 1. A vaccine composition comprising myostatin and an adjuvant formulation, the adjuvant formulation being present in an amount of at least 50% v / v of the composition; an oil phase; an immunostimulatory oligonucleotide; and a) a polycationic carrier; or b) MPL-A or an analogue thereof; A vaccine composition comprising any one of the following: [Aspect 23] 23. The composition of embodiment 22 comprising MPL-A or an analog thereof, wherein the formulation comprises less than 0.5 μg of sterol per 50 μl of the composition. [Aspect 24] Use of a vaccine according to any one of aspects 22 to 23 for reducing the amount of myostatin in an animal. [Aspect 25] 25. The use according to aspect 24, wherein the animal is a poultry animal. [Aspect 26] A vaccine composition comprising a Trueperella pyogenes antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the composition, an immunostimulatory oligonucleotide, and a polycationic carrier. [Aspect 27] 27. Use of a vaccine composition according to embodiment 26 for the treatment or prevention of an infection caused by Trueperella pyogenes. [Aspect 28] 1. A vaccine composition comprising at least one of an E. coli antigen, a BRV antigen or a BCV antigen, and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the vaccine composition, an immunostimulatory oligonucleotide, and at least one of a polycationic carrier and an aluminum source. [Aspect 29] a. the E. coli antigen, if present, is selected from the group consisting of E. coli K99, E. coli F41, and combinations thereof; b. The BRV antigen, if present, is selected from the group consisting of BRV G6, BRV G10, and combinations thereof; 29. A vaccine composition according to embodiment 28. [Aspect 30] 30. The vaccine composition of any one of aspects 28-29, wherein the polycationic carrier, if present, is dextran DEAE; and the immunostimulatory oligonucleotide is CpG. [Aspect 31] 31. The vaccine composition according to any one of aspects 28 to 30, comprising an aluminium source which is an aluminium hydroxide gel. [Aspect 32] 32. The vaccine composition according to aspect 31, wherein the aluminium source is present in an amount of 5% to 20% v / v. [Aspect 33] 33. Use of a vaccine composition according to any one of aspects 28 to 32 for the treatment or prevention of enteritis caused by E. coli, BCV or BRV in a bovine animal. [Aspect 34] 34. The use according to aspect 33, wherein said vaccine induces immunity to said antigen(s) for at least 6 months. [Aspect 35] 1. A vaccine composition comprising a Rhipicephalus microplus antigen and an adjuvant, the adjuvant comprising: a) an aqueous adjuvant comprising an immunostimulatory oligonucleotide, a saponin, a sterol, a quaternary amine, a polyacrylic polymer, and a glycolipid; and b) an oil-based adjuvant comprising an oil phase present in an amount of at least 50% v / v of said vaccine composition, said oil-based adjuvant comprising an immunostimulatory oligonucleotide and a polycationic carrier; A vaccine composition selected from the group consisting of: [Aspect 36] 36. The vaccine composition of embodiment 35, wherein the saponin is Quil A, the sterol is cholesterol, the quaternary amine is DDA, the glycolipid is N-(2-deoxy-2-L-leucylamino-bD-glucopyranosyl)-N-octadecyldodecanoylamide or a salt thereof, and the immunostimulatory oligonucleotide is CpG. [Aspect 37] 36. The vaccine composition of embodiment 35, wherein the polycationic carrier is dextran DEAE and the immunostimulatory oligonucleotide is CpG. [Aspect 38] 38. Use of a vaccine composition according to any one of aspects 35 to 37 for the treatment or prevention of an infection caused by Rhipicephalus microplus. [Aspect 39] A vaccine composition comprising a foot and mouth disease (FMD) antigen and an adjuvant formulation, the adjuvant formulation comprising an oil phase present in an amount of at least 50% v / v of the vaccine composition, an immunostimulatory oligonucleotide, and a polycationic carrier. [Aspect 40] 1. A vaccine composition comprising a foot-and-mouth disease (FMD) antigen and an adjuvant formulation, said vaccine composition comprising an oil phase present in an amount of at least 36% v / v of said vaccine composition, an immunostimulatory oligonucleotide, and a polycationic carrier, and further wherein said vaccine composition is a water-in-oil emulsion. [Aspect 41] 41. The vaccine composition of embodiment 39 or 40, wherein the immunostimulatory oligonucleotide is CpG and the polycationic carrier is DEAE dextran. [Aspect 42] 42. Use of a vaccine composition according to any one of aspects 39 to 41 for the treatment or prevention of FMD in cattle. [Aspect 43] A vaccine composition comprising a Streptococcus uberis (S. uberis) antigen and an adjuvant formulation, the adjuvant formulation being present in an amount of at least 50% v / v of the composition, an oil phase, a polycationic carrier, and a) immunostimulatory oligonucleotides; b) a combination comprising a saponin, a sterol, and a quaternary amine; or c) any combination thereof; 23. A vaccine composition comprising: [Aspect 44] 44. Use of a vaccine according to embodiment 43 for the treatment or prevention of an infection caused by B. suberis.

Claims

1. 1. A method for producing a diagnostic or therapeutic antibody, comprising: immunizing a non-human source animal with an antigen and an adjuvant formulation comprising a water-in-oil emulsion, DEAE-dextran and a CpG-containing immunostimulatory oligonucleotide, followed by extracting a source of said antibody from said source animal and, optionally, purifying said antibody; [0023] The method of claim 1, the antigen is FeLVgp70, The method, wherein the source animal is a rat, mouse, guinea pig, hamster, bovine animal, goat, rabbit, horse, porcine animal, or sheep.

2. 2. The method of claim 1, wherein the source animal is a goat.

3. The method of claim 1 or 2, wherein the source of the antibody is serum or milk.

4. The method of claim 3, wherein the antibody is a polyclonal antibody.

5. 5. The method of any one of claims 1-4, wherein said feeder animal induces serum titers to at least 1000 antigens in said antibody source.

6. 6. The method of claim 5, wherein said source animal induces a serum titer against an antigen in said antibody source of at least 10,000.

7. 6. The method of claim 5, wherein said source animal induces a serum titer against an antigen in said antibody source of at least 100,000.

8. The method of claim 4, wherein the source of the antibody is serum.

9. The method of claim 1 or 2, wherein the source of the antibody is the spleen.

10. The method of claim 9, wherein the antibody is a monoclonal antibody.

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