WO2015042449A2 - Novel streptococcus vaccines - Google Patents

Abstract

The instant invention provides various formulations comprising combinations of immunostimulating oligonucleotides, polycationic carriers, sterols, saponins, quaternary amines, TLR-3 agonists, glycolipids, and MPL-A or analogs thereof in oil emulsions, use thereof in preparations of immunogenic compositions and vaccines, and use thereof in the treatment of animals, most particularly in regard of protection and treatment of bovines against Streptococcus uberis.

Description

NOVEL STREPTOCOCCUS VACCINES

FIELD OF THE INVENTION

This invention relates generally to novel adjuvant formulations for enhancing the immune response to antigens for use in immunogenic and vaccine compositions. This invention also relates to methods of preparation and use of the adjuvant, immunogenic, and vaccine compositions.

The present invention further provides vaccine compositions comprising Streptococcal antigens capable of raising protective and therapeutic host immune responses and for use as vaccines to protect against and/or reduce instances of Streptococcus infections.

BACKGROUND OF THE INVENTION

Bacterial, viral, and parasitic infections are wide spread in humans and animals. Diseases caused by these infectious agents are often resistant to antimicrobial pharmaceutical therapy, leaving no effective means of treatment. Consequently, a vaccinology approach is increasingly used to control infectious disease. A whole infectious pathogen can be made suitable for use in a vaccine formulation after chemical inactivation or appropriate genetic manipulation. Alternatively, a protein subunit of the pathogen can be expressed in a recombinant expression system and purified for use in a vaccine formulation. Vaccines can be made more efficacious by including an appropriate adjuvant in the composition.

The term ^"adjuvant^' generally refers to any material that increases the humoral or cellular immune response to an antigen. Adjuvants are used to accomplish two objectives: They slow the release of antigens from the injection site, and they enhance stimulation of the immune system. Traditional vaccines are generally composed of a crude preparation of inactivated or killed or modified live pathogenic microorganisms. The impurities associated with these cultures of pathological microorganisms may act as an adjuvant to enhance the immune response. However, the immunity invoked by vaccines that use homogeneous preparations of pathological microorganisms or purified protein subunits as antigens is often poor. The addition of certain exogenous materials such as an adjuvant therefore becomes necessary. Further, in some cases, synthetic and subunit vaccines may be expensive to produce. Also, in some cases, the pathogen cannot be grown on a commercial scale, and thus, synthetic/subunit vaccines represent the only viable option. The addition of an adjuvant may permit the use of a smaller dose of antigen to stimulate a similar immune response, thereby reducing l the production cost of the vaccine. Thus, the effectiveness of some injectable medicinal agents may be significantly increased when the agent is combined with an adjuvant.

Many factors must be taken into consideration in the selection of an adjuvant. An adjuvant should cause a relatively slow rate of release and absorption of the antigen in an efficient manner with minimum toxic, allergenic, irritating, and other undesirable effects to the host. To be desirable, an adjuvant should be non-viricidal, biodegradable, capable of consistently creating a high level of immunity, capable of stimulating cross protection, compatible with multiple antigens, efficacious in multiple species, non-toxic, and safe for the host (eg, no injection site reactions). Other desirable characteristics of an adjuvant are that it is capable of micro-dosing, is dose sparing, has excellent shelf stability, is amenable to drying, can be made oil-free, can exist as either a solid or a liquid, is isotonic, is easily manufactured, and is inexpensive to produce. Finally, it is highly desirable for an adjuvant to be configurable so as 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.

The choice of an adjuvant depends upon the needs for the vaccine, whether it be an increase in the magnitude or function of the antibody response, an increase in cell mediated immune response, an induction of mucosal immunity, or a reduction in antigen dose. A number of adjuvants have been proposed, however, none 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 a water-in-oil emulsion and extracts of mycobacterium. Unfortunately, FCA is poorly tolerated and it can cause uncontrolled inflammation. Since the discovery of FCA over 80 years ago efforts have been made to reduce the unwanted side effects of adjuvants.

Some other materials that have been used as adjuvants include metallic oxides (e.g., aluminum hydroxide), alum, inorganic chelates of salts, gelatins, various paraffin-type oils, synthesized resins, alginates, mucoid and polysaccharide compounds, caseinates, and blood-derived substances such as fibrin clots. While these materials are generally efficacious at stimulating the immune system, none has been found to be entirely satisfactory due to adverse effects in the host (e.g., production of sterile abcesses, organ damage, carcinogenicity, or allergenic responses) or undesirable pharmaceutical properties (e.g., rapid dispersion or poor control of dispersion from the injection site, or swelling of the material). Streptococcus uberis is an important mastitis-causing pathogen, responsible for a large proportion of both clinical and sub-clinical cases of mastitis in many parts of the world. The pathogen affects cattle, goats and sheep; however, infection of cattle is of primary importance due to its impact on the dairy industry, through welfare issues for affected animals, and also because of the significant financial impact on producers. As a species, 5. uberis is highly-heterogenous; it is biochemically and physiologically ill-defined, and is serologically heterogenous (Hardie, J.M. 1986 Other streptococci. In Sergey's Manual of Systematic Bacteriology Vol. 2, ed. Sneath, P.H.A., Mair, N.S., Sharp, M.E. & Holt, J.G. pp. 1068-1071. Baltimore: Williams & Wilkins). Furthermore, there is evidence of genetic heterogeneity, driven predominantly through horizontal gene transfer (Coffey T.J., Pullinger G.D., Urwin R., Jolley K.A., Wilson S.M., Maiden M.C., Leigh J.A. 2006. First insights into the evolution of Streptococcus uberis: A multilocus sequence typing scheme that enables investigation of its population biology.

Originally, DNA-DNA hybridization studies suggested the existence of two related 5. uberis genotypes (designated types I and I I) (Garvie & Bramley 1979; Collins et a/. 1984), both associated with the bovine host and a cause of mastitis. Type I I 5. uberis was later re-classified as Streptococcus parauberis (Williams AM, Collins M D. 1990. Molecular taxonomic studies on Streptococcus uberis types I and II . Description of Streptococcus parauberis sp. J. Appl. Bacteriol. 68: 485-490), which is also increasingly found as a cause of disease in fish; significantly, phylogenetic studies have since shown the two species to be related (Nho SW, Hikima J, Cha IS, Park SB, Jang HB, del Castillo CS, Kondo H, Hirono I, Aoki T, Jung TS. 2011. Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis. Journal of Bacteriology.193: 3356-3366), and hence they are likely to share conserved portions of genome. It is, in part, due to the heterogeneity of the 5. uberis population that efforts to develop an effective vaccine have been unsuccessful. This is because 5. uberis antigens which have been shown to have promise as vaccines have not always been found to be conserved amongst the broader population. The present invention is directed to the provision of novel vaccine compositions that employ conserved S. uberis antigens, thereby maximizing the ability of such compositions to prevent or treat infections throughout the world caused by the wide variety of genetically divergent Streptococcus species and S. uberis strains. SUMMARY OF INVENTION

The instant invention provides novel vaccine compositions and adjuvant formulations useful for vaccines.

In the first aspect, the invention provides an adjuvant formulation comprising an oily phase and an aqueous phase, wherein the oily phase comprises at least 50% of the formulation v/v, wherein said formulation comprises at least one of monophosphoryl lipid A (MPL-A) or an analog thereof and an immunostimulatory oligonucleotide, with provisos that a) if said immunostimulatory oligonucleotide is absent, then the formulation comprises a poly l :C, a glycolipid, and, optionally, a quaternary amine; or a polycationic carrier; and b) if said monophosphoryl lipid A (M PL-A) or the analog thereof is absent, then the formulation comprises a source of aluminum, and, optionally, a polycationic carrier.

In different embodiments, the oily phase may comprise an oil and, optionally, an oil-soluble emulsifier. In some embodiments, both said monophosphoryl lipid A (MPL-A) or the analog thereof are present in the adjuvant formulation. In these embodiments, the formulation further comprises a sterol (e.g., cholesterol), a poly l :C, or a combination thereof.

In certain set of embodiments, in addition to the oil and the optional emulsifier(s), the adjuvant formulations include 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 comprise poly l :C ("TCMYO") and/or a saponin ("QTCMO" or "QTCMYO", respectively).

In yet further alternative embodiments, in addition to the oil and the optional emulsifier(s), the adjuvant formulations also include a combination of a quaternary amine, a glycolipid, M PL-A or an analog thereof, and poly l :C ("ODYRM").

In yet further set of embodiments, in addition to the oil and the optional emulsifier(s), the adjuvant formulations also include a combination of a saponin, a sterol, a quaternary amine, and a polycationic carrier ("Q.CDXO").

In further embodiments, in addition to the oil and the optional emulsifier(s), the adjuvant may include the immunostimulatory oligonucleotide, a source of aluminum, and, optionally, a polycationic carrier ("TO A" and "TXO-A", respectively).

In a second aspect, the adjuvant formulation according any of the embodiments recited above, may include an antigen component, thus forming a vaccine composition. In additional aspects of the invention, different combinations of the antigen compound and the adjuvant formulations are provided. Preferably, the antigen component provides one or more macromolecule components from Streptococcus uberis, including any strains thereof, to include proteins, nucleic acids, lipds, glyoclipids, lipopolysacchairdes, polysaccharides, all and the like. In an additional embodiment of the invention, the antigen component is provided from any Streptococcus species, as long as the antigen is cross reactive against Streptococcus, and is protective or immunizing, owing to combination with the efficacious adjuvants of the present invention.

Accordingly, in preferred examples, there are provided vaccine compositions comprising an effective amount of Streptococcus antigen and an adjuvant formulation, wherein the antigen is selected from any one, two, three or four of the following: Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ I D NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP- 002563137); wherein the adjuvant formulation comprising an oily phase and an aqueous phase, wherein the oily phase comprises at least 50% of the formulation v/v, wherein said formulation comprises at least one of monophosphoryl lipid A (M PL-A) or an analog thereof and an immunostimulatory oligonucleotide, with provisos that:

a) if said immunostimulatory oligonucleotide is absent, then the formulation comprises:

i. a poly l:C, a glycolipid, and, optionally, a quaternary amine; or ii. a polycationic carrier;

b) if said monophosphoryl lipid A (MPL-A) or the analog thereof is absent, then the formulation comprises a source of aluminum.

There is additionally provided a vaccine composition comprising an adjuvant formulation wherein

the immunostimulatory oligonucleotide, if present, is a CpG or an oligoribonucleotide; the polycationic carrier, if present, is selected from the group consisting of dextran, dextran DEAE (and derivatives thereof), PEGs, guar gums, chitosan derivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC) polyethylenimene, poly aminos; and the quaternary amine, if present, is selected from the group consisting of DDA and avridine.

Thefore, in a further preferred example, vaccine compositions are provided in which the glycolipid component of the adjuvant formulation, if present, comprises a compound of formula I

Formula I

wherein, R¹ and R² are independently hydrogen, or a saturated alkyl radical having up to 20 carbon atoms; X is -CH₂-, -0- or -NH-; R² is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵ are independently hydrogen, -S0₄ ^2~, -P0 ^2~, -COCi_i₀ alkyl; R⁶ is L- alanyl, L-alpha-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-phenyalany, L-prolyl, L-seryl, L- threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers.

As will be seen below, the specific Streptococcus antigens used in the practice of the present invention are sufficiently conserved among Streptococcus uberis strains, and additionally among strains of other Streptococcus species that they permit the development of a wide variety of vaccine compositions that employ a wide variety of adjuvants, and which are useful in preventing or treating Streptococcus infection and disease. Accordingly, in an important embodiment of the invention there is provided a vaccine composition comprising an effective amount of 1, 2, 3, or 4 Streptococcus antigens and an adjuvant, wherein the 1, 2, 3 or 4 antigens are selected from the group consisting of Streptococcus uberis ferrichrome binding protein (SEQ. I D NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ, ID NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137). In a further aspect of the invention, specific pairs of Streptococcus antigens are used with an adjuvant to form a vaccine composition, wherein the resultant combined antigen provided in the composition is either (a) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776) and Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947), or (b) Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276), and Streptococcus uberis serine protease (SEQ I D NO 11, locus tag SUB1868, accession number YP-002563137).

In a further aspect of the invention, the combination of 1, 2, 3 or 4 Streptococcus antigens that are used with an adjuvant to form a vaccine composition of the invention are thus selected from the group consisting of (a) to (k) as follows:

(a) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ I D NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ I D NO 11, locus tag SUB1868, accession number YP-002563137);

(b) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); and Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276);

(c) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); and Streptococcus uberis serine protease (SEQ I D NO 11, locus tag SUB1868, accession number YP-002563137);

(d) . Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137); (e) Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP- 002561947); Streptococcus uberis lipoprotein (SEQ ID NO : 8, locus tag SUB0950, accession number YP- 002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137)

(f) Streptococcus uberis ferrichrome binding protein (SEQ I D NO: 2, locus tag SUB0423, accession number YP-002561776); and Streptococcus uberis elongation factor Tu (SEQ ID NO: 5, locus tag SUB0604, accession number YP-002561947);

(g) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); and Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276);

(h) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137);

(i) Streptococcus uberis elongation factor Tu (SEQ ID NO: 4, locus tag SUB0604, accession number YP- 002561947); and Streptococcus uberis lipoprotein (SEQ ID NO : 6, locus tag SUB0950, accession number YP-002562276);

(j) Streptococcus uberis elongation factor Tu (SEQ ID NO: 5, locus tag SUB0604, accession number YP- 002561947); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137); and

(k) Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP- 002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137).

In connection with selecting combinations of antigens for use in the vaccine compositions of the invention, it will be appreciated that greater overall protection of the vaccinated animal from challenge by any of a wide variety of Streptocccus strains will be achieved using preferably 2, more preferably 3, and most preferably all 4 of the above-identified antigens, or immunologically active fragments of any thereof. This is particularly important taking into account the natural evolution of additional strains that infect herds, in order to provide a vaccine composition that remains protecting and commercially relevant for as long as possible. Additionally, as shown in the Examples below, should it be desired to use less that 4 antigen components per vaccine dose (i.e. 1, 2 or only 3 antigens) then it is appropriate to increase the dosage of the remaining antigens so that the total micrograms of antigen delivered per dose is not lessened compared to a 4-way antigen combination.

Brief Description of the Drawin s

Figure 1 shows a map of the pFLEXlO vector, as a representative construct

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"About" or "approximately," when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater, unless about is used in reference to time intervals in weeks where "about 3 weeks," is 17 to 25 days, and about 2 to about 4 weeks is 10 to 40 days.

"Adjuvant" means any substance that increases the humoral or cellular immune response to an antigen. Adjuvants are generally used to accomplish two objectives: the controlled release of antigens from the injection site, and the stimulation of the immune system.

"Adjuvant formulation" refers to formulations having adjuvanting properties.

"Alkyl" refers to both straight and branched saturated hydrocarbon moieties.

"Amine" refers to a chemical compound containing nitrogen. Amines are a group of compounds derived from ammonia by substituting hydrocarbon groups for the hydrogen atoms. "Quaternary amine" refers to an ammonium based compound with four hydrocarbon groups.

"Antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the 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 the constant regions.

"Antigen" or "immunogen" refers to any substance that is recognized by the animal's immune system and generates 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 extract, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, individually or in any combination thereof. The term antigen also includes antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

"Bacterin" means a suspension of one or more killed bacteria which may be used as a component of a vaccine or immunogenic composition.

"Buffer" means a chemical system that prevents change in the concentration of another chemical substance, e.g., proton donor and acceptor systems serve as buffers preventing marked 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 (conjugate acid).

"Cellular immune response" or "cell mediated immune response" is one mediated by T- lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by activated T-cells, white blood cells, or both; or a T lymphocyte or other immune cell response that kills an infected cell.

"Companion animals" refers to dogs, cats and equines.

"Consisting essentially" as applied to the adjuvant formulations refers to formulation which does not contain unrecited additional adjuvanting or immunomodulating agents in the amounts at which said agent exert measurable adjuvanting or immunomodulating effects.

"Delayed type hypersensitivity (DTH)" refers to an inflammatory response that develops 24 to 72 hours after exposure to an antigen that the immune system recognizes as foreign. This type of immune response involves mainly T cells rather than antibodies (which are made by B cells).

"Dose" refers to a vaccine or immunogenic composition given to a subject. A "first dose" or "priming vaccine" refers to the dose of such a composition given on Day 0. A "second dose" or a "third dose" or an "annual dose" refers to an amount of such composition given subsequent to the first dose, which may or may not be the same vaccine or immunogenic composition as the first dose.

The term "emulsifier" is used broadly in the instant disclosure. It includes substances generally accepted as emulsifiers, e.g., different products of TWEEN^® or SPAN^® product lines (fatty acid esters of polyethoxylated sorbitol and fatty-acid-substituted sorbitan surfactants, respectively), and different solubility enhancers such as PEG-40 Castor Oil or another PEGylated hydrogenated oil. "Humoral immune response" refers to one that is mediated by antibodies.

"Immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. Immune responses can usually be determined using standard immunoassays and neutralization assays, which are known in the art.

"Immunologically protective amount" or "immunologically effective amount" or "effective amount to produce an immune response" of an antigen is an amount effective to induce an immunogenic response in the recipient. The immunogenic response may be sufficient for diagnostic purposes or other testing, or may be adequate to prevent signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a disease agent. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of an animal to an immunogenic composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain, whereas the protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and overall health and performance of the subject. The immune response may comprise, without limitation, induction of cellular and/or humoral immunity.

"Immunogenic" means evoking an immune or antigenic response. Thus an immunogenic composition would be any composition that induces an immune response.

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

"Lipids" refers to any of a group of organic compounds, including the fats, oils, waxes, sterols, and triglycerides that are insoluble in water but soluble in nonpolar organic solvents, are oily to the touch, and together with carbohydrates and proteins constitute the principal structural material of living cells.

"Pharmaceutically acceptable" refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use. The term "Poly l:C" refers to naturally occurring polymers of polyinosinic:polycytadylic acids as well as synthetic forms thereof, e.g., with stabilized backbone and preferably having TLR-3 agonist activity.

"Reactogenicity" refers to the side effects elicited in a subject in response to the administration of an adjuvant, an immunogenic, or a vaccine composition. It can occur at the site of administration, and is usually assessed in terms of the development of a number of symptoms. These symptoms can include inflammation, redness, and abscess. It is also assessed in terms of occurrence, duration, and severity. A "low" reaction would, for example, involve swelling that is only detectable by palpitation and not by the eye, or would be of short duration. A more severe reaction would be, for example, one that is visible to the eye or is of longer duration.

"Room Temperature" means a temperature from 18 to 25°C.

"Saponin" refers to a group of surface-active glycosides of plant origin composed of a hydrophilic region (usually several sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure.

"Steroids" refers to any of a group of organic compounds belonging to biochemical class of lipids, which are easily soluble in organic solvents and slightly soluble in water. Steroids comprise a four-fused ring system of three fused cyclohexane (six-carbon) rings plus a fourth cyclopentane (five- carbon) ring.

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

"Subject" refers to any animal for which the administration of an adjuvant composition is desired. It includes mammals and non-mammals, including primates, livestock, companion animals, laboratory test animals, captive wild animals, aves (including in ova), reptiles, and fish. Thus, this term includes but is not limited to monkeys, humans, swine; cattle, sheep, goats, equines, mice, rats, guinea pigs, hamsters, rabbits, felines, canines, chickens, turkeys, ducks, other poultry, frogs, and lizards. "TCID50" refers to "tissue culture infective dose" and is defined as that dilution of a virus required to infect 50% of a given batch of inoculated cell cultures. Various methods may be used to calculate TCI D50, including the Spearman-Karber method which is utilized throughout this specification. For a description of the Spearman-Karber method, see B. W. Mahy & H. 0. Kangro, Virology Methods Manual, p. 25-46 (1996).

"Therapeutically effective amount" refers to an amount of an antigen or vaccine that would induce an immune response in a subject receiving the antigen or vaccine which is adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus or a bacterium. Humoral immunity or cell- mediated immunity or both humoral and cell-mediated immunity may be induced. The immunogenic response of an animal to a vaccine may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and overall health and performance of the subject. The amount of a 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 skilled in the art.

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

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

"Triterpeniods" refers to a large and diverse class of naturally occurring organic molecules, derived from six five-carbon isoprene (2-methyl-l,3-butadiene) units, which can be assembled and modified in thousands of ways. Most are multicyclic structures which differ from one another in functional groups and in their basic carbon skeletons. These molecules can be found in all classes of living things.

"Vaccine" refers to a composition that includes an antigen, as defined herein. Administration of the vaccine to a subject results in an immune response, generally against one or more specific diseases. The amount of a vaccine that is therapeutically effective may vary depending on the particular antigen used, or the condition of the subject, and can be determined by one skilled in the art. Adjuvant formulations and methods of making

The instant application discloses several adjuvant formulations suitable for the instant invention. The common feature of these adjuvants is the presence of oil and one or more emulsifiers, wherein the oily phase comprises more than 50% of the vaccine composition encompassing the adjuvant formulations disclosed therein.

Multiple oils and combinations thereof are suitable for use of the instant invention. These oils include, without limitations, animal oils, vegetable oils, as well as non-metabolizable oils. Non-limiting examples of vegetable oils suitable in the instant invention are corn oil, peanut oil, soybean oil, coconut oil, and olive oil. Non-limiting example of animal oils is squalane. Suitable non-limiting examples of non-metabolizable oils include light mineral oil, straight chained or branched saturated oils, and the like.

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

Typically, the oily phase is present in an amount from 50% to 95% by volume; preferably, in an amount of greater than 50% to 85%; more preferably, in an amount from greater than 50% to 60%, and more preferably in the amount of greater than 50-52% v/v of the vaccine composition. The oily phase includes oil and emulsifiers (e.g., SPAN^® 80, TWEEN^® 80 etc), if any such emulsifiers are present.

The volume of the oily phase is calculated as a sum of volumes of the oil and the emulsifier(s). Thus, for example, if the volume of the oil is 40% and the volume of the emulsifier(s) is 12% of a composition, then the oily phase would be present at 52% v/v of the composition. Similarly, if the oil is present in the amount of about 45% and the emulsifier(s) is present in the amount of about 6% of a composition, then the oily phase is present at about 51% v/v of the composition. It also should be understood that since the adjuvants of the instant invention form only a part of the vaccines of the instant invention, oily phase is present in an amount from 50% to 95% by volume; preferably, in an amount of greater than 50% to 85%; more preferably, in an amount from 50% to 60%, and more preferably in the amount of 50-52% v/v of each of the adjuvants of the instant invention.

In a subset of embodiments, applicable to all adjuvants/vaccines of the instant invention, the volume percentage of the oil and the oil-soluble emulsifier together is at least 50%, e.g., 50% to 95% by volume; preferably, in an amount of greater than 50% to 85%; more preferably, in an amount from 50% to 60%, and more preferably in the amount of 50-52% v/v of the vaccine composition. Thus, for example and without limitations, the oil may be present in the amount of 45% and the lipid-soluble emulsifier would be present present in the amount of greater than 5% v/v. Thus, the volume percentage of the oil and the oil-soluble emulsifier together would be at least 50%.

In yet another subset, applicable to all vaccines of the invention, volume percentage of the oil is over 40%, e.g., 40% to 90% by volume; 40% to 85%; 43% to 60%, 44-50% v/v of the vaccine composition.

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

In additional embodiments, the emulsifiers used herein do not include lecithin, or use lecithin in an amount which is not immunologically effective.

Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants (commercially available under the name SPAN^® or ARLACEL^®), fatty acid esters of polyethoxylated sorbitol (TWEEN^®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR^®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL^® M-53), polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL^®), polyoxyethylene fatty alcohol ethers (BRIJ^®); polyoxyethylene nonphenyl ethers (TRITON^® N), polyoxyethylene isooctylphenyl ethers (TRITON^® X). Preferred synthetic surfactants are the surfactants available under the name SPAN^® and TWEEN^®, such as TWEEN^®-80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN^®-80 (sorbitan monooleate).

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

Additional ingredients present in the instant adjuvant formulations include cationic carriers, immunostimulatory oligonucleotides, monophospholipid A and analogs thereof (MPL-A), Polyinosinic:polycytidylic acid (poly l :C), saponins, quaternary ammoniums, sterols, glycolipids, a source of aluminum (e.g., REHYDRAGEL^® or VAC 20^® wet gel) and combinations thereof.

Suitable cationic carriers include, without limitations, dextran, dextran DEAE (and derivatives thereof), PEGs, guar gums, chitosan derivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC) polyethylenimene, poly aminos like polylysine and the like.

Suitable immunostimulatory oligonucleotides include ODN (DNA-based), ORN (RNA-based) oligonucleotides, or chimeric ODN-ORN structures, which may have modified backbone including, without limitations, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl- modifications), and phosphodiester modifications. In some embodiments, poly inosinic -cytidylic acid or derivative thereof (poly l:C) may be used.

CpG oligonucleotides are a recently described class of pharmacotherapeutic agents that are characterized by the presence of an unmethylated CG dinucleotide in specific base-sequence contexts (CpG motif). (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229-232, which is incorporated herein by reference). These CpG motifs are not seen in eukaryotic DNA, in which CG dinucleotides are suppressed and, when present, usually methylated, but are present in bacterial DNA to which they confer immunostimulatory properties. In selected embodiments, the adjuvants of the instant invention utilize a so-called P-class immunostimulatory oligonucleotide, more preferably, modified P- class immunostimulatory oligonucleotides, even more preferably, E-modified P-class oligonucleotides. P-class immunostimulatory oligonucleotides are CpG oligonucleotides characterized by the presence of palindromes, generally 6-20 nucleotides long. The P-Class oligonucleotides have the ability to spontaneously self-assemble into concatamers either in vitro and/or in vivo. These oligonucleotides are, in a strict sense, single-stranded, but the presence of palindromes allows for formation of concatamers or possibly stem-and-loop structures. The overall length of P- class immunostimulatory oligonucleotides is between 19 and 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.

In one aspect of the invention the immunostimulatory oligonucleotide contains a 5' TLR activation domain and at least two palindromic regions, one palindromic region being a 5' palindromic region of at least 6 nucleotides in length and connected to a 3' palindromic region of at least 8 nucleotides in length either directly or through a spacer.

The P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E-modification refers to ethyl-modified nucleotide(s). Thus, E-modified P-class immunostimulatory oligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5' nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazol, O- Methylation, modification with proynyl-dU, inosine modification, 2-bromovinyl attachment (preferably to uridine).

The P-class immunostimulatory oligonucleotides may also contain a modified internucleotide linkage including, without limitations, phosphodiesther linkages and phosphorothioate linkages. The oligonucleotides of the instant invention may be synthesized or obtained from commercial sources. P-Class oligonucleotides and modified P-class oligonucleotides are further disclosed in published PCT application no. WO2008/068638, published on Jun. 12, 2008. Suitable non-limiting examples of modified P-class immunostiumulatory oligonucleotides are provided below ("*" refers to a phosphorothioate bond and "_" refers to a phosphodiester bond).

SEQ. ID NO: 13 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: 14 5' 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: 15 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: 16 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: 17 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: 18 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: 19 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: 20 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: 21 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: 22 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: 23 5' -UUGUUGUUGUUGUUGUUGUU-3'

SEQ ID NO: 24 5' -UUAUUAUUAUUAUUAUUAUU-3'

SEQ ID NO: 25 5' - A A ACG CU C AG C C AA AG C AG - 3 '

SEQ ID NO: 26 clTdCdGdTdCdGdTdTdTdTrGrUrUrGrUrGrUdTdTdTdT-3'

The amount of P-class immunostimulatory oligonucleotide for use in the adjuvant compositions depends upon the nature of the P-class immunostimulatory oligonucleotide used and the intended species.

Suitable analogs of MPL-A include, without limitations can be bacterial derived natural LPS altered or unaltered in structure or synthetic, Glucopyranosyl Lipid Adjuvant (GLA), pertactin , varying substitutions at 3-O-position of the reducing sugar, synthetic forms of lipid A analog with low endotoxicity.

Sterols share a common chemical core, which is a steroid ring structure^], having a hydroxyl (OH) group, usually attached to carbon-3. The hydrocarbon chain of the fatty-acid substituent varies in length, usually from 16 to 20 carbon atoms, and can be saturated or unsaturated. Sterols commonly contain one or more double bonds in the ring structure and also a variety of substituents attached to the rings. Sterols and their fatty-acid esters are essentially water insoluble. In view of these chemical similarities, it is thus likely that the sterols sharing this chemical core would have similar properties when used in the vaccine compositions of the instant invention. Sterols are well known in the art and can be purchased commercially. For example cholesterol is disclosed in the Merck Index, 12th Ed., p. 369. Suitable sterols include, without limitations, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

Suitable saponins include triterpenoid saponins. These triterpenoids a group of surface-active glycosides of plant origin and share common chemical core composed of a hydrophilic region (usually several sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure. Because of these similarities, the saponins sharing this chemical core are likely to have similar adjuvanting properties. Triterpenoids suitable for use in the adjuvant compositions can come from many sources, either plant derived or synthetic equivalents, including but not limited to, Quillaja saponaria, tomatine, ginseng extracts, mushrooms, and an alkaloid glycoside structurally similar to steroidal saponins.

If a saponin is used, the adjuvant compositions generally contain an immunologically active saponin fraction from the bark of Quillaja saponaria. The saponin may be, for example, Quil A or another purified or partially purified saponin preparation, which can be obtained commercially. Thus, saponin extracts can be used as mixtures or purified individual components such as QS-7, QS-17, QS- 18, and QS-21. In one embodiment the Quil A is at least 85% pure. In other embodiments, the Quil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure..

Quaternary amine compounds are ammonium based compounds with four hydrocarbon groups. In practice, hydrocarbon groups are generally limited to alkyl or aryl groups. In a set of embodiments, the quaternary amine compounds are composed of four alkyl chains, two of which are C10-C20 alkyls and the remaining two are C1-C4 alkyls. In one set of embodiments, the quaternary amine is Dimethyldioctadecylammonium bromide, chloride or pharmaceutically acceptable counterion (DDA).

Suitable glycolipids are generally those which activate the Th2 response. The glycolipids include, without limitations, those encompassed by Formula I and that are generally described in US Publication 20070196384 (Ramasamy et al).

Formula I

In the structure of Formula I, R¹ and R² are independently hydrogen, or a saturated alkyl radical having up to 20 carbon atoms; X is -CH₂-, -O- or -NH-; R² is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵ are independently hydrogen, -S0₄ ^2", -PO4^2", -COCi_ 10 alkyl; R⁶ is L-alanyl, L-alpha-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-phenyalany, L- prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers.

In a set of embodiments, the suitable glycolipid is N-(2-Deoxy-2-L-leucylamino-b-D- glucopyranosyl)-N-octadecyldodecanoylamide or an acetate thereof.

Aluminum is a known adjuvant or a component of adjuvant formulations and is commercially available in such forms as Reheis, Inc, Brentag alhydrogel REHYDRAGEL^® or VAC 20^® wet gel. REHYDRAGEL^® is a crystalline aluminum oxyhydroxide, known mineralogically as boehmite. It is effective in vaccines when there is a need to bind negatively charged proteins. The content of Al₂0₃ ranges from 2% to 10% depending on grade, and its viscosity is 1000-1300 cP. Generally, it may be described as an adsorbent aluminum hydroxide gel. VAC^® 20 wet gel is a white or almost white, translucent, viscous colloidal gel. In certain embodiments, the content of Al₂0₃ is about 2% w/v.

In other embodiments, the source of aluminum can also be prepared by precipitated aluminum hydroxide processes.

In certain set of embodiments, in addition to the oil and the optional one or more emulsifiers, the adjuvant formulations also comprise (or consist essentially, or consist) a combination of monophosphoryl lipid A (MPL-A) or an analog thereof, a sterol, and an immunostimulatory oligonucleotide. The adjuvants containing these ingredients are referred to as "TCMO". The TCMO adjuvant formulation may also optionally include poly l:C ("TCMYO") and/or a saponin. Thus, adjuvant formulations comprising, or consisting essentially of, or consisting of a combination of monophosphoryl lipid A (MPL-A) or an analog thereof, a sterol, and an immunostimulatory oligonucleotide and saponin are referred to as "QTCMO." In addition, the adjuvant formulations may also include poly l:C. Such adjuvants are referred to as "QTCMYO".

In a set of embodiments, TCMO adjuvants comprise light mineral oil in the amount of 40% to 50% v/v of the total volume of the vaccine composition. The emulsifiers include TWEEN-80 and SPAN- 80, total amount 0.1% to 40% v/v of the total volume of the vaccine composition, provided that sorbitan monooleate and oil together comprise about 50.5% to 52% v/v of the composition. The immunostimulatory oligonucleotide is an ODN, preferably, a palindrome containing ODN, optionally, with a modified backbone. In certain embodiments, one dose of TCMO contains between about 1 ug and about 400 ug of the immunostimulating oligonucleotide, between about 1 ug and about 1000 ug of the sterol, between about 0.1 ug and 500 ug MPL-A or the analog thereof.

The amounts of other compounds per dose are selected based on the subject species.

For example, in some embodiments suitable for cattle, sheep or adult swine, one dose of TCMO would contain between about 50 and 400 ug (e.g., 50-300, or 100-250 ug, or about 50 to about 100 ug for adult pigs and about 100 to about 250 ug for cattle) of the immunostimulatory oligonucleotide, between about 100 and about 1000 ug (e.g., 200-1000, 250-700 ug, or about 400-500 ug) of the sterol, such as cholesterol, and between about 5 and about 500 ug (e.g., 5-100 ug, or 5-50 ug, or 10-25 ug) of MPL-A or the analog thereof.

In some embodiments suitable for companion animals or piglets, one dose of TCMO would contain between about 5 and 100 ug (e.g., 10-80, or 20-50 ug) of the immunostimulatory oligonucleotide, between about 5 and 100 ug (e.g., 10-80, or 20-50 ug) of the sterol such as cholesterol, and between about 0.5 and about 200 ug (e.g., 1-100 ug, or 5-50 ug, or 5-20 ug) of MPL-A or the analog thereof.

In some embodiments suitable for poultry, one dose of TCMO adjuvant would contain between about 0.1 and about 5 ug (e.g., 0.5-3 ug, or 0.9-1.1 ug) of immunostimulatory oligonucleotide, between about 0.5 and about 50 ug (e.g., 1-20 ug, or 1-10 ug) of the sterol such as cholesterol, and between about 0.1 to 10 ug (e.g., 0.5 - 5 ug, or 1-5 ug) of MPLA or the analog thereof. ). MPL-A is present in the amount of 0. lug/dose to 2,000 ug/dose.

In certain embodiments, TCMO adjuvants are prepared as follows:

a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in light mineral oil. The resulting oil solution is sterile filtered;

b) The immunostimulatory oligonucleotide and Polyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous solution;

c) The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation TCMO.

In TCMYO adjuvants, the cholesterol, oil, optional emulsifiers, MPL-A, and the immunostimulatory oligonucleotides are present as in the TCMO adjuvant formulation for the respective species. Poly l:C may be present generally in the amount between about 1 ug and about 100 ug per dose. More specifically, poly l:C may be present in the amount of 5-100 ug per dose (e.g., 5-50 ug, or 10-30 ug) in certain embodiments suitable for cattle, adult swine, or sheep. In certain embodiments suitable for companion animals or piglets, one dose of TCMYO contains between about 1 and about 50 ug (e.g., 5-50 ug, or 10-20 ug) of poly l :C. In certain embodiments suitable for poultry vaccines, one dose of TCMYO contains between about 1 and about 10 ug (e.g., 1-5 ug, or 3-5 ug) of poly l:C.

In certain embodiments, TCMYO adjuvants are prepared similarly to the TCMO adjuvants, and the poly l:C is added to the aqueous solution.

In a set of embodiments, in QTCMO adjuvants, the cholesterol, oil, optional emulsifiers, MPL-A, and the immunostimulatory oligonucleotides are present as in the TCMO adjuvant formulation for the respective species. A saponin is preferably Quil A or a purified fraction thereof, and may be present in the amounts of between about 0.1 ug and about 1000 ug per dose.

More specifically the saponin may be present in the amount of of 0.1 to 5 ug per 50 ul of the vaccine composition (e.g., 0.5 - 30 ug per 50 ul of the composition, or more preferably 1 - 10 ug) per dose in certain embodiments suitable for poultry vaccines. In certain embodiments suitable for applications in companion animals and piglets, the saponin, e.g., Quil A or a purified fraction thereof is present in the amounts between about 10 and about 100 ug per dose (e.g., 10-50 ug or 20-50 ug per dose). In certain embodiments suitable for cattle, adult swine, or sheep, the saponin, such as Quil A or a purified fraction thereof, is present in the amount of between about 100 and about 1000 ug per dose (e.g., 200-800 ug, or 250-500 ug per dose).

In certain embodiments, QTCMO adjuvants are prepared similarly to TCMO adjuvants, and the saponin is added to the aqueous solution.

In a set of embodiments, in QTCMYO adjuvants, the saponin is present as in QTCMO adjuvant, and the rest of the ingredients are present as in TCMYO, for the respective species.

In certain embodiments, QTCMYO adjuvants are prepared similarly to TCMYO adjuvants, and the saponin is added to the aqueous solution.

In alternative embodiments, in addition to the oil and the optional emulsifier(s), the adjuvant formulations also comprise (or consist essentially of, or consist of) a combination of monophosphoryl lipid A (MPL-A) or an analog thereof and a polycationic carrier. These adjuvants are referred to as "XOM". In a set of embodiments, in XOM adjuvants for companion animals or piglets, the polycationic carrier is present in the amount of 1-50 mg per dose (e.g., 1-25 mg per dose, or 10-25 mg per dose), and the M PL-A or the analog thereof is present in the amount of between about 1-50 ug per dose (e.g., 1-25 ug per dose, or 10-25 ug per dose).

In certain embodiments suitable for cattle, sheep and adult pigs, the polycationic carrier is present in the amount of between about 5 and about 500 mg per dose (e.g., 10-500 mg, or 10-300 mg, or 50-200 mg per dose) and the MPL-A or the analog therof is present in the amount ofbetween about 1 and about 100 ug per dose (e.g., 5-100 ug, or 5-50 ug, or 10-30 ug).

In certain embodiments suitable for companion animals and piglets, the polycationic carrier is present in the amount of between about 1 and about 50 mg per dose (e.g., 1-25 mg per dose, or 10-25 mg per dose), and MPL-A or the analog thereof is present in the amount of between about 0.5 and about 200 ug (e.g., 1-100 ug, or 5-50 ug, or 5-20 ug) per dose.

In certain embodiments suitable for poultry vaccines, the polycationic carrier is present in the amount of between 0.5 and 25 mg per dose (e.g., 1-20 mg, or 1-10 mg or 5-10 mg), and the M PL-A or the analog thereof is present in the amount between about 0.5 and 10 ug per dose (e.g., 1-10 ug, or 1- 5 ug, or 2-5 ug).

In certain embodiments, XOM adjuvants are prepared as follows:

a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in light mineral oil. The resulting oil solution is sterile filtered;

b) Dextran DEAE and Polyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous solution;

c) The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation XOM.

In additional alternative embodiments, in addition to the oil and the emulsifier(s), the adjuvant formulations also comprise (or consist essentially of, or consist of) a combination of an immunostimulatory oligonucleotide and a polycationic carrier, with a proviso that if said polycationic carrier is dextran DEAE, then the antigen is not E coli J-5 bacterin. These adjuvants are referred to as "TXO". In a set of embodiments, the TXO adjuvants may also include a source of aluminum, such as AI(OH)₃ gel. The TXO adjuvants with aluminum are referred to as "TXO-A". In a set of embodiments, in TXO adjuvants, the immunostimulatory oligonucleotide, preferably an ODN, preferably containing a palindromic sequence, and optionally with a modified backbone, may be present in the amount of 0.5-400 ug per dose, and the polycationic carrier may be present in the amount of 0.5-400 mg per dose. The dosages wary depending on the subject species.

For example, in certain embodiments suitable for cattle, sheep or adult swine, one dose of TXO would comprise between about 50 and 400 ug (e.g., 50-300, or 100-250 ug, or about 50 to about 100 ug for adult pigs and about 100 to about 250 ug for cattle) of the immunostimulatory oligonucleotide, and the polycationic carrier may be present in the amount of between about 5 and about 500 mg per dose (e.g., 10-500 mg, or 10-300 mg, or 50-200 mg per dose).

In certain embodiments suitable for companion animals or piglets, one dose of TXO would comprise between about 5 and 100 ug (e.g., 10-80 ug, or 20-50 ug) of the immunostimulatory oligonucleotide, while the polycationic carrier may be present in the amount of 1-50 mg per dose (e.g., 1-25 mg per dose, or 10-25 mg per dose).

In certain embodiments suitable for poultry, one dose of TXO adjuvant would between about 0.1 and about 5 ug (e.g., 0.5-3 ug, or 0.9-1.1 ug) of immunostimulatory oligonucleotide, and the polycationic carrier may be present in the amount of between 0.5 and 25 mg per dose (e.g., 1-20 mg, or 1-10 mg or 5-10 mg).

In certain embodiments, TXO adjuvants are prepared as follows:

Sorbitan monooleate is dissolved in light mineral oil. The resulting oil solution is sterile filtered; The immunostimulatory oligonucleotide, Dextran DEAE and Polyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous solution; and

The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation TXO.

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

In certain embodiments, TXO-A adjuvants are prepared similarly to TXO adjuvants, and the source of aluminum is added to the aqueous solution. TXO and TXO-A adjuvants are preferred in regard of Streptococcus vaccines. In additional embodiments, the adjuvants of the instant invention contain the oil, optional emulsifier(s), the immunostimulatory oligonucleotide and the source of aluminum. These compounds are present in the ranges disclosed for TXO-A adjuvant, except that the polycationic carrier is absent in TOA. TOA adjuvant is prepared similarly to TXO adjuvant, except the aqueous phase contains the source of aluminum rather than DEAE dextran.

In certain embodiments, in addition to the oil and the emulsifier(s), the adjuvant formulations also comprise (or consist essentially of, or consist of) a combination of a polycationic carrier and a source of Aluminum. This adjuvant is referred to as AXO. These compounds may be present in amounts similar to those present in an adjuvant TXO-A for the respective species, and adjuvant AXO may be prepared similarly to TXO-A, but without addition of the immunostimulating oligonucleotide. In certain other embodiments, in addition to the oil and the emulsifier(s), the adjuvant formulations also comprise (or consist essentially of, or consist of) a combination of a saponin and sterol. This adjuvant is referred to as Q.CO. The nature and the amounts of the ingredients of Q.CO are similar to the amounts of the saponin, the sterol, the oil and the emulsifier(s) in adjuvant QTCMO. QCO may be prepared by adding an aqueous solution comprising the saponin the sterol and, preferably, the water soluble emulsifier into an oily phase, comprising the oil and, preferably, the oil-soluble emulsifier under continuous homogenization.

In yet further alternative embodiments, in addition to the oil and the emulsifier(s), the adjuvant formulations also comprise (or consist essentially of, or consist of) a combination of a quaternary amine, a glycolipid, MPL-A or an analog thereof, and poly l :C. These adjuvants are referred to as "ODYRM".

In ODYRM adjuvants, the oil is generally a mixture of phospholipids such as phosphatidyl cholines. AMPHIGEN^® is a suitable example of such oil, and would be present in the amount similar to the amount of oil, as described above.

In a set of embodiments, in ODYRM adjuvants, the quaternary amine, e.g., DDA, is present in the amount of between about 1 ug and about 200 ug per dose, poly l:C is present in the amount of between about 0.5 ug and 100 ug per dose, the glycolipid is present in the amount of between about 0.5 ug and about 2000 ug per dose, and the MPL-A or the analog thereof is present in the amount of between about 0.5 ug and 100 ug per dose.

More specifically, in certain embodiments suitable for administration to cattle, adult swine, or sheep, the quaternary amine may be present in the amount of between about 50 ug and about 200 ug per dose (e.g., 50-150 ug, or about 100 ug), poly l :C may be present in amounts of between about 1 ug and about 100 ug per dose (e.g., 1-50 ug or 5-50 ug), the glycolipid may be present in the amount of between about 500 ug and about 2000 ug per dose (e.g., 500-100 ug or about 1000 ug), and MPLA or the analog thereof may be present in the amount of between about 5 ug and about 100 ug per dose (e.g., 5-50 ug, or 10-50 ug).

In certain embodiments suitable for administration to companion animals and piglets, the quaternary amine may be present in the amount between about 5 and about 500 ug per dose (e.g., 10- 100 ug per dose, or 20-50 ug per dose), the poly l :C may be present in the amount of between about 5 ug and about 25 ug per dose (e.g., 50-20 ug, or about 10 ug), the glycolipid may be present in the amount of between about 10 and about 100 ug per dose (e.g., 20-100 ug or 25-50 ug), and the M PL-A or the analog thereof may be present in the amount of between about 5 and about 50 ug per dose (e.g., 5-20 ug, or 10-20 ug).

In certain other embodiments, suitable for poultry vaccines, one dose would contain between about 1 ug and about 10 ug of the quaternary ammonium compound (e.g., 5-10 ug, or about 5 ug), between about 0.5 and about 10 ug of poly l:C (e.g., 1-10 ug or 1-5 ug), between about 0.5 and 10 ug of the glycolipid (e.g., 1-10 ug or 5-10 ug or 1-5 ug), and between about 0.5 ug and about 5 ug of MPL-A or the analog thereof (e.g., 0.5-5 ug or 1-5 ug).

In certain embodiments, ODYRM adjuvants are prepared as follows:

Sorbitan monooleate, MPL-A are dissolved in light mineral oil. The resulting oil solution is sterile filtered and dispersed in water with some surfactant, ethanol and acetic acid;

Polyoxyethylene (20) sorbitan monooleate, quaternary amine, e.g., DDA, and poly l :C are dissolved in aqueous phase, thus forming the aqueous solution; and

The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation ODYRM.

In yet further set of embodiments, in addition to the oil and the emulsifier(s), the adjuvant formulations also comprise (or consist essentially of, or consist of) a combination of a saponin, a sterol, a quaternary amine, a polycationic carrier, with a proviso that if said polycationic carrier is dextran DEAE, then the antigen is not E coli J-5 bacterin. These adjuvants are referred to as "Q.CDXO" .

In QCDXO adjuvants, in certain embodiments, the saponin, e.g., Quil A may be present in the amounts of between about 0.1 ug and about 1000 ug per dose, the sterol, e.g., cholesterol, is present between about 1 ug and about 1000 ug per dose, the quaternary amine, e.g., DDA, is present in the amount of between about 1 ug and about 200 ug per dose, and the polycationic carrier may be present in the amount of 0.5-400 mg per dose. The dosages wary depending on the subject species.

In certain embodiments suitable for cattle, sheep, and adult swine, the saponin is present in the amount of between about 100 and about 1000 ug per dose (e.g., 200-800 ug, or 250-500 ug per dose), sterol is present in the amounts between about 100 and about 1000 ug (e.g., 200-1000, 250-700 ug, or about 400-500 ug), the quaternary amine may be present in the amount of between about 50 ug and about 200 ug per dose (e.g., 50-150 ug, or about 100 ug), and the polycationic carrier may be present in the amount of between about 5 and about 500 mg per dose (e.g., 10-500 mg, or 10-300 mg, or 50- 200 mg per dose).

In certain embodiments suitable for applications in companion animals and piglets, the saponin, e.g., Quil A or a purified fraction thereof is present in the amounts between about 10 and about 100 ug per dose (e.g., 10-50 ug or 20-50 ug per dose), the sterol is present in the amounts between about 5 and 100 ug (e.g., 10-80, or 20-50 ug), the quaternary amine may be present in the amount between about 5 and about 500 ug per dose (e.g., 10-100 ug per dose, or 20-50 ug per dose), and and the polycationic carrier may be present in the amount of 1-50 mg per dose (e.g., 1-25 mg per dose, or 10- 25 mg per dose.

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

In certain embodiments, Q.CDXO adjuvants are prepared as follows:

Sorbitan monooleate is dissolved in oil. The resulting oil solution is sterile filtered;

Polyoxyethylene (20) sorbitan monooleate, quaternary amine, e.g., DDA, the polycationic carrier, the sterol and the saponin are dissolved in aqueous phase, thus forming the aqueous solution; and The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation QCDXO.

Antigens and Diseases

The compositions can contain one or more antigens. The antigen can be any of a wide variety of substances capable of producing a desired immune response in a subject, including, without limitations, one or more of viruses (inactivated, attenuated, and modified live), bacteria, parasites, nucleotides (including, without limitation nucleic-acid based antigens, e.g., DNA vaccines), polynucleotides, peptides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including tumor cells), tissues, polysaccharides, carbohydrates, fatty acids, teichioc acid, peptidoglycans, lipids, or glycolipids, individually or in any combination thereof.

The antigens used with the adjuvants of the invention also include immunogenic fragments of nucleotides, polynucleotides, peptides, polypeptides, that can be isolated from the organisms referred to herein.

Live, modified-live, and attenuated viral strains that do not cause disease in a subject have been isolated in non-virulent form or have been attenuated using methods well known in the art, including serial passage in a suitable cell line or exposure to ultraviolet light or a chemical mutagen. Inactivated or killed viral strains are those which have been inactivated by methods known to those skilled in the art, including treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), sterilizing radiation, heat, or other such methods.

Two or more antigens can be combined to produce a polyvalent composition that can protect a subject against a wide variety of diseases caused by the pathogens. Currently, commercial manufacturers of vaccines, as well as end users, prefer polyvalent vaccine products. While conventional adjuvants are often limited in the variety of antigens with which they can be effectively used (either monovalently or polyvalently), the adjuvants described herein can be used effectively with a wide range of antigens, both monovalently and polyvalently. Thus, the antigens described herein can be combined in a single composition comprising the adjuvants described herein.

Some examples of bacteria which can be used as antigen combinations with the adjuvant compositions include, but are not limited to, 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 spp., Chromobacterium viscosum, Erysipelothrix rhusiopathieae, Listeria monocytogenes, Ehrlichia canis, Escherichia coli, Haemophilus influenzae, Haemophilus somnus, Helicobacter suis, Lawsonia intracellulars, Legionella pneumophilia, Moraxellsa sp., Mycobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasma mycoides subsp. mycoides LC, Clostridium perfringens, Odoribacter denticanis, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida, Photorhabdus luminescens, Porphyromonas gulae, Porphyromonas gingivalis, Porphyromonas salivosa, Propionibacterium acnes, Proteus vulgaris, Pseudomonas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKWl, Pseudomonas fragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp Bll-1, Alcaliges eutrophus, Psychrobacter immobilis, Rickettsia prowazekii, Rickettsia rickettsia, Salmonella enterica all serovars, including for example: 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 suis, Streptomyces exfoliates, Streptomyces scabies, Sulfolobus acidocaldarius, Syechocystis sp.. Vibrio cholerae, Borrelia burgdorferi, Treponema denticola, Treponema minutum, Treponema phagedenis, Treponema refringens, Treponema vincentii, Treponema palladium, Trueperella pyogenes and Leptospira species, such as the known 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 any and all combinations thereof. In the presence of the adjuvants of the invention, combinations involving one or more antigens from Streptococcus uberis are preferred.

Both inactivated viruses and attenuated live viruses may be used in the adjuvant compositions. Some examples of viruses which can be used as antigens include, but are not limited to, Avian herpesviruses, Bovine herpesviruses. Canine herpesviruses, Equine herpesviruses. Feline viral rhinotracheitis virus, Marek's disease virus, Ovine herpesviruses, Porcine herpesviruses, Pseudorabies virus, Avian paramyxoviruses, Bovine respiratory syncytial virus, Canine distemper virus, Canine parainfluenza virus, canine adenovirus, canine parvovirus, Bovine Parainfluenza virus 3, Ovine parainfluenza 3, Rinderpest virus, Border disease virus, Bovine viral diarrhea virus (BVDV), BVDV Type I, BVDV Type II, Classical swine fever virus. Avian Leukosis virus. Bovine immunodeficiency virus. Bovine leukemia virus, Bovine tuberculosis, 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 encephalomyletitis virus. Porcine parvovirus, Porcine Circovirus (PCV) Type I, PCV 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, Rhinoviruses, 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. Antigens from any of the above viruses can be combined with bacterial antigens (whether whole or subunit components) using the adjuvant formulations of the present invention, in which case preferred bacterial antigens include those provided from Streptococcus uberis.

Examples of peptide antigens include Bordetella bronchiseptica p68, GnRH, IgE peptides, Fel dl, and cancer antigens, and combinations thereof. Examples of other antigens include nucleotides, carbohydrates, lipids, glycolipids, peptides, fatty acids, lipoteichoic and teichoic acid, and peptidoglycans, and combinations thereof.

Some examples of parasites which can be used as antigens 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 (Barber pole worm)Ostertagia ostertagi(stomach worm), 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 external parasites including, but not limited to, ticks, including Ixodes, Rhipicephalus, Dermacentor, Amblyomma, Boophilus, Hyalomma, and Haemaphysalis species, and combinations thereof.

Additionally, in regard of the aforementioned antigens including those from parasites, the vaccine composition preferably also contains antigens derived from Streptococcus uberis, again in the presence of the vaccine formulations of the invention.

The amount of antigen used to induce an immune response will vary considerably depending on the antigen used, the subject, and the level of response desired, and can be determined by one skilled in the art. For vaccines containing modified live viruses or attenuated viruses, a therapeutically effective amount of the antigen generally ranges from about 10² Tissue Culture Infective Dose (TCID)₅₀ to about 10¹⁰ TCID₅₀, inclusive. For many such viruses, a therapeutically effective dose is generally in the range of about 10² TCID₅₀ to about 10⁸ TCID₅₀, inclusive. In some embodiments, the ranges of therapeutically effective doses are about 10³ TCID₅₀ to about 10⁶ TCID₅₀, inclusive. In some other embodiments, the ranges of therapeutically effective doses are about 10⁴ TCID₅₀ to about 10⁵ TCID₅₀, inclusive.

For vaccines containing inactivated viruses, a therapeutically effective amount of the antigen is generally at least about 100 relative units per dose, and often in the range from about 1,000 to about 4,500 relative units per dose, inclusive. In other embodiments, the therapeutically effective amount of the antigen is in a range from about 250 to about 4,000 relative units per dose, inclusive, from about 500 to about 3,000 relative units per dose, inclusive, from about 750 to about 2,000 relative units per dose, inclusive, or from about 1,000 to about 1,500 relative units per dose, inclusive.

A therapeutically effective amount of antigen in vaccines containing inactivated viruses can also be measured in terms of Relative Potency (RP) per mL. A therapeutically effective amount is often in the range from about 0.1 to about 50 RP per mL, inclusive. In other embodiments, the therapeutically effective amount of the antigen is in a range from about 0.5 to about 30 RP per mL, inclusive, from about 1 to about 25 RP per mL, inclusive, from about 2 to about 20 RP per mL, inclusive, from about 3 to about 15 RP per mL, inclusive, or from about 5 to about 10 RP per mL, inclusive.

The number of cells for a bacterial antigen administered in a vaccine ranges from about lxlO⁵ to about 5xl0¹⁰ colony forming units (CFU) per dose, inclusive. In other embodiments, the number of cells ranges from about lxlO⁷ to 5xl0¹⁰ CFU/dose, inclusive, or from about lxlO⁸ to 5xl0¹⁰ CFU/dose, inclusive. In still other embodiments, the number of cells ranges from about lxlO² to 5xl0¹⁰ CFU/dose, inclusive, or from about lxlO⁴ to 5xl0⁹ CFU/dose, inclusive, or from about lxlO⁵ to 5xl0⁹ CFU/dose, inclusive, or from about lxlO⁶ to 5xl0⁹ CFU/dose, inclusive, or from about lxlO⁶ to 5xl0⁸ CFU/dose, inclusive, or from about lxlO⁷ to 5xl0⁹ CFU/dose, inclusive.

The number of cells for a parasite antigen administered in a vaccine ranges from about lxlO² to about lxlO¹⁰ per dose, inclusive. In other embodiments, the number of cells ranges from about lxlO³ to about lxlO⁹ per dose, inclusive, or from about lxlO⁴ to about lxlO⁸ per dose, inclusive, or from about lxlO⁵ to about lxlO⁷ per dose, inclusive, or from about lxlO⁶ to about lxlO⁸ per dose, inclusive.

It is well known in the art that with conventional adjuvants, a substantially greater amount of inactivated viruses than modified live or attenuated viruses is needed to stimulate a comparable level of serological response. However, it has been surprisingly found that with the adjuvant compositions described herein, approximately the same amounts of inactivated virus and modified live virus stimulate similar levels of serological response. In addition, smaller amounts of modified live, attenuated, and inactivated virus are needed with the adjuvants described herein when compared with conventional adjuvants to achieve the same level of serological response. These unexpected findings demonstrate conservation of resources and reduction of cost during preparation of immunogenic and vaccine compositions. For vaccines with wide utility, the manufacture of millions of doses per year is required, so these savings can be substantial.

Administration of the Compositions

Dose sizes of the compositions typically range from about 1 mL to about 5 mL, inclusive, depending on the subject and the antigen. For example, for a canine or feline, a dose of about 1 mL is typically used, while in cattle a dose of about 2-5 mL is typically used. However, these adjuvants also can be formulated in microdoses, wherein doses of about 100 .mu.L can be used.

The routes of administration for the adjuvant compositions include parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, intravenous administration and in ova. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan.

Use of the Compositions One of the requirements for any vaccine adjuvant preparation for commercial use is to establish the stability of the adjuvant solution for long periods of storage. Provided herein are adjuvant formulations that are easy to manufacture and stable for at least 18 months. In one embodiment, the formulations are stable for about 18 months. In another embodiment, the formulations are stable for between about 18 to about 24 months. In another embodiment the formulations are stable for about 24 months. Accelerated testing procedures also indicate that the formulations described herein are stable.

An advantageous feature of the present adjuvant compositions is that they can be safely and effectively administered to a wide range of subjects. In the art, it is expected that combinations of adjuvants will demonstrate more reactogenicity than the individual components. However, the compositions described herein show decreased reactogenicity when compared to compositions in which any one or two of the components are used, while the adjuvant effect is maintained. It has also been surprisingly found that the adjuvant compositions described herein demonstrate safety improvements when compared with other adjuvant compositions.

The adjuvant compositions described herein are useful for inducing a desired immune response in a subject.

The adjuvants described herein can be used to show serological differentiation between infected and vaccinated animals. Thus, they can be used in a marker vaccine in which the antigen in the vaccine elicits in the vaccinated animals a different antibody pattern from that of the wild-type virus. A marker vaccine is generally used in conjunction with a companion diagnostic test which measures the difference in antibody patterns and demonstrates which animals have been vaccinated and which animals are infected with the wild-type virus. Such technology is useful in the control and eradication of viruses from a subject population. The invention will be further described in the following non- limiting examples.

Specific additional non-limiting embodiments are as follows:

In a first embodiment, the invention provides an adjuvant formulation comprising an oily phase and an aqueous phase, wherein the oily phase comprises at least 50% of the formulation v/v, wherein said formulation comprises at least one of monophosphoryl lipid A (MPL-A) or an analog thereof and an immunostimulatory oligonucleotide, with provisos that: a) if said immunostimulatory oligonucleotide is absent, then the formulation comprises:

I. a poly l:C, a glycolipid, and, optionally, a quaternary amine; or ii. a polycationic carrier;

b) if said monophosphoryl lipid A (MPL-A) or the analog thereof is absent, then the formulation comprises a source of aluminum.

In a second such embodiment, the invention provides the adjuvant formulation of the first embodiment, wherein the immunostimulatory oligonucleotide, if present, is a CpG or an oligoribonucleotide; the polycationic carrier, if present, is selected from the group consisting of dextran, dextran DEAE (and derivatives thereof), PEGs, guar gums, chitosan derivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC) polyethylenimene, poly aminos; and the quaternary amine, if present, is selected from the group consisting of DDA and avridine.

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

In the fourth such embodiment, the inv ention provides the adjuvant formulation according to any one of first through third embodiments, wherein the glycolipid, if present, comprises a compound of formula I

Formula I

wherein, R¹ and R² are independently hydrogen, or a saturated alkyl radical having up to 20 carbon atoms; X is -CH₂-, -0- or -NH-; R² is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵ are independently hydrogen, -S0₄ ^2~, -P0₄ ^2~, -COCi_i₀ alkyl; R⁶ is L- alanyl, L-alpha-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-phenyalany, L-prolyl, L-seryl, L- threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers.

In the fifth such embodiment, the invention provides the adjuvant formulation of the fourth embodiment, wherein the glycolipid is N-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N- octadecyldodecanoylamide or a salt thereof.

In the sixth such embodiment, the invention provides the adjuvant formulation of the fifth embodiment, wherein the salt is an acetate.

In a seventh such embodiment, the invention provides the adjuvant formulation of any one of fists thought fourth embodiments, comprising both said monophosphoryl lipid A (MPL-A) or the analog thereof, and further comprising at least one of a sterol and a poly l :C.

In an eight such embodiment, the invention provides the adjuvant formulation according to the seventh embodiment, comprising the sterol and further comprising a saponin.

In a ninth such embodiment, the invention provides the adjuvant formulation of any one of the senventh and the eighth embodiments, 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.

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

In an eleventh further such embodiment, the invention provides the adjuvant formulation according to the seventh embodiment, comprising the poly l:C, and further comprising at least one of the quaternary amine and the glycolipid.

In a twelfth such embodiment, the invention provides the adjuvant formulation of any one of the first-eleventh embodiments, comprising the MPL-A or the analog thereof in the amount of 0.5 - 100 ug per dose.

In a 13th such embodiment, the invention provides the adjuvant formulation according to the twelfth embodiment, wherein the MPL-A or the analog thereof is present in the amount of 5-50 ug per dose, or 5-20 ug per dose, or 1-5 ug per dose.

In a 14th such embodiment, the invention provides the adjuvant formulation of any one of the fisrt-thirteenth embodiments, comprising the immunostimulatory oligonucleotide in the amount of 0.5 to 400 ug per dose. In a 15 such embodiment, the invention provides the adjuvant formulation of the fourteenth embodiment, wherein the immunostimulatory oligonucleotide is present in the amount of about 100 to about 250 ug per dose or about 20 to about 50 ug per dose, or about 1 ug per dose.

In the sixteenth such embodiment, the invention provides the adjuvant formulation of any one of first through fifteenth embodiments, comprising the polycationic carrier in the amount of between about 0.5 and about 400 mg per dose.

In a 17th such embodiment, the invention provides the adjuvant formulation of the sixteenth embodiment, wherein said polycationic carrier is present in the amount of 50-300 mg per dose or 1-25 mg per dose, or 1-10 mg per dose.

In an 18th such embodiment, the invention provides the adjuvant formulation of any one of the first-seventeenth embodiment, comprising the glycolipid in the amount of between about 0.5 and about 2000 ug per dose.

In a 19^th such embodiment, the invention provides the adjuvant formulation of the eighteenth embodiment, wherein the glycolipid is present in the amount of about 1000 ug per dose, or 25-50 ug per dose, or 1-10 ug per dose.

In a 20th such embodiment, the invention provides the adjuvant formulation of any one of the first-nineteenth embodiments, comprising the sterol in the amount of between about 0.1 and about 1000 ug per dose.

In a 21st such embodiment, the invention provides the adjuvant formulation according to the twentieth embodiment, wherein the sterol is present in the amount of 250-500 ug per dose, or 20-50 ug per dose, or 1-10 ug per dose.

In a 22nd such embodiment, the invention provides the adjuvant formulation of any one of first through twenty-first embodiment, comprising the saponin in the amount of between 0.1 and 1000 ug per dose.

In a 23^rd such embodiment, the invention provides the adjuvant formulation of the twenty- second embodiment, wherein the saponin is present in the amount of 250-500 ug per dose, or 20-50 ug per dose, or 1-10 ug per dose.

In a 24th such embodiment, the invention provides the adjuvant formulation of any one of first through twenty-third embodiment, comprising the poly l:C is in the amount of between about 0.5 and about 100 ug per dose. In a 25th such embodiment, the invention provides the adjuvant formulation of the twenty- fourth embodiment, wherein the poly l:C is present in the amount of 5-50 ug per dose, or 5-20 ug per dose, or 1-5 ug per dose.

In a 26th embodiment, the invention provides the adjuvant formulation of any one of first through twenty-fifth embodiment, comprising the source of aluminum, which is an aluminum hydroxide gel.

In a 27^th embodiment, the invention provides the adjuvant formulation of twenty-sixth embodiment, wherein said source of aluminum is present in the amount of 5%-20% v/v of the formulation.

In a 28th such embodiment, the invention provides the adjuvant formulation of the twenty- seventh embodiment, wherein said source of aluminum is present in the amount of 10% v/v of the formulation.

In the twenty-ninth embodiment, the invention provides The adjuvant formulation of any one of the first through twenty-eighth embodiment, wherein the oily phase comprises an oil and an oil- soluble emulsifier.

In the thirtieth such embodiment, the invention provides the adjuvant formulation of any one of the first through the twenty-ninth embodiment, wherein said oily phase is present in the amount of up to 85% v/v.

In the thirty-first such embodiment, the invention provides the adjuvant formulation according to the thirtieth embodiment, wherein said oily phase is present in the amount of 51%.

In the thirty-second such embodiment, the invention provides the adjuvant formulation of any one of the twenty-ninth through the thirty-first embodiments, wherein the oil comprises 40-84% v/v of the formulation, and the oil-soluble emulsifier comprises 1-11% v/v of the formulation.

In the thirty-third such embodiment, the invention provides the adjuvant formulation of the thirty-second embodiment, wherein the oil comprises 45% v/v of the formulation, and the oil-soluble emulsifier comprises 6% v/v of the formulation.

In the thirty-fourth embodiment, the invention provides the adjuvant formulation according to any one of the first through thirty-third embodiment, wherein said oil is selected from the group consisting of squalane, vegetable oils, triglycerides, non-metabolizable straight- chain alkane oils, and any combination thereof. In the thirty-fifth such embodiment, the invention provides the adjuvant formulation according to the thirty-fourth embodiment, wherein said oil is a light mineral oil.

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

In the thirty-seventh such embodiment, the invention provides a vaccine composition comprising an effective amount of an antigen and an adjuvant formulation comprising an oily phase and an aqueous phase, wherein the oily phase comprises at least 50% of the formulation v/v, a polycationic carrier, and

a. a combination of a saponin and a sterol, and optionally, a quaternary amine; with provisos that if said adjuvant formulation consists essentially of DEAE dextran, Quil A, Cholesterol, and DDA, the antigen is not E coli J-5 bacterin; or

b. an immunostimulatory oligonucleotide.

In the thirty-eighth such embodiment, the invention provides he 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 thereof), PEGs, guar gums, chitosan derivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC) polyethylenimene, poly aminos, and the quaternary amine, if present, is selected from the group consisting of DDA and avridcine.

In the thirty-ninth such embodiment, the invention provides the 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.

In the fourtieth such embodiment, the invention provides the vaccine composition of any one of thirty-seventh though thirty-ninth embodiments, wherein the immunostimulatory oligonucleotide is a CpG.

In the fourty-first such embodiment, the invention provides the vaccine composition of any one of thirty-seventh through fourtieth embodiment, wherein said polycationic carrier is present in the amount of between about 0.5 and about 400 mg per dose. In the fourty-second such embodiment, the invention provides the vaccine composition of the fourty-first embodiment, wherein said polycationic carrier is present in the amount of 50-300 mg per dose or 1-25 mg per dose, or 1-10 mg per dose.

In the fourty-third such embodiment, the invention provides the vaccine composition of any one of thirty-seventh through fourty-second embodiments, comprising the saponin in the amount of between about 0.1 and about 1000 ug per dose.

In the fourty-fourth such embodiment, the invention provides the vaccine composition of the fourty-third embodiment, wherein the saponin is present in the amount of 250-500 ug per dose, or 20- 50 ug per dose, or 1-10 ug per dose.

In the fourty-fifth such embodiment, the invention provides the vaccine composition of any one of thirty-seventh through fourty-fourth embodiments, comprising the sterol in the amount of between about 0.1 and about 1000 ug per dose.

In the fourty-sixth such embodiment, the invention provides the vaccine composition of the fourty-fifth embodiment, wherein the sterol is present in the amount of 250-500 ug per dose, or 20-50 ug per dose, or 1-10 ug per dose.

In the fourty-seventh such embodiment, the invention provides the vaccine composition of any one of thirty-seventh through fourty-sixth embodiments, comprising the quaternary amine in the amount of between about 1 and about 200 ug per dose.

In the fourty-eighth such embodiment, the invention provides the vaccine composition of fourty-seventh embodiment, wherein the quaternary amine is present in the amount of about 100 ug per dose or between about 10 and about 100 ug per dose or about 5 ug per dose.

In the fourty-ninth such embodiment, the invention provides the vaccine composition of any one of of thirty-seventh through fourty-eighth embodiments, comprising the immunostimulatory oligonucleotide in the amount of between about 0.5 ug and about 400 ug per dose.

In the fiftieth such embodiment, the invention provides the vaccine composition of the fourty- ninth embodiment, wherein the immunostimulatory oligonucleotide is present in the amount of 100- 250 ug per dose, or 20-50 ug per dose or about 1 ug per dose.

In the fifty-first such embodiment, the invention provides the vaccine composition of any one of thirty-seventh through fiftieth embodiments, wherein the oily phase comprises an oil and an oil- soluble emulsifier. In the fifty-second such embodiment, the invention provides the vaccine composition of any one of thirty-seventh through fifty-first embodiments, wherein said oily phase is present in the amount of up to 85% v/v.

In the fifty-third such embodiment, the invention provides the vaccine composition of the fifty- second embodiment, wherein said oily phase is present in the amount of 51% v/v.

In the fifty-fourth embodiment, the invention provides the vaccine composition of any one of fifty-first through fifty-third embodiments, wherein the oil comprises 40-84% v/v of the vaccine composition, and the oil-soluble emulsifier comprises 1-11% v/v of the vaccine composition.

In the fifty-fifth such embodiment, the invention provides the vaccine composition of the fifty- third embodiment, wherein the oil comprises 45% v/v of the formulation, and the oil-soluble emulsifier comprises 6% v/v of the formulation.

The invention also provides a vaccine composition comprising a Streptococcus uberis antigen and an adjuvant comprising an oily phase, said oily phrase beuing preferably present at at least 50% v/v of the composition, and also a polycationic carrier, wherein said composition may further comprise an immunostimulatory oligonucleotide, a saponin, a sterol, and a quartenary amine, and any combinations thereof. The invention further provides provides a vaccine composition comprising a Streptococcus uberis (S. uberis) antigen and an adjuvant formulation comprising an oily phase, said oily phase being present in the amount of at least 50% v/v of the composition; a polycationic carrier; and an immunostimulatory oligonucleotide;

a combination comprising a saponin, a sterol, and a quaternary amine; or

a combination thereof.

Streptocccus uberis antigens

The present invention is also based upon the identification of a number of antigens derived from a species of the genus Streptococcus, which can be used to raise immune responses in animals - particularly those animals susceptible or predisposed to infection by (or with) the Streptococcus species. The antigens provided by this invention may be exploited to provide compositions and vaccines for raising protective immune responses in animals - the protective immune responses serving to reduce, prevent, treat or eliminate certain Streptococcus infections as well as diseases and/or conditions caused or contributed to thereby. In one aspect, the present invention provides one or more Streptococcus uberis antigen(s) or a fragment or fragments thereof, for use in raising an immune response in an animal.

Despite the fact that Streptococcus uberis is a highly-heterogeneous species, the inventors have discovered antigens which are conserved in different 5. uberis strains. That is to say, while the antigens of this invention may be derived or obtained from a single 5. uberis strain, they may protect animals against infections and/or diseases caused or contributed to different (heterologous) Streptococcus uberis strains. Moreover, the inventors have noted that the antigens provided by this invention may also be used to protect animals against infections (or diseases) caused or contributed to by other Streptococcus spp., including, for example, infections, diseases and/or conditions caused or contributed to by including (but not limited to) 5. parauberis, 5. agalactiae and 5. dysgolactiae. Similalry, the antigens described herein may find application in the diagnosis of infections, diseases and/or conditions with a streptococcal (including, but not limited to, 5. uberis, S. parauberis, S. agalactiae and 5. dysgalactiae) aetiology. For convenience, each of the specific Streptococcus species and strains relevant to this invention shall be collectively referred to under the general term "Streptococcus", "streptococci", or "streptococcal". Moreover, it should be understood that references to 5. uberis include all related 5. uberis strains and variants.

An immune response which protects against infection by/with a pathogen or against certain diseases or conditions, may be a referred to as a 'protective response'. Therefore, in the context of this invention, the immune responses elicited by the antigens described herein may be regarded as 'protective' immune responses.

The antigens provided by this invention are immunogenic or antigenic in that they elicit host immune responses; the precise nature of the response (humoral and/or cellular for example) may depend on the formulation of the antigen, its route of administration and/or the presence or absence of adjuvant.

The effectiveness of any immune response elicited by the antigens of this invention may be assessed relative to the prevalence or rate of the relevant Streptococcus based infection/disease among a population of animals not exposed to, contacted with or administered antigens of this invention. One of skill will appreciate that animals not exposed to, contacted with or administered an antigen of this invention may lack a protective immune response and are therefore more susceptible to Streptococcus infections and/or diseases.

A second aspect of this invention provides a composition, immunogenic composition or vaccine composition comprising, consisting essentially of, or consisting of, two or more of the Streptococcus uberis antigens described herein, for use in raising an immune response in an animal. I n one embodiment, the immune response is a protective response.

In a third aspect, the invention provides the use of one or more Streptococcus uberis antigens or a fragment(s) thereof for the manufacture of a medicament or vaccine for use in the treatment and/or prevention of a Streptococcus uberis infection and/or a disease or condition caused thereby or associated therewith.

In a fourth aspect, the invention provides a method of raising an anti-Sfreprococcus uberis immune response in an animal, said method comprising the step of administering to an animal, an amount of one or more Streptococcus antigen(s) or fragment(s) thereof, sufficient to induce an anti- Streptococcus uberis immune response.

The term "animal" also encompasses any animal known to be susceptible to a Streptococcus infection, disease or condition. For example, any animal susceptible to infections, diseases or conditions caused or contributed to by S. uberis, S. parauberis, S. agalactiae and/or 5. dysgalactiae (or indeed any strains of any of these) is encompassed under the general term "animal" as used herein. For example, the term "animal" may include humans and animals collectively known as avian (birds), piscine (e.g. fish), porcine (e.g. pig), bovine (e.g. cattle), caprine (e.g. goats) and/or ovine (e.g. sheep) animals.

Diseases and/or conditions caused or contributed to by Streptococcus (including for example S. uberis and 5. dysgalactiae) include clinical and sub-clinical cases of mastitis. As such, the antigens provided by this invention may be used to raise immune responses in animals which are susceptible, predisposed or prone to developing (streptococcal based) mastitis, said responses being protective against the development of mastitis. For example, the invention may be used to raise immune responses in human, porcine, bovine, caprine and/or ovine animals, said responses being protective against the development of mastitis.

In view of the above, this invention provides:

(i) Streptococcus uberis vaccines and medicaments comprising the same; and

(ii) methods exploiting one or more antigens derived from Streptococcus uberis;

for use in raising immune responses in human, avian, piscine, bovine, porcine, caprine and/or ovine animals.

It should be understood that all references to "antigen" encompass immunogenic components, proteins or peptides derived from Streptococcus. The antigens may comprise cell-surface antigens and/or intracellular antigens. The antigens of this invention may be prepared using recombinant technology (as described later) but may be obtained or purified from 5. uberis cells and cell cultures. For example, S. uberis cell-surface antigens may be isolated from 5. uberis cell-wall preparations.

Specifically, the term "antigen" encompasses the exemplary Streptococcus uberis antigens identified and listed as (1) ferrichrome binding protein (See SEQ I D NOS: 1,2 and 3, locus tag SUB0423, and accession number YP-002561776); (2) elongation factor Tu (See SEQ ID NOS: 4, 5 and 6, locus tag SUB0604, and accession number YP-002561947; (3) a lipoprotein (See SEQ ID NOS: 7, 8 and 9, locus tag SUB0950, and accession number YP-002562276; and (4) a serine protease (See SEQ ID NOS: 10, 11 and 12, locus tag SUB1868, and accession number YP-002563137). Further information concerning identification and function of these proteins is available from PromBase, based on a March 23, 2009 download from the National Center for Biotechnology I nformation of the USNIH, Bethesda, MD, USA (see also NCBI records indicating database deposit of these Strain 0140J sequences in 2008-2009 by the Sanger Institute Wellcome Trust, Cambridge, UK and initial characterization of the uberis genome (Ward et al., BMC Genomics, 2009, v. 10 , p.54-70. See also B.R. Shome et al, Trop. Anim. Health Prod (2012) v 44, pp. 1981-1992.

The 5. uberis antigens identified in Table 1 are those identified by mass spectrometric analysis of cell-wall sub-cellular fractions of 20 5. uberis strains, and further found (by PCR analysis to determine carriage of the antigen-encoding genes) among a larger panel of S. uberis strains. Consequently, one or more of these antigens may be exploited in this invention and used in the methods, vaccines and/or compositions described herein., as they are conserved among strains, making such antigens widely applicable vaccine candidates.

In view of the above, the term "antigen" as used herein, encompasses antigens encoded by or comprising/ consisting (essentially of) the sequences deposited under each of the accession numbers identified in Table 1. It should be noted that the sequences deposited under each of the accession numbers identified above are derived from a 5. uberis strain designated 0140J (accession number NC 012044). Thus, while the invention encompasses antigens derived from this S. uberis strain, it also encompasses the identical, homologous or othologous antigens present in other Streptococci and/or 5. uberis strains.

The inventors have recombinantly prepared four S. uberis antigens and the sequences of these cloned antigens are provided in Table 1 below. One of skill will appreciate that recombinant sequences may comprise sequences which differ from any corresponding wild type or reference sequences and may comprise, for example sequences which encode protein or peptide tags. Recombinant sequences may be modified by, for example, the deletion of signal peptide sequences. The sequences presented in Table 1 may have a 5'-nucleotide sequence encoding a 6 x histidine tag - a tag of this type may be used for purification purposes. Also, some of the sequences presented in Table 1 have been further modified to lack sequences encoding secretion signal peptides present in the corresponding wild type sequences.

Table 1

Recombinant gene sequence Translated product

rSUB0423 ferrichrome binding protein (SEQ I D NO: 1 and 2)

Seq ID NO: 1 Seq ID NO: 2

ATGGGCAGCAGCCATCATCATCATCATCACAGCA MGSSHHHHHHSSGLVPRGSHMLEMSQSTKQEDHK GCGGCCTGGTGCCGCGCGGCAGCCATA TKLSQMPKISGFTYKGKVPENPKRVV

TGCTCGAGATGTCACAAAGCACAAAGCAAGAAGA SLSSTYTGYLAKLDIPLVGITSYDHKNPVLKKYI TCATAAAACAAAACTATCACAAATGC KDAKVVSATDLESITALEPDLI IVGSN

CAAAGATCTCTGGTTTTACCTATAAAGGGAAGGT EENI SQLAEI PLI SIEYRKHDYLQVFSDFGKVF ACCAGAAAACCCTAAAAGAGTAGTTA NKTKETDKWLQEWKTKT SFESDVKA

GTTTATCTTCAACCTACACCGGTTATTTGGCAAA VTGNNATFTIMGLYEKDIYLFGKDWGRGGEI IHQ GCTCGATATCCCACTAGTTGGAATCA AFQYQAPEKVKMEVFPKGYLSI SQEV

CTTCTTATGATCACAAAAATCCCGTCTTAAAGAA LPDYIGDYVVVAAEDEKTGSSLYESDLWKNIPAV ATACATCAAGGATGCTAAAGTTGTCT QKNHVINVNANTFYFTDPLSLEYELK

CTGCAACCGACCTAGAAAGCATTACGGCCTTGGA TLTDAILTQKTHN ACCTGATTTAATTATTGTGGGTTCAA

ATGAAGAAAATATCAGTCAATTAGCTGAAATCGC TCCCCTTATTTCCATTGAATACCGCA

AACATGACTATTTACAGGTATTCTCAGATTTTGG TAAAGTCTTTAACAAAACCAAAGAAA

CCGACAAATGGTTACAGGAATGGAAAACAAAAAC AGCTTCTTTTGAAAGTGACGTTAAAG

CAGTTACAGGTAATAATGCTACCTTTACCATAAT GGGATTATATGAGAAAGATATCTATC

TTTTCGGTAAAGATTGGGGTCGTGGTGGTGAAAT CATTCACCAAGCCTTCCAATATCAAG

CTCCAGAAAAAGTAAAAATGGAGGTTTTCCCAAA AGGCTATTTGTCCATTTCACAAGAAG

TTCTTCCAGATTATATTGGTGATTATGTCGTTGT CGCTGCAGAGGATGAAAAAACAGGTT

CTTCTCTTTATGAAAGTGACCTTTGGAAAAATAT ACCAGCCGTTCAAAAAAATCATGTCA

TAAATGTTAATGCGAATACCTTTTATTTCACTGA CCCTCTGTCATTAGAGTATGAATTAA

AAACCTTAACGGATGCTATCTTGACTCAGAAAAC TCACAAC A rSUB0604 elongation factor Tu (SEQ ID NO: 4 and 5)

Seq I D NO:4 Seq ID NO:5

TGGGCAGCAGCCATCATCATCATCATCACAGCAG MGSSHHHHHHSSGLVPRGSHMLEMAKEKYDRSKP CGGCCTGGTGCCGCGCGGCAGCCATA HVNIGTIGHVDHGKTTLTAAITTVLA

TGCTCGAGATGGCAAAAGAAAAATACGATCGTAG RRLPTSVNQPKDYASIDAAPEERERGITINTAHV TAAACCCCACGTTAACATTGGTACAA EYETETRHYAHIDAPGHADYVKNMIT

TTGGACACGTTGACCACGGTAAAACTACTTTGAC GAAQMDGAILVVASTDGPMPQTREHILLSRQVGV AGCTGCAATTACAACTGTACTTGCTC KHLIVFMNKIDLVDDEELLELVEMEI

GTCGCTTACCAACTTCAGTTAACCAACCAAAAGA RDLLSEYDFPGDDLPVIQGSALKALEGDSKYEDI TTACGCTTCTATCGATGCTGCTCCAG IMELMKTADEYI PEPERDTDKPLLLP

AAGAGCGCGAACGCGGAATCACTATCAACACTGC VEDVFS ITGRGTVASGRIDRGTVRVNDEIEIVGI ACACGTTGAGTACG AACTG AACTC KEETKKAVVTGVEMFRKQLDEGLAGD

GTCACTATGCCCACATTGATGCCCCAGGACACGC NVGILLRGVQRDEIERGQVIAKPGSINPHTKFKG GGACTATGTTAAAAACATGATCACTG EVYILSKDEGGRHTPFFNNYRPQFYF

GTGCTGCCCAAATGGACGGAGCTATCCTTGTTGT RTTDVTGS IELPAGTEMVMPGDNVTI SVELIHPI TGCATCAACTGATGGACCAATGCCAC AVEQGTTFSIREGGRTVGSGIVSEIE

AAACTCGTGAGCACATCCTTCTTTCACGCCAAGT TGGTGTTAAACACCTTATCGTTTTCA

TGAACAAAATCGACCTTGTTGACGATGAAGAATT GCTTGAATTAGTTGAAATGGAAATCC

GTGACCTTCTTTCAGAATACGATTTCCCAGGTGA TGACCTACCAGTTATCCAAGGTTCAG

CTCTTAAAGCTCTTGAAGGTGATTCTAAATACGA AGACATCATCATGGAATTGATGAAAA

CTGCTGATGAGTATATTCCAGAACCAGAACGTGA TACAGACAAACCATTACTTCTTCCAG

TCGAAGACGTATTCTCAATCACAGGTCGTGGTAC TGTAGCTTCAGGACGTATCGATCGTG

GTACTGTTCGTGTCAACGACGAAATTGAAATCGT TGGTATCAAAGAAGAAACTAAAAAAG

CAGTTGTTACTGGTGTTGAAATGTTCCGTAAACA ACT GACGAAGGTCTTGCAGGAGATA

ACGTAGGTATCCTTCTTCGTGGTGTTCAACGTGA CGAAATCGAACGTGGACAAGTTATTG

CTAAACCAGGTTCAATCAACCCACACACTAAATT CAAAGGTGAAGTTTACATCCTTTCTA

AAGATGAAGGTGGACGTCATACTCCATTCTTCAA CAACTACCGTCCTCAATTCTATTTCC

GTACAAC GACGTAAC GGTTC ATCGAACTTCC AGCTGGTACTGAAATGGTAATGCCTG

GTGATAACGTGACAATCAGCGTTGAGTTGATCCA CCCAATCGCCGTTGAACAAGGTACTA

CTTTCTCAATCCGTGAAGGTGGACGTACTGTTGG TTCAGGTATTGTTTCAGAAATCGAAG

CTTAA rSUB0950 lipoprotein (SEQ ID NO: 7 and 8)

Seq I D NO: 7 Seq ID NO: 8

ATGGGCAGCAGCCATCATCATCATCATCACAGCA MGSSHHHHHHSSGLVPRGSHMLEMDSKDAKTDLK GCGGCCTGGTGCCGCGCGGCAGCCAT AAIVTDTGGVDDKSFNQSAWEGLEA

ATGCTCGAGATGGATAGCAAAGATGCTAAAACAG WGKENGLKKGAGFDYFQSNSESEYATNLDTAVSS ATTTAAAAGCTGCTATTGTTACTGAT GYNVVYGIGFALKDAI DKAAGDNSDVN

ACAGGTGGTGTTGATGATAAATCATTTAACCAAT YIIVDDVIEGKDNVASVTFADNEAAYLAGIAAAK CTGCTTGGGAAGGTTTAGAAGCTTGG TTKTKVVGFVGGMEGTVI TRFEKGFE

GGTA AGA A TGGGCTT A A AGGTGCTGGTT AGVKSVDDSIQIKVDYAGSFGDAAKGKTI AAQY TCGACTACTTCCAATCAAATAGTGAA AGGADVIYQAAGGTGAGVFNEAKAVN

TCAGAATATGCTACTAATCTTGACACTGCTGTCT EKKDEADKVWVIGVDRDQKEEGKYTSKDGKESNF CAAGTGGTTATAACGTAGTATATGGA VLASSIKQVGKSVQLINKLVTDKKFP

ATCGGATTTGCCCTTAAAGATGCAATTGATAAAG GGKTTVYGLKDGGVDIATTNLSDDAIKAVKEAKE CTGCTGGTGACAATAGTGATGTTAAC KIISGDVKVPEK

TATATTATCGTTGACGATGTCATCGAAGGAAAAG ATAATGTTGCAAGTGTAACTTTTGCG

GATAACGAAGCTGCTTATCTTGCTGGTATTGCTG CAGCTAAAACTACAAAAACTAAAGTA

GTAGGTTTTGTAGGTGGTATGGAAGGTACTGTTA TCACTCGTTTTGAAAAAGGTTTTGAG

GCGGGAGTGAAATCAGTTGATGATTCTATCCAAA TCAAAGTTGACTACGCTGGATCATTT

GGTGATGCTGCTAAAGGTAAAACAATTGCCGCAG CTCAATATGCAGGTGGTGCTGACGTT

ATTTATCAAGCCGCTGGTGGTACTGGAGCAGGTG TCTTCAATGAAGCTAAAGCTGTAAAT

GAGAAAAAAGATGAAGCTGATAAAGTTTGGGTAA TCGGTGTAGACCGTGACC AAAAG G

GAAGGTAAATACACTTCAAAAGACGGTAAAGAAT CTAACTTTGTTCTAGCATCTTCAATT

AAACAAGTTGGTAAATCTGTACAACTGATTAACA AACTTGTTACTGATAAAAAATTCCCT

GGTGGAAAAACAACTGTTTATGGATTAAAAGATG GTGGTGTTGATATTGCAACAACAAAC

CTTTCTGATGATGCTATAAAAGCTGTTAAAGAAG CTAAAGAAAAAATTATTTCTGGCGAT

GTAAAAGTTCCTGAAAAATAA rSUB1868 serine protei nase (SEQ I D NO: 10 and 11)

Seq I D NO: 10 Seq I D NO : 11

ATGGGCAGCAGCCATCATCATCATCATCACAGCA MGSSHHHHHHSSGLVPRGSHMTNLNNPTTTSKVT GCGGCCTGGTGCCGCGCGGCAGCCAT YKNTTNTTKAVKVIQDAWSVVNYQK

ATGACAAATCTTAATAACCCAACGACGACAAGTA NDSLNSAMDIFSQGDSSTKENDGLSIYSEGSGVI AAGTAACCTATAAAAATAC ACTAAT YKKDGDSAYLVTNNHVIDKAERIEI I

ACGACTAAAGCTGTTAAAGTGATTCAAGATGCAG LADGSKVVGKLIGADTYSDLAWKISSDKIKTVA TTGTTTCTGTAGTTAACTATCAAAAA QFADSSKINIGEVAIAIGSPLGTEYA

AATGATTCTTTAAACTCAGCCATGGATATTTTTA NSVTEGIVSSLSRTVTLKNEEGQTVSTNAIQTDA GTCAAGGTGATTCATCAACTAAAGAG AINPGNSGGPLINIEGQI IGINSSKI

AATGATGGACTTTCTATTTATAGTGAAGGATCAG SQSKSSGNAVEGMGFAIPANDVIKI INQLESKGE GTGTTATATACAAAAAAGATGGTGAT VVRPALGI SMVNLSDLSTNALDQLKV

TCTGCATATCTGGTAACCAACAACCACGTAATAG PKNVTSGIWAKWDNMP SGKLEQYD11TEI DG ACAAAGCTGAAAGAATTGAAATTATT EEVSSTSDLQSILYGHDINDTVKVTF

TTAGCTGATGGTTCAAAAGTTGTTGGGAAATTAA YRGNDKKSTTIELTKTTKDLEK TTGGTGCTGACACTTATTCTGACCTG

GCTGTTG AAAA TTCTTC GAC AAATTAAGA CTGTAGCTCAGTTTGCAGATTCTTCC

AAAATAAACATAGGTGAAGTTGCAATTGCAATTG GTAGTCCTCTTGGAACAGAATATGCT

AATTCCGTAACTGAAGGAATTGTTTCAAGTTTAA GTAGAACAGTAACTTTAAAAAATGAA

GAAGGACAAACTGTTTCAACTAATGCCATTCAAA CAGATGCTGCTATTAACCCTGGAAAC

TCTGGCGGACCTTTAATTAATATTGAAGGACAAA T A TGGAATAAACTCTAGCAAAATC

TCACAGTCTAAATCA CTGGAAATGCAGTCGAAG GAATGGGATTTGCAATTCCAGCTAAT

GACGTTATTAAAATTATTAACCAACTTGAAAGCA AAGGCGAAGTAGTTCGACCTGCATTA

GGTATTTCAATGGTTAATCTAAGTGATTTATCAA CAAATGCCCTTGATCAGCTCAAAGTT

CCAAAAAATGTTACTAGTGGTATCGTAGTTGCTA AAGTCGTAGACAATATGCCTGCCTCA

GGAAAACTTGAACAATATGATATTATCACTGAAA TTGATGGGGAAGAAGTGAGCAGTACA

AGTGATTTACAAAGTATTCTGTATGGGCATGATA TTAATGATACCGTAAAAGTCACTTTT

TATAGAGGTAATGATAAGAAATCTACTACTATTG AATTAACTAAAACTACC

AAAGATTTAGAAAAATAA

As such, this invention relates to one or more recombinant forms of any of the Streptococcus uberis antigens identified in this specification - the recombinant sequences being optionally modified (relative to the corresponding wild type sequence) to include sequences encoding tagging or labelling moieties and or through the deletion of one or more wild type sequences - such as, for example, a sequence encoding a signal peptide. The invention may further relate to the equivalent or corresponding antigens present in other Streptococcal species, including for example the identical, homologous or orthologous antigens present in S. agalactiae, S. parauberis and 5. dysgalactiae. One of skill will appreciate that sequences encoding the antigens of this invention derived from other strains of 5. uberis may differ in nucleic acid sequence. These sequence differences may arise as a result of the natural variation, mutation(s) and/or (single or multiple) nucleotide polymorphisms that often exist between the genomes of related species. Where the biological function of a particular protein is reliant on a particular amino acid sequence, selective pressure will tend to ensure that only mutations which introduce silent or conservative changes to the encoded protein are retained within the microbial population. Consequently, one of skill will recognise that despite divergence in gene coding sequences, retention of biological function through retention of functional epitopes may occur, even though the sequence of less-important regions of a protein coding sequence may vary greatly between strains within a species and between species.

In order to raise an immune response that interferes with/abrogates the function of wild-type proteins produced by 5. uberis during infection, and in so doing reduce the infective capacity of the pathogen. It therefore follows that the same antigens, or derivatives thereof, may also be used to induce equivalent protective immune responses against pathogens other than S. uberis. Indeed, an immune response raised by any of the antigens described herein may offer protection against any pathogen (in particular any other Streptococcus species) expressing those antigens. As is well known in the art, the degeneracy of the genetic code permits substitution of one or more bases in a codon without changing the primary amino acid sequence. Consequently, although the nucleic acid sequences described in this application are known to encode 5. uberis antigens which elicit immune responses in animals, the degeneracy of the code may be exploited to yield variant nucleic acid sequences which encode the same or similar primary amino acid sequences. Additionally or alternatively, the invention further encompasses sequences which have been codon optimised, perhaps for expression in certain cellular (for example bacterial) systems. As such, the term

"antigen" encompasses nucleic acid sequences which encode the amino acid sequences of the S. uberis antigens described herein including for example described in Tables 1 and 2. The invention may further extend to cDNA generated from messenger RNA encoding any of the antigens of this invention. It should be understood that this invention further extends to fragments or portions of the various antigens and sequences disclosed herein. The fragments and/or portions of these antigens and/or sequences may themselves provide or encode antigens which are antigenically/immunogenically similar to the complete or whole Streptococcus uteris antigens disclosed herein. Thus the fragments and/or portions of this invention are capable of eliciting an immune response which is substantially identical, or similar, to an immune response elicited by the complete antigen from which the fragment is derived. The terms "fragments" and "portions" as applied to the S. uberis antigens of this invention, encompass immunogenic and/or antigenic fragments and/or portions, which fragments and/or portions can be used to raise immune responses in animals. Fragments or portions of any of the antigens disclosed herein may elicit protective immune responses in animals and may comprise epitopes capable of eliciting protective immune responses.

A fragment or portion of an antigen provided by this invention may comprise any number of amino acid/nucleic acid residues from about 5 to about 10 residues to about n-1 residues, wherein "n" is the total number of (amino acid or nucleic acid) residues of a 5. uberis antigen described herein. For example, a fragment or portion of a S. uberis antigen may comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300 residues - the upper limit (n-1) depending upon the size (n) of the nucleic acid encoding the complete antigen or the number (n) of amino acid residues comprising the primary sequence of the antigen.

In view of the above, the antigen fragments or portions provided by this invention include fragments and portions of any of the sequences identified in Tables 1 above.

The term "antigen" may further encompass antigens which exhibit a degree of identity and/or homology to the antigens and/or antigen sequences described herein. By way of example, a homologous or identical sequence provided by this invention may exhibit at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology or identity to the various sequences provided herein - including, for example, those sequences identified by accession number in Table 1. Preferably such homology is 80% or higher, most preferably 90%, or optimally 95% or higher.

The degree of (or percentage) "homology" between two or more (amino acid or nucleic acid) sequences may be determined by aligning the sequences and determining the number of aligned residues which are identical or which are not identical but which differ by redundant nucleotide substitutions (the redundant nucleotide substitution having no effect upon the amino acid encoded by a particular codon, or conservative amino acid substitutions). Homology may assessed by using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, Basic local alignment search tool, J. Mol. Biol. V. 215, pp 403-410.)

A degree (or percentage) "identity" between two or more (amino acid or nucleic acid) sequences may also be determined by aligning the sequences and ascertaining the number of exact residue matches between the aligned sequences and dividing this number by the number of total residues compared - multiplying the resultant figure by 100 would yield the percentage identity between the sequences. Preferably, such identity is 80% or higher, most preferably 90%, or optimally 95% or higher.

As with the antigenic fragments and/or portions provided by this invention, any antigens encoded by or comprising/consisting (essentially of) sequences which homology and/or identity to the sequences described in this application may be immunogenic and suitable for raising immune responses in animals, wherein the immune responses are protective against S. uberis infection and/or diseases and/or conditions caused or contributed to thereby.

A variant, derivative or mutant antigen of this invention may comprise or be encoded by, a nucleic acid or amino acid sequence which comprises one or more nucleotide and/or amino acid substitutions, inversions, additions and/or deletions relative to a reference sequence. A reference sequence may be any of the sequences described in this application. The term "substitution" may encompass one or more conservative substitution(s). One of skill in this field will understand that the term "conservative substitution" is intended to embrace the act of replacing one or more amino acids of a protein or peptide with an alternate amino acid with similar properties and which does not substantially alter the physico-chemical properties and/or structure or function of the native (or wild- type) protein. Examples of such conservative substitutions are presented in Table 2. Table 2 Conservative amino acid substitutions

Residue Abbreviation Conservative substitutions

Alanine Ala Ser

Arginine Arg Lys

Asparagine Asn Gin, His

Aspartic acid Asp Glu

Cysteine Cys Asn

Glutamic adic Glu Ser

Glutamine Gin Asp

Glycine Gly Pro

Histidine His Asn, Gin

Isoleucine lie Leu, Val

Leucine Leu lie, Val

Lysine Lys Arg, Gin

Methionine Met Leu, lie

Phenylalanine Phe Met, Leu, Tyr

Proline Pro

Serine Ser Thr, Gly

Threonine Thr Ser, Val

Tryptophan Trp Tyr

Tyrosine Tyr Trp, Phe

Valine Val lie, Leu One of skill will appreciate that the antigens described herein may comprise domains and regions (or epitopes) which represent the immunogenic or active parts. The immunogenic of active parts of an antigen of this invention are those domains, regions or epitopes which are capable of inducing an immune response in the relevant animal. Therefore, antigens for use in this invention may comprise synthetic or recombinant constructs or fusions which comprise these immunogenic or active regions, domains or epitopes. For example, antigens for use in this invention may comprise sequences which exhibit at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity (as defined above) to the sequences of the active or immunogenic (epitope containing) domains or regions of the antigens described herein, including, for example, those sequences identified by accession number in Table 1.

In the context of this invention, a variant, derivative or mutant 5. uberis antigen may comprise or be encoded by a variant, derivative or mutant sequence which, when compared to a reference sequence (such as for example a wild-type S. uberis sequence or a sequence encoding any of the specific 5. uberis antigens presented above, is found to contain one or more amino acid/nucleotide substitutions, additions, deletions and/or inversions.

An antigen which may be regarded as a derivative of the 5. uberis antigens described herein may further comprise one or more features (for example epitopes or domains) of an 5. uberis antigen fragment or mutant, variant or derivative described herein, optionally in combination with one or more modifications to the structure of the antigen or one or more of the amino acid residues thereof.

As with the antigenic fragments and/or portions provided by this invention, the mutant, variant and/or derivative sequences described herein may be immunogenic and suitable for raising immune responses in animals, wherein the immune responses are protective against 5. uberis infection and/or diseases and/or conditions caused or contributed to thereby.

(a) antigens as specifically named above

(b) antigens encoded by sequences comprising, consisting or consisting essentially of sequences which exhibit at least about 60% identity to the sequences described in this application (that is, for example, the sequences disclosed by reference to deposited accession numbers); (c) antigens encoded by or comprising, consisting or consisting essentially of sequences which represent variant , derivative or mutant sequences of those sequences encoding the S. uberis antigens described herein;

(d) antigens comprising sequences corresponding to the immunogenic domains of the antigens described herein (for example, the antigens presented in Table 2). Antigens of this type may comprise sequences which share about 60% to about 100% sequence identity with the immunogenic domains of any of the antigens described herein; and

(e) antigens (typically proteins) which antigens or proteins comprise, consist essentially of, or consist of, one or more immunogenic fragments or domains derived from one or more of the antigens as specifically named above.

It will be appreciated that the S. uberis antigens described in this application may be obtained by direct purification from 5. uberis cultures and/or protein/membrane preparations thereof. Additionally or alternatively, the antigens of this invention may be generated recombinantly.

PCR techniques may be exploited to selectively amplify the appropriate antigen (for example 5. uberis antigen) gene sequences from a variety of sources including, for example, stored Streptococcus and/or S. uberis isolates, clinical isolates, diseased material and the like. Cloned antigen sequences may be introduced into a vector (such as a plasmid or expression cassette). I n one embodiment, the vector may further comprise a nucleotide sequence of a tag or label to assist in protein purification procedures.

A host cell may be transformed or transfected with a vector and maintained under conditions suitable to induce expression of and antigen (for example a S. uberis antigen) gene sequence and production of recombinant antigen. Prokaryotic or eukaryotic cells, such as, for example bacterial, plant, insect, mammalian and/or fungal dcells, may be transformed or transfected with one or more of the vectors described herein. One of skill in this field will be familiar with the techniques used to introduce heterologous or foreign nucleic acid sequences, such as expression vectors, into cells and these may include, for example, heat-shock treatment, use of one or more chemicals (such as calcium phosphate) to induce transformation/transfection, the use of viral carriers, microinjection and/or techniques such as electroporation. Further information regarding transformation/transfection techniques may be found in Current Protocols in Molecular Biology, Ausuble, F.M ., ea., John Wiley & Sons, N .Y. (1989) which is incorporated herein by reference. In one embodiment, the host cell is a bacterial cell such as, for example, an Escherichia coli cell.

Techniques used to purify recombinant proteins generated in this way are known and, where the recombinant protein is tagged or labelled, these may include the use of, for example, affinity chromatography techniques.

In view of the above, this invention may provide expression vectors comprising S. uberis antigen gene sequence(s) and host cells transformed therewith.

For convenience all of the antigens, including the 5. uberis antigens (both purified and/or recombinant forms) described herein, shall hereinafter be collectively referred to as "antigens" or "S. uberis antigens". Moreover, references to specific antigens and/or 5. uberis antigens should be taken to include (immunogenic) fragments or portions derived therefrom (as described above) as well as any mutants, variants and derivatives thereof and/or antigens exhibiting a degree of homology/identity thereto.

The inventors have discovered that animals (in particular bovine, porcine, caprine and/or ovine animals) administered one or more of the 5. uberis lipoprotein (acc No: YP_002562276), 5. uberis serine proteinase (acc No: YP_002563137), 5. uberis ferrichrome binding protein: (acc No: YP_002561776) and/or S. uberis elongation factor Tu: (acc No: YP_002561947) antigens elicit particularly effective protective immune responses.

As such, this invention may provide immunogenic and/or vaccine compositions comprising one or more of the 5. uberis lipoprotein (acc No: YP_002562276), 5. uberis serine proteinase (acc No: YP_002563137), 5. uberis ferrichrome binding protein: (acc No: YP_002561776) and/or S. uberis elongation factor Tu : (acc No: YP_002561947).

A vaccine of this invention may comprise, consist essentially of or consist of a 5. uberis ferrichrome binding protein, 5. uberis elongation factor Tu, S. uberis lipoprotein, 5. uberis serine proteinase, an immunogenic fragment of any of these or a combination thereof optionally in combination with an adjuvant for use in (i) treating masititis or (ii) raising an immune response which is protective against mastitis. The vaccine composition may provide only one of the above antigens, although it is preferred that at least 2 antigens be used, more preferably 3, and most preferably that all 4 antigens be used, taking into account that the definition of antigen includes any immunologically active fragment of said antigen. The vaccine may be further exploited in methods of treating subjects in need thereof, the method comprising administering an immunologically effective amount of the vaccine to the subject. The subject may be a bovine subject or any subject (bovine or otherwise) predisposed or susceptible to contracting or developing mastitis. The subject may be suffering from mastitis. The subject may be predisposed or susceptible to contracting or developing a 5. uberis infection or a 5. uberis associated disease and/or condition.

In one embodiment, any of the antigens or 5. uberis antigens described herein may be admixed with another component, such as another polypeptide and/or an adjuvant, diluent or excipient. Additionally, or alternatively, vaccines or vaccine compositions provided by this invention may, for example, contain viral, fungal, bacterial or other parasite antigens used to control other diseases/infections or infestations. For example, the vaccine or vaccine composition may be included within a multivalent vaccine, which includes antigens against other ovine or bovine pathogens/diseases. The antigens of this invention may independently (or together) be used with additional (for example non-S. uberis) polypeptides. For example, the antigens of this invention may be fused, bound or otherwise conjugated to the 5. uberis antigens described herein. Thus this invention further encompasses fusions comprising the 5. uberis antigens described herein. The fusions may be internal fusions where a polypeptide, peptide or protein is embedded into the amino acid sequence of an antigen of this invention. Additionally or alternatively, the fusions may comprise C- or N-terminal fusions in which a polypeptide, peptide or protein is fused to the N- and/or C-terminal portion of a 5. uberis antigen of this invention. I n some cases, the 5. uberis antigens (or the fragments thereof) of this invention may take the form of haptens - that is to say they are small molecules which elicit immune responses only when attached to a large carrier such as a peptide or protein. In some cases, the carier protein or peptide may not elicit an immune response by itself. Where the 5. uberis antigen is a hapten, it may be fused, combined, bound or conjugated with or to a carrier protein or peptide so as to enhance or increase its ability to raise an immune response in an animal. In a further aspect, the present invention provides an animal population treated, vaccinated and/or immunised with an antigen or antigen(s), vaccine or composition of this invention. For example, the invention provides treated, vaccinated or immunised human, avian, piscine,bovine, porcine, caprine and/or ovine populations; for example, the invention may provide farmed populations of birds, fish, cattle, pigs sheep and/or goats which have been treated, vaccinated and/or immunised with an antigen or antigen(s), vaccine or composition described herein. As stated, a vaccine or composition of this invention may comprise one or more of the 5. uberis antigens described herein optionally in combination with one or more other antigens and/or adjuvants.

The compositions, including the vaccine compositions, provided by this invention may be formulated as sterile pharmaceutical compositions comprising one or more of the antigens described herein and a pharmaceutical excipient, carrier or diluent. These composition may be formulated for oral, topical (including dermal and sublingual), intramammary, parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal and/or mucosal administration.

The (vaccine) compositions described herein, may comprise a discrete dosage unit and may be prepared by any of the methods well-known in the art of pharmacy. Methods typically include the step of bringing into association one or more of the S. uberis antigens described herein with liquid carriers or finely-divided solid carriers or both.

Compositions (the term "composition" including immunogenic and vaccine compositions of this invention), suitable for oral administration , wherein the carrier is a solid, are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of one or more of the 5. uberis antigens of this invention. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound (for example one or more 5. uberis antigen(s)) in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, for example in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

Compositions suitable for oral administration include controlled release dosage forms, for example tablets wherein an active compound (for example one or more S. uberis a ntigens) is formulated in an appropriate release-controlling matrix, or is coated with a suitable release-controlling film. Such compositions may be particularly convenient for prophylactic use.

Composition and vaccine compositions formulated for parenteral administration include sterile solutions or suspensions of an active compound (for example one or more 5. uberis antigens) in aqueous or oleaginous vehicles. Compositions of this invention, including vaccine and/or immunogenic compositions, may comprise, or further comprise cryoprotectant compounds or compositions, preservative(s), antibiotics, adjuvants and the like.

Injectable compositions and vaccines may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers, which are sealed after introduction of the formulation until required for use. Alternatively, an active compound (for example one or more 5. uberis antigens) may be in powder form that is constituted with a suitable vehicle, such as sterile, pyrogen-free water or phosphate buffered saline PBS before use.

Compositions comprising one or more antigens or 5. uberis antigens of this invention may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g. subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. They may also include preparations or adjuvants known to enhance the affinity and/or longevity of an animal (for example bovine, ovine or caprine) immune response, such as single or double emulsions of oil in water. Such long-acting compositions are particularly convenient for prophylactic use. Compositions suitable (or formulated) for mucosal administration include compositions comprising particles for aerosol dispersion, or dispensed in drinking water. When dispensed, such compositions should desirably have a particle diameter in the range 10 to 200 microns to enable retention in, for example, the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable compositions include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.

It should be understood that, in addition to the carrier ingredients mentioned above, the various compositions described herein may include an appropriate one or more additional (pharmaceutically acceptable) carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. Compositions suitable for topical formulation may be provided, for example, as gels, creams or ointments.

Compositions for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water-soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus, particularly suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) (for example one or more 5. uteris antigens) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to, for example, animal feed - perhaps by way of an intermediate premix, or diluted in animal drinking water.

Liquid concentrates of this invention suitably contain one or more antigens or S. uberis antigens and may optionally further include an acceptable water-miscible solvent for veterinary use, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals.

In addition to providing 5. uberis antigens for use in raising immune responses in animals, the present invention may also provide polyclonal and/or monoclonal antibodies (or antigen binding fragments thereof) that bind (or have affinity or specificity for) any of the antigens or S. uberis antigens provided by this invention. Production and isolation of polyclonal/monoclonal antibodies specific for protein/peptide sequences is routine in the art, and further information can be found in, for example "Basic methods in Antibody production and characterisation" Howard & Bethell, 2000, Taylor & Francis Ltd. Such antibodies may be used in diagnostic procedures, to, for example detect or diagnose S. uberis infection/infestations in animal (for example bovine, caprine or ovine) species, as well as for passive immunisation

The present invention further provides a vaccine for use in preventing or controlling Streptococcus infections and/or associated diseases and/or conditions. The vaccines may find particular application in preventing or controlling mastitis (namely mastitis with a streptococcal aetiology). The vaccines provided by this invention may be used to prevent or control 5. uberis infections and/or associated diseases. The vaccines may also be used to prevent or control streptococcal infections and/or associated diseases in human, avian, piscine, bovine, porcine, ovine and/or caprine hosts. The vaccine may be a polypeptide or polynucleotide vaccine - the polypeptides and/or polynucleotides providing, or encoding, one or more of the (5. uberis) antigens described herein.

The invention further provides a method for immunising an animal against an infection or disease caused or contributed to by a Streptococcus species, said method comprising the step of administering a vaccine of the invention to a subject in need thereof. Subjects in need thereof may be animals suffering (or suspected of suffering from) a streptococcal infection or disease and/or animals regarded as susceptible or predisposed to such diseases and/or infections. For example, the invention provides methods of immunising a human, an avian, a piscine, a caprine, a porcine, an ovine and/or bovine subject against 5. uberis infection/infestation and associated diseases, said method comprising the step of administering a vaccine of the invention to the human, avian, piscine, caprine, porcine, ovine or bovine subject.

The antigens and/or vaccines of this invention may be exploited in the immunisation of animals against mastitis, wherein the mastitis results from, is associated with or is caused or contributed to by, a Streptococcus species (for example 5. uberis or S. dysgalactioe) or infection therewith.

The antigens of this invention may find further application as the basis of diagnostic or identification/detection tests for various Streptococcus species/strains including any species or strain which expresses any of the antigens described herein. For exa mple, the antigens provided by this invention may be applied to the detection, identification and/or diagnosis of S. uberis, S. agalactiae, S. dysgalactioe, and S. parauberis infections, and/or diseases caused thereby or associated therewith. The antigens may also be used in methods for detecting the presence of streptococci in samples.

Thus, an aspect of this invention may provide an {in vitro) method of detecting, identifying and/or diagnosing a streptococcus spp., 5. uberis, S. agalactiae, S. dysgalactioe and/or S. parauberis infection or a disease caused thereby or associated therewith, the method comprising detecting a level of one or more of the antigens described herein in a sample and/or detecting a level of an antibody with specificity for one or more of the antigens described herein, in a sample.

A sample may comprise any biological fluid or tissue including, for example, whole blood; red blood cells, serum; plasma; saliva; sweat; semen; milk, (disease) tissue biopsy; tissue scraping; tissue/organ wash/lavage; and/or faecal preparations. The term "sample" may further extend to samples of animal feed/drink, bedding or field samples such as soil and grass.

Methods of detecting levels of antigen or antibody in a sample may comprise immunological methods. For example, methods of detecting levels of antigen or antibody may exploit enzyme-linked immunosorbant assay (ELISA) techniques. By way of example, one or more of the antigens described herein may be immobilised to a substrate and the immobilised antigen(s) used to probe a sample for the presence of antibodies reactive thereto. After a suitable period of incubation between the immobilised antigen and the sample, the presence or absence of antibodies may be detected by means of a secondary binding agent (for example antibody) optionally conjugated to a detectable moiety, with specificity for antibodies generated by the relevant species - for example bovine, caprine or ovine antibodies. The presence of antibodies may indicate the presence of a Streptococcus (for example S. uberis) and/or an infection therewith or a Streptococcus (for example an 5. uberis) associated disease or condition.

Alternatively, binding agents or antibodies with specificity to one or more of the antigens described herein may be immobilised onto a substrate. The substrate may then be used to probe a sample for the presence of one or more antigens to which the binding agents or antibodies bind. After a suitable period of incubation between the sample and the immobilised antibody, the substrate may be contacted with a secondary binding agent or antibody with specificity for the relevant antigens. The secondary binding agent or antibody may be conjugated to a detectable moiety. Alternatively, the immobilised binding agent:antigen:binding agent complexes may be further probed with a tertiary binding agent or antibody capable of binding to the secondary binding agent. The tertiary binding agent may be conjugated to a detectable moiety.

Other immunological techniques such as immunohistochemical staining may exploit binding agents (for example antibodies/ conjugated antibodies) with specificity for one or more of the antigens described herein, to detect the presence or absence of Streptococci (for example 5. uberis) antigens in a sample, for example a tissue sample.

Molecular methods may also be used to detect the presence of any of the antigens described herein (for example 5. uberis antigens) in a sample. For example, primer sequences designed to amplify sequences encoding one or more of the (5. uberis) antigens of this invention may be used to probe samples for the presence of the relevant (for example 5. uberis) nucleic acid. Further information regarding these (PCR-based) techniques may be found in, for example, PCR Primer: A Laboratory Manual, Second Edition Edited by Carl W. Dieffenbach & Gabriela S. Dveksler: Cold Spring Harbour Laboratory Press and Molecular Cloning: A Laboratory Manual by Joseph Sambrook & David Russell: Cold Spring Harbour Laboratory Press.

The present invention also extends to kits comprising reagents and compositions suitable for identifying or detecting the presence or absence of Streptococci (including 5. uberis, S. dysgalactiae and/or S. parauberis) and/or diagnosing or detecting streptococci (including 5. uberis, S. dysgalactiae and/or S. parauberis) infections or diseases. For example, depending on whether or not the kits are intended to be used to identify or detect streptococci and/or levels of antigen or antibodies thereto in samples, the kits may comprise substrates having antigens of this invention or agents capable of binding the same, bound thereto. In addition, the kits may comprise agents capable of binding the relevant (for example 5. uberis) antigens. Thus, where the kit is to be used to identify levels of S. uberis antigen in samples, the kit may comprise an agent capable of binding the relevant S. uberis antigen. The kit may comprise specifically raised polyclonal antibodies or monoclonal antibodies - the antibodies having specificity for antigens provided by this invention. Where the kits are intended to diagnose or detect streptococci or streptococcal diseases in specific animals (for example human, avian, porcine, piscine, bovine, caprine and/or ovine animals) the kits may comprise binding agents or antibodies capable of binding immunoglobulin from the relevant species. The antibodies may be conjugated to detectable moieties. Kits for use in detecting the expression of genes encoding any of the antigens of this invention may comprise one or more oligonucleotides/primers for detecting/amplifying/probing the relevant antigen-encoding sequences. The kits may also comprise other reagents to facilitate, for example, sequencing, PCR and/or RFLP analysis. All kits described herein may further comprise instructions for use.

Adjuvant formulations that are less than 50% oily phase

Sometimes, it is impossible or impracticable to concentrate the antigen, and low- concentrations of antigen solutions have to be used. Thus in some embodiments, the vaccine compositions of the instant invention comprise the adjuvants as described above, wherein the content of the oily phase in these adjuvant formulations is diluted and wherein the vaccine composition comprises a water-in-oil emulsion. In practice, it is possible to create a water-in-oil emulsion wherein the oily phase is less than 50%.

Briefly, first, the adjuvant formulation of the instant invention is prepared as described above. In said adjuvant formulaiton, the oily phase comprises over 50% v/v of the adjuvant formulation. The amounts of ingredients other than oil and emulsifiers a re scaled up respectively, based on the final target concentration and desired dilution. For example, if one aims to prepare a vaccine composition where the adjuvant formulation comprises 80% v/v, the amounts of ingredients other than the oil are scaled up by the factor of 1.25 (1/0.8). The amounts of emulsifiers, if any (e.g., TWEEN^®80 and/or SPAN^®80) do not need to be scaled up, but preferably, the volume ratio between the oil and the emulsifier(s) is kept the same in the adjuvant formulation and in the final vaccine composition. Antigen solution is then added to the adjuvant formulation.

Water-in-oil emulsion's integrity can be maintained as long as the dispersed spherical water droplets are not present in a more concentrated form than the maximum packing fraction for random packing of monodisperse droplets, i.e. : 0.64. See Tadros, Emulsion Formation, Stability and Rheology, 1^st ed. 2013, Wiley-VCH GmbH & Co KGaA. This implies that we can maintain a water-in-oil emulsion as long as the total volume fraction occupied by the aqueous droplets does not exceed 0.64, i.e. : 64% v/v. Conversely this implies that the oily phase should not drop below 36% v/v.

Accordingly, in different embodiments of this aspect of the invention vaccine formulations are provided, comprising the antigen compound, and the diluted adjuvant formulation according to the previously described embodiments, wherein the oily phase comprises over 36% of the vaccine composition v/v, and wherein the vaccine composition is a water-in-oil emulsion. Without limitations, 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 oily phase is, in different embodiments, 37% 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.

At a minimum, the concentration of oily phase should be sufficiently high to create a depot effect and protect the antigen and immunomodulator(s) from a rapid degradation by the host's immune system, preferably 20% or more v/v of the vaccine composition.

Accordingly, in another aspect, in the vaccine compositions of the instant invention, the amounts of the oily phase in the adjuvant formulations are diluted such that the vaccine formulation is an oil-in- water emulsion, with the oily phase comprising 20% or more v/v of the vaccine composition. Again, the amounts of ingredients other than oil and emulsifiers are scaled up respectively, based on the final target concentration and desired dilution. For example, if one aims to prepare a vaccine composition where the adjuvant formulation comprises 33.3% v/v, the amounts of ingredients other than the oil are scaled up by the factor of 3 (1/0.333). The amounts of emulsifiers, if any (e.g., TWEEN^®80 and/or SPAN^®80) do not need to be scaled up, but preferably, the volume ratio between the oil and the emulsifier(s) is kept the same in the adjuvant formulation and in the final vaccine composition. In different embodiments, the vaccine composition is an oil-in-water emulsion, wherein the oily 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.

Again, adjuvant formulations suitable for this aspect of the invention include TCMO, TCMYO, Q.TCMO, QTCMYO, XOM, TXO, TXO-A, TAO, AXO, QCO, ODYRM, QCDXO, with a proviso that the oily phase in the adjuvant formulation may be below 50% v/v, but above 20% v/v of the final vaccine composition.

Alternative Adjuvant Formulations

Although the present invention is described in terms of particularly highly preferred adjuvant components, the inherent immunological efficacy of the antigens permits the use of additional well known adjuvant systems. Such alternatively useful adjuvants are described as follows, wherein the processes of producing such formulations, and selecting amounts of antigen and adjuvant per dose are generally recognized by those skilled in the art. I mmunogenic compositions of the present invention can include one or more adjuvants. Adjuvants include, but a re not limited to any of the following, individually or in combination:

Triterpenoids suitable for use in the adjuvant compositions can come from many sources, either plant derived or synthetic equivalents, including but not limited to, Quillaja saponaria, tomatine, ginsing extracts, mushrooms, and an alkaloid glycoside structurally similar to steroidal saponins. Thus, triterpenoids suitable for use in the adjuvant compositions include saponins, squalene, and lanosterol. If a saponin is used, the adjuvant compositions generally contain an immunologically active saponin fraction from the bark of Quillaja saponaria. The saponin may be, for example, Quil A or another purified or partially purified saponin preparation, which can be obtained commercially. Thus, saponin extracts can be used as mixtures or purified individual components such as QS-7, QS-17, Q.S-18, and QS-21.

Sterols suitable for use in the adjuvant compositions include β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. These sterols are well known in the art and can be purchased commercially. For example cholesterol is disclosed in the Merck Index, 12th Ed., p. 369. The adjuvant compositions can further include one or more immunomodulatory agents such as, e.g., quaternary ammonium compounds (e.g., DDA), and interleukins, interferons, or other cytokines. These materials can be purchased commercially.

The adjuvant compositions can further include one or more polymers such as, for example, DEAE Dextran, polyethylene glycol, and polyacrylic acid and polymethacrylic acid (eg, CARBOPOL^®; Lubrizol Corp., Wickliffe, OH). Such material can be purchased commercially. Another specific example is polyacrylic acid (e.g., the CARBOPOL^® polymers), which has an average equivalent weight of 76. They are produced from primary polymer particles of about 0.2 to 6.0 microns in average diameter. The CARBOPOL^® polymers swell in water up to 1000 times their original volume and ten times their original diameter to form a gel when exposed to a pH environment greater than the pKa of the carboxylate group. At a pH greater than the pKa of carboxylate group, the carboxylate groups ionize resulting in repulsion between the negative charges, which adds to the swelling of the polymer.

The adjuvant compositions can further include one or more Th2 stimulants such as, for example. Bay R1005^® and aluminum. Bay R1005^®, a glycolipid, can be synthesized according to the procedure found in Lockhoff, 0. (Angew. Chem. Int. Ed. Engl. 30:1611-1620; 1991). It is recommended that it is stored at 2-8° C in an airtight container. Its chemical or physical properties are that it is slightly hygroscopic, does not form polymorphs, is chemically stable in air and light at temperatures up to 50° C and in aqueous solvents at pH 2-12 at ambient temperature. It is an amphiphilic molecule which forms micelles in aqueous solution.

M ultiple oils and combinations thereof are suitable for use. These oils include, without limitations, animal oils, vegetable oils, as well as non-metabolizable oils. Non-limiting examples of vegetable oils suitable in the instant invention are corn oil, peanut oil, soybean oil, coconut oil, and olive oil. Non-limiting example of animal oils is squalane. Suitable non-limiting examples of non- metabolizable oils include light mineral oil, straight chained or branched saturated oils, and the like. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique. The term is synonymous with "liquefied paraffin", "liquid petrolatum" and "white mineral oil." The term is also intended to include "light mineral oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, J. T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred mineral oil is light mineral oil commercially available under the name DRAKEOL^® (Penreco; Karns City, PA).

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

Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention include sorbitan-based non-ionic surfactants, e.g. fatty-acid-substituted sorbitan surfactants, commercially available under the name SPAN^® (Sigma-Aldrich; St. Louis, MO) or ARLACEL^® (Croda; East Yorkshire, England); fatty acid esters of polyethoxylated sorbitol (TWEEN^®; Croda), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR^®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL^® M-53; Seppic, Puteaux, France), polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL^®; Sigma-Aldrich), polyoxyethylene fatty alcohol ethers (BRIJ^®; Sigma-Aldrich); polyoxyethylene nonphenyl ethers (TRITON^® N; Dow Chemical Co., Midland, Ml), polyoxyethylene isooctylphenyl ethers (TRITON^® X; Sigma-Aldrich). Preferred synthetic surfactants are the surfactants available under the name SPAN^® and TWEEN^®, such as TWEEN^®-80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN^®-80 (sorbitan monooleate).

Suitable cationic carriers may be included in the adjuvant formuation, and can include, without limitations, dextran, dextran DEAE (and derivatives thereof), PEGs, guar gums, chitosan derivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC) polyethylenimene, poly aminos like polylysine and the like.

Immunostimulatory oligonucleotides may be present in the aduvant, and can include ODN (DNA-based), ORN (RNA-based) oligonucleotides, or chimeric ODN-ORN structures, which may have modified backbone including, without limitations, phosphorothioate modifications, halogenations, alkylation (e.g., ethyl- or methyl- modifications), and phosphodiester modifications. In some embodiments, poly inosinic -cytidylic acid or derivative thereof (poly I :C) may be used.

CpG oligonucleotides are a recently described class of pharmacotherapeutic agents that are characterized by the presence of an unmethylated CG dinucleotide in specific base-sequence contexts (CpG motif). (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229-232, which is incorporated herein by reference). These CpG motifs are not seen in eukaryotic DNA, in which CG dinucleotides are suppressed and, when present, usually methylated, but are present in bacterial DNA to which they confer immunostimulatory properties.

Suitable oligonucleotides can include P-class immunostimulatory oligonucleotides, also modified P- class immunostimulatory oligonucleotides, as well as E-modified P-class oligonucleotides. P-class immunostimulatory oligonucleotides are CpG oligonucleotides characterized by the presence of palindromes, generally 6-20 nucleotides long. The P-Class oligonucleotides have the ability to spontaneously self-assemble into concatamers either in vitro and/or in vivo. These oligonucleotides are, in a strict sense, single-stranded, but the presence of palindromes allows for formation of concatamers or possibly stem-and-loop structures. The overall length of P- class immunostimulatory oligonucleotides is between 19 and 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.

The immunostimulatory oligonucleotide contains a 5' TLR activation domain and at least two palindromic regions, one palindromic region being a 5' palindromic region of at least 6 nucleotides in length and connected to a 3' palindromic region of at least 8 nucleotides in length either directly or through a spacer. P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E-modification refers to ethyl-modified nucleotide(s). Thus, E-modified P-class immunostimulatory oligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5' nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazol, O-Methylation, modification with proynyl-dU, inosine modification, 2-bromovinyl attachment (preferably to uridine). P-class immunostimulatory oligonucleotides may also contain a modified internucleotide linkage including, without limitations, phosphodiesther linkages and phosphorothioate linkages. The oligonucleotides of the instant invention may be synthesized or obtained from commercial sources. P- Class oligonucleotides and modified P-class oligonucleotides are further disclosed in published PCT application no. WO2008/068638, published on Jun. 12, 2008.

Aluminum is a known adjuvant, or a component of adjuvant formulations, and is commercially available in such forms as REHYDRAGEL^® (General Chemical; Parsippany, New Jersey). REHYDRAGEL^® is a crystalline aluminum oxyhydroxide, known mineralogically as boehmite. It is effective in vaccines when there is a need to bind negatively charged proteins.

A number of cytokines or lymphokines have been shown to have immune-modulating activity, and thus may be used as adjuvants. These can include, but not be limited to, the interleukins 1-a, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., US 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms), the interferons-a, β and γ, granulocyte-macrophage colony stimulating factor (see, for example, US 5,078,996, and ATCC Accession Number 39900), macrophage colony stimulating factor, granulocyte colony stimulating factor, GSF, and the tumor necrosis factors a and β. Still other adjuvants useful in this invention include chemokines, including without limitation, MCP-1, ΜΙΡ-Ια, ΜΙΡ-Ιβ, and RANTES. Adhesion molecules, such as a selectin, e.g., L-selectin, P-selectin, and E-selectin may also be useful as adjuvants. Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM-1; a member of the integrin family such as LFA-1, VLA-1, Mac-1 and pl50.95; a member of the immunoglobulin superfamily such as PECAM, ICAMs (e.g., ICAM-1, ICAM-2 and ICAM- 3), CD2 and LFA-3; co-stimulatory molecules such as CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL- 1, and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor. Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still another adjuvant molecule includes Caspase (ICE).

Other adjuvants can include the RIBI adjuvant system (Ribi I nc.; Hamilton, MT), Freund's complete and incomplete adjuvants. Block copolymer (CytRx; Atlanta, GA), SAF-M (Chiron; Emeryville, CA), AMPHIGEN⁸ adjuvant (described in US 5,084,269 and US 6,814,971), Avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, or muramyl dipeptide. Also useful is MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094, and hereby incorporated by reference. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate (AGP) compounds, or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in US 6,113,918, hereby incorporated by reference.

Examples

Example 1. Addition of Aluminum to TXO results in an improved stability

A preferred TXO blend formulation may contain 50 mg/ml of DEAE Dextran. Dextran, when present at high concentrations in subcutaneous injections, can cause injection site reactions in the animals. Hence it is proposed to try varying concentrations for DEAE Dextran to check if safety and good therapeutic value can be obtained without compromising the stability of the vaccine formulation. Characterization and stability tests are important as they inform us whether this vaccine can be formulated consistently and with a good shelf life for manufacturing. Viscosity tests are performed at a range of shear rates in order to look for shear thinning (drop in apparent viscosity as shear rate goes up) or shear thickening (increase in apparent viscosity as shear rate goes up), which is a flow characteristic of Non-Newtonian fluids. Syringe force tests are performed to ensure that the vaccine will be easy to draw out and easy to administer over a large number of doses in the field.

Since the immunostimulating oligonucleotide is not expected to alter the stability of the formulation, it was not added to the adjuvant mixtures in this example. AXO (Aluminum + Dextran + Oil) blends of varying REHYDRAGEL^® (5% to 16%) and DEAE Dextran (50 mg/ml - 10 mg/ml) concentrations are formulated tested for viscosity, syringe force and settling using an XO (Dextran + Oil) blend as a control. The tested compositions were as follows: Approximately 10 ml of sample was filled into each of five 15-ml Corning centrifuge tubes and left still over a week in order to observe an accelerated settling effect on the emulsions due to the tubes' narrow dimensions and conical bottom. The samples were also tested for syringeability and viscosity. The results are shown below. In Table 3

Table 3

These data indicate that upon subjection to accelerated settling in the centrifuge tubes, the blend with 16% REHYDRAGEL^® is the most stable. Further, from previous work by the inventors, it was known that higher DEAE Dextran concentration is associated with higher viscosities and possible shear thinning. The results of these experiments indicate that the addition of REHYDRAGEL^® more than compensates for anticipated loss in the shear thinning (pseudoplastic) properties afforded by DEAE Dextran. It was also observed that even though the 16% REHYDRAGEL^® formulation had a higher syringe force, it was not noticeably harder to inject (syringe force for water is 3N), see Table 4.

Table 4

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

Example 2 -Bacterial strains , culture conditions and expression

The reference strain, Streptococcus uberis 0140J (ATCC BAA-854) was used in this study, in addition to a further 71 5. uberis clinical isolates derived from cases of bovine and ovine mastitis from distinct farms within the UK, Italy and the USA, and comprised strains which either persisted or were cured following antibiotic therapy. For routine culture, bacteria were propagated in Brain Heart Infusion (BHI) broth or agar. Bacteria for inclusion in proteomic analyses were propagated in a defined medium to provide a suitable growth environment lacking medium-derived peptides that would interfere with a mass spectrometric approach. Irrespective of the medium used, cultures were incubated static at 37°C.

Analysis of S. uberis cell-wall and cell-wall-associated proteins

Twenty 5. uberis strains, including the reference strain, were assessed in a proteomic analysis to identify putatively conserved proteins. Bacteria were propagated in 50 ml volumes to late exponential growth-phase; growth curves for each strain had been generated in a prior experiment, whereby growth was measured in the defined medium over time (data not shown). Bacterial cells were harvested by centrifugation at 30,000 χ g for 20 m and washed twice with ice-cold PBS. Subsequently, in microcentrifuge tubes, the bacterial pellets were carefully re-suspended in 0.5 ml of PBS containing 40% (w/v) sucrose, ImM DTT and 20μg sequencing grade modified trypsin (Promega). Proteolytic digestion of cells to liberate cell-wall and cell-wall-associated proteins was carried out for 2 h at 37°C with gentle shaking. Subsequently, the digestion mixtures were centrifuged at 30,000 χ g for 10 m to pellet cells, and each supernatant was transferred to a fresh microcentrifuge tube. Incubation of supernatants was continued overnight at 37°C, then each was filtered through a 0.45 μιη Spin-X centrifuge tube filter (Corning) and stored in a refrigerator until required.

Mass spectrometric analysis

Peptide mixtures were cleaned using a C5-Reversed Phase HPLC column. Subsequently, filtered samples were subjected to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. Liquid chromatography was performed using an Ultimate 3000 nano-HPLC system (Dionex) comprising a WPS-3000-well plate micro auto-sampler, a FLM-3000 flow manager and column compartment, a UVD-3000 UV detector, an LPG-3600 dual-gradient micro-pump and an SRD- 3600 solvent rack controlled by Chromeleon chromatography software (Dionex: http://wwwl.dionex.com). A micro-pump flow rate of 246 μΙ min ¹ was used in combination with a cap-flow splitter cartridge, affording a s2 flow split and a final flow rate of 3 μΙ min ¹ through a 5 cm x 200 μιη ID monolithic reversed-phase column (Dionex/LC Packings) maintained at 50°C. Samples of 1-4 μΙ were applied to the column by direct injection. Peptides were eluted by the application of a 15 min linear gradient from 8-45% solvent B (80% (w/v) acetonitrile, 0.1% (v/v) formic acid) and directed through a 3 nl UV detector flow cell. LC was interfaced directly with an Esquire HCTplus™ 3-D high capacity ion trap mass spectrometer (Bruker Daltonics) via a low-volume (50 μΙ min^"1 maximum) stainless steel nebuliser (Agilent) and ESI . Parameters for tandem MS analysis were set as previously described (Batycka et al., 2006).

Deconvoluted MS/MS data was submitted to an in-house server running MASCOT (MATRIX SCIENCE), and searched against the fully-annotated S. uberis 0140J genome sequence (NC 012004) using the MASCOT search algorithm. To this end, the fixed- and variable-modifications selected were carbamidomethyl (C) and oxidation (M) respectively, and mass tolerance values for MS and MS/MS were set at 1.5Da and 0.5Da respectively. Molecular weight search (MOWSE) scores attained for individual protein identifications were inspected manually and considered significant only if two or more peptides were matched for each protein and identified peptide contained an unbroken "b" or "y" ion series of a minimum of four amino acid residues. An in-house software programme was used to process raw MASCOT data and generate non-redundant lists of proteins identified in each of the cell- wall sub-cellular fractions of the 20 5. uberis strains. Proteins which were present in 50% or greater of the 20 strains (Table 4a) were considered putative candidate antigens for vaccine development and were subjected to further study to confirm conservation.

Assessing carriage of candidate antigen-encoding genes among a larger panel of strains

To further appraise the conservation of proteins identified by mass spectrometry, PCR was used to determine the presence/absence of protein-coding sequences within the genomes of the larger panel of S. uberis strains, which included those 20 strains originally subjected to mass spectrometric analysis. Genomic DNA of 72 S. uberis strains was extracted from overnight cultures using the DNeasy Blood & Tissue Extraction Kit (Qiagen) as per the manufacturer's instructions for 'hard to lyse' Gram- positive bacteria. Oligonucleotide primer pairs were designed to allow PCR amplification of each of the target genes. PCR was conducted using Taq PCR MasterMix Kit (Qiagen), as per the manufacturer's instructions. Following PCR, amplicons were visualised over UV light following electrophoresis through 1% (w/v) agarose gels containing GelRed. In all cases a PCR product was observed, indicative of the antigen encoding gene being present in each of the analysed strains. Subsequently, selected PCR products were analysed further by sequencing to confirm the identity of the amplified sequences. Based upon the results of proteomic and genomic screening, 4 conserved targets were identified and were chosen for further assessment as candidate vaccine antigens.

Each of the 4 genes were amplified by PCR (as previously) from S. uberis 0140J genomic DNA using oligonucleotide primers designed to include appropriate restriction endonuclease recognition sites to facilitate in-frame cloning into the expression plasmid. For genes predicted to contain a secretion signal peptide-encoding sequence (as determined using SignalP V.3.0; Bendtsen et al. 2004), each forward primer was designed to anneal, in-frame, immediately after the predicted signal peptide- encoding sequence. PCR amplicons were initially cloned using the TOPO TA Cloning Kit (Life Technologies Corp.). Subsequently, the 5. uberis genes were cleaved from the TOPO vector using the primer-encoded restriction endonuclease sites; digests were electrophoresed through 1% (w/v) agarose gels, and the desired fragments were excised and purified using the QIAquick Gel extraction Kit (Qiagen). Finally, each 5. uberis gene was cloned into pET-15b expression plasmid (Novagen), to allow expression of each protein with an N-terminal 6 ^χ histidine (his) residue tag to facilitate downstream purification

Thus in regard of 4 proteins conserved among isolates selected by proteomic analysis.

SUB0423 ferrichrome binding protein SEQ ID NO:2

SUB0604 elongation factor Tu SEQ ID NO:5

SUB0950 lipoprotein SEQ ID NO:8

SUB1868 serine protease SEOJD NO:ll

Target genes are listed according to their locus tag within the S. uberis 0140J genome (accession number NC 012004), and the name and accession number of the products they encode.

Preliminary appraisal of the expression of various 4 proteins was conducted . Following electrophoresis through polyacrylamide gels, recombinant products were excised and subjected to MALDI-ToF MS. Mass spectrometric data was searched against the fully-annotated 5. uberis 0140J genome sequence (NC 012004) using the MASCOT search algorithm to confirm the identities of the recombinant proteins (data not shown). Subsequently, 4 were chosen (based on the level of expression) for assessment in a preliminary vaccination experiment.

Scaled expression of candidate antigens

The 4 antigens chosen for further study were (N.B. 'r' prefix denotes recombinant product) rSUB423 (ferrichrome binding protein), rSUB604 (elongation factor Tu), rSUB950 (lipoprotein) and rSUB1868 (serine protease). The coding sequences of each of the 4 antigens, and the corresponding translated amino acid sequences are presented in Table 1 above.

Starter cultures of Escherichia coli BL21(DE3) containing each of the 4 recombinant plasmids were propagated in Lysogenic Broth (LB) containing 50 μg/ml of carbenicillin, overnight at 37°C with shaking. These were then used to inoculate 1 1 volumes of LB (containing 50 μg/ml of carbenicillin) in 5 I conical flasks. Cultures were shaken at 37°C until an OD₅₀onm of 0.6 was reached. Expression of the recombinant genes was induced by supplementation of cultures with IPTG to a final concentration of 1 mM; incubation was then continued as before for a further 1 h, then rifampicin was added to a final concentration of 150 μg/ml and incubation was continued for a further 3 h. Cells were harvested by centrifugation at 12,000 χ g for 15 m at 4°C, a nd cell pellets containing recombinant proteins were retained. Two of the recombinant proteins (rSUB950 and rSUB1868) remained soluble during expression and could be purified under native conditions. In contrast, the remaining proteins (rSUB423 and rSUB604) formed inclusion bodies during extraction and required purification under denaturing conditions.

Protein purification under native conditions

The cell pellets containing rSUB950 and rSUB1868 were re-suspended in 20 ml each of lysis buffer (lx BugBuster protein extraction reagent (Merck Millipore), 50 mM Tris HCI pH 8.0, 500 mM NaCI, 10 mM imidazole, 25 U/ml Benzonase enzyme (Merck Millipore) and lx Complete EDTA-free protease inhibitor cocktail (Roche Applied Science)) and incubated at 37°C for 60 min to allow cell lysis and degradation of nucleic acids. Cell lysates were centrifuged at 22,000 χ g for 30 m at 4°C to pellet cell debris. Then, recombinant proteins in the clarified supernatants were recovered by immobilized metal ion affinity chromatography (IMAC) using Ni-CAM resin (Sigma). For each protein, 2 χ 12 ml Eco- Pac Chromatography Columns (Bio-Rad) were loaded with 2 ml (bed volume) resin and washed by gravity flow with 10 ml of Equilibration Buffer (50 mM Tris HCI pH 8.0, 500 mM NaCI, 10 mM imidazole, lx Complete EDTA-free protease inhibitor cocktail). The outlet of each column was sealed, and for each clarified lysate, 10 ml was added to each of 2 columns prior to sealing the inlet of each column with Parafilm (VWR). Columns were incubated on a tube rotator, overnight at 4°C. After incubation, columns were drained by gravity flow, and washed with 8 ^χ 5 ml of Wash Buffer (50 mM Tris HCI pH 8.0, 500 mM NaCI, 10 mM imidazole, lx Complete EDTA-free protease inhibitor cocktail). The recombinant protein in each column, bound to the Ni-CAM resin via the N-terminal 6 x his tag, was eluted in 5 x 2 ml of Elution Buffer (50 mM Tris HCI pH 8.0, 500 mM NaCI, 250 mM imidazole, lx Complete EDTA-free protease inhibitor cocktail). Subsequently, the eluate of equivalent proteins was pooled, and each protein was concentrated using Amicon Ultra-15, 10 kDa M_r cut-off, centrifugal filter units, as per the manufacturer's instructions.

Purification under denaturing conditions

Cell pellets containing inclusion bodies were initially treated according to the native extraction protocol. Subsequently, cell pellets containing inclusion bodies were suspended in 20 ml of native lysis buffer. Lysozyme was added to a final concentration of 1 kU/ml and digestion was carried out at room temperature for 15 min. After incubation, an equal volume of BugBuster reagent (diluted in distilled water) was added to the suspensions, which were mixed by vortexing for 1 min and centrifuged at 5,000 x g for 15 min at 4°C to collect inclusion bodies. Inclusion body pellets were then washed a further 3 times with 1:10 diluted BugBuster, as previously. Finally, each inclusion body was dissolved in 20 ml of 8 M urea (pH 8.0) at room temperature for 15 m, and then centrifuged at 5,000 ^χ g for 15 m at room temperature to pellet (remove) insoluble material. Subsequently, purification of each protein was performed using Ni-CAM resin, as described elsewhere except that the column buffers used for native purification were replaced with: Equilibration Buffer (0.1 M sodium phosphate, 8 M urea pH 8.0), Wash Buffer (0.1 M sodium phosphate, 8 M urea pH 6.3), and Elution Buffer (0.1 M sodium phosphate, 8 M urea pH 4.5).

Size exclusion high-performance liquid chromatography (HPLC)

Size exclusion HPLC was performed using a Superose 12 10/300GL column (GE Healthcare) pre- equilibrated with lx PBS, pH6.8 (for native, soluble proteins) or 8 M urea in 1 x PBS, pH6.8 (for denatured, insoluble proteins). I ndividual injections of 200 ml were applied to the column and proteins were resolved over a period of 60 m at a flow rate of 0.5 ml/m. The proteins that eluted from the column were monitored spectrophotometrically at a wavelength of 280nm. For each protein, fractions of 1 ml were collected, and those corresponding to peaks of UV-absorbent material were examined for the presence of protein by SDS-PAGE and Coomassie Brilliant Blue staining. For each protein, eluted fractions observed to contain an enriched source of the protein of interest were pooled and concentrated by centrifugation with amicon ultra filters (lOkDa cut off).

Each of the 4 recombinant proteins was subjected to MALD-TOF MS, and mass spectrometric data was searched against the fully-annotated 5. uberis 0140J genome sequence (NC 012004) using the MASCOT search algorithm to confirm that the identities of the recombinant proteins were as expected (data not shown).

Example 3-Formulation of the Streptococcus uberis vaccine composition with the TXO adjuvant.

The TXO is a water in mineral oil emulsion that is used as a vaccine adjuvant. The preparation of this formula begins by the preparation of the oily phase which is comprised of Span-80 surfactant added into mineral oil that was pre-warmed to 37 degrees C to facilitate solubilization of the surfactant. This material is prepared fresh and then sterile filtered through a 0.2 um absolute sterilizing grade filter and stored at room temperature until the next step of the vaccine preparation. The mixing is achieved by using a magnetic stirring bar on small stir plate.

The second step of this vaccine preparation is the making of the aqueous phase. The aqueous phase is prepared at room temperature by addition of components to a specified quantity of PBS (which is the vaccine extender) in a mixing container. Thus, DEAE-Dextran, CpG and antigen are added consecutively to achieve the specified concentrations. Subsequently, the Tween 80 surfactant is added and allowed to fully dissolve. It should be noted that different mixing orders is also operable. The mixing is achieved by using a magnetic stirring bar on small stir plate. The preferred CpG used herein is as follows although numerous other species exemplified herein can be used.

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: 20 )

The third and final step is to form the emulsion by adding the aqueous phase into the oily phase. The oily phase is placed into a beaker under a laminar airflow hood and a Silverson

homogenizer utilizing a rotor /stator combination setup is used to achieve the mixing and proper shear. The stator screen is the high shear round hole design. The aqueous phase is fed directly into the already mixing oily phase at ~30 mL/in with a recirculation speed of 10,000 to 11,000 RPM . The resulting mixture should be an opaque white stable water in oil emulsion.

Therefore, based on preparation of a representative batch of vaccine composition providing 2mL doses, there is used 0.9 mL of mineral Oil 5LT NF (available as Drakeol LT Mineral Oil N.F., white mineral oil, CAS 8042-47-5, also known as Drakesol® 260-AT, from Penreco, Woodlands, TX, USA) provided from 100% stock solution, per 2 mL dose. Each vaccine antigen is provided to a final concentration of 37.5 units/mL or 75 units/dose, in this case from stock solutions as follows: uberis 0423, 760 microgram/mL, volume added 0.0987 mL; uberis 00604, 700 microgram/mL, volume added 0.1071 mL; uberis 0950, 1400 microgram/mL, volume added 0.0536 mL; and uberis 1868, 680 microgram/mL, volume added 0.1103 mL. A 2 ML dose also contains 0.1260 mL of a 100% stock solution of Span 80, and 0.0290 mL of a 100% stock solution of Tween 80. CpG "23877" (SEQ ID NO:20 as above) is added as 0.0125 mL of a 20mg/mL stock solution, thereby providing 250 micrograms per 2mL dose (it should be noted the amount of CpG can be varied still with good vaccine effect, such as by providing about 10-1000 micrograms CpG per 2 mL dose, preferably 50-500 micrograms per 2 mL dose, and most preferably about 100-250 micrograms CpG per 2 mL dose, which may be higher or lower based on the CpG species selected. Each 2 mL dose further comprises 0.1 gram of DEAE dextran (added as 0.5 mL of a 0.2 g/mL solution). A typical 2M L dose is made up to total volume using lOmM PBS, which typically requires about 0.0628 mL/2mL dose, with trace merthiolate also added.

Example 4 - Challenge Model in Dairy Cows

The objective of the study was to evaluate sub0423, sub0604, sub0950 and subl868 as potential antigen candidates for Streptococcus uberis vaccine development in a vaccination-challenge model. Animals were allotted to treatment groups (Table 5 below) and vaccinated on Study Days 0 and 28 in accordance with this allotment. T01 received a dose of saline, T02 received sub0423 & sub0604, T03 received sub0950 & subl868 and T04 received sub0423, sub0604, sub0950 & subl868. On Study Day 70, approximately 21 days post-calving, all animals were challenged with a 5 mL intramammary infusion of 10 CFU / mL of a heterologous 5. uberis strain in PBS. Injection sites were observed and volumes calculated for analysis of least squares mean (LSM) values to assess the safety of these formulations. Overall, the LSM injection site volumes for all treatments at all time points were all <123.4 cm³. Injection sites volumes greater than 200 cm³ were considered to be larger than the clinically acceptable threshold. Injection site swellings in five animals, one in treatment group T02 and two animals each in treatment groups T03 and T04, exceeded the threshold of 200 cm³. The largest injection site volume recorded was for an animal in T04 on Study Day 35. Efficacy was evaluated in this model for both intramammary infection (I M I) and clinical mastitis (CM). The study was considered a valid test as 85.7% of animals in treatment group T01 developed both I M I and CM. The treatment group with the lowest frequency of IMI and CM was T04 (LSM 50.0 for both). In addition, duration of CM was shortest for animals in the T04 treatment group, an indicator for reduction of severity of clinical disease. The investigational vaccine, T04, which contains all four antigens, is sufficiently safe and efficacious to warrant further evaluation.

All vaccines were administered subcutaneously on Study Days 0 and 28. There were four randomized blocks with two animals per treatment group in two of the blocks (8 animals per block) and three animals per treatment group in the other blocks (12 animals per block).

Table 5. Study Design

The study was conducted according to the protocol with the following exceptions. There was minimal impact on the study outcome associated with these exceptions.

Due to insufficient quantity of IVP for treatment groups T03 & T04, three animals received less than 2.0 mL of IVP as indicated in Table 6. Data for Animal 534 was flagged from the summary and analysis from Study Days 27-84.

Table 6. List of Animals Receiving Less Than 2.0 mL IVP

On Study Day 28 for Group 4, approximately 0.05 mL of IVP leaked from the needle during the administration of IVP to Animal 536, treatment group T02. As this was a very minimal amount, impact on vaccine efficacy or safety is minimal. All details regarding calving outcome were to have been recorded on the Calving Outcome form. To facilitate data entry for data to be summarized, calving outcome and number of offspring were recorded. Calf gender, length of calf in inches and birth details were recorded on the Fresh Cow Checklist form as these data are not summarized. Calves were not weighed during this study. In addition, calving outcome was recorded as: l=Live calf, 2=Still birth, 3=Death by dystocia, 4=Other, 5=Did not calve; rather than 0=Live calf, l=Still birth, 2= abortion, 3=Death by dystocia, 4=Twins or other, and 5=Did not calve. Heparinized blood samples collected on 10 Jul 12 with sa mple IDs of H0013 (Animal 521), H0015 (Animal 516) and H0023 (Animal 513) contained a clot. .

Stages

Challenge Phase was corrected from twice daily observations to once daily observations on Study Day 34 to reflect Section 8.4.1 General Health Observations (GHO) which states that GHO will be observed and recorded daily. In addition, the Challenge Phase calendar was updated to include injection site observations on Challenge Phase Day {-)!. When an abnormal clinical observation score was recorded for an animal, rectal temperature and physical examination were completed and results recorded in a "Note-to-File" instead of DVMax as was noted in the protocol. I njection site evaluations were conducted for all cows on Study Day 14. However, data for Animals 526, 529, 530, 531 and 532 in Group 3 and Animals 533, 537 and 539 in Group 4 did not save in FREDe. Injection sites for these animals were re-evaluated and recorded in FREDe on Study Day 15. Data from these two days of observations were combined for summary and analysis. Clinical observation, milk appearance and udder evaluation scores were inadvertently not saved in FREDe for animals in Block 1 on AM, Challenge Phase Day 9. These data are missing from the dataset. Clinical observations, milk appearance and udder evaluation scores were inadvertently not completed on Challenge Study Day 14 PM, for all animals in Block 1. For subsequent blocks the aforementioned observations were removed from the study calendar. However, these observations were inadvertently collected on Challenge Day 14 PM, for all animals in Block 4, data for these animals was flagged from the summary and analysis. Due to insufficient quantities of the recombinant antigens, reagents were not available to conduct S. uberis serologic total IgG, IgGl and lgG2 antibody titer assays, milk total IgG antibody titer assay and interferon gamma responses; therefore, these assays were not completed. Samples were however collected and have been stored for potential future assay. Formulations were then provided as follows (Table 7, see also Example 3). Note that each antigen is provided at 75 micrograms/dose, so the total amount of antigen where 4 antigens are used is twice the protein level provided by the use of 2 antigens.

Table 7

* WFI = Water For Injection

5. uberis challenge strain 29048 culture was stored at approximately -70° C in Trypticase soy broth (TSB) containing 10% glycerol. The day before challenge, 50 mL of pre-warmed Todd Hewitt broth was inoculated with 50 μί of thawed stock. After 15 to 18 hours of incubation at 37° C, 1 mL of the overnight culture was used to inoculate 50 mL of pre-warmed Todd Hewitt broth and incubated for ~7 hours at 37° C, or until the broth reached the pre-determined optical density. After incubation, the flask grown suspension was diluted ten-fold in sterile, cold PBS (pH 7.4) to reach a concentration of approximately 100 colony-forming units (CFU) / mL. This final suspension was further diluted in cold, sterile PBS to achieve a final concentration of 50 CFU / 5 mL dose. Challenge material was maintained on ice prior to and during challenge of all animals. Aliquots were retained in the Laboratory and serial dilutions were performed at beginning and end of challenge for viable plate counts. Beginning and final challenge material concentrations are presented as shown below (Table 8).

Table 8 Challenge Concentrations (Unit = CFU / Dose)

Calculated from Diluted Stock

Date (Dose Target = 50 CFU / Dose)

Start End

First day 48.5 48.0

First day plus 14 44.0 50.0 Table 8 Challenge Concentrations (Unit = CFU / Dose)

Animals were placed into thestudy as follows. Animals arrived at the farm site, and the number of days that animals were acclimated is presented by block as shown below. Animals were initially housed in Building 608 and then were moved with their co-hort to the winter feed lots (building 305 and 422) for calving. Moving of animals to the winter feed lots ranged from Study Day 0 post vaccination to Study Day 10. When an animal calved it was transported back to Building 608. Approximately one week prior to challenge, all animals within the block were transported to Building 609 for the challenge phase of the study. All animals met inclusion criteria as defined in the protocol. Animal details at the time of enrollment were documented,see also Table 9, as to acclimation times.

Table 9. Number of Days Animals were Acclimated (Day 0)

However, eight animals (Table 10) were removed from the study for reason unlrelated to IVP administration or challenge.

Table 10.

Study Day 0 and the Calendar of Events for each block are provided in the study file. No animals enrolled in the study were sold. Milk from all animals was discarded. Disposition of all laboratory samples and IVP / CP was in accordance with the protocol requirements. Unused challenge material was mixed into a bleach solution and discarded into a drain. Appropriate documentation is provided in the study file. Clinical mastitis (CM) was defined as a challenged quarter having a clinical score of at least two for both milk and udder score at the same time, or a score of three for either milk or udder and isolation of S. uberis (>100 cfu / mL) from the quarter within 5 days. Mean scores and duration are presented in the tables immediately below.

Table 1 1A. Frequency of Cows with at Least One Clinical Mastitis Infection

Table 1 1 B. Summary of Duration of Clinical Mastitis (Unit = Number of Days)

Intramammary infection (IMI) was defined as the isolation of S. uberis (>100 cfu / mL) from two samples within a 5-day period and data are presented in Table 12.

Table 12. Frequency of Cows with at Least One Intramammary Infection

Frequency of abnormal milk appearance, and udder evaluation and general health scores are presented in Table 13 and 14. Animals in treatment group T04, demonstrated the lowest frequency of abnormal milk appearance and udder evaluation scores. Table 13. Frequency Distributions of Whether Animal Ever had at Least One Quarter with Appearance Score >=2 by Treatment

Table 14. Frequency Distribution of Whether Animal Ever had at Least One Quarter with an Udder Score >=2 by Treatment

Efficacy was evaluated in this model for both IMI and CM. The study was considered a valid test as 85.7% of animals in treatment group T01 developed both IMI and CM. The treatment group with the lowest frequency of IMI and CM was T04 (LSM 50.0 for both). In addition, duration of CM was shortest for animals in the T04 treatment group, an indicator for reduction of severity of clinical disease. This investigational vaccine, T04, which contains all four antigens, is sufficiently safe and efficacious.

It should also be noted that serum antibody titers for all four proteins were significantly higher for animals in treatment group T04 compared to T01 on Study Day 70. Further, least square mean values for animals in both treatment groups T01 and T04 on Study Day 0 were not different indicating no previous exposure to these antigens. Milk antibody titer least square mean values for all four proteins were significantly higher for animals in treatment group T04 compared to T01 on Study Day 70, as well.

Examples 5-8 below alternative protocols for the purification of the preferred uberis proteins *h yield. Example 5

Expression and Purification of the Recombinant Streptococcus uberis 423 Ferrichrome Binding Protein

The gene construction included a pFLEX 30 vector (see below), which expressed the protein as a soluble intracellular protein with a molecular weight of 34,164.70 Daltons and an isoelectric point of 6.055. After fermentation and expression in a 500 mL shake flask, the cells were centrifuged to a hard pellet, prior to cell breakage with an Avestin homogenizer. The expressed protein was in the soluble fraction after centrifugation, and it failed to bind to Q-Sepharose Fast Flow anion exchange

chromatographic medium (Pharmacia, Uppsala) in 5 mM sodium phosphate, 1 mM EDTA, pH 8.5. The resulting protein was of reasonable purity by SDS- PAGE. After concentration of the SUB423 protein in Centricon centrifugal concentrators, the sample contained a total protein concentration of 2.89 mg/mL, in a 50 mL volume, for a total protein content of 144.5 mg. This protein sample was sterile filtered into sterile 50 mL tubes, assigned the lot number 1024-RLG-4, and frozen at -50°C until needed.

The complete amino acid sequence of the 5. uberis recombinant SUB423 Ferrichrome Binding Protein is shown below. The amino acids added by the pFLEX vector are shown in bold.

JPVLKKYI K

DAKVVSATDLESITALEPDLI IVGSNEENISQLAEIAPLISIEYRKHDYLQVFSDFGKVFNKTKETDKWLQEWKTKTASFES DVKAVTGNNATFTI MGLYEKDIYLFGKDWGRGGEIIHQAFQYQAPEKVKM EVFPKGYLSISQEVLPDYIGDYVV VAAEDEKTGSSLYESDLWK IPAVQKNHVINVNANTFYFTDPLSLEYELKTLTDAILTQ.KTHN (see SEQ ID NO:3)

Fermentation Conditions

The fermentation seed was prepared in 10 mL of o/n culture in Teknova product Kan50 (Catalog # L8950, Lot # L895002J1201). For the seed culture, the agitation was set at 200 RPM, the temperature was 33°C, and the pH was 7.0. The batch fermentation was conducted in 500 mL of Terrific Broth (Gibco) with 25 mL o/n culture in a 2 L flask. The fermentation conditions are shown in Table 15, below.

Table 15. Fermentation conditions for cell growth and expression of the SUB0423

Ferrichrome Binding Protein

Acid Base

EFT Time OD(¾600nm Temp Tot Tot DH RPM Feed Notes

0.00 7:30AM 0.01 33°C 0.0 0.0 7.00 200

Reduce

3.00 TO 2.80 42°C 0.0 0.0 NT 200 temp@1 1 :00AM

4.00 T1 5.50 39°C 0.0 0.0 NT 200 -

5.00 T2 6.60 39°C 0.0 0.0 NT 200 -

6.00 T3 7.00 39°C 0.0 0.0 NT 200 Harvest

For Purification, the cell paste resulting from centrifugation of the fermentation broth was suspended in 200 mL of a buffer containing 5 mM Tris-HCI, I mM EDTA, pH 8.0 using a tissue homogenizer. The suspension then was passed through the Avestin homogenizer, followed by centrifugation at 9000 RPM for 20 minutes. After SDS PAGE indicated that the SUB423 Ferrichrome Binding Protein was in the soluble supernatant (data not shown); this soluble fraction was submitted to chromatography on a 2.5 X 4.5 cm column of Q. Sepharose Fast Flow equilibrated with 5 mM sodium phosphate, 1 mM EDTA, pH 8.0 buffer. The sample was titrated to pH 8.5 with dilute NaOH prior to application to the column. The column load and spent fraction contained 200 mL; an additiona l 200 mL of 5 mM sodium phosphate, 1 mM EDTA, pH 8.0 buffer was utilized to provide an additional wash of the column. These two eluted fractions were combined, thus the spent and wash fractions contained a total of 400 mL. The column then was eluted successively with 200 mL each of equilibration buffer containing 100 mM NaCI, 200 mM NaCI and 300 mM NaCI. All of these fractions were analyzed by SDS PAGE. SDS-PAGE was then conducted on Novex NuPAGE 4-12% Bis- Tris polyacrylamide gels using Novex NuPAGE MES SDS Running Buffer and Mark 12 protein standards to assess protein purity and expression. The gel was stained with I nvitrogen Simply Blue SafeStain and destained against deionized water. The Invitrogen prestained protein standards were used for SDS-PAGE of fermentation fluids. The concentration of the resulting protein solution was determined using the Pierce-Thermo Fisher Bradford Coomassie Blue colorimetric protein assay, using bovine serum albumin as a standard. The protocol that came with the kit was followed.

Certain of the fermentation results are shown in Table 15, above. The cell density increased from 2.8 at TO to 7.0 at the time of harvest, 2.5 hours post-induction. After harvest, the cells were centrifuged to a hard pellet, which was stored at -20°C until the protein was extracted and isolated. After induction of the fermentation flask by temperature change, the amount of the expressed

SUB423 Ferrichrome Binding Protein increased until harvest at three hours post- induction.

Purification

The SUB423 Ferrichrome Binding Protein was expressed as a soluble intracellular protein which was released from the cells upon cell breakage in the Avestin homogenizer. A dilute buffer containing 5 mM sodium phosphate, 1 mM EDTA, pH 8.0 was used to suspend the cells and in cell breakage, and the resulting supernatant after cell breakage and centrifugation was adjusted to pH 8.5 with dilute NaOH. These procedures were followed so that the supernatant could be applied directly to a Q-Sepharose Fast Flow column in the same dilute buffer. SDS-PAGE analysis of the eluted fractions (Data not shown) indicated that the SUB423 protein did not bind to the Q-Sepharose column under the conditions of chromatography. The SUB423 protein eluted in the column spent and wash fraction, which contained a total of 400 mL and a protein concentration of 0.43 mg/mL. The sample was concentrated in Millipore Centriprep centrifugal concentrators with a molecular weight cutoff of 50,000. The resulting

concentrated protein solution had a protein concentration of 2.89 mg/mL in a volume of 50 mL, for a total protein content of 144.5 mg. The resulting protein was soluble in aqueous buffer, and was sterile filtered into sterile 50 mL centrifuge tubes prior to storage at -50°C. The lot number for this protein sample was 1024-RLG-4, which also was the notebook reference for the protein.

SDS-PAGE of the fermentation broth showed a continuous increase in the SUB423 protein over time after induction of tf. coli cells by means of a temperature shift. After breakage of the cells, the SUB423 protein was in the soluble fraction, and it failed to bind to Q-Sepharose Fast Flow anion exchanger in the presence of 5 mM sodium phosphate, 1 mM EDTA, pH 8.5. This was surprising, in that the isoelectric point of the protein is 6.055, and it should have a negative charge at pH 8.5. Another 5. uberis recombinant protein, the SUB950 Lipoprotein, did bind to Q-Sepharose under the same conditions (1). The isoelectric point of the SUB950 Lipoprotein is somewhat lower, at 4.815. The reason for the Ferrichrome Binding Protein not adhering to the Q- Sepharose column may have been that the protein was in a high molecular weight soluble aggregate form; we have seen lack of binding to anion exchangers of protein aggregates in the past. The Ferrichrome Binding Protein was of reasonable purity after Q-Sepharose chromatography. The SUB950 Lipoprotein, which did bind to this anion exchanger, was of higher purity. A total of 144.5 mg of the Ferrichrome Binding Protein was prepared and it was in 50 mL and a protein concentration of 2.89 mg/mL. The resulting protein was sterile filtered into sterile tubes and stored at -50°C until needed.

The pFLEX vector system (see also Figure 1) is further described as follows. A series of vectors designed for the flexible expression of recombinant proteins in E. coli has been developed. These plasmids are based upon a colEl (pUC) origin of replication, which allows them to be maintained at a high copy number. They also contain the M13 origin of replication, which allows for rescue of single- stranded plasmid DNA when cells containing this plasmid are transfected with a helper phage. The gene of interest (GOI) is cloned into a multiple cloning site (MCS) downstream of the P_L promoter. Also encoded on the pFLEX plasmids is the λ c/857 temperature-sensitive repressor; this prevents expression from the λ P_L promoter at 30°C. At 42°C, the repressor is inactive, thereby allowing a high level of transcription and subsequent translation to occur from the P_L promoter. For a selectable marker, these plasmids contain the kanamycin resistance gene. The following functional elements and their derivatives have been constructed for incorporation into the pFLEX vectors:

For PFE- This cassette encodes for 3 different elements:

Protective Peptide (P)

Encoded is a 10 amino acid peptide containing 6 threonine residues; this peptide has been shown to increase protein stability when fused to the amino terminus.

FLAG (F)

This element encodes for the FLAG monoclonal antibody binding site; this can be used for detection and purification purposes.

Enterokinase (E)

The third element encodes an enterokinase recognition sequence; this allows for specific removal of the PFE cassette via enterokinase cleavage.

A variant of the PFE cassette has been generated which encodes only for the Protective Peptide (P). The PFE cassette is inserted within the MCS of the pFLEX vectors. In-frame cloning of the GOI into one of the restriction sites downstream of the PFE cassette will result in a PFE-GOI fusion protein. Integrity of the fusion junctions can be determined by sequencing with the EEL-Forward and M13- Forward primers.

For IR This cassette contains the genes encoding for two tRNAs; the codons that these tRNAs recognize are used infrequently in E. coli. The first (I) recognizes the codon AUA, which adds isoleucine to the growing polypeptide chain; the second (R) recognizes the codons AGA and AGG, and inserts arginine. The IR cassette, when present in any of the pFLEX vectors, is inserted at the Kas\ site.

pFLEX30 uses a P element, and a representative general construct appears as Figure 1 (shown for FLEX 10) which uses a PFE element

Example 6

Expression and Purification of the Streptococcus uberis Serine Protease SUB0950 Lipoprotein

Conditions of fermentation to express the protein showed continuous expression on SDS-PAGE following induction of the recombinant E. coli culture with increased temperature. The SUB950 Lipoprotein was expressed as an intracellular soluble protein. After cell breakage and centrifugation, the soluble recombinant SUB950 protein was applied to a Q-Sepharose anion exchange colum n, and it adhered to the column. The SUB950 protein then was eluted with a buffer containing 50 mM NaCI in equilibration buffer. The resulting purified protein appeared relatively pure on SDS-PAGE. After concentration in a centrifugal concentrator, the protein content was 0.66 mg/mL in 166 mL, for a total protein content of 109.6 mg.

The gene for the SUB950 Lipoprotein was cloned into E. coli strain BL21 using a pFLEX 30 vector. The complete amino acid sequence of the resulting protein is shown below. The expressed protein should have a molecular weight of 34,978.86 Daltons and an isoelectric point of 4.815. The complete amino acid sequence of the 5. uberis recombinant SUB950 Lipoprotein is shown below. The amino acid sequence from the structural gene is shown in black, the amino acids added by the pFLEX vector are shown in bold.

MGTTTTTTSLHMGSKDAKTDLKAAIVTDTGGVDDKSFNQSAWEGLEAWGKENGLKKGAGFDYFQSNSESEYATNLDTAVS

SGYNVVYGIGFALKDAIDKAAGDNSDVNYIIVDDVIEGKDNVASVTFADNEAAYLAGIAAAKTTKTKVVGFVGGMEGTVI

TRFEKGFEAGVKSVDDSIQI KVDYAGSFGDAAKGKTIAAAQYAGGADVIYQAAGGTGAGVFNEAKAVNEKKDEADKVWVI

GVDRDQKEEGKYTSKDGKESNFVLASSI KQVGKSVQLINKLVTDKKFPGGKTTVYGLKDGGVDIATTNLSDDAIKAVKEA

KEKIISGDVKVPEK. (SEQ ID NO:9)

Fermentiation Conditions

The fermentation seed was prepared in 10 mL of o/n culture in Teknova Kan~^ (Catalog # L8950, Lot U L895002J 1201). For the seed culture, the agitation was set at 200 RPM, the temperature was 37°C, and the pH was 7.0. The batch fermentation was conducted in 500 mL of Terrific Broth with 25 mL o/n culture in a 2 L flask. The fermentation conditions are shown in Table 16, below. A total of 6 grams of cell pellet were recovered.

Table 16. Fermentation conditions for cell growth and expression of the SUB950

Lipoprotein.

Acid Base

EFT Time OD(¾600nm Tem Tot Tot PH RPM Feed Notes

0.00 7:30AM 0.01 33°C 0.0 0.0 7.0 200 0

3.00 TO 2.8 42°C 0.0 0.0 NT 200 0 Heat induction

4.00 T1 3.6 39°C 0.0 0.0 NT 200 0

5.00 T2 5.35 39°C 0.0 0.0 NT 200 0

6.00 T3 5.5 39°C 0.0 0.0 NT 200 0 Harvest

Purification

A sample of cell paste containing 6 grams was suspended in 200 mL of a buffer containing 5 mM Tris-HCI, 1 mM EDTA, pH 8.0 using a tissue homogenizer. The suspension then was passed through the Avestin homogenizer, followed by centrifugation at 9000 RPM for 20 minutes. After SDS-PAGE indicated that the SUB950 Lipoprotein was in the soluble fraction (data not shown), the soluble fraction then was subjected to chromratography on a 2.5 X 6.0 cm column of Q-Sepharose Fast Flow equilibrated in 5 mM sodium phosphate, 1 mM EDTA, pH 8.0 buffer. After application of the soluble supernatant, the column was washed with the equilibration buffer, and a total of 225 mL was collected. After this, an additional 250 mL ofequilibration buffer plus 50 mM NaCI was applied to the column, followed by another 225 mL of 50 mM NaCI in equilibration buffer. The column then was eluted with two 100 mL aliquots of 100 mM NaCI in equilibration buffer, two 100 mL aliquots of 200 mM NaCI in equilibration buffer, and one 225 mL volume of 500 mM NaCI in equilibration buffer. Chromatography was at room temperature in an open column, and the column was run without a pump nor an Akta chromatography instrument. Eluted samples were subjected to SDS-PAGE. SDS- PAGE was conducted on Novex NuPAGE 4-12% Bis-Tris polyacrylamide gels using Novex NuPAGE MES SDS Running Buffer and Mark 12 protein standards. The gel was stained with I nvitrogen Simply Blue SafeStain and destained against deionized water. The Invitrogen prestained protein standards were used for SDS-PAGE of fermentation fluids. The concentration of the resulting protein solution was determined using the Pierce-Thermo Fisher Bradford Coomassie Blue colorimetric protein assay, using bovine serum albumin as a standard. The protocol that came with the kit was followed Results

The cell density increased from 2.8 at TO to 7.0 at harvest. After harvest, the cells were centrifuged to a pellet. The wet weight of the cell pellet was 6 grams. After induction of the fermentation flasks with a temperature shift, the amount of the expressed SUB950 Lipoprotein increased until harvest at 3 hours post induction. For purification, The SUB950 Lipoprotein was expressed as a soluble intracellular protein which was released from the cells upon cell breakage in the Avestin homogenizer. After centrifugation, the supernatant was titrated to pH 8.0 with dilute NaOH and applied directly to a Q-Sepharose Fast Flow column which had been equilibrated with a buffer containing 5 mM sodium phosphate, 1 mM EDTA, pH 8.0. The protein adhered to the column when applied in the equilibration buffer, and eluted with application to the column of 50 mM NaCI in equilibration buffer. SDS-PAGE showed that the SUB950 protein bound to the column upon application, and did not elute in the initial elution with equilibration buffer. Continued elution with 50 mM NaCI in equilibration buffer resulted in elution of the SUB950 protein. The protein was of reasonable purity after this single chromatographic step. Further elution of the column with increasing concentrations of NaCI resulted in elution of protein impurities but little if any SUB950 lipoprotein. The column rich product was concentrated from 250 mL to 160 mL using Millipore Centriprep centrifugal concentrators. The Bradford total protein assay indicated that the protein content of the protein after concentration was 0.66 mg/mL. The volume of the concentrated rich fraction was 160 m L, therefore the total protein amount was 109.6 mg. The protein was sterile filtered into sterile 50 mL centrifuge tubes, and then stored at -50°C. This sample of SUB950 Lipoprotein was designated as lot # 1024-RLG-2, which also is the notebook reference. Accordingly, we have cloned the gene for Strep, uberis SUB950 Lipoprotein into recombinant E. coli using a pFLEX 30 vector. The resulting protein was expressed in fermentation after heat induction. The SUB950 Lipoprotein was expressed as a soluble, intracellular protein which was purified by chromatography on the anion exchanger Q-Sepharose Fast Flow. After concentration using centrifugal concentrators, the resulting protein appeared on SDS PAGE as one major band. From 500 mL of fermentation medium in a shake flask we recovered 109.6 mg of purified protein, as 166 mL of a 0.66 mg/mL protein solution. Example 7

Expression and Purification of the Streptococcus uberis elongation factor Tu SUB0604

We have also expressed and purified the Streptococcus uberis Elongation Factor Tu protein in recombinant E. coli using a construct which includes a pET15 vector (Novagen) to place a hexa- histidiniyl tag near the N-terminus of the protein. The theoretical molecular weight of the protein is 46,450.56 Daltons, and its isoelectric point should be 5.203. A 500 mL shake flask was utilized to express the protein, after which the insoluble and soluble fractions were separated by centrifugation after disrupting the cells in an Avestin homogenizer. SDS-PAGE indicated that the majority of the Elongation Factor Tu protein was in the insoluble fraction. The insoluble fraction was centrifuged to a pellet, after which it was resuspended in 4% Tergitol 15-S-7 in water and centrifuged again, followed by two water washes and centrifugation after each water wash. The resulting protein fraction showed a major protein band on SDS-PAGE with an approximate apparent molecular weight in the 46,000 Dalton range, plus several very minor bands. After solubilization of the isolated protein in 6M urea in Dulbecco's phosphate buffered saline, Bradford total protein analysis indicated a protein concentration of 0.95 mg/mL, after concentrating the protein sample from 100 mL to 55 mL. The total amount of protein isolated was ~52 mg. The sample was sterile filtered into sterile 50 mL tubes and stored at -50°C

The gene for the S. uberis Elongation Factor Tu protein was cloned into E. coli using a pET15 vector, which places a hexahistidinyl tag near the N-terminus of the protein. The molecular weight of the protein should be 46,450.56 Daltons, and the isoelectric point should be 5.203. The compelte amino acid sequence of the protein is shown below containing a hexahistidinyl tag (in bold).

MGSSHHHHHHSSGLVPRGSHM LEMAKEKYDRSKPHVNIGTIGHVDHGKTTLTAAITTVLARRLPTSVNQPKDYASI D AAPEERERGITINTAHVEYETETRHYAHI DAPGHADYVKN MITGAAQMDGAILVVASTDGPM PQTREHILLSRQVGV KHUVFMNKI DLVDDEELLELVEM EIRDLLSEYDFPGDDLPVIQGSALKALEGDSKYEDII M ELMKTADEYI PEPERDTDK PLLLPVEDVFSITGRGTVASGRIDRGTVRVNDEI EIVGIKEETKKAVVTGVEM FRKQLDEGLAGDNVGILLRGVQRDEIE RGQVIAKPGSINPHTKFKGEVYILSKDEGGRHTPFFNNYRPQFYFRTTDVTGSIELPAGTEMVMPGDNVTISVELI HPIA VEQGTTFSIREGGRTVGSGIVSEIEA (SEQ ID NO: 6 )

Fermentation Conditions

The fermentation seed was prepared in lOOmL of o/n culture in Teknova Amp^"*^"^ (Catalog # L8950, Lot U L895002J1201). For the seed culture, the agitation was set at 200 RPM, the temperature was 37°C, and the pH was 7.0. The batch fermentation was conducted in 500 mL of Terrific Broth with 12.5 mL o/n culture in a 2 L flask. The fermentation conditions are shown in Table 17, below.

Table 17. Fermentation conditions for growth and expression of the S. uteris

Elongation Factor Tu protein

Acid Base

EFT Time OD®600nm Temp Tot Tot PH RPM Feed Notes

0.00 7:00AM 0.01 37°C 0.0 0.0 7.00 200

3.00 TO 3.00 37°C 0.0 0.0 NT 200 1 mM IPTG

4.00 T1 3.60 37°C 0.0 0.0 NT 200

5.00 T2 3.50 37°C 0.0 0.0 NT 200

6.00 T3 3.50 37°C 0.0 0.0 NT 200 Harvest

Isolation of the Protein

After harvest of the shake flask fermentation, the cells were centrifuged to a hard pellet and frozen at - 20°C. The frozen cells were thawed in 200 mL of 50 mM Tris-HCI, 10 mM EDTA, pH 8.0, using a tissue homogenizer. The cells then were lysed by use of an Avestin homogenizer, followed by centrifugation. SDS-PAGE was performed on the lysate, the soluble supernatant, and the inclusion body protein pellet. When the product protein was found to be in the insoluble pellet, the pellet was resuspended in 4% Tergitol 15-S-7 in water. The resuspended pellet then was recentrifuged, resuspended in water, centrifuged again, and then resuspended in water and centrifuged again. The final pellet was solubilized in 6M urea in Dulbecco's phosphate buffered saline. SDS-PAGE was conducted on Novex NuPAGE 4-12% Bis-Tris polyacrylamide gels using Novex NuPAGE MES SDS Running Buffer and Mark 12 protein standards. The gel was stained with Invitrogen Simply Blue SafeStain and destained against deionized water. The Invitrogen prestained protein standards were used for SDS-PAGE of fermentation fluids. The concentration of the resulting protein solution was determined using the Pierce-Thermo Fisher Bradford Coomassie Blue colorimetric protein assay, using bovine serum albumin as a standard. The protocol that came with the kit was followed.

Fermentation Results

The cell density increased from an OD of 3.0 at TO, the time of induction with 1 mM I PTG, to 3.5 at T3 when the cells were harvested. The cells continued to grow after induction, and the wet weight of the centrifuged cell pellet was 4.0 grams. SDS-PAGE of samples taken during the shake flask fermentation showed that the Elongation Factor Tu protein was present at induction, and increased over time after induction. SDS-PAGE indicated that Elongation Factor Tu was in the insoluble fraction after lysis of the E. coli cells post harvest, and that the purity of the protein increased after washing in the detergent Tergitol 15-S-7 followed by a water wash, in each case followed by centrifugation. The major band from SDS-PAGE was in the range of ~46,000, and there were only minor protein bands in addition to the major band. After washing and centrifuging the insoluble protein, the Elongation Factor Tu was dissolved in 100 mL of 6M urea in Dulbecco's phosphate buffered saline, after which it was subjected to concentration in Centriprep centrifugal concentrators to reduce the volume to 55 mL. The apparent total protein concentration from the Bradford reaction was 0.54 mg/mL prior to concentration and 0.95 mg/mL after concentration. The total amount of protein in the prep after concentration was ~52 mg. The protein still is in 6M urea in Dulbecco's phosphate buffered saline. After concentration, the sample was sterile filtered into sterile 50 mL tubes and stored at -50°C. Accordingly, We have expressed and isolated the recombinant Streptococcus uberis Elongation Factor Tu protein using an E. coli construct containing a pET15 vector which will put a hexa- histidinyl tag near the N-terminus of the protein. After performing a 500 mL shake flask fermentation, the product protein was expressed as an insoluble inclusion body protein which was isolated by centrifugation followed by washing the inclusion body pellet with a solution containing 4% Tergitol 15-S-7 in water followed by centrifugation. The resulting pellet was washed twice more with water, followed by a final centrifugation. The fina l pellet was dissolved in 6M urea in Dulbecco's phosphate buffered saline, followed by concentration in a Centriprep centrifugal concentrator. The isolated protein showed a major band on SDS-PAGE corresponding to

approximately 46,000 Daltons, the approximate theoretical molecular weight of the protein. Because of the purity of the protein on SDS- PAGE, we chose not to purify this protein further by nickel chelate affinity chromatography, taking advantage of the hexahistidinyl tag. Bradford analysis indicated that we prepared 52 mg of total protein, as 55 mL of a solution containing 0.95 mg/mL total protein. After purification, we sterile filtered the sample into sterile 50 mL tubes and stored it at -50°C until it is needed.

Example 8

Expression and Purification of the Streptococcus uberis Serine Protease SUB1868

We expressed and purified recombinant Streptococcus uberis SUB1868 serine protease in an E. coli culture as follows. This E. coli culture utilized strain BL21(DE3) and contained a pET15b vector, (Novagen) leading to attachment of a hexahistidinyl tag near the N-terminus of the serine protease.

After lysis and centrifugation of the harvested E. coli cells, SDS-PAGE indicated that the serine protease was in the soluble as well as the insoluble fraction. When applied to a nickel chelate affinity chromatographic column, very little of the soluble fraction adhered to the column. A small fraction of the applied material bound and was eluted with 25 mM imidazole. It is more customary for hexahistidinyl containing proteins to bind to nickel chelate affinity chromatography, even in the presence of 25 mM imidazole, and then to elute with application of 100 mM imidazole to the column.

When 6M urea was added to the material which did not bind to the nickel column, and that material was reapplied to a nickel column, this time the majority of the serine protease bound to the column.

The majority of the bound serine protease eluted with 25 mM imidazole, but some was still bound and was eluted with 100 mM imidazole. The insoluble inclusion body material also was made 6 M in urea and applied to a nickel chelate affinity chromatography column. The majority of serine protease in this fraction bound to the column and was eluted with 25 mM imidazole. The serine protease purified from the insoluble inclusion body fraction was more heterogeneous than the serine protease purified from the soluble fraction. We were able to purify 1.5 mg of serine protease from the initial chromatography of the soluble fraction, and another 59 mg of serine protease from

rechromatography of the soluble fraction in the presence of 6 M urea.

The gene for the SUB1868 serine protease was cloned into E. coli strain BL21(DE3) using a pET15b vector. The complete amino acid sequence of the resulting protein, inclusing a

hexahistidinyl tag near the N-terminus, is shown below. The resulting molecular weight should be

40,512.30 Daltons, and the expected isoelectric point is 5.603.

MGSSHHHHHHSSGLVPRGSHMTNLNNPTTTSKVTYKNTTNTTKAVKVIQDAVVSVVNYQKNDSLNSAMDIFSQGDSS TKENDGLSIYSEGSGVIYKKDGDSAYLVTNNHVIDKAERIEIILADGSKVVGKLIGADTYSDLAVVKISSDKIKTVAQFADSSK INIGEVAIAIGSPLGTEYANSVTEGIVSSI-SRTVTLKNEEGQTVSTNAIQTDAAINPGNSGGPLINIEGQIIGINSSKISQSKSS GNAVEGMGFAIPANDVIKIINQ.LESKGEVVRPALGISMVNL5DL5TNALDQLKVPKNvTSGIVVAKVVDNMPAS GKLEQYDIITEIDGEEVSSTSDLQSILYGHDINDTVKvTFYRGNDKKSTTIELTKTTKDLEK (SEQ ID NO: 12 ).

Fermentation Conditions

The fermentation seed was prepared in 50 mL of o/n culture in Teknova Amp^"*^

(Catalog # 8950). Agitation was at 200 RPM, the temperature was 37°C, and the pH was 7.0.

The batch fermentation (see Table 18) was conducted in 500 mL of "Terrific Broth" with 25 mL o/n culture in a 2 Liter flask. Growth was conducted at 37°C until TO, when the culture was induced at an O.D. of ~1.5 with 1 mM IPTG. The culture continued to grow after induction.

Good expression resulted. The final centrifuged cell pellet weighed 5.0 grams (wet weight).

Table 18. Fermentation conditions for cell growth and expression of the

SUB1868 serine protease.

Acid Base

EFT Time OD(5 600nm Temp Tot Tot PH RPM Feed Notes

0.00 7:00 AM 0.01 30°C 0.0 0.0 7.00 200

4.00 TO 1.50 30°C 0.0 0.0 NT 200 ImMIPTG

5.00 Tl 1.90 30°C 0.0 0.0 NT 200

6.00 T2 3.20 305C 0.0 0.0 NT 200

7.00 T3 4.40 30°C 0.0 0.0 NT 200 Harvest

Purification

A sample of centrifuged cell paste containing 5.0 grams was dissolved in 200 mL of chilled 50 mM Tris-HCI, 10 mM EDTA, pH 8.0. Initially the sample was homogenized with a tissue homogenizer, a VWR Power Max Advanced Homogenizing System, AHS-250, using a setting of 3. All homogenization was conducted on ice. The setting then was increased to 6, after which the cells were lysed by passage through an Avestin homogenizer. The sample then was centrifuged at 9000 RPM using a Sorvall RC6+ centrifuge at 4°C. The supernatant and the pellet were subjected to SDS-PAGE. When the serine protease was found in both the soluble supernatant and the pellet, both fractions were subjected to nickel chelate affinity chromatography.

The clarified supernatant was dialyzed against two 4-Liter changes of 500 mM NaCI, 50 mM sodium phosphate, pH 8.7, prior to loading onto a 2.5 X 8.0 cm column of QJAGEN Ni-NTA Superflow nickel chelate affinity chromatographic medium, using a BioRad low pressure open ended chromatography column without a flow adaptor, a three-way stopcock, and plastic tubing. The dialyzed supernatant contained a total of 200 mL. The column had been

equilibrated with the same NaCI-sodium phosphate buffer as was used in sample dialysis. The column was run manually, without the use of an Akta chromatography instrument, and fractions also were collected manually. Column flow was under gravity, not controlled by a pump. Chromatography was conducted at room temperature.

After loading the sample, equilibration buffer was used to wash the column until a total of 500 mL of column effluent, the spent/wash fraction was collected. After this elution, a total of 200 mL of 25 mM Imidazole in equilibration buffer, pH 8.0, was used to elute the column.

After this, 100 mM Imidazole, pH 7.0, was used to elute the rich fraction, a total of seven 30 mL rich fractions were collected.

The column feed, spent, 25 mM Imidazole and 100 mM Imidazole fractions were

examined by SDS-PAGE prior to pooling the rich fractions. When the majority of the product was found in the column spent fraction, it was made 6 M in urea and subjected to ultrafiltration and diafiltration in a Millipore Labscale TFF system using a BioMax PES 10,000 molecular weight cutoff membrane. The volume was reduced to ~100 mL, followed by diafiltration against 4 diavolumes of a buffer containing 6 M urea, 500 mM NaCI, D-PBS, pH 8.7. This sample was applied to a 2.5 X 5.5 mL column of nickel chelate affinity chromatography packing, followed by elution with the same 6 M urea buffer. A total of 800 mL of spent/wash was collected. After this, 130 mL of 25 mM imidazole in equilibration buffer was applied to the column. Finally, 100 mM imidazole in equilibration buffer was used to elute the column, seven 30 mL fractions were collected. All of the eluted fractions were subjected to SDS- PAGE. A small amount of the serine protease was found in the 25 mM imidazole elution fraction after chromatography of the soluble fraction. This 25 mM imidazole fraction was collected, after which it was concentrated in CentriPrep centrifugal concentrators with a 10,000 molecular weight cutoff membrane.

The insoluble inclusion body fraction after cell lysis was made 4% in Tergitol 15-S-7, after which it subjected to disruption with a tissue homogenizaer, and it was again centrifuged. This pellet from centrifugation was resuspended in purified water, subjected to disruption with a tissue homogenizer, and recentrifuged. The inclusion body pellet was dissolved in 100 mL of a buffer containing 6 M urea in Dulbecco's phosphate buffered saline, pH adjusted to 8.5 with dibasic sodium phosphate. This sample was applied to a 2.5 X 3.0 cm nickel chelate affinity chromatography column, after which it was eluted with an additional 100 mL of 6 M urea in 500 mM sodium chloride plus D- PBS pH adjusted to 8.5. The column then was eluted with 32.5 mL of 25 mM imidazole in 6 M urea equilibration buffer, after which the column was eluted with 100 mM imidazole in equilibration buffer. Seven fractions containing 10 mL each were collected. All eluted fractions were subjected to SDS-PAGE.

SDS-PAGE was conducted on Novex NuPAGE 4-12% Bis-Tris polyacrylamide gels using Novex NuPAGE MES SDS Running Buffer and Mark 12 protein standards. The gel was stained with I nvitrogen Simply Blue SafeStain and destained against deionized water. The Invitrogen prestained protein standards were used for SDS-PAGE of fermentation fluids. The concentration of the resulting protein solution was determined using the Pierce-Thermo Fisher Bradford Coomassie Blue colorimetric protein assay, using bovine serum albumin as a standard. The protocol that came with the kit was followed.

Results

The cell density increased from 1.5 at TO to 4.4 at harvest (Table 1) After harvest, the cells were centrifuged to a pellet. The wet weight of the cell pellet was 5.0 grams. SDS-PAGE of the fermentation broth showed a continuous increase in the SUB1868 serine protease over time. The SUB1868 E. coli cell lysate gave a strong band for SUB1868, as did the soluble supernatant after centrifugation of the cell lysate. There was a protein band for SUB1868 in the insoluble inclusion body fraction, but not as strong as that of the soluble fraction. The apparent molecular weight of the SUB1868 band appeared larger than the expected 40,512 Daltons, which is the theoretical molecular weight.

SDS-PAGE of the column fractions from nickel chelate affinity chromatography of the SUB1860 soluble fraction from the cell lysate showed that the majority of the SUB1860 did not bind to the column. A small amount of SUB1860 did bind to the column and was eluted with 25 mM imidazole. There was virtually no SUB1860 which bound to the column and eluted with 100 mM imidazole. The small amount of serine protease which eluted in the 25 mM imidazole fraction initially contained 200 mL, the volume was reduced to 10 mL by use of centrifugal concentrators. The protein content of the concentrated fraction was 0.15 mg/mL, consequently there was a total of 1.5 mg in that fraction. We do not know how much protein was lost over concentration. The lot number assigned to this concentrated sample was 1024-RLG-15A.

The soluble fraction which did not bind to nickel chelate affinity chromatography was made 6M in urea, followed by ultrafiltration and diafiltration. The resulting sample was reapplied to a nickel chelate affinity chromatographic column which was equilibrated and eluted using buffers which were 6M in urea. Urea was included in all elution buffers. The results, as shown in Figure 6, indicated that the majority of the serine protease bound to the column and eluted with application of 25 mM imidazolel to the column. The first two fractions eluted with 100 mM imidazole also contained the serine protease, but at a lower concentration than the fraction eluting with 25 mM imidazole. The 25 mM imidazole fraction and the first two 100 mM imidazole fractions were combined, this pool contained 190 mL. After reduction of the volume of this pool to ~80 mL by use of centrifugal concentrators, the total protein content of this pool, lot # 1024-RLG-15B, was 0.738 mg/mL. This pool therefore contained a total protein content of 59 mg.

We also solubilized the inclusion body fraction in 6 M urea and applied it to a nickel chelate affinity chromatographic column (Figure 7). The majority of the serine protease bound to the column and eluted with application of 25 mM imidazole to the column. This eluted fraction was not as pure as the 25 mM imidazole fraction from initial chromatography of the soluble fraction. We chose not to use the serine protease from inclusion bodies in further studies. The serine protease initially purified from the soluble protein fraction, and also after 6M urea to the soluble protein fraction. Both of these fractions appear reasonably pure on SDS-PAGE. We assigned lot of numbers of 1024-RLG15A and 1024- RLG-15B, respectively, to these two fractions. They were sterile filtered into sterile 50 mL centrifuge tubes, and they are in storage at -50°C. Accordingly, we have expressed the recombinant Streptococcus uberis SUB1868 serine protease utilizing an E. coll culture obtained from the Moredun Group in Scotland. This culture is strain BL21(DE3) and contains the pET15b vector, and places a hexahistidinyl tag near the N-terminus of the protein, allowing for purification by nickel chelate affinity chromatography. Following harvest of the shake flask fermentation, after the E. coli cells were lysed and the lysate centrifuged, the SUB1868 serine protease was seen on SDS-PAGE in the soluble as well as the insoluble fraction. When the soluble fraction was subjected to nickel chelate affinity chromatography, only a small portion of the serine protease bound to the column, after which it was eluted with 25 mM imidazole. Usually, 100 mM imidazole is required to elute hexahisitindyl containing recombinant proteins from nickel chelate affinity resin. This fraction which eluted with 25 mM imidazole contained very little protein, but it was very pure. The majority of the protein did not bind to the column. This unbound material was made 6M in urea and applied to a nickel chelate affinity column in which 6M urea was included in all of the buffers. Under these conditions nearly all of the serine protease bound to the column, and the majority of it eluted with application of 25 mM imidazole to the column, a small amount was eluted with 100 mM imidazole. This material also was of reasonable purity.

In addition to the soluble intracellular fraction, SDS-PAGE also indicated that the insoluble inclusion body fraction contained the SUB1868 serine protease, although there was less of this protein in the insoluble fraction as compared to the soluble fraction, and the insoluble protein was not as pure. After solubilization in a buffer containing 6M urea, the protein in the insoluble inclusion body fraction was applied to a nickel chelate affinity column utilizing buffers containing 6M urea. The majority of the serine protease bound to the column and was eluted from the column with 25 mM imidazole. This column rich fraction was not as pure as that seen with the soluble protein, there were multiple minor bands on SDS-PAGE indicating proteins larger and smaller than the serine protease.

Claims

1. A vaccine composition comprising an effective amount of Streptococcus antigen and an adjuvant formulation, wherein the antigen is selected from any one, two, three or four of the following: Streptococcus uberis ferrichrome binding protein (SEQ. I D NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ. I D NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ ID NO : 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ I D NO 11, locus tag SUB1868, accession number YP-002563137); wherein the adjuvant formulation comprising an oily phase and an aqueous phase, wherein the oily phase comprises at least 50% of the formulation v/v, wherein said formulation comprises at least one of monophosphoryl lipid A (MPL-A) or an analog thereof and an immunostimulatory oligonucleotide, with provisos that:

c) if said immunostimulatory oligonucleotide is absent, then the formulation comprises:

j. a poly l:C, a glycolipid, and, optionally, a quaternary amine; or

ii. a polycationic carrier;

d) if said monophosphoryl lipid A (MPL-A) or the analog thereof is absent, then the formulation comprises a source of aluminum.

2. The vaccine composition of claim 1 comprising an adjuvant formulation wherein

the immunostimulatory oligonucleotide, if present, is a CpG or an oligoribonucleotide; the polycationic carrier, if present, is selected from the group consisting of dextran, dextran DEAE (and derivatives thereof), PEGs, guar gums, chitosan derivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC) polyethylenimene, poly aminos; and

the quaternary amine, if present, is selected from the group consisting of DDA and avridine.

3. The vaccine composition of any one of claims 1 or 2, wherein the glycolipid component of the uvant formulation, if present, comprises a compound of formula I

Formula I

wherein, R¹ and R² are independently hydrogen, or a saturated alkyl radical having up to 20 carbon atoms; X is -CH₂-, -0- or -NH-; R² is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵ are independently hydrogen, -S0₄ ^2", -P0₄ ^2~, -COC_{l w} alkyl; R⁶ is L- alanyl, L-alpha-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-phenyalany, L-prolyl, L-seryl, L- threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers.

4. The vaccine composition of claim 3, wherein the glycolipid component of the adjuvant formulation comprisies N-(2-Deoxy-2-L-leucylamino-p-D-glucopyranosyl)-N-octadecyldodecanoylamide or a salt thereof.

5. The vaccine composition of any one of claims 1-4, wherein the adjuvant formulation comprises both said monophosphoryl lipid A (MPL-A) or the analog thereof, and further comprises at least one of a sterol and a poly l:C.

6. The vaccine composition of claims 5, wherein the adjuvant formulation comprises a sterol and further comprising a saponin.

7. The vaccine composition of any one of claims 1-6, wherein the adjuvant formulation thereof comprises e poly l:C, and further comprising at least one of the quaternary amine and the glycolipid.

8. The vaccine composition of any one of claims 1-6, wherein the adjuvant formulation thereof comprises a source of aluminum, which is an aluminum hydroxide gel.

9. A vaccine composition comprising an effective amount of an antigen and the adjuvant formulation according to any one of claims 1-8, wherein the oily phase of the composition is at least 50%.

10. A vaccine composition comprising an effective amount of any one, two, three, or four antigens selected from the group consisting of Streptococcus uberis ferrichrome binding protein (SEQ. ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ ID NO : 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ I D NO 11, locus tag SUB1868, accession number YP-002563137); and an adjuvant formulation comprising an oily phase and an aqueous phase, wherein the oily phase comprises at least 50% of the formulation v/v, a polycationic carrier, and

a. a combination of a saponin and a sterol, and optionally, a quaternary amine; or b. an immunostimulatory oligonucleotide

11. The vaccine composition of claim 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 a CpG.

12. Use of the vaccine composition according to any one of claims 1-10 for treatment or prevention of infections caused by. Streptococcus.

13. A vaccine composition comprising 1, 2, 3, or 4 antigens selected from the group consisting of Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ ID NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137), and any combination of 1, 2, 3, or 4 members thereof; and an adjuvant, said adjuvant being selected from the group consisting of:

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 oily phase present in the amount of at least 50% v/v of the vaccine composition and comprising an immunostimulatory oligonucleotide and a polycationic carrier.

14. The vaccine composition of claim 13, wherein the saponin is Quil A, the sterol is cholesterol, the quaternary amine is DDA, the glycolipid is N-(2-Deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N- octadecyldodecanoylamide or a salt thereof, and the immunostimulatory oligonucleotide is a CpG.

15. The vaccine composition of claim 14, wherein the polycationic carrier is dextran DEAE and the immunostimulatory oligonucleotide is a CpG.

16. A use of the vaccine composition according to any one of claims 13-15 for treatment or prevention of an infection caused by Streptococcus uberis.

17. A vaccine composition according to Claim 13 comprising an adjuvant and an adjuvant formulation, said adjuvant formulation comprising an oily phase present in the amount of at least 50% v/v of said vaccine composition, an immunostimulatory oligonicleotide and a polycationic carrier.

18. The vaccine composition of claim 17, wherein the immunostimulatory oligonucleotide is a CpG, and the polycationic carrier is DEAE dextran.

19. A vaccine composition comprising a Streptococcus uberis (5. uberis) antigen and an adjuvant formulation comprising an oily phase, said oily phase being present in the amount of at least 50% v/v of the composition; a polycationic carrier; and

a) an immunostimulatory oligonucleotide; and optionally

b) a combination comprising a saponin, a sterol, and a quaternary amine; or

c) a combination thereof.

20. A vaccine composition comprising an effective amount of Streptococcus antigen and an adjuvant formulation, wherein the adjuvant formulation comprising an oily phase and an aqueous phase, wherein the oily phase comprises at least 50% of the formulation v/v, wherein said formulation comprises at least one of monophosphoryl lipid A ( PL-A) or an analog thereof and an immunostimulatory oligonucleotide, with provisos that: e) if said immunostimulatory oligonucleotide is absent, then the formulation comprises:

k. a poly l:C, a glycolipid, and, optionally, a quaternary amine; or ii. a polycationic carrier;

f) if said monophosphoryl lipid A (MPL-A) or the analog thereof is absent, then the formulation comprises a source of aluminum.

21. The vaccine composition of claim 20 comprising an adjuvant formulation wherein

the immunostimulatory oligonucleotide, if present, is a CpG or an oligoribonucleotide; the polycationic carrier, if present, is selected from the group consisting of dextran, dextran DEAE (and derivatives thereof), PEGs, guar gums, chitosan derivatives, polycellulose derivatives like hydroxyethyl cellulose (HEC) polyethylenimene, poly aminos; and

the quaternary amine, if present, is selected from the group consisting of DDA and avridine.

22. A vaccine composition comprising an effective amount of 1, 2, 3, or 4 Streptococcus antigens and an adjuvant, wherein the 1, 2, 3 or 4 antigens are selected from the group consisting of Streptococcus uberis ferrichrome binding protein (SEQ. I D NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137).

23. The vaccine composition of Claim 22, wherein the resultant combined antigen provided in the composition is selected from the group consisting of (a) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776) and Streptococcus uberis elongation factor Tu (SEQ ID NO: 5, locus tag SUB0604, accession number YP-002561947), and (b) Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276), and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP- 002563137).

24. The vaccine composition of Claim 22, wherein the resultant combined antigen provided in the composition is selected from the group consisting of:

(a) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); Streptococcus uberis lipoprotein (SEQ I D NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ I D NO 11, locus tag SUB1868, accession number YP-002563137);

(b) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); and Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276);

(c) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP-002561947); and Streptococcus uberis serine protease (SEQ I D NO 11, locus tag SUB1868, accession number YP-002563137);

(d) . Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137);

(e) Streptococcus uberis elongation factor Tu (SEQ I D NO: 5, locus tag SUB0604, accession number YP- 002561947); Streptococcus uberis lipoprotein (SEQ ID NO : 8, locus tag SUB0950, accession number YP- 002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137)

(f) Streptococcus uberis ferrichrome binding protein (SEQ I D NO: 2, locus tag SUB0423, accession number YP-002561776); and Streptococcus uberis elongation factor Tu (SEQ ID NO: 5, locus tag SUB0604, accession number YP-002561947); (g) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); and Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP-002562276);

(h) Streptococcus uberis ferrichrome binding protein (SEQ ID NO: 2, locus tag SUB0423, accession number YP-002561776); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137);

(i) Streptococcus uberis elongation factor Tu (SEQ ID NO: 5, locus tag SUB0604, accession number YP- 002561947); and Streptococcus uberis lipoprotein (SEQ ID NO : 8, locus tag SUB0950, accession number YP-002562276);

(j) Streptococcus uberis elongation factor Tu (SEQ ID NO: 5, locus tag SUB0604, accession number YP- 002561947); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137); and

(k) Streptococcus uberis lipoprotein (SEQ ID NO: 8, locus tag SUB0950, accession number YP- 002562276); and Streptococcus uberis serine protease (SEQ ID NO 11, locus tag SUB1868, accession number YP-002563137).

25. A vaccine composition comprising one or more Streptococcus uberis antigens selected from :

(a) one or more of:

(i) 5. uberis elongation factor Tu: (acc No: YP_002561947);

(ii) S. uberis lipoprotein (acc No: YP_002562276);

(iii) S. uberis serine proteinase (acc No: YP_002563137); and

(iv) S. uberis ferrichrome binding protein: (acc No: YP_002561776);

(b) immunogenic fragments of any one of proteins (i)-(iv) above;

(c) antigens comprising, consisting or consisting essentially of sequences which exhibit at least about 60% homology or identity to the sequences of proteins (i)-(iv) above; (d) antigens comprising, consisting or consisting essentially of sequences which represent variant, derivative or mutant sequences of proteins (i)-(iv) above;

(e) antigens encoded by nucleic acid sequences encoding each of proteins (i)-(iv) above; and

(f) antigens comprising sequences of or corresponding to, the immunogenic domains or epitopes of the proteins given as (i) to (iv) above.

26. A vaccine for use in a method of raising an immune response protective against mastitis in cattle, said vaccine comprising, consisting essentially of or consisting of one or more of the proteins selected from the group consisting of:

S. uberis:

(i) elongation factor Tu: (acc No: YP_002561947);

(ii) lipoprotein (acc No: YP_002562276);

(iii) serine proteinase (acc No: YP_002563137);

(iv) ferrichrome binding protein: (acc No: YP_002561776); and

(v) an immunogenic fragment of any on of (i)-(iv).