AU746127B2 - Saponin adjuvant composition - Google Patents

Description

WO 99/27959 PCT/A U98/00990 SAPONIN ADJUVANT COMPOSITION TECHNICAL FIELD This invention relates to adjuvant compositions for stimulating an immune response to an antigenic substance 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 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 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 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 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 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 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/A U98/00990 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 (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 containing purified mycobacterial component such as Nacetylmuramyl-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 (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.

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 polyelectrolyte immunoadjuvant such as DEAE dextran in the form of an emulsion having the polycationic polyelectrolyte dissolved in the aqueous phase. The two-component immuncadjuvant is said to overcome the rapid decline in the immune response associated with polycationic 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 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/A U98/00990 -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 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 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 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 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.

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 antigenic substance, comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; and an immunoadjuvant oil.

According to a second aspect of the present invention there is provided a vaccine for administration to an animal, comprising: an antigenic substance; and l WO 99/27959 PCT/A U98/00990 4 an adjuvant composition comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; 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: providing said antigenic substance; providing an adjuvant composition for stimulating an effective immune response to said antigenic substance, comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; and an immunoadjuvant oil; and challenging said animal with said antigenic substance and said adjuvant composition.

According to a fourth aspect of the present invention there is provided the use of an adjuvant composition comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; and 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 there is provided the use of an adjuvant composition comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; and 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/A U98/00990 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 structure. In particular, an extract from the South American tree Quillaja saponarioa shows good adjuvant activity and is now denoted "Quil While the precise chemical composition of Quil A is not known, the sugar moieties detected in the mixture include rhamnose, fucose, 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 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 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 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 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 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 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 mineral oil and 15% mannide monooleate as an 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 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 for example, more preferably 60:40 to 40:60 Such a broad range of ratios of oil 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 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.

Preferably, vaccines including adjuvant compositions in accordance with the present invention contain the saponin component at a concentration greater than and the polycationic polyelectrolyte at a concentration of greater than 1mg/ml. More preferably, they contain saponins in a concentration of greater than 100gg/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/A U98/00990 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 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 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 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 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) 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 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, 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/A U98/00990 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 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 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 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 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 fetuinphomopsin conjugate, phomopsin A conjugated to ovalbumin, 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 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, 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 WO 99/27959 PCT/A U98/00990 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 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 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: 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.lml of 0.1% sheep red blood cells/well coated plate; Figure 3 shows the antibody response to phomopsins in cattle in an ELISA assay on 50 ng phomopsins/0.lml/well coated plate; Figure 4 shows the antibody response to ovalbumin in cattle in an ELISA assay on 50 ng ovalbumin/0.lml/well coated plate; Figure 5 shows the antibody response to phomopsins in wethers in an ELISA assay on 50ng phomopsins/0.lml/well coated plate; Figure 6 shows the antibody response to foetal calf serum in wethers in an ELISA assay on 50ng foetal calf serum/0.lml/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.lml/well coated plate.

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/A U98/00990 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 component was 85% mineral oil and 15% mannide monooleate (incomplete Freund's adjuvant, ICF) in all formulations.

In one formulation the adjuvant comprised a of DEAE dextran as the cationic polymer in incomplete Freund's oil (10mg DEAE/ICF). In another the adjuvant was 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 Freund's oil (0.5mg Quil A/5mg DEAE/ICF). A primary and one booster injection were administered 16 weeks apart. Antiphomopsin antibody titres (measured by ELISA) were not detectable prior to vaccination. Table 1 shows antiphomopsin antibody titres 2 and 8 weeks after the booster injection.

TABLE 1 Adjuvant Dose Sheep Titre Titre formulation volume Nos. Booster plus 2 Booster plus 8 weeks weeks DEAE/ ICF Iml 12 55,000 8,000 Quil A/ Iml 12 23,000 2,000

ICF

Quil A/ iml 12 99,000 37,000

DEAE/ICF

The results show that the trivalent adjuvant prepared according to this invention gave a significantly higher 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 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 7 WO 99/27959 PCT/A U98/00990 11 longer duration of effect and the synergistic response achieved.

Example 2 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 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 DEAE/ Iml 12 8,000 2,000

ICF

Quil Iml 12 9,000 1,000 A/ ICF Quil iml 12 44,000 14,000 A/

DEAE/ICF

Once again the adjuvant formulated according to this invention demonstrates an enhanced, synergistic, longlasting effect when compared to two component formulations in which one of the three components specified in this invention is missing.

Example 3 In this example a comparison was made between two trivalent adjuvant formulations incorporating a triterpinoid (Quil 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 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 Quil A/5mg lml 11 20,000 16,000 DEAE/ ICF Quil A/5mg Iml 11 40,000 25,000 DEAE/ Montanide 888___ 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 number of available products can give advantage for particular applications.

Example 4 Animal species: Rabbit 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 microlitres of antigen (Img 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 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.

Freund's incomplete/DEAE dextran. 1 mg VLP/NS2 was resuspended in 1ml of 15% DEAE-dextran and added to iml 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 This invention. Five 0.lml 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 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.

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 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 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.

0 'o Results.

The antibody titres achieved for each protocol are given in Table 4.

TABLE 4 Rabbit Day 1 Day Day 44 Day 61 Day 72 Day 89 Day 96 Day Day 123 109 This 0 1000 5000 >50000 >50000 >50000 >50000 >50000 25000 invention This 0 25000 25000 >50000 >50000 >50000 25000 5000 1000 invention Freund's 0 0 5000 5000 25000 >50000 25000 25000 1000 Freund's 0 0 1000 1000 5000 25000 >50000 5000 5000 ICF 0 0 5000 25000 50000 25000 5000 5000 1000 ICF 0 1000 5000 5000 5000 >50000 25000 25000 5000 PBS 0 0 1000 5000 1000 1000 5000 1000 1000 PBS 0 0 1000 50000 5000 5000 5000 1000 0 The results demonstrate the effectiveness of an adjuvant encompassed by compared to Freund's complete adjuvant, ICF and the antigen injected control.

this invention in PBSA as a '0 00 0 \0 WO 99/27959 PCTAU98/00990 Example 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 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 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, 1977).

Hybrid white leghorn chickens eggs (Ex SPF Unit) were 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 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 added to 0.15 ml of phosphate buffered saline and emulsified with 0.25 ml of Complete Freund's adjuvant 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.

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 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 i-:l WO 99/27959 PCT/A U98/00990 16 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.

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 Chickens sera were incubated at 56°C for 30 minutes to inactivate complement. Fifty .l 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 .l of heat inactivated sera, before and after the immunisation, were added to wells of the first row. Two fold serial dilutions were performed across the plates.

Fifty p. 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 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 p4 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 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 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 were performed across the plates. After 2 hours incubation at room temperature, the plates were washed four times and 100 .l of 1/20,000 anti-chicken IgG, developed in rabbit, WO 99/27959 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, 5, tetramethylbenzidine (Sigma Cat.

T2885) substrate was added and incubated for a further minutes before the stopping solution was added. Titres were expressed as the reciprocal of the dilution resulting in optical density of the wells.

The results of the experiment are shown in Figure 1 and 2 and Tables 5 to 7.

Table 5. Adjuvant experiment.in chickens (hybrid white leghorn) Adjuvant Isolator Pink No. Sex Weight (gm) Tissue reaction Tissue reaction Remark Before 2 weeks efter Before 2 weeks after 8 weeks after 2 weeks after 2 weeks after 8 weeks after prime prime boost I boost i boost I prime boost I boost I 4 41 Female 310 500 680 909 1372 4 42 Female 315 529 722 940 1399 4 43 Female 285 484 617 845 1298 (OulIlN 4 44 Male 336 584 794 1156 1875 DEAEI 4 45 Male 433 715 955 1322 2137 888 il) 4 48 Male 345 586 738 1084 1663 3 47 Female 287 475 645 841 977 Head pecked by others 3 48 Female 361 590 767 941 1290 3 49 Female 309 408 646 831 1162 3 50 Male 398 651 895 1159 1585 3 51 Male 390 675 946 1307 1832 3 52 Male 439 728 1047 1303 1980 Mean 351 575 786 1053 1548 SD 54 103 143 192 356 Counts 12 12 12 12 12 SE 18 30 41 55 103 Adjuvant Isolator Yellow No. Sex Weight (am Tissue reaction Tissue reaction Tissue reaction Before 2 weeks after Before 2 weeks after 8 weeks after 2 weeks after 2 weeks after 8 weeks after prme prme boost I boost I boost I prime boost I boost i 4 1 Female 313 527 700 953 1435 4 2 Female 310 482 610 843 1184 4 3 Female 308 511 680 940 1318 4 4 Male 354 638 834 1185 1904 Lump on left leg Lump on left lea 4 5 Male 323 573 755 1092 1884 CFIICF 4 6 Male 368 664 830 1160 2006 3 7 Female 317 518 680 902 970 Head pecked by others 3 8 Female 315 518 736 958 1397 3 9 Female 348 557 752 988 1416 3 10 Male 415 708 1002 1325 1985 3 11 Male 420 724 1033 1356 1957 3 12 Male 279 463 714 931 1458 Head pecked by others Mean 339 573 777 1052 1575 SD 44 88 128 169 352 Courts 12 12 12 12 12 SE 13 25 37 49 102 WO 99/27959 19 Table 6. Adjuvant experiment in chickens (hybrid white leghorn) PCT/AU98/00990 Adjuvant Isolator Pink Sex Titre using haemagglutination assay (reciprocal dilution) no. 2 weeks after Before 2 weeks after 8 weeks after prime boost 1 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 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 1 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 SD 6 9 17 19 Counts 12 12 12 12 SE 2 3 5 v-s C T -iv WO 99/27959 Table 7. Adjuvant experiment in chickens (hybrid white leghorn) PCT/AU98/00990 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 42 888 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 3 50 Male 34 42 191 27 3 51 Male 20 14 42 3 52 Male 60 32 191 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 Sprime 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 4 5 Male 6 19 22 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 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.

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 primary injection. The animals in one group received an injection of 100 tg phomopsin A conjugated to 336 tg 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 were injected with 100 tg phomopsin A conjugated to 336 jg 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 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 at the injection sites were inspected and 10 ml blood was collected from jugular vein. A booster injection of 100 jg phomopsin A conjugated to 440 ig foetal calf serum was given subcutaneously as in primary injection. In the case of the comparison group this was formulated in Incomplete 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 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 p. in carbonate coating buffer pH 9.6 and 100 .l 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 .l of 0.1 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.

After 2 hours incubation at room temperature, the plates were washed four times and 100 p.l 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, 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 of the dilution resulting in 0.5 optical density of the wells.

ELISA on 50 ng ovalbumin/well coated microtitre plates 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 Tables 8 to 12.

WO 99/27959 WO 9927959PCT/A U98/00990 23 Table 8. Adjuvant trial in cattle Adjuvant Animal no. Sex Animal no. Weight (kg) Left Right Before Before Before 8 weeks rim boost 1 boost 1 after boost 1 Female 373 148 164 186 210 210 6 Male 9721 183 203 240 282 282 14 Male 9701 173 188 226 256 256 Male 9713 153 171 194 230 230 (Quil A/ 20 Female 998 138 152 169 194 1 194 DEAEI 24 Female 285 160 174 194 221 1 221 888 oil) 25 Male 342 135 152 182 202 1 202 28 Female 9723 184 200 227 256 256 Female 9710 160 180 209 224 224 31 Male 9732 157 176 209 230 230 34 Male 631 155 164 177 210 210 Female 9720 165 178 183 216 216 37 Female 425 176 186 211 241 241 Mean 1 160 176 201 229 229 Counts 1 13 13 13 13 1 S D 1 15 16 22 25 2 S E 4 4 6 77 Adjuvant Animal no. Sex Animal no. Weight (kg) Left Right Before Before 8 weeks prime Iboost I after boost 1 4 Male 9726 143 148 1l62 199 199 8 Female 9709 140 150 167 185 185 Female 9705 155 168 185 205 205 13 Male 979 157 175 187 215 215 CFIICF 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 2 14 232 232 32 1Female 1 466 160 178 '199 22m 222 36 1 Male 1 418 116- 124 143 174 1 174 Mean 155 169 184 213 213 Counts 11 11 11 11 11 SD 19 23 26 2626 SE 6 7 8 8 8 .AV-XL>.V -VU.XTy;*Xt~rVfl-" WO 99/27959 WO 9927959PCT/A U98/00990 24 Table 9. Adjuvant trial in cattle Adjuvant Animal no. Sex Animal no. Tissue reactions to phomopsins-ovalbumn conjugate (2 mUdose) Left Right 2 weeks after Before 2 weeks after 8 weeks after prime boost 1 boost 1 boost I Female 373 6 Male 9721 3x3 cm 14 Male 1 9701 Male 9713 (Owl A/ 20 Female 998 DEAEI 24 Female 285 888 oil) 25 Male 342 28 Female 9723 x cm 3x4 cm Female 9710 31 Male 9732 7x5 cm 7x6 cm US6 cm 3x3 cm 34 Male 631 3x3 cm Female 9720 37 Female 425 Adjuvant Animal no. Sex Animal no. ITissue reactions to phomopsins-ovalbumin conjugate (2 mI/dose) Left Right 2 weeks after Before 2 weeks after 8 weeks after prime boost I boost 1 boost 1 4 Male 9726 -3x3 cm 8 Female 9709 Ox5 cm 6x4 cm 8X6 cm Female J_9705 10x3 cm 6x5 cm Ux4 cm 13 Male 979 CF/IC F 17 Male 983 12x6 cm 8x6 cm 2W cm 19 Female 215 8x6 cm Ux8 cm 4x5 cm 22 Female 377 23 Female 263 6x7 cm 3x3 cm 26 Male 9730 7x6 cm 2 Female 466 8x8 cm 4x5 cm 36 Male 418 10x6 cm -7x8 cm 7x6 cm WO 99/27959 WO 9927959PCT/A U98/00990 25 Table 10. Adjuvant trial in cattle Adjuvant Animal no. Sex Animal no. Tissue reactions to 5 in I vaccine (4 mldose) Left Right 4 weeks after Before 2 weeks after 8 weeks after prime boost 1 boost 1 boost 1 Female 373 6 M ale 9721 14 Male 9701_____ -Male 9713 2x-cm- 2x3-cm 3x5 cm (Gull A/ 20 Female 998 4x3 cm 4x4 cm lxi xlcm DEAE/ 24 Female 285 888 oil) 25 Male 342 :2x2:cm8xm xc 28 Female 9723 Female 9710 5xcxcm 31 Male 9732 3x4 cm 34 Male 631 ~c m Female 9720 3x2:cm::. :::3x2 7x- _37 Female 425 4~m 3~m Adjuvant Anmlno. -Sex An imal no. wks Tissue reactions to 5 in 1 vaccie (4 mldose) LeftRigt 4wees afer efoe 2weeks after r8 weeks after eI Igh prime Iboast iI boost I boost I 4 8 13 17 19~ ,Male. 9726 22cm FemalIe 9709 .c Female 1 705 LAV** I Q. I 4: 5x5cm

CFACF

Mae 983 1 5x3:cm:: Female 215 FFemale 377 .6x3:cm O::x4cm 7x6cm '3x4+1OXIG cm .::3x4zm__ 3x3 cm 1)12 t~~i~i 263 F 5:X4cm 26 Mae 9730 Fe ae 466 I i I WO 99/27959 PCT/AU98/00990 26 Table 11. Adjuvant trial in cattle Adjuvant Animal No. Sex _Anti-phomopsin IgG titre (x100) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost 1 after boost 1 Female 16 38 120 6 Male 7 17 134 14 Male 26 170 514 158 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 Female 16 33 86 46 31 Male 29 81 261 156 34 Male 5 23 94 59 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 (x100) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost I after boost 1 4 Male 7 17 60 129 8 Female 12 64 291 299 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 .;11 WO 99/27959 WO 9927959PCT/A U98/00990 27 Table 12. Adjuvant trial in cattle Adjuvant Animal No. Sex ______Anti-ovalbumin lgG titre (xI0O) 2 weeks Before 2 weeks 8 weeks prime boost I after boost I after boost.I Female 4 5 8 6 Male 6 8 78 43 14 Male 2 40 110 63 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 28 Female 1 19 35 43 Female 5 5 32 24 31 Male 2 5 29 34 Male 1 2 9 6 Female 0 4 25 51 37 Female 2 4 49 19 Mean 2 9 45 31 Counts 13 13 13 13 SID 2 1 10 32 16 ISE 0 1 3 9- 4 Adjuvant Animal No. Sex Anti-ovalbumin lgG titre (xIOO) 2 weeks Before 2 weeks 8 weeks prime boost 1 after boost I after boost I 4 Male 2 3 10 29 8 Female 2 23 84 113 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 SD1 12 50 92 ISE 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 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 to 12 weeks old lambs. Twenty four, ten to twelve weeks old lambs, weighing between 10 to 20 kg, were weighed and ml blood was collected from jugular vein. They were divided into two equal groups randomised according to weight. One group was injected with 50 jig phomopsin A conjugated to 220 pig foetal calf serum, 0.5 mg Quil A and 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 ig conjugated to foetal calf serum 220 jig dissolved in 1 ml of sterile water, and emulsified with 1 ml of Complete Freund's 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.

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 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 jg conjugated to 220 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.

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 p.1 in carbonate coating buffer pH 9.6 and 100 p.1 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 1l of 0.1 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.

After 2 hours incubation at room temperature, the plates were washed four times and 100 p.1 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, 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 of the dilution resulting in 0.5 optical density of the wells.

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 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 U WO 99/27959 WO 9927959PCT/AU98/00990 30 Tables 13 to Table 13. Adjuvant trial in wethers Adjuvant Animal no. Weight (kg) Before 2 weeks Before 2 weeks 8 weeks after prime boost I after boost 1 after boost 1 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 28.2 131.0 26.5 13.0 19.5 26.4 28.0 23.5 96 13.5 20.2 34.2 37.5 34.5 Mean 174.0 19.8 28.3 30. 7' 26.8 S D 1.65 1.70 4.11 4.80 4.86 Counts 12 12 12 12 1 12 ISE 0.48 0.49: 1.19 1.39 1.40 Adjuvant Animal no. Weight Left Before 2 weeks Before 2 weeks 8 weeks _____prime after prime boost I after boost I after boosti1 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 I196 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 I 1.6 By eight weeks after boost 1 all sheep lost body weight due to the drought and lack of food ZUt... WO 99/27959 -31 Table 14. Adjuvant trial in wethers PCT/A U98/00990 Adjuvant Animal No. ______Anti-phomopsin lgG titre (xl 000) 2 weeks Before 2 weeks 8 weeks _______afterprime boost I after boost I after boosti1 21 5 95 57 86 14 22 281 145 87 26 7 68 33 88 20 10 59 72 (Quit 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 15 8 84 .56 96 7 24 98 62 Mean 23 33 172 158 Counts 12 12 12 12 SID 1 12 58 178 210 ISE 4 17 51 61 Adjuvant Animal No. Anti-phom psin lgG titre xlOOO) 2 weeks Before 2 weeks 8 weeks after prime boost I after boost I after boosti1 145 9 49 211 62 146 11 50 120 51 147 21 144 274 219 148 14 51 105 83 CFIICF 149 22 219 256 140 150 45 203 298 351 151 15 48 121 152 36 107 365 494 153 22 56 151 191 154 20 188 197 165 P 155 22 49 252 109 156 11 134 173 252 Mean 21 108 210 183 Counts 12 12 12 12 rso 11 1 67 81 132 SE 3 19 23 38 WO 99/27959 PCT/A U98/00990 32 Table 15. Adjuvant trial in wethers Adjuvant Animal No. Anti-foetal calf serum 1gG titre 100) 2 weeks Before 2 weeks 8 weeks after prime boost 1 after boost I after boost I 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 93 24 26 135 141 94 25 58 164 207 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 1gG titre (xl000) 2 weeks Before 2 weeks 8 weeks after prime boost I after boost I after boost 1 145 6 23 246 130 146 4 42 173 59 147 9 143 395 164 148 4 29 156 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 84 301 173 156 9 97 994 382 Mean 12 136 758 323 Counts 12 12 12 12 SD 8 106 535 240 SE 2 31 154 69 WO 99/27959 PCT/A U98/00990 33 Example 8 In this example the immunogen was ovalbumin. It was injected into rats in complete Freund's adjuvant for the 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 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 pg ovalbumin, 40 pg Quil A 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 pg ovalbumin was dissolved in 0.16 ml of phosphate buffered saline and emulsified with 0.16 ml of Complete Freund's 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 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 ml blood was collected from tail vein. For the booster 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 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/A U98/00990 34 vein.

Four weeks after the booster injection the rats were weighed and tissue reactions at the injection sites were inspected.

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 Ovalbumin was diluted as 50 ng/0.1 ml in carbonate 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 (Nunc-Immuno plate, F 96 Cert.maxisorp, Cat. 439454). After overnight incubation at 4 0 C, the plates were washed four times with 0.05 tween 20 in saline.

After the washing, 100 p.l 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 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 p1 of 1/16,000 anti-rat IgG, developed in goat, conjugated to peroxidase (Sigma Cat. A 9037) was added and incubated for a further 2 hours. After washing the plate four times 3, 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 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 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 1 after boost 1 after boost1 Cagel/0 197 208 212 214 219 224 222 Ca/gell 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 DEAE/ Cage 2/1 207 222 226 221 228 229 233 888 oil) Cage2/2 202 211 214 215 222 225 226 Cage 2/3 234 224 230 231 234 241 247 Cage 3/1 189 170 198 219 232 215 211 Cae 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 CaQe 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 nD 15 18 113 13 12 12 11 SE 4 5 3 4 3 3 3 Adjuvant Cagel 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 1 after boost 1 after boosti 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 Ca e 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 611 207 223 254 233 236 235 240 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 -Z77, T, -c WO 99/27959 36 Table 17. Adjuvant trial in Long-Evans female rats PCT/AU98/00990 Adjuvant Cagel 5 days 11 days 1 1 week 2 weeks 8 weeks Animal No. after prime after primel after boost 1 after boost I after boost I Cagel/0 Cagel/1 1+ Cagel/2 1+ Cagell/3 1+ 1+ (Quil A/ Cage 2/0 1+ DEAEI Cage 2/1 888 oil) Cage 2/2 1 1+ Cage 2/3 I_ 1+ Cage 3/1 Cage 3/2 1+ Cage 3/3 Cage 7/1 Cage 7/2 Adjuvant Cagel 5 days 11 days I week 2 weeks 8 weeks I Animal No. after prime lafter prime after boost 1 after boost 1 after boost I Cage 4/0 14++ 1 1 2++ Cage 4/1 1 1 1+ 2++ Cage 4/2 1+ 1. I 2++ Cage 4/3 1 1 2 2 CF/ICF Cage 5/0 1 24++ 24++ Cage 5/1 24++ 2 2+ 1 ,1 Cage5/2 1+ 1+ Cage 5/3 1+ Cage 6/0 1 opened 1 2+ 2+ Cage 6/1 1 opened 1 1 opened 1 2+ Cage 6/2 114 1 11 1 1+ ,1 Cage 6/3 1 2++ small lump (1-2 mm) 1 and 2 numbers of lumps Medium lump (3-5 mm) Big lump (more than 5 mm) WO 99/27959 PCT/A U98/00990 Table 18. Adjuvant trial in Long-Evans female rats Adjuvant (Quil A/

DEAE/

888 oil) Cage/ Animal No.

Titre 2 weeks after prime 1 Before boost 1 33 2 weeks after boost 1 155 8 weeks after boost 1 247 CaaellO Cagel/1 1 38 68 156 Cagel/2 1 13 28 96 Cagel/3 0 9 29 77 Cage 2/0 1 36 93 126 Cage 2/1 1 26 85 92 Cage 2/2 0 12 23 49 Cage 2/3 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 Caae 7/2 150 Mean 1 35 95 148 Counts 13 13 13 13 SD 3 22 59 71 SE 1 6 16 Adjuvant Cage/ Titre (xlOOO) Animal No. 2 weeks Before 2 weeks 8 weeks after prime boost I after boost I after boost I 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 CF/ICF Cage 510 2 39 113 69 Cage 5/1 2 46 67 Cage 5/2 1 2 27 34 Cage 513 2 68 87 Cage 6/0 11 89 174 128 Cage 6/1 0 62 126 87 Cage 6/2 4 7 27 36 Caae 6/3 3 66 95 87 Mean 3 45 89 67 Counts 12 12 12 12 kSD 3 29 44 27 SE 1 8 13 8 WO 99/27959 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 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 range of infectious diseases and against natural products of the human and animal body such as hormones.

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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 substance, comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; and an immunoadjuvant oil.

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.

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

An adjuvant composition as claimed in any one of claims 1 to 4 wherein the immunoadjuvant oil is a 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: an antigenic substance; and an adjuvant composition comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; 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 Quil A. A vaccine as claimed in any one of claims 7 to 9 wherein the polycationic polyelectrolyte is diethylaminoethyl dextran.

WO 99/27959 PCT/AU98/009 40

11. A vaccine as claimed in any one of claims 7 to 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 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

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

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

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 steps of: providing said antigenic substance; providing an adjuvant composition comprising: a saponin with immune stimulating activity; a polycationic polyelectrolyte with immune stimulating activity; and an immunoadjuvant oil; and challenging said animal with said antigenic substance and said adjuvant composition.

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

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

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

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

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