AU1551499A - Saponin adjuvant composition - Google Patents

Description

WO 99/27959 PCT/AU98/00990 SAPONIN ADJUVANT COMPOSITION TECHNICAL FIELD This invention relates to adjuvant compositions for stimulating an immune response to an antigenic substance 5 when co-administered to an animal with said antigenic substance, and to vaccines containing said adjuvant composition. BACKGROUND ART Vaccination against disease has a long history. In 10 general terms the technique involves injection of an antigenic substance, or antigen, into an animal whereby the presence of the antigenic substance generates an immune response in the animal. Classical vaccination techniques involve the injection of killed organisms but more recently 15 vaccines comprising attenuated live organisms or antigenic components of an organism have been developed. It is frequently found with killed vaccines and, more particularly, with vaccines comprising a component of an organism that the immune response is substantially less 20 than the response to natural infection. However, the effectiveness of such vaccines can be considerably enhanced by the co-administration of a suitable adjuvant composition with the antigenic substance. Adjuvants, while not necessarily being antigenic themselves, potentiate or 25 enhance an animal's immune response to the antigenic substance with which it is challenged. There are many adjuvants known and used but there is an ongoing need to identify new and effective adjuvants which are inexpensive, which produce minimal injection site irritation and 30 discomfort and which are widely applicable and effective. A common formulation for vaccines is to present the antigen(s) in an aluminium hydroxide gel. While this is effective in some cases and is reasonably benign, in many cases this adjuvant fails to induce a sufficiently 35 protective response. It is also well known that antigens emulsified in a mineral oil vehicle together with whole mycobacterial cells (Freund's complete adjuvant, FCA) can produce a generally effective immune response against a WO 99/27959 PCT/AU98/00990 2 wide range of antigens. However, this formulation is unacceptable for routine use because of the inflammation, granulomas, ulceration and other lesions which can be formed at the injection site. Mineral oils alone 5 (frequently referred to as Freund's Incomplete Adjuvant, FIA or Incomplete Freund's, ICF) are less damaging but are also less effective. Neutral oils (such as miglyol) and vegetable oils (such as arachis oil), ISCOMS and liposomes have also been used. Also effective are adjuvants 10 containing purified mycobacterial component such as N acetylmuramyl-L-alanyl-D-isogulutamine (MDP) or its analogues in aqueous or oil formulations. Among other adjuvants which have been or are currently used are the saponins, particularly triterpenoid mixtures such as Quil A 15 (a purified extract from the bark of the tree Quillaja saponarioa) in aqueous solution or in the form of a matrix with cholesterol. Polycations such as diethylaminoethyldextran (DEAE dextran) can also be effective as adjuvants in some cases. 20 There have also been proposals to use a combination of two adjuvants substances in an adjuvant composition. For example, Australian patent no. 602348 describes an immunoadjuvant comprising an immunoadjuvant oil substantially free of mycobacteria and a polycationic 25 polyelectrolyte immunoadjuvant such as DEAE dextran in the form of an emulsion having the polycationic polyelectrolyte dissolved in the aqueous phase. The two-component immunoadjuvant is said to overcome the rapid decline in the immune response associated with polycationic 30 polyelectrolyte adjuvants on the one hand and, on the other, the weak initial response associated with immunoadjuvant oils. Accordingly, the two-component adjuvant is said to fill the gap in the prior art between those adjuvants inducing high peak/short life antibody 35 responses and those inducing low peak/long life responses. International application no. 88/07547 is primarily concerned with a novel peptide nevertheless, it also discloses the use of a novel adjuvant comprising DEAE WO 99/27959 PCT/AU98/00990 3 dextran and a saponin or aluminium hydroxide and notes an improved antibody titre when the two-component immunoadjuvants are used. In particular, solutions of DEAE dextran and saponin in phosphate buffered saline are used 5 but there is no suggestion of the incorporation of an immunoadjavent oil into such compositions. Australian patent no. 640414 discloses a solid vaccine composition comprising an antigenic substance capable of inducing the generation of antibodies on parenteral 10 administration to an animal, a saponin and a polycationic adjuvant. The essence of the invention is that the vaccine is formulated as solid to be implanted in the animal to thereby induce a long-lasting immune response. There is no suggestion of the presence of an immunoadjuvants oil in the 15 composition and, indeed, the specification teaches away from the use of an oil as it is critical to the invention that this formulation be solid. In the present invention it has been found, surprisingly, that combinations of certain adjuvants 20 enhance the effectiveness of an antigenic substance in stimulating an immune response to a much greater extent than the sum of the profiles that would be obtained by the use of the components separately or through the use of a two-component immunoadjuvant. 25 DISCLOSURE OF THE INVENTION According to a first aspect of the present invention there is provided an adjuvant composition for stimulating an effective immune response in an animal to an antigenic substance when co-administered to said animal with said 30 antigenic substance, comprising: (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune stimulating activity; and (c) an immunoadjuvant oil. 35 According to a second aspect of the present invention there is provided a vaccine for administration to an animal, comprising: (1) an antigenic substance; and WO 99/27959 PCT/AU98/00990 - 4 (2) an adjuvant composition comprising: (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune stimulating activity; 5 (c) an immunoadjuvant oil. According to a third aspect of the present invention there is provided a method of stimulating an effective immune response in an animal to an antigenic substance, comprising the steps of: 10 (1) providing said antigenic substance; (2) providing an adjuvant composition for stimulating an effective immune response to said antigenic substance, comprising: (a) a saponin with immune stimulating activity; 15 (b) a polycationic polyelectrolyte with immune stimulating activity; and (c) an immunoadjuvant oil; and (3) challenging said animal with said antigenic substance and said adjuvant composition. 20 According to a fourth aspect of the present invention there is provided the use of an adjuvant composition comprising: (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune 25 stimulating activity; and (c) an immunoadjuvant oil to stimulate an effective immune response in an animal challenged with an antigenic substance. According to a fifth aspect of the present invention 30 there is provided the use of an adjuvant composition comprising: (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune stimulating activity; and 35 (c) an immunoadjuvant oil in the preparation of a medicament for administration to an animal, wherein said medicament further comprises an antigenic substance.

WO 99/27959 PCT/AU98/00990 5 The saponins are common secondary constituents of plants and typically are glycosides composed of several (hydrophilic) sugars in association with a (hydrophobic) molecule, which can be either a steroid or triterpenoid 5 structure. In particular, an extract from the South American tree Quillaja saponarioa shows good adjuvant activity and is now denoted "Quil A". While the precise chemical composition of Quil A is not known, the sugar moieties detected in the mixture include rhamnose, fucose, 10 arabinose, xylose, galactose, glucose, apiose and glucuronic acid and the hydrophobic moiety has a triterpenoid structure. The nature of Quil A is discussed as length in Australian patent application no. 10777/95, the disclosure of which is incorporated herein by 15 reference. Preferably, the saponin is a triterpenoid compound or a mixture of triterpenoid compounds. More preferably, the saponin is Quil A or the extract disclosed in Australian application no. 10777/95, or compounds obtainable from 20 these extracts. Still more preferably, the saponin is Quil A. As used throughout the description and claims the term "polycationic polyelectrolyte" refers to polymer or oligomers, natural or synthetic, that, by virtue of their 25 chemical structure, acquire a plurality of discrete positive charges in aqueous solution under appropriate pH conditions. Suitable polycationic polyelectrolytes are DEAE dextran, polyethyleneimine, ethoxylated polyethyleneimine, epichlorhydrin-modified 30 polyethyleneimine, diethylaminoethyl ester and amide derivatives of acrylate polymers, copolymers and the like. The most preferable polycationic polyelectrolyte is DEAE dextran, which is a polycationic derivative of dextran (average molecular weight 10000 to 1000000, preferably 35 200000 to 750000, most preferably 500000) containing diethylamino ethyl groups linked to glucose in a 1:3 ratio. Typically the polycationic polyelectrolyte is in aqueous solution, for example, phosphate buffered saline.

WO 99/27959 PCT/AU98/00990 - 6 While the invention embraces a wide range of immunoadjuvant oils, mineral oils are preferred. More preferred are those mineral oils already known in the art for use as adjuvants and including substances such as 5 Drakeol, Markol, squalene, squalane and the like but the preferred mineral oil is Montanide oil. Mineral oil immunoadjuvants are frequently referred to as Freund's incomplete adjuvant and this adjuvant typically comprises 85% mineral oil and 15% mannide monooleate as an 10 emulsifier. Typically the adjuvant composition of the present invention takes the form of an emulsion with the polycationic polyelectrolyte dissolved in the aqueous phase and the mineral oil forming the non-aqueous phase. It is 15 well known that immunoadjuvant emulsions of individual oils used separately can be formulated with oil to water phase ratios extending over a broad range and embracing the ratios 80:20 to 20:80 (v/v) for example, more preferably 60:40 to 40:60 (v/v). Such a broad range of ratios of oil 20 phase to aqueous phase also applies in the present invention except that the aqueous phase will always comprise a polycationic polyelectrolyte solution and the composition will also include a saponin. While not wishing to be bound by theory, the saponin is amphiphilic and which 25 may partition itself between the phases with the hydrophilic sugar residues in the aqueous phase and the hydrophobic triterpenoid structure in the non-aqueous phase. Accordingly, the saponin may serve to stabilise the emulsion. 30 Preferably, vaccines including adjuvant compositions in accordance with the present invention contain the saponin component at a concentration greater than 50.g/ml and the polycationic polyelectrolyte at a concentration of greater than 1mg/ml. More preferably, they contain 35 saponins in a concentration of greater than 100pg/ml and the polycationic polyelectrolyte component in a concentration of greater than 1.5mg/ml. The upper limits of concentration of the saponin component and the WO 99/27959 PCT/AU98/00990 - 7 polycationic polyelectrolyte are essentially determined by economic considerations since these components are expensive, but the saponin may be present in concentrations up to 10mg/ml, typically up to 1mg/ml, and the polycationic 5 polyelectrolyte may be present in concentrations up to 200mg/ml, typically 150mg/ml. The emulsifiers used to form the novel compositions of the invention are those known in the art such as mannide monooleate, Arlacela A, Arlacela 80 and Tween 80. It will 10 be recognised by those skilled in the art that the adjuvant composition can be used in virtually any vaccine including any antigenic substance, although it will be recognised that many factors other than the nature of the adjuvant composition will influence the nature of and level of the 15 antibody response to the vaccine. The adjuvant composition is particularly useful when used in conjunction with a whole cell killed vaccine or killed viral vaccine or a vaccine comprising a proteinaceous substance, which may or may not be 20 glyocosylated or otherwise chemically modified, alone or as a carrier for a low molecular weight compound. In general, the antigenic substance will give rise to an immune response against a disease-causing agent but may also give rise to antibodies against an agent (such as a hormone) 25 which does not normally give rise to a disease. The disease causing agent may be a structural component or toxin of a virus, bacteria or other microbe. Examples of virally-caused diseases which may be controlled by vaccines including the adjuvant composition of the present invention 30 include infectious bursal disease virus, Newcastle disease, infectious bronchitis virus, pseudorabies, parvovirus, classical swine fever, equine influenza, bovine viral diarrhoea virus and canine corona virus. Examples of bacterially-caused diseases include atrophic rhinitis, 35 loptospirosis, clostridial infections, bordetella brochisepticum infections in cats, coryza in poultry, fowl chloera, Mycoplasma gallisepticum infections in poultry, pleuropmeumonia and rabies. The adjuvant composition may WO 99/27959 PCT/AU98/00990 -8 also be used in conjunction with sub-unit vaccines produced using recombinant DNA technology such as in a sub-unit vaccine against cattle ticks. The antigenic substance may also comprise a target low 5 molecular weight compound conjugated to a carrier selected so as not to be recognised by the organism as "self" and thereby to generate an immune response against the low molecular weight compound. Suitable carriers include fetuin, ovalbumin, bovine serum albumin, foetal calf serum 10 and human serum albumin. Alternatively, the carrier may be keyhole limpet haemocyanin or beta-galactosidase, among others. The low molecular weight compound may be conjugated to the carrier by any convenient means. Suitable conjugators include glutaraldehyde, toluene 15 diisocyanate, carbodiimide, or any other suitable conjugator. The small molecules which may be conjugated to a character include toxins such as phomopsin or other substances such as mammaliam hormones or steroids against 20 which it may be desirable to raise an immune response. Other antigens which may be employed include red blood cells and virus like particles, particularly VLP/NS2. Preferably, the antigenic substance is a fetuin phomopsin conjugate, phomopsin A conjugated to ovalbumin, 25 phomopsin A-fetal calf serum conjugate, a virus-like particle, particularly VLP/NS2 (a VLP comprising a blue tongue virus antigen encoded by a recombinant baculovirus vector), sheep red blood cells, or ovalbumin. In the method of the invention the antigenic substance 30 and adjuvant composition are conveniently mixed prior to administration. Typically, the antigenic substance is in aqueous solution, such as phosphate-buffered saline. The polycationic polyelectrolyte may also be in aqueous solution, such as in solution in phosphate-buffered saline, 35 and together these components form the aqueous phase of the emulsion. However, it will be appreciated that the antigenic substance and adjuvant may be administered sequentially, and even that the various components of the WO99/27959 PCT/AU98/00990 - 9 adjuvant composition may be administered sequentially rather than simultaneously provided that they undergo a physiological interaction in vivo. Throughout the specification, except where the context 5 requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", ie. the features specified may be associated with further features in various embodiments of the invention and are not to be construed, necessarily, as the 10 only features of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The present invention will now be further described, by way of example only, with reference to the accompanying drawings, in which: 15 Figure 1 shows the antibody response to sheep red blood cells in chickens in a Haemagglutination assay; Figure 2 shows the antibody response to sheep red blood cells in chickens in an ELISA assay on 0.1ml of 0.1% sheep red blood cells/well coated plate; 20 Figure 3 shows the antibody response to phomopsins in cattle in an ELISA assay on 50 ng phomopsins/0.1ml/well coated plate; Figure 4 shows the antibody response to ovalbumin in cattle in an ELISA assay on 50 ng ovalbumin/0.1ml/well 25 coated plate; Figure 5 shows the antibody response to phomopsins in wethers in an ELISA assay on 50ng phomopsins/0.1ml/well coated plate; Figure 6 shows the antibody response to foetal calf 30 serum in wethers in an ELISA assay on 50ng foetal calf serum/0.1ml/well coated plate; and Figure 7 shows the antibody response to ovalbumin in Long-Evans hooded rats using an ELISA assay on 50 ng ovalbumins/0.1ml/well coated plate. 35 MODES OF CARRYING OUT THE INVENTION Example 1 In this example a fetuin-phomopsin conjugate was employed as the antigen in a comparison of three adjuvant WO 99/27959 PCT/AU98/00990 - 10 formulations including one formulation conforming to the invention described in this specification. Sheep (12 per group) were the animal species used. The dose volume (iml) and antigen concentration were kept constant and the oil 5 component was 85% (v/v) mineral oil and 15% (v/v) mannide monooleate (incomplete Freund's adjuvant, ICF) in all formulations. In one formulation the adjuvant comprised a 10mg/dose of DEAE dextran as the cationic polymer in incomplete 10 Freund's oil (10mg DEAE/ICF). In another the adjuvant was 0.5mg Quil A as a triterpenoid component in incomplete Freund's oil (0.5mg Quil A/ICF) and in the third, representative of this invention, 5mg of DEAE and 0.5mg of Quil A were mixed together in incomplete 15 Freund's oil (0.5mg Quil A/5mg DEAE/ICF). A primary and one booster injection were administered 16 weeks apart. Anti phomopsin antibody titres (measured by ELISA) were not detectable prior to vaccination. Table 1 shows anti phomopsin antibody titres 2 and 8 weeks after the booster 20 injection. TABLE 1 Adjuvant Dose Sheep Titre - Titre formulation volume Nos. Booster plus 2 Booster plus 8 weeks weeks 10mg DEAE/ ICF iml 12 55,000 8,000 0.5mg Quil A/ iml 12 23,000 2,000 ICF 0.5mg Quil A/ iml 12 99,000 37,000 5mg DEAE/ICF The results show that the trivalent adjuvant prepared according to this invention gave a significantly higher 25 antibody titre two weeks after the booster injection than either of the divalent formulations lacking one component of the trivalent formulation. The titre obtained with the trivalent adjuvant was also greater than the sum of the titres produced by the two divalent formulations 30 demonstrating an unexpected synergism between the components. Eight weeks after the booster injection the trivalent vaccine was out-performing the divalent formulations by an increased margin demonstrating the WO 99/27959 PCT/AU98/00990 - 11 longer duration of effect and the synergistic response achieved. Example 2 5 In another comparison of two, two-component mixtures and a three component mixture representative of this invention, three groups of 12 sheep were injected with a fetuin-phomopsin conjugate antigen formulated in an adjuvant mixture of DEAE dextran in incomplete Freund's 10 adjuvant (ICF) or Quil A in ICF or a mixture of DEAE dextran and Quil A in ICF. The antibody titres achieved are shown in Table 2. TABLE 2 Adjuvant Dose Sheep Titre - Titre formulation volume Nos. Booster plus 2 Booster plus 8 weeks weeks 10mg DEAE/ iml 12 8,000 2,000 ICF 0.5mg Quil iml 12 9,000 1,000 A/ ICF III 0.5mg Quil iml 12 44,000 14,000 A/ 5mg DEAE/ICF 15 Once again the adjuvant formulated according to this invention demonstrates an enhanced, synergistic, long lasting effect when compared to two component formulations 20 in which one of the three components specified in this invention is missing. Example 3 In this example a comparison was made between two 25 trivalent adjuvant formulations incorporating a triterpinoid (Quil A), a cationic polymer (DEAE dextran) and two different commercially available oils (ICF or Montanide 888). Both formulations incorporated a commercial preservative, Thimerosal, and the antigen was a phomopsin 30 fetuin conjugate. The results are shown in Table 3 WO 99/27959 PCT/AU98/00990 - 12 TABLE 3 Adjuvant Dose Sheep Anti-phomopsin Anti-phomopsin formulation volume Nos. titre - Booster titre - Booster plus 2 weeks plus 8 weeks 0.5mg Quil A/5mg 1ml 11 20,000 16,000 DEAE/ ICF 0.5mg Quil A/5mg lml 11 40,000 25,000 DEAE/ Montanide 888 5 The results demonstrate the high titres obtained with this invention and the longevity of the effect with both formulations. A better response is seen with the Montanide oil under the conditions used demonstrating that careful selection of the oil component of the invention from a 10 number of available products can give advantage for particular applications. Example 4 Animal species: Rabbit 15 The antigen: Virus-like particles (VLPs). Adjuvant formulations: This invention. 2.5mg Quil A and 50mg DEAE-dextran in 3ml of PBSA was filtered through a 0.2pm filter. Six hundred microlitres of this solution was added to 200 20 microlitres of antigen (1mg VLP/NS2, a VLP comprising a blue tongue virus antigen encoded by a recombinant baculovirus vector, pelleted and resuspended in 200 microlitres PBSA). 1.2 ml of Montanide ISA 50V was then added to this combined solution. The mixture was sonicated 25 and emulsified to form a viscous liquid. Freund's complete. iml Freund's Complete adjuvant was added to 1 ml VLP/NS2 (1mg) in PBSA. This solution was sonicated and emulsified. An extremely viscous, almost solid emulsion was formed. 30 Freund's incomplete/DEAE dextran. 1 mg VLP/NS2 was resuspended in Iml of 15% DEAE-dextran and added to 1ml of Freund's incomplete adjuvant. This solution was sonicated and emulsified.

WO 99/27959 PCT/AU98/00990 - 13 PBSA. VLP/NS2 was dissolved in PBSA at a concentration of 1mg per ml. Vaccination protocols 5 This invention. Five 0.1ml intradermal injections per rabbit were given for the primary vaccination and 0.3 ml in each hind leg were given for the booster. Freund's complete. Four intradermal injections of 0.1ml were given as the primary vaccination and 0.4ml 10 intramuscular injection per hind leg was given as a booster. Freund's incomplete/DEAE dextran. Five intradermal injections were given per rabbit were given as the primary vaccination and 0.3ml per hind leg were given as a booster. 15 PBSA. A 0.5ml intramuscular injection was given to each hind legs for both the primary and booster injections. The rabbits were bled on day 1 and injections were given on day 5, day 54, and day 78. This example demonstrates that the adjuvant 20 composition of the present invention performs very well when compared to Freund's adjuvant. In the data shown in Table 4 it will be apparent that the immune response using the adjuvant of the present invention begins earlier than the immune response when Freund's adjuvant is used and is 25 stronger and more long lasting. In addition, the adjuvant does not induce the formation of lesions at the injection site as Freund's adjuvant can.

WO 99/27959 PCT/AU98/00990 14 (C4 CO 0 0 0 0 0 0 0 0 S 0 0 0 0 0 0 00 1 0 0 0 0 0 0 H r-i LO r-I L H ( " - m 0 0 0 0 0 0 0 C0 mn 0 0 0 0 0 0 0 0 0 0 0 L A LA L n L H r-- jH 4 m m a Hm 4 A 0 oo %D- CD C C) CD0 0 0O S 0 0 0 0 0 o t~ C) C ) C cc 'I rt o a 0 0 o a , go C 0 0 0 0 0 0 co S0 0 0 0 0 0 co C 0 0 a 0a 00 -m A AA m S0 0 0 0 0 0 0 0 00 . 0 0 0 00 0 M ~L LAC LA LA L 0 00) W- L r Q)A A AA HL 4 oO U)l 0 00 J 0 0 0 0 0 0 00 r 0 0 0 0 0 0 r 0 C 0 0 0 0 0 0 00 0 LA LA L L 0 00 0 _ L - r-I - ) r 1Q A A 0 040 "i C) ' 0 0 0 0 0 00 > 0C 0 0 0 0 0 0)0 4 s 0 0 0 0 0 0 00 C H O0 LA LA LA H- LA LA) LA 0 -4 14-4 0 tL 1NC) 00 0 0 0 00 >) H q H 020 U) 024 o a4.02000 0U U ~mm ::1 H H a4 P4 j . 4. ) P) o IX E-qE- U U WO99/27959 PCT/AU98/00990 - 15 Example 5 In this example a comparison was made between the immune response and injection site reaction of chickens to sheep red blood cells. The red blood cells were 5 administered either in Freund's complete adjuvant with a booster injection, in incomplete Freund's adjuvant or in an adjuvant system typifying this invention for both primary and booster injections. Sterile sheep blood (100 ml) was collected and 1 10 volume of blood was added immediately into 1.2 volume of Alsever's solution (Methods in Immunology and Immunochemistry, vol 4, 41, Eds: Williams, C.A. and Chase, M.W., 1977). Hybrid white leghorn chickens eggs (Ex SPF Unit) were 15 set on 2/10/97 and chickens were hatched 21 days later. Five week old chickens were weighed and divided into two groups of 12. 0.5 to iml blood was collected from the wing vein of each chicken prior to vaccination. For the primary injections formulated using an adjuvant typifying 20 that described in this invention, 0.1 ml of sheep red blood cells were added to 0.1 ml of phosphate buffered saline containing 62.5 pg Quil A, 1.25 mg DEAE-dextran and emulsified with 0.3 ml of Montanide 888 oil (60 %). In the comparison group, 0.1 ml of sheep red blood cells were 25 added to 0.15 ml of phosphate buffered saline and emulsified with 0.25 ml of Complete Freund's adjuvant (50 %). In both groups, the total volume injected was 0.5 ml per dose. It was administered in equal volumes to the thigh muscles of both legs. 30 After two weeks the chickens were weighed, tissue reactions at the injection sites were inspected and 0.5 to 1 ml blood was collected from wing vein. After a further 13 days the chickens were weighed once again, tissue reactions at the injection sites were 35 inspected and 0.5 to 1 ml blood was collected from wing vein. After the inspection a booster injection of 0.1 ml of sheep red blood cells was given intra muscularly in both adjuvants as for the primary injection but incomplete WO 99/27959 PCT/AU98/00990 - 216 Freund's adjuvant was employed in the comparison group. Two weeks later the chickens were weighed, tissue reactions at the injection sites were inspected and 0.5 to 1 ml blood was collected from wing vein. 5 Eight weeks after the booster injection the chickens were weighed, tissue reactions at the injection sites were inspected and 0.5 to 1 ml blood was collected from wing vein. Haemagglutination Assay 10 Chickens sera were incubated at 56 0 C for 30 minutes to inactivate complement. Fifty pl of phosphate buffered saline was added to all wells of row 1 to 12 of 96 wells, U-shaped bottom, microtest plates (Sarstedt, Australia). Fifty p1l of heat inactivated sera, before and after the 15 immunisation, were added to wells of the first row. Two fold serial dilutions were performed across the plates. Fifty p1l of 2 % sheep red blood cells suspension was added to all wells. The plates were shaken for 1 minute, covered and incubated at 4 0 C for 2 hours. Titres were expressed as 20 the reciprocal of the highest dilution resulting in complete agglutination. ELISA on sheep red blood cells coated microtitre plates Sheep red blood cells were diluted as 0.1 % in carbonate coating buffer pH 9.6 and 100 p1l of the solution 25 was added to all wells of row 2 to 12 of 96 wells, flat bottom, microtitre plates (Nunc-Immuno plate, F 96 polysorp, Cat. 475094). After overnight incubation at 4 0 C, the plates were washed four times with 0.05 % tween 20 in saline. After the washing, 100 l of 0.1 % gelatine in 30 phosphate buffered saline was added to all wells of row 2 to 12 of microtitre plates. Which was followed by the addition of the reference serum and sera for testing, diluted 1/100 in 0.1 % gelatine in phosphate buffered saline, to the wells of row 2. Two fold serial dilutions 35 were performed across the plates. After 2 hours incubation at room temperature, the plates were washed four times and 100 p.il of 1/20,000 anti-chicken IgG, developed in rabbit, WO99/27959 PCT/AU98/00990 - 17 conjugated to peroxidase (Sigma Cat. A 9046) was added and incubated for a further 1 hour. After washing the plate four times 3, 3', 5, 5'- tetramethylbenzidine (Sigma Cat. T2885) substrate was added and incubated for a further 15 5 minutes before the stopping solution was added. Titres were expressed as the reciprocal of the dilution resulting in 0.5 optical density of the wells. The results of the experiment are shown in Figure 1 and 2 and Tables 5 to 7.

WO 99/27959 PCT/A U98/00990 -0 * 0 0 0 .0 X0. 4) 4) 4 IC, 0~ 0 00 CC to N 0c 0N C4 0 m o '0 .0 E E *o 0 0 F- 1,0P.0"to0 m0 f,( (oWQNo(Dr ' m I.- m (N (0 - Nf O r- 10 o N1,ID; GNn 0 N 0 P-c *0 0 -! lcf ;- ;; 4 f(0l.0(l0 W M-q - W"w OO . I O ( J C4 f. m 20 ~ ~ ~ ~ ~ ~ ~ (((U-( j--~- 0- -mnc n q c vc )c qm f 5m. EIG U E EE 'EE' ' ',u' 0 ,U. U - Lf L. E 'C *' 0 00 00 * - -I I .0 0' > U it_ _ __ _ _ __ _ __ _ ___-(l( WO 99/27959 PCT/AU98/00990 - 19 Table 6. Adjuvant experiment in chickens (hybrid white leghorn) Adjuvant Isolator Pink Sex Titre using haemagglutination assay (reciprocal dilution) no. 2 weeks after Before 2 weeks after 1 8 weeks after prime boost I boost 1 boost 1 4 41 Female 32 4 16 8 4 42 Female 16 8 64 8 4 43 Female 8 8 64 16 (Quil A/ 4 44 Male 32 8 32 4 DEAE/ 4 45 Male 16 4 16 4 888 oil) 4 46 Male 16 8 16 4 3 47 Female 64 16 64 2 3 48 Female 64 32 128 16 3 49 Female 8 8 64 8 3 50 Male 64 8 32 4 3 51 Male 16 8 32 4 3 52 Male 64 16 128 16 Mean 33 11 55 8 SD 24 8 39 5 Counts 12 12 12 12 SE 7 2 11 2 Adjuvant Isolator Yellow Sex Titre using haemagglutination assay (reciprocal dilution) no. 2 weeks after Before 2 weeks after 8 weeks after prime boost I boost 1 boost 1 4 1 Female 4 4 8 8 4 2 Female 8 2 4 4 4 3 Female 4 2 8 4 4 4 Male 4 8 32 32 4 5 Male 16 16 16 4 CF/ICF 4 6 Male 4 4 8 4 3 7 Female 16 8 32 4 3 8 Female 16 4 16 32 3 9 Female 16 16 64 64 3 10 Male 4 32 32 8 3 11 Male 8 8 8 4 3 12 Male 2 2 16 8 Mean 9 9 20 15 SD 6 9 17 19 Counts 12 12 12 12 SE 2 3 5 5 WO 99/27959 PCT/AU98/00990 - 20 Table 7. Adjuvant experiment in chickens (hybrid white leghorn) Adjuvant Isolator Pink Sex Titre using ELISA (x 1000) no. 2 weeks after Before 2 weeks after 8 weeks after prime boost 1 boost 1 boost 1 4 41 Female 35 12 85 11 4 42 Female 22 9 115 14 4 43 Female 36 20 43 21 (Quil A/ 4 44 Male 36 18 189 12 DEAE/ 4 45 Male 19 13 42 15 888 oil) 4 46 Male 106 35 431 17 3 47 Female 54 24 250 21 3 48 Female 36 15 82 33 3 49 Female 5 4 34 10 3 50 Male 34 42 191 27 3 51 Male 20 14 42 10 3 52 Male 60 32 191 20 Mean 38 20 141 18 SD 26 11 117 7 Counts 12 12 12 12 SE 8 3 34 2 Adjuvant Isolator Yellow Sex Titre using ELISA (x 1000) no. 2 weeks after Before 2 weeks after 8 weeks after prime boost 1 boost 1 boost 1 4 1 Female 1 3 9 4 4 2 Female 2 2 5 6 4 3 Female 2 2 7 8 4 4 Male 1 5 17 10 4 5 Male 6 19 22 5 CF/ICF 4 6 Male 3 4 12 8 3 7 Female 2 3 12 6 3 8 Female 2 4 5 6 3 9 Female 2 9 15 14 3 10 Male 1 15 29 6 3 11 Male 2 9 13 8 3 12 Male 0 1 4 3 Mean 2 6 12 7 SD 1 6 7 3 Counts 12 12 12 12 SE 0 2 2 1 WO 99/27959 PCT/AU98/00990 - 21 Example 6 In this example a comparison was made between cattle injected with phomopsin A conjugated to ovalbumin in Freund's complete adjuvant with a booster injection of the 5 conjugate antigen in incomplete Freund's adjuvant or with the same antigen delivered in an adjuvant prepared according to this invention. Twenty four, five months old cattle were weighed and two 10 ml samples of blood were collected from each animal. 10 All the cattle also received 4 ml of five in one vaccine, injected subcutaneously to the left side of the back of the neck. Two weeks later the cattle were weighed and divided into two randomised groups. The animals were then given a 15 primary injection. The animals in one group received an injection of 100 pg phomopsin A conjugated to 336 pg ovalbumin, 1 mg Quil A and 10 mg DEAE-dextran dissolved in 0.8 ml of sterile water and emulsified with 1.2 ml of Montanide 888 oil (60 %). The animals in the other group 20 were injected with 100 pg phomopsin A conjugated to 336 tg ovalbumin dissolved in 1 ml of sterile water, and emulsified with 1 ml of Complete Freund's adjuvant (50 %). In both groups, immunogens were injected subcutaneously as a total volume of 2 ml to the right side of the back of the 25 neck, below the ear. Thirteen days later tissue reactions at the injection sites were inspected and 10 ml blood was collected from jugular vein. After four weeks cattle were weighed, tissue reactions 30 at the injection sites were inspected and 10 ml blood was collected from jugular vein. A booster injection of 100 pg phomopsin A conjugated to 440 pg foetal calf serum was given subcutaneously as in primary injection. In the case of the comparison group this was formulated in Incomplete 35 Freund's Adjuvant. A second dose of five in one vaccine was also injected subcutaneously to the left side of the back of the neck.

WO 99/27959 PCT/AU98/00990 - 22 Two weeks later tissue reactions at the injection sites were inspected and 10 ml blood was collected from jugular vein. Eight weeks after the booster injection cattle were 5 weighed and tissue reactions at the injection sites were inspected and 10 ml blood was collected from jugular vein. ELISA on 50 ng phomopsins/well coated microtitre plates Phomopsins were diluted as 50 ng/100 pl in carbonate coating buffer pH 9.6 and 100 pl of the solution was added 10 to all wells of row 2 to 12 of 96 wells, flat bottom, microtitre plates (Sarstedt, Australia). After overnight incubation at 400C, the plates were washed four times with 0.05 % tween 20 in saline. After the washing, 100 pl of 0.1 % gelatine in phosphate buffered saline was added to all 15 wells of microtiter plates. Reference serum and sera for testing, diluted 1/100 in 0.1 % gelatine in phosphate buffered saline, were then added to the wells of row 2. Two fold serial dilutions were performed across the plates. After 2 hours incubation at room temperature, the plates 20 were washed four times and 100 pl of 1/15,000 anti-bovine IgG, developed in rabbit, conjugated to peroxidase (Sigma Cat.B 1520) was added and incubated for a further 2 hour. After washing the plate four times 3, 3', 5, 5' tetramethylbenzidine (Sigma Cat. T2885) substrate was added 25 and incubated for a further 20 minutes before the stopping solution was added. Titres were expressed as the reciprocal of the dilution resulting in 0.5 optical density of the wells. ELISA on 50 ng ovalbumin/well coated microtitre plates 30 ELISA was also performed on 50 ng ovalbumin/well coated plates as above in phomopsins 50 ng/well coated plates except 96 wells, flat bottom, Nunc-Immuno maxisorp microtitre plates (Cat. 439454) were used for the assay. The results obtained are shown in Figures 3 and 4 and 35 Tables 8 to 12.

WO 99/27959 PCT/AU98/00990 - 23 Table 8. Adjuvant trial in cattle Adjuvant Animal no. Sex Animal no. Weight (kg) Left Right Before Before Before 8 weeks Prime boost I boost I after boost 1 5 Female 373 148 164 186 210 210 6 Male 9721 183 203 240 282 282 14 Male 9701 173 188 226 256 256 15 Male 9713 153 171 194 230 230 (Quil N 20 Female 998 138 152 169 194 194 DEAE/ 24 Female 285 160 174 194 221 221 888 oil) 25 Male 342 135 152 182 202 202 28 Female 9723 184 200 227 256 256 30 Female 9710 160 180 209 224 224 31 Male 9732 157 176 209 230 230 34 Male 631 155 164 177 210 210 35 Female 9720 165 178 183 216 216 37 Female 425 176 186 211 241 241 Mean 160 176 201 229 229 Counts 13 13 13 13 13 SD 15 16 22 25 25 SE 4 4 6 7 7 Adjuvant Animal no. Sex Animal no. Weight (k) Left Right Before Before 8 weeks Prime boost I after boost 1 4 Male 9726 143 148 162 199 199 8 Female 9709 140 150 167 185 185 10 Female 9705 155 168 185 205 205 13 Male 979 157 175 187 215 215 CF/ICF 17 Male 983 183 204 221 252 252 19 Female 215 168 193 220 255 255 22 Female 377 167 173 169 200 200 23 Female 263 146 160 162 206 206 26 Male 9730 176 188 214 232 232 32 Female 466 160 178 199 222 222 36 Male 418 116 124 143 174 174 Mean 155 169 184 213 213 Counts 11 11 11 11 11 SD 19 23 26 26 26 SE 6 7 8 8 8 WO 99/27959 PCT/AU98/00990 - 24 Table 9. Adjuvant trial in cattle Adjuvant Animal no. Sex Animal no. Tissue reactions to phomopsins-ovalbumin conjugate (2 ml/dose) Left Right 2 weeks after Before 2 weeks after 8 weeks after prime boost 1 boost 1 boost 1 5 Female 373 6 Male 9721 3x3 cm 14 Male 9701 15 Male 9713 (Quil A/ 20 Female 998 DEAE/ 24 Female 285 888 oil) 25 Male 342 28 Female 9723 3x5 cm 3x4 cm 30 Female 9710 31 Male 9732 7x5 cm 7x5 cm 4x5 cm 3x3 cm 34 Male 631 3x3 cm 35 Female 9720 37 Female 425 Adjuvant Animal no. Sex Animal no. Tissue reactions to phomopsins-ovalbumin conjugate (2 ml/dose) Left Right 2 weeks after Before 2 weeks after 8 weeks after prime boost I boost I boost 1 4 Male 9726 3x3 cm 8 Female 9709 8x5 cm 6x4 cm 8x6 cm 10 Female 9705 10x3 cm 6x5 cm 3x4 cm 13 Male 979 CF/ICF 17 Male 983 12x6 cm 8x6 cm 2x2 cm 19 Female 215 8x5 cm 4x8 cm 4x5 cm 22 Female 377 23 Female 263 6x7 cm 3x3 cm 26 Male 9730 7x5 cm 32 Female 466 8x8 cm 4x5 cm 36 Male 418 10x6 cm 7x8 cm 7x6 cm WO 99/27959 PCT/AU98/00990 - 25 Table 10. Adjuvant trial in cattle Adjuvant Animal no. Sex Animal no. Tissue reactions to 5 in 1 vaccine (4 mUidose) Left Right 4 weeks after Before 2 weeks after 8 weeks after prime boost I boost I boost 1 5 Female 373 .: .. 6 Male 9721 . ... 14 M ale 9701 . ... . . . 15 Male 9713 2x2cm 2x3cm , 3x5cm , (Quil A/ 20 Female 998 4x3 cm 4x4 cm lxi cm DEAE/ 24 Female 285 8c 5x5 .cm 888 oil) 25 Male 342 2x2cm 8xcm 5x5c 28 Female 9723 ... . . 30 Female 9710 .... 5x4cm xS cm 31 Male 9732 . ..

5x5cm 3x4:cm 34 Male 631 ...... ____ 4x4cm 3x3 cm .... _____•__ 35 Female 9720 73xxccmm _-._7m

.

. 37 Female 425 I.4x2:c ..... 3x3:cm: .. . Adjuvant Animal no. Sex Animal no. Tissue reactions to 5 in 1 vaccine (4 mildose) Left Right 4 weeks after Before 2 weeks after 8 weeks after prime boost 1 boost 1 boost 1 ______ - x cm .. 3x3 cm 4 Male 9726 2x2 cm 6x3m 3x3cm 8 Female 9709 W .. 2x2 cm 3x5lxlc:cm: m 10 Female 9705 "_ ' 6x4cm 3x5cm 13 Male 979 :_- _7x6 cm CFACF 17 Male 983 5x3 cm 5x5 cm 3x4+10xlOcm , 19 Female 215 ._.: ' . • . . . :. . 22 Female 377 .... . .3x3 cm. 23 Female 263 5x4 cm . ... 3x3 cm .::: . 26 Male 9730 3x2 cm

-

3x4cm 3x3 cm 32 Female 466 = = .== 36 Male 418 3x5n cmn___ WO 99/27959 PCT/AU98/00990

-

26 Table 11. Adjuvant trial in cattle Adjuvant Animal No. Sex Anti-phomopsin IgG titre (xl00) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 5 Female 16 38 120 95 6 Male 7 17 134 60 14 Male 26 170 514 158 15 Male 12 20 99 51 (Quil A/ 20 Female 4 22 119 74 DEAE/ 24 Female 17 121 286 92 888 oil) 25 Male 11 17 50 24 28 Female 26 162 340 116 30 Female 16 33 86 46 31 Male 29 81 261 156 34 Male 5 23 94 59 35 Female 14 74 200 191 37 Female 8 50 237 231 Mean 15 64 195 104 Counts 13 13 13 13 SD 8 55 131 63 SE 2 15 36 17 Adjuvant Animal No. Sex Anti-phomopsin IgG titre (xI00) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 4 Male 7 17 60 129 8 Female 12 64 291 299 10 Female 2 27 186 173 13 Male 10 45 186 93 CF/ICF 17 Male 8 74 299 142 19 Female 5 19 162 28 22 Female 11 104 595 128 23 Female 4 45 205 128 26 Male 9 41 256 302 32 Female 10 70 231 240 36 Male 3 19 94 82 Mean 7 48 233 158 Counts 11 11 11 11 SD 3 28 141 88 SE 1 8 43 27 WO 99/27959 PCT/AU98/00990 - 27 Table 12. Adjuvant trial in cattle Adjuvant Animal No. Sex Anti-ovalbumin IgG titre (xl00) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 5 Female 4 5 8 15 6 Male 6 8 78 43 14 Male 2 40 110 63 15 Male 2 3 13 28 (Quil A/ 20 Female 2 9 77 36 DEAE/ 24 Female 3 9 78 22 888 oil) 25 Male 2 3 37 35 28 Female 1 19 35 43 30 Female 5 5 32 24 31 Male 2 5 29 15 34 Male 1 2 9 6 35 Female 0 4 25 51 37 Female 2 4 49 19 Mean 2 9 45 31 Counts 13 13 13 13 SD 2 10 32 16 SE 0 3 9 4 Adjuvant Animal No. Sex Anti-ovalbumin g19G titre (x100) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 4 Male 2 3 10 29 8 Female 2 23 84 113 10 Female 1 6 19 16 13 Male 2 4 60 37 CF/ICF 17 Male 4 15 147 181 19 Female 2 5 51 34 22 Female 3 15 63 73 23 Female 1 15 85 237 26 Male 2 6 52 173 32 Female 5 42 178 285 36 Male 2 8 70 77 Mean 3 13 75 114 Counts 11 11 11 11 SD 1 12 50 92 SE 0 3 15 28 WO 99/27959 PCT/AU98/00990 - 28 Example 7 In this example a comparison was made between sheep injected with a phomopsin A- fetal calf serum conjugate in Freund's complete adjuvant with a booster injection given 5 in incomplete Freund's adjuvant and the same antigen injected in an adjuvant formulation prepared according to this invention. Primary injections were started during marking of 10 to 12 weeks old lambs. Twenty four, ten to twelve weeks 10 old lambs, weighing between 10 to 20 kg, were weighed and 10 ml blood was collected from jugular vein. They were divided into two equal groups randomised according to weight. One group was injected with 50 4g phomopsin A conjugated to 220 pg foetal calf serum, 0.5 mg Quil A and 5 15 mg DEAE-dextran dissolved in 0.8 ml of sterile water and emulsified with 1.2 ml of Montanide 888 oil (60 %). The second group was injected with phomopsin A 50 pg conjugated to foetal calf serum 220 4g dissolved in 1 ml of sterile water, and emulsified with 1 ml of Complete Freund's 20 adjuvant. Both groups were injected subcutaneously with 2 ml to the right side of the back of the neck, below the ear. In addition all lambs received an injection of 2 ml of six in one plus selenium vaccine, injected subcutaneously at a separate site. 25 Two weeks after the primary injection lambs were weighed, tissue reactions at the injection sites were inspected and 10 ml blood was collected from jugular vein. A second dose of six in one plus selenium vaccine was injected subcutaneously at the back of the neck behind the 30 ear. Three months after the primary injection lambs were weighed, tissue reactions at the injection sites were inspected and 10 ml blood was collected from jugular vein. A booster injection of phomopsin A 50 4g conjugated to 220 35 jg foetal calf serum was given subcutaneously as in primary injection. In the case of the comparison group Incomplete Fruend's Adjuvant was used as the adjuvant in place of WO 99/27959 PCT/AU98/00990 - 29 Freund's complete adjuvant. Two weeks after the booster injection lambs were weighed, tissue reactions at the injection sites were inspected and 10 ml blood was collected from jugular vein. 5 Eight weeks after the booster injection lambs were weighed and tissue reactions at the injection sites were inspected and 10 ml blood was collected from jugular vein. ELISA on 50 ng phomopsins/well coated microtitre plates Phomopsins were diluted as 50 ng/100 pil in carbonate 10 coating buffer pH 9.6 and 100 pl of the solution was added to all wells of row 2 to 12 of 96 wells, flat bottom, microtitre plates (sarstedt, Australia). After overnight incubation at 40 0 C, the plates were washed four times with 0.05 % tween 20 in saline. After the washing, 100 pl of 0.1 15 % gelatine in phosphate buffered saline was added to all wells of microtiter plates. Reference serum and sera for testing, diluted 1/100 in 0.1 % gelatine in phosphate buffered saline, were then added to the wells of row 2. Two fold serial dilutions were performed across the plates. 20 After 2 hours incubation at room temperature, the plates were washed four times and 100 pil of 1/14,000 anti-sheep IgG, developed in donkey, conjugated to peroxidase (Sigma Cat. A 3415) was added and incubated for a further 2 hour. After washing the plate four times 3, 3', 5, 5' 25 tetramethylbenzidine (Sigma Cat. T2885) substrate was added and incubated for a further 20 minutes before the stopping solution was added. Titres were expressed as the reciprocal of the dilution resulting in 0.5 optical density of the wells. 30 ELISA on 50 ng foetal calf serum/well coated microtitre plates ELISA was also performed on 50 ng foetal calf serum/well coated plates as above in phomopsins 50 ng/well coated plates except 96 wells, flat bottom, Nunc-Immuno 35 polysorp microtitre plates (Cat. 475094) were used and coating was done at 4 0 C. The results obtained are shown in Figures 5 and 6 and WO 99/27959 PCT/AU98/00990 - 30 Tables 13 to 15. Table 13. Adjuvant trial in wethers Adjuvant Animal no. eight (kg) Before 2 weeks Before 2 weeks 8 weeks prime after prime boost I after boost 1 after boost 1 85 11.5 18.7 28.6 31.0 28.0 86 14.0 18.3 28.4 30.0 26.5 87 15.5 20.2 20.2 19.0 15.5 88 14.5 21.4 32.4 35.5 29.5 (Quil A/ 89 14.5 19.1 32.0 33.5 30.5 DEAE/ 90 16.5 22.6 23.8 33.5 29.5 888 oil) 91 15.0 20.2 31.0 30.0 24.0 92 11.5 16.1 24.0 26.5 23.0 93 16.0 21.6 30.6 33.0 30.0 94 12.5 19.6 28.2 31.0 26.5 95 13.0 19.5 26.4 28.0 23.5 96 13.5 20.2 34.2 37.5 34.5 Mean 14.0 19.8 28.3 30.7 26.8 SD 1.65 1.70 4.11 4.80 4.86 Counts 12 12 12 12 12 SE 0.48 0.49 1.19 1.39 1.40 Adjuvant Animal no. Weight (kg) 8Iek Left Before 2 weeks Before 2 weeks 8 weeks prime after prime boost I afterboost 1 after boost 1 145 15.0 20.4 29.8 34.0 31.5 146 13.0 18.2 28.6 32.5 29.5 147 10.5 13.5 15.6 17.0 12.5 148 12.5 16.3 27.2 30.0 27.0 CF/ICF 149 16.0 21.4 32.4 35.5 30.5 150 17.5 22.0 36.2 36.0 31.0 151 13.5 17.3 26.2 28.0 24.5 152 18.0 24.2 37.2 38.5 34.5 153 17.5 22.4 31.2 30.5 28.0 154 16.0 20.4 27.4 30.0 25.0 155 15.5 19.5 32.2 35.0 31.0 156 14.0 19.7 25.2 27.5 25.0 Mean 14.9 19.6 29.1 31.2 27.5 SD 2.28 2.93 5.67 5.62 5.64 Counts 12 12 12 12 12 SE 0.66 0.85 1.64 1.62 1.63 By eight weeks after boost 1 all sheep lost body weight due to the drought and lack of food WO 99/27959 PCT/AU98/00990 - 31 Table 14. Adjuvant trial in wethers Adjuvant Animal No. Anti-phomopsin IgG titre (xl000) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 85 21 5 95 57 86 14 22 281 145 87 26 7 68 33 88 20 10 59 72 (Quil A/ 89 50 215 328 565 DEAE/ 90 31 15 165 71 888 oil) 91 37 40 662 640 92 28 9 75 56 93 19 10 92 54 94 8 27 61 86 95 15 8 84 56 96 7 24 98 62 Mean 23 33 172 158 Counts 12 12 12 12 SD 12 58 178 210 SE 4 17 51 61 Adjuvant Animal No. Anti-phomopsin IgG titre (xl000) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 145 9 49 211 62 146 11 50 120 51 147 21 144 274 219 148 14 51 105 83 CF/ICF 149 22 219 256 140 150 45 203 298 351 151 15 48 121 80 152 36 107 365 494 153 22 56 151 191 154 20 188 197 165 155 22 49 252 109 156 11 134 173 252 Mean 21 108 210 183 Counts 12 12 12 12 SD 11 67 81 132 SE 3 19 23 38 WO 99/27959 PCT/AU98/00990 - 32 Table 15. Adjuvant trial in wethers Adjuvant Animal No. Anti-foetal calf serum g19G titre (xl00) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 85 6 7 120 127 86 17 41 403 176 87 20 15 148 53 88 13 25 132 "123 (Quil A/ 89 71 238 656 343 DEAE/ 90 14 24 125 68 888 oil) 91 39 47 2290 1915 92 10 15 116 65 93 24 26 135 141 94 25 58 164 207 95 10 41 288 230 96 20 67 228 131 Mean 22 50 400 298 Counts 12 12 12 12 SD 18 62 616 516 SE 5 18 178 149 Adjuvant Animal No. Anti-foetal calf serum g19G titre (x1000) 2 weeks Before 2 weeks 8 weeks after prime boost I afterboost 1 afterboost 1 145 6 23 246 130 146 4 42 173 59 147 9 143 395 164 148 4 29 156 45 CF/ICF 149 15 332 1453 195 150 15 77 1015 317 151 32 277 990 806 152 9 141 855 636 153 12 97 676 473 154 22 289 1837 496 155 7 84 301 173 S 156 9 97 994 382 Mean 12 136 758 323 Counts 12 12 12 12 SD 8 106 535 240 SE 2 31 154 69 WO99/27959 PCT/AU98/00990 - 33 Example 8 In this example the immunogen was ovalbumin. It was injected into rats in complete Freund's adjuvant for the 5 primary injection and incomplete Freund's for a booster injection. The antibody response obtained was compared with that of sheep given the same antigen formulated in an adjuvant prepared according to this invention. Female Long-Evans hooded rats were kept in cages of 4 10 rats/cage. Twenty five, twelve weeks old Long-Evans rats were weighed and divided into two groups. 0.2 to .5 ml blood was collected from tail vein prior to vaccination. For the primary injection using an adjuvant formulation typical of this invention, 65 gg ovalbumin, 40 pg Quil A 15 and 0.8 mg DEAE-dextran were dissolved in 0.128 ml of phosphate buffered saline and emulsified with 0.192 ml of Montanide 888 oil (60 %). In the comparison group 65 p.g ovalbumin was dissolved in 0.16 ml of phosphate buffered saline and emulsified with 0.16 ml of Complete Freund's 20 adjuvant (50 %). Both groups were injected subcutaneously as a total volume of 0.32 ml divided into two sites at the back. Two weeks after the primary injection the rats were weighed, tissue reactions at the injection sites were 25 inspected and 0.2 to 0.5 ml blood was collected from tail vein. Twelve days later the rats were re-weighed, tissue reactions at the injection sites were inspected and 0.2 to 0.5 ml blood was collected from tail vein. For the booster 30 injection, 65 pg ovalbumin was given subcutaneously as in primary injection. Incomplete Freund's Adjuvant was used in place of complete Freund's adjuvant for the comparison group. One week after the booster injection and then again 35 two weeks after the booster injection the rats were weighed again, tissue reactions at the injection sites were inspected and 0.2 to 0.5 ml blood was collected from tail WO 99/27959 PCT/AU98/00990 - 34 vein. Four weeks after the booster injection the rats were weighed and tissue reactions at the injection sites were inspected. 5 Eight weeks after the booster injection the rats were weighed, tissue reactions at the injection sites were inspected and 0.2 to 0.5 ml blood was collected from tail vein. ELISA on 50 ng ovalbumin/well coated microtitre plates 10 Ovalbumin was diluted as 50 ng/0.1 ml in carbonate coating buffer pH 9.6 and 100 p1l of the solution was added to all wells of row 2 to 12 of 96 wells, flat bottom, microtitre plates (Nunc-Immuno plate, F 96 Cert.maxisorp, Cat. 439454). After overnight incubation at 4 0 C, the plates 15 were washed four times with 0.05 % tween 20 in saline. After the washing, 100 p.1 of 0.1 % gelatine in phosphate buffered saline was added to all wells of microtiter plates. This was followed by the addition of the reference serum and sera for testing, diluted 1/100 in 0.1 % gelatine 20 in phosphate buffered saline, to the wells of row 2. Two fold serial dilutions were performed across the plates. After 2 hours incubation at room temperature, the plates were washed four times and 100 p1l of 1/16,000 anti-rat IgG, developed in goat, conjugated to peroxidase (Sigma Cat. A 25 9037) was added and incubated for a further 2 hours. After washing the plate four times 3, 3', 5, 5' tetramethylbenzidine (Sigma Cat. T2885) substrate was added and incubated for a further 20 minutes before the stopping solution was added. Titres were expressed as the reciprocal 30 of the dilution resulting in 0.5 optical density of the wells. The results are shown in Figure 7 and Tables 16 to 18.

WO 99/27959 PCT/AU98/00990 - 35 Table 16. Adjuvant trial in Long-Evans female rats Adjuvant Cage/ Body weight (gm) Animal No. Before 2 weeks Before 1 week 2 weeks 4 weeks 8 weeks prime after prime boost 1 after boost 1 after boost I after boost 1 after boost Cage1/0 197 208 212 214 219 224 222 Cage1/1 199 212 220 210 237 234 230 Cagel/2 182 189 196 192 198 202 204 Cagel/3 203 201 208 216 234 217 222 (Quil A/ Cage 2/0 192 207 217 215 220 203 232 DEAEl Cage 2/1 207 222 226 221 228 229 233 888 oil) Cage 2/2 202 211 214 215 222225 226 Cage 2/3 234 224 230 231 234 241 247 Cage 3/1 189 170 198 219 232 215 211 Cage 3/2 205 190 230 240 233 221 234 Cage 3/3 198 184 207 211 213 238 234 Cage 7/1 223 224 223 233 234 232 235 Cage 7/2 228 229 227 238 238 235 235 Mean 205 205 216 220 226 224 228 Counts 13 13 13 13 13 13 13 SD 15 18 11 13 12 12 11 SE 4 5 3 4 3 3 3 Adjuvant Cage/ _ __ Body weight (gm wm) Animal No. Before 2 weeks Before 1 week 2 weeks 4 weeks 8 weeks prime after prime boost I after boost I after boost I after boost I after boost Cage 4/0 205 214 219 222 222 231 246 Cage 4/1 186 198 209 204 207 221 222 Cage 4/2 198 207 206 206 210 220 225 Cage 4/3 200 211 230 224 227 233 236 CF/ICF Cage 5/0 198 216 220 222 232 233 235 Cage 5/1 194 208 210 206 218 229 221 Cage 5/2 196 206 213 215 222 226 230 Cage 5/3 205 213 218 224 234 237 241 Cage 6/0 191 204 221 218 221 225 253 Cage 6/1 207 223 254 233 236 235 240 Cage 6/2 215 234 241 236 241 240 248 Cae 6/3 182 203 212 208 210 210 212 Mean 198 211 221 218 223 228 234 Counts 12 12 12 12 12 12 12 SD 9 10 14 11 11 8 12 SE 3 3 4 3 3 2 4 WO 99/27959 PCT/AU98/00990 36 Table 17. Adjuvant trial in Long-Evans female rats Adjuvant Cage/ 5 days 11 days 1 week 2 weeks 8 weeks Animal No. after prime after prime after boost 1 after boost I after boost 1 Cagel/0 Cagel/1 1+ Cagel/2 1+ Cagel/3 1++ 1+ (Quil A/ Cage 2/0 1+ DEAE/ Cage 2/1 888 oil) Cage 2/2 1 +++ 1++ 1+ Cage 2/3 1+ Cage 3/1 Cage 3/2 1+ Cage 3/3 Cage 7/1 SCage 7/2 Adjuvant cage/ 5 days 11 days 1 week 2 weeks 8 weeks Animal No. after prime after prime after boost I after boost I after boost 1 Cage 4/0 1 ++ 1 ++ 1 + 2 ++ Cage 4/1 1 + 1+ 1 +++ 2 ++ Cage 4/2 1 ++ 1 ++ 1 ++ 2 ++ Cage4/3 1+ 1+ 2++ 2++ CF/ICF Cage 5/0 1 ++ 1 ++ 2 ++ 2 ++ Cage 5/1 2++ 2 ++ 2+ 1 +, 1 ++ Cage5/2 1+ 1+ 1+,1 +++ 1 +,1++ Cage 5/3 1 + 1 + , 1 +++ Cage 6/0 1 +++ opened 1 ++ 2+ 2+ 1 +, 1 ++ Cage 6/1 1 ++ opened 1 ++ 1 +++ opened 1 .+++ 2+ Cage 6/2 1 + 1+++ 1 +, 1 +++ 1 +, 1 +++ Cage 6/3 1 ++ 1 ++ 2 ++ 2 ++ + = small lump (1-2 mm) ++ = Medium lump (3-5 mm) +++ = Big lump (more than 5 mm) 1 and 2 = numbers of lumps WO 99/27959 PCT/AU98/00990 37 Table 18. Adjuvant trial in Long-Evans female rats Adjuvant Cage/ I Titre (x1000) Animal No. 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 Cagel/O 1 33 155 247 Cagel/1 1 38 68 156 Cagel/2 1 13 28 96 Cagel/3 0 9 29 77 (Quil Al Cage 2/0 1 36 93 126 DEAE/ Cage 2/1 1 26 85 92 888 oil) Cage 2/2 0 12 23 49 Cage2/3 0 33 78 173 Cage 3/1 2 55 155 68 Cage 3/2 0 28 68 195 Cage 3/3 10 91 164 246 Cage 7/1 1 57 210 252 Cage 7/2 0 21 80 150 Mean 1 35 95 148 Counts 13 13 13 13 SD 3 22 59 71 SE 1 6 16 20 Adjuvant Cage/ Titre (xl000) Animal No. 2 weeks Before 2 weeks 8 weeks after prime boost I after boost I after boost 1 Cage 4/0 0 18 94 57 Cage 4/1 0 12 38 33 Cage 412 4 63 120 81 Cage 4/3 4 67 98 66 CFIICF Cage 5/0 2 39 113 69 Cage 5/1 2 46 67 65 Cage 5/2 1 2 27 34 Cage 5/3 2 68 87 55 Cage 6/0 11 89 174 128 Cage 6/1 0 62 126 87 Cage 6/2 4 7 27 36 Cage 6/3 3 66 95 87 Mean 3 45 89 67 Counts 12 12 12 12 SD 3 29 44 27 SE 1 8 13 8 WO99/27959 PCT/AU98/00990 - 38 Examples 5 to 8 demonstrate that the newly invented adjuvant stimulates the immune system of a variety of animal species against a range of antigens with an efficacy similar to or better than the benchmark Freund's adjuvant 5 but without the injection site reactions induced by the latter. INDUSTRIAL APPLICABILITY The adjuvant compositions of the present invention are applicable to the preparation of vaccines against a wide 10 range of infectious diseases and against natural products of the human and animal body such as hormnnones.

Claims (22)

1. An adjuvant composition for stimulating an effective immune response to an antigenic substance when co administered to an animal with said antigenic 5 substance, comprising: (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune stimulating activity; and (c) an immunoadjuvant oil. 10

2. An adjuvant composition as claimed in claim 1 wherein the saponin is a triterpenoid compound or a mixture of triterpenoid compounds.

3. An adjuvant composition as claimed in claim 2 wherein the saponin is Quil A. 15

4. An adjuvant composition as claimed in any one of claims 1 to 3 wherein the polycationic polyelectrolyte is diethylaminoethyl dextran.

5. An adjuvant composition as claimed in any one of claims 1 to 4 wherein the immunoadjuvant oil is a 20 mineral oil.

6. An adjuvant composition as claimed in claim 5 wherein the mineral oil is Freund's incomplete adjuvant or a Montanide oil.

7. A vaccine for administration to an animal, comprising: 25 (1) an antigenic substance; and (2) an adjuvant composition comprising: (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune stimulating activity; 30 (c) an immunoadjuvant oil.

8. A vaccine as claimed in claim 7 wherein the saponin is a triterpenoid compound or a mixture of triterpenoid compounds.

9. A vaccine as claimed in claim 8 wherein the saponin is 35 Quil A.

10. A vaccine as claimed in any one of claims 7 to 9 wherein the polycationic polyelectrolyte is diethylaminoethyl dextran. WO 99/27959 PCT/AU98/00990 - 40

11. A vaccine as claimed in any one of claims 7 to 10 wherein the immunoadjuvant oil is a mineral oil.

12. A vaccine as claimed in claim 11 wherein the mineral oil is Freund's incomplete adjuvant or a Montanide 5 oil.

13. A vaccine according to any one of claims 7 to 13 wherein the saponin is present in a concentration of between 50pm/ml and 10mg/ml.

14. A vaccine according to claim 13 wherein the saponin is 10 present in a concentration between 100pm/ml and 1mg/ml.

15. A vaccine as claimed in any one of claims 7 to 14 wherein the polycationic polyelectrolyte is present in a concentration between 1mg/ml and 200mg/ml. 15

16. A vaccine as claimed in claim 15 wherein the polycationic polyelectrolyte is present in a concentration between 1.5mg/ml and 150mg/ml.

17. A method of stimulating an effective immune response in an animal to an antigenic substance, comprising the 20 steps of: (1) providing said antigenic substance; (2) providing an adjuvant composition comprising: (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune 25 stimulating activity; and (c) an immunoadjuvant oil; and (3) challenging said animal with said antigenic substance and said adjuvant composition.

18. The use of an adjuvant composition comprising: 30 (a) a saponin with immune stimulating activity; (b) a polycationic polyelectrolyte with immune stimulating activity; and (c) an immunoadjuvant oil to stimulate an effective immune response in an animal 35 challenged with an antigenic substance.

19. The use of an adjuvant composition comprising: (a) a saponin with immune stimulating activity; WO 99/27959 PCT/AU98/00990 - 41 (b) a polycationic polyelectrolyte with immune stimulating activity; and (c) an immunoadjuvant oil in the preparation of a medicament for administration 5 to an animal, wherein said medicament further comprises an antigenic substance.

20. An adjuvant composition substantially as hereinbefore described with reference to the Examples.

21. A vaccine substantially as hereinbefore described with 10 reference to the Examples.

22. A method of vaccinating animals substantially as herein described with reference to the Examples.