AU694774B2 - An adjuvant - Google Patents

Description

WO 95/30436 PCT/AU95/00261 AN ADJUVANT The present invention relates generally to adjuvants which comprise a combination of at least two cytokines or functional derivatives thereof. More particularly, the present invention is directed to an adjuvant such as a vaccine adjuvant comprising at least two cytokines or functional derivatives thereof wherein the cytokines are selected from IL-1 P and TNFa or IL-1 and GM-CSF. The present invention is further directed to genetic adjuvants encoding at least two cytokines or derivatives thereof either separately or fused together. The present invention also contemplates a method for enhancing an immune response to an antigen comprising the administration of at least two cytokines which act in synergy to enhance an immune response to said antigen. The present invention is particularly useful in pharmaceutical vaccines and genetic vaccines in humans and livestock animals.

Bibliographic details of the publications referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The increasing sophistication of molecular biological and protein chemical techniques has, and continues to, revolutionise vaccine development. Frequently, however, recombinant and synthetic molecules exhibit less immunogenicity relative to the whole organism or cell from which they are derived. In order to increase the efficiency of such vaccines, considerable research has been directed to the development of immunological adjuvants.

J-IS11~U WO 95/30436 PCT/AU95/00261 -2- Adjuvants promote the immune response in a number of ways (East et al, 1993). First, some adjuvants maintain a depot of antigen at the site of injection, as demonstrated by the correlation between persistence of antigen at the site of injection and maintenance of serum antibody levels. Second, adjuvants are capable of promoting accumulation of immunoreactive cells at the site of injection and in the draining lymph nodes, which ensures optimal exposure of specific immunoresponsive cells to the antigen. Adjuvants also modify the activities of cells that are concerned with generating and maintaining the immune response. Additionally, adjuvants modify the presentation of antigen to the immune system.

Cytokines such as interleukins (IL-1 to 13), colony stimulating factors (CSFs), tumour necrosis factors (TNF-a and p) and interferons (IFN-a, P and y) are the hormones of the immune system which control and determine the nature of the immune response (Balkwill and Burke, 1989). Adjuvants with the capacity for immune modulation achieve their activity primarily by modification of cytokine production, probably by direct action upon T cells or macrophages (Cox and Coulter, 1992). Cytokines regulate various aspects of the immune response to vaccination including cellular traffic at the site of vaccination (Zimmerman et al., 1992), antigen presentation (Valle et al., 1991), the phenotype of the T helper cell response (Romagnani, 1992), maturation and differentiation of the B cell response (Snapper and Mond, 1993) and differentiation of non-specific killer cells such as eosinophils and mast cells (Clutterbuck et al, 1989).

Recombinant cytokines not only have the potential to enhance the immune response to a vaccine antigen but also alter the immune response leading to different effector mechanisms.

As an alternative to chemical adjuvants, the use of cytokines as natural adjuvants is attracting considerable interest (reviewed by Heath and Playfair, 1992). Various cytokines have been shown to be effective immunological adjuvants in a variety of model systems, enhancing protection induced by bacterial, viral and parasitic antigens.

Some of these molecules have already been shown to enhance the immune response of immunodepressed animals to antigens delivered by viral vectors (Ramshaw et al., 1987), or when incorporated into the emulsion to enhance the antibody response to inactivated

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WO 95/30436 PCT/AU95100261 -3vaccines (Good, 1988; Blecha, 1990). Furthermore, in the murine model, cytokines such as IL-1, IL-2 and IFN-y have been shown to have adjuvant effects for both cellular and humoral response (Staruch and Wood, 1983; Nunberg et al., 1989; Playfair and deSouza, 1987). Limited adjuvanticity of TNF has been reported by Ghiara et al, (1987).

The adjuvant effects of some cytokines have been studied in domestic livestock. Yilma et al., (1985) reported that bovine IFN-y enhanced the humoral responses of cattle vaccinated with a viral glycoprotein and Reddy et al (1990) demonstrated increased neutralising antibody titres and cytotoxic cell responses to bovine herpes virus-1 when recombinant bovine IL-P1 (rBoIL-1p) was included in the vaccine regimen.

Recombinant human IL-2 has been administered to pigs vaccinated with Actinobacillus pleuropneumonia bacterin (Anderson et al., 1987; Nunberg et al., 1988) or a pseudorabies subunit vaccine (Kawahima and Platt, 1989) with increased levels of protection and neutralising antibodies respectively. Recently, Nash et al (1993) reported that the incorporation of recombinant ovine IL-p1 (rOvIL-1p) in a alum based vaccine resulted in significantly higher antibody levels to their model antigen, ovalbumin.

Despite the potential of using single cytokines as adjuvants in vaccine preparations, little work has been reported on adjuvants consisting of two or more cytokines. This may, in part, have been due to the high cost in preparing recombinant cytokines.

Furthermore, cytokines also regulate the activity of other cytokines and, frequently, this involves a down regulation. As a result, it is not readily apparent which combinations of cytokines may work in concert. Heath and Playfair (1992) have suggested the possibility of using combinations of cytokines as adjuvants, however, their proposition contemplates a vast number of combinations and permutations of cytokines with little success in the combinations actually tried.

In the past, substantial technical difficulties have delayed development of vaccines against commercially significant livestock parasites such as gastrointestinal nematodes.

In work leading up to the present invention, the inventors sought to improve the immunogenecity of antigens such as nematode antigens by incorporating specific combinations of recombinant cytokines into vaccine formulations. Furthermore, WO 95/30436 PCT/AU95/00261 -4alternative adjuvants are required for human use.

There is a need to identify particular synergistic combinations of cytokines which are capable of acting as adjuvants useful, for example, in the preparation of vaccines.

Accordingly, the present invention provides an adjuvant, said adjuvant comprising at least two cytokine activities acting in synergy to enhance an immune response to an antigen. More particularly, the present invention is directed to an adjuvant, said adjuvant comprising a first polypeptide having IL-i 13 activity and a second polypeptide having TNF-a activity, said first and second polypeptides acting in synergy to enhance an immune response to an antigen.

In a related embodiment, there is provided a first polypeptide having IL-113 activity and a second polypeptide having GM-CSF activity, said first and second polypeptides acting in synergy to enhance an immune response to an antigen.

The adjuvants of the present invention may also comprise an antigen. Preferred antigens are those from parasites or other pathogenic agents which infect animals or birds and in particular humans or livestock animals. Examples of parasites and pathogenic agents include intracellular and extracellular bacterial and other prokaryotic organisms, eukaryotic organisms such as yeasts and fungi, protooan organisms, metazoan organisms and ameoba, parasites such as nematodes, for example, species of Haemonchus, kTrichostrongylus or Ostertagia or a cestode such as Taenia ovis. The present invention extends to any antigen from any pathogen or parasite of animals or birds. The present invention also extends to antigens from non-pathogens such as pollen grains and other irritants for which an immune response is desirable.

The first and second polypeptides may be recombinant molecules or isolated, naturally occurring molecules. The term "polypeptide" is used in its broadest sense and includes a protein or a peptide having the desired activity. The first and second polypeptides of the present invention each exhibits one or more of the identifying characteristics of one of IL-1 3, TNFa or GM-CSF but each may not necessarily exhibit the full complement WO 95/30436 PCTIAU95/00261 of activities exhibited by the corresponding naturally occurring cytokine. The principal requirement is that the first and second polypeptides exhibit sufficient IL-1 TNFa or GM-CSF activity such that the combined immune enhancing effect of the first and second polypeptides is greater than either the first or second polypeptides alone.

In a most preferred embodiment, the first polypeptide is a recombinant or isolated, naturally occurring IL-lp and the second polypeptide is a recombinant or isolated, naturally occurring TNF-a or GM-CSF or a functional mutant, derivative, part, fragment, analogue or homologue thereof. The IL-1 TNFa and GM-CSF are generally of animal origin such as from a human, livestock animal sheep, cow, pig, horse, goat or donkey), laboratory test animal mouse, rat, guinea pig or rabbit), a domestic animal dog or cat), avian species chicken or other poultry bird) or a captive animal kangaroo, deer or wild boar). More preferably, the cytokines are of human or livestock animal origin. .Preferred livestock animals include ovine, bovine and porcine species.

As stated above, the first and second polypeptides may be IL-1p and TNFPx or IL-1p and GM-CSF proteins, respectively. Reference herein to "IL-lp", "TNFa" and "GM- CSF" includes reference to the naturally occurring molecules or to any amino acid substitutions, deletions and/or additions to either molecule but which retains the synergistic properties when in combination with the other molecule.

Amino acid insertions include amino acid and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino or carboxyl terminal fusions, of the order of 1 to 4 residues. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein.

Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 1.

i 1 WO 95/30436 PCT/AU95/00261 -6- Original Residue Ala Arg Asn Asp Cys Gin Glu Gly His Ile Leu Lys Met Phe Ser Thr Trp Tyr Val TABLE 1 Exemplary Substitutions Ser Lys Gin; His Glu Ser Asn Asp Pro Asn; Gin Leu; Val Ile; Val Arg; Gin; Glu Leu; Ile Met; Leu; Tyr Thr Ser Tyr Trp; Phe Ile; Leu Generally amino acids are replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains, etc.

Amino acid substitutions are typically of single residues; insertions usually will be in the order of about 1-10 amino acid residues; and deletions will range from about 1-20 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues.

WO 95/30436 PCT/AU95/00261 -7- Amino acid variants of IL-1p, TNFa and GM-CSF may also readily be made using peptide synthetic techniques well known in the art, such Ps solid phase peptide syntheses (Merrifield, 1964) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example using M13 mutagenesis. The manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art and are described for example in Maniatis etal (1982).

Other examples of recombinant or synthetic mutants and derivatives of the cytokines of the present invention include single or multiple substitutions, deletions and/or additions i to any molecule associated with the cytokine such as carbohydrates, lipids and/or proteins or polypeptides.

The terms "analogue" and "derivatives" also extend to any amino acid derivative of IL- TNFa or GM-CSF.

Analogues of the cytokines contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or derivatising the molecule and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitroenzylation of amino groups with 2, 4, 6 trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed by reduction with NaBH 4 I WO 95/30436 PCT/AU95/00261 S-8- The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a coresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4chloromercuribenzoate, 4-chloromercuriphenylsulphononicacid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with Nbromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-aminobutyric acid, 4-amino-3acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine,4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.

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WO 95/30436 PCT/AU95/00261 -9- Crosslinkers can be used, for example, to stabilise 3D conformations using homobifunctional crosslinkers such as the bifunctional imido esters having (CH 2 )n spacer groups with n 1 to n 6, glutaraldehyde, N-hydroxysuccinimide estrs aind heterobifunctional reagents which usually contain an amino-reactive moity such as Nhydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide In addition, peptides could be conformationally constrained by, for example, incorporation of C a and Na-methylamino acids, introduction of double bonds between Ca and Cp atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention, therefore, extends to peptides or polypeptides and amino acid hybrid molecules and/or chemical analogues thereof haing the identifying characteristics of either IL-l1p and/or TNFa 'and/or GM-CSF as broadly described herein, and/or to regions parts or fragments thereof capable of, or responsible for, the synergistic adjuvant properties when both cytokines are in combination.

Examples of human and animal diseases for which vaccination may be appropriate include but are not limited to Parkinsons, hepatitis, cancer, AIDS or AIDS related conditions, liver cirrhosis, asthma, chicken pox, influenza, diphtheria, common cold, Legionnaire's disease, malaria, measles, meningitis, mumps, pneumonia, ring worm, scarlet fever, syphilis, tetanus, tuberculosis, typhoid fever, diarrhoea, pept:c ulcer, schistomaniasis, whooping cough, muscular dystrophy, rheumatoid arthritis, sore throat.

The antigens will include gpl20 (HIV), urease from Helicobacterpyloris. Vaccines may also be applicable for contraceptive use in humans or animals such as livestock animals and companion animals. Examples of specific livestock diseases include, but are not limited to, mastitis, babesiosis, anaplasmosis, pink-eye, Johne's disease, bovine ephemereal fever, cattle tick, ostertagia, disease caused by taenis hydatigena, pastivirus, tuberculosis, thielierosis, foot and mouth disease, blow-fly strike, footrot, ostertagia, haemonchosis, trichostrongylosis, chlamydia, salmonella abortus, caseous lymphadenitis, i L WO 95/30436 PCT/AUJ95/00261 flecerot, disease caused by rotavirus, E. coli, Actinobacillus pleuropneumonia, classical swine fever, African swine virus, Newcastle's disease. Examples of diseases of companion animals include but are not limited to canine distemper, heart worm, rabies, hepatitis, cat flu, triple antigen.

The first and second polypeptides of the present invention may also exist as fusion proteins with other molecules or with each other. Examples of molecules which may be fused to either the first or second polypeptide include immune enhancing molecules, other cytokines or enzymes or components thereof such as glutathione-S-transferase (GST). Where the first and second polypeptides are fused together, there will generally be a spacer peptide between both polypeptides. The fusion must not, however, effect the synergistic adjuvant properties of both polypeptides. The present invention contemplates other forms of fusions in addition to an amide bond between the Nterminus of one polypeptide and the C-terminus of the other polypeptide such as the 15 formation of disulphide bridges between the first and second polypeptides or either polypeptide and a further molecule.

The present invention further contemplates genetic adjuvants comprising one or more nucleic acid molecules encoding separately or as fusion molecules, polypeptides having either IL-lp3 and TNFca activities or IL-lp3 and GM-CSF activities. The genetic adjuvants may also comprise a genetic sequence encoding an antigen. In the latter case, the composition is known as a genetic vaccine.

M The present invention is predicated, in part, on the surprising discovery that the combinations IL- Ip and TNFa or IL-Ip and GM-CSF exhibit a synergistic co-adjuvant effect. The term "adjuvant" is used in its broadest sense as any substance which enhances, increases, upwardly modulates or otherwise facilitates an immune response to an antigen. The immune response may be measured by any convenient means such as antibody titre or level of cell-mediated response. Although the present invention is particularly exemplified by reference to an immune response to a recombinant fusion protein from Taenia ovis or from the Haemonchus contortus antigen tropomyosin, it is clear that the subject invention extends to any antigen or gro,. L otigens capable of WO 95/30436 PCTAU95/00261 11 stimulating a humoral and/or cell mediated immune response in animals or birds and in particular humans or livestock animals.

Particularly preferred antigens are from livestock nematodes such as from Haemonchus, Trichostrongylus or Ostertagia.

Furthermore, the cytokine combinations preferably modulate a T helper (Th) subset response such as a Thl or Th2 response to induce either a cell-mediated and/or humoral respoise. The Thl response is associated with enhanced macrophage activation, increased cytotoxic T cell activity and delayed-type hypersensitivity responses. The Th2 response is associated with increased production of antibodies such as IgGI or IgE leading to increased mastocytosis and eosinophilia, hallmarks of nematode parasite infection.

The adjuvant composition of the present invention may be packaged for sale with each cytokine contained in a separate compartment with instructions to combine two or more of the cytokines with a desired antigen prior to use. The package may instead contain the adjuvant composition in a 'single compartment with instructions to combine the adjuvant with a desired antigen prior to use. Alternatively, the adjuvant composition may be administered subsequently or prior to administration of the antigen.

*Accordingly, another aspect of the present invention contemplates a method of enhancing, increasing, upwardly modulating or otherwise facilitating an immune response in an animal or a bird to an antigen, said method comprising administering to said animal or bird an immune-enhancing effective amount of an adjuvant composition which comprises a first polypeptide having IL-1P activity and a second polypeptide having TNFa activity. Alternatively, the first and second polypeptides have IL-1p and GM-CSF activities, respectively. Preferably, the animal is a human or livestock animal.

The method may further comprise the administration of an antigen such as hereinbefore described or may comprise the administration of a genetic adjuvant or genetic vaccine as described above.

WO 95/30436 PCT/AU95/00261 12- An animal or bird as contemplated by this aspect of the present invention is as hereinbefore defined in relation to the source of cytokine molecules. Where the animal or bird is to be treated with IL-10, TNFa or GM-CSF from the same species, this is referred to as a "homologous" system. Where one or more of the cytokines are from a different animal or bird to the animal or bird being treated, then this is referred to as a "heterologous" system. Both homologous and heterologous systems are contemplated.

by the present invention.

In an alternative embodiment, the method comprises the sequential administration to said animal or bird of a polypeptide having one of IL- P activity or TNFa/GM-CSF activity followed by a polypeptide having the other of said IL-1 activity or TNFa/GM-CSF activity.

The polypeptides contemplated above are preferably as hereinbefore defined and include fusion polypeptides.

In a most preferred embodiment, the method contemplated by' the present invention comprises administering to an animal human or livestock animal) or bird a vaccine composition comprising an antigen, and a first and second polypeptide as contemplated above. The present invention, therefore, is further directed to such a vaccine composition. In a most preferred embodiment, the vaccine composition is a vaccine against nematode infection in livestock animals. The vaccine composition may also be a genetic vaccine comprising one or more nucleic acid molecules encoding the cytokine or like molecules with or without an antigen and encoded separately or fused together.

The active ingredients of the vaccine composition of the present invention comprising one or more antigens or active immunogenic fragments thereof and an adjuvant composition comprising IL-11P and TNFa or IL-1P and GM-CSF as hereinbefore defined are contemplated to exhibit excellent activity in enhancing an immune response in an animal or bird when administered in an amount which depends on the particular case.

The variation depends, for example, on the animal or bird, the antigen and/or the source of first and second polypeptides. For example, from about 0.5 ug to about 20 mg of WO 95/30436 PCT/AU95/00261 -13 antigen or combined total of antigen per kilogram of body weight per day may be administered. Furthermore, for 10gg to 10 mg of IL-lp, TNFa and/or GM-CSF each may be administered per dose. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The active compound may be, administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active ingredients which comprise one or more antigen and first and second polypeptides as hereinbefore defined may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of the active ingredients may allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer the vaccine by other than parenteral administration, the vaccine composition will be coated by, or administered with, a material to prevent its inactivation. For example, the vaccine composition may be co-administered with enzyme inhibitors or in liposomes. The vaccine composition may also contain other compounds such as aluminium hydroxide, resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFP) and trasylol. Liposomes include water-in-oilin-water emulsions as well as conventional liposomes. The vaccine may also comprise Freund's Complete, Freund's Incomplete Adjuvant or aluminum hydroxide.

The active compounds may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

WO 95/30436 PCT/AU95I00261 -14 The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chiorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions caii be brought about i y the use in the compositions of agents delaying absorption, for example.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected as described above, the vaccine may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet For WO 95/30436 PCT/AU95/00261 oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of antigen. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in the vaccine compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it maycontain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be doated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. Where the vaccine is intended for animal use, the vaccine is conveniently administrable with the animal feed, such as grain. The vaccine composition may also be incorporated into a grain base or may be topically applied to feed grain.

As used herein carriers and/or diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifuangal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the vaccine compositions 1f~ WO 95/30436 PCT/AU95/00261 16is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The latter is particularly contemplated as far as the present invention extends to multivalent vaccines or multi-component vaccines.

Genetic vaccines may be administered as naked nucleic acid or via viral, insect, bacterial or yeast vectors, or by direct injection into target cells or tissue.

The present invention is further described by reference to the following non-limiting Figures and the Examples.

0 The following abbreviations are used in the Figure legends and Examples set forth hereinafter: IL-p TNFat

GM-CSF

rOvIL-1 p rOvTNFa rGM-CSF A1OH or Alum

GST

PBS

IFA

i/m

EIA

OD

PBST

TMB

PHA

Interleukin-1 P Tumour Necrosis Factor-a Granulocyte-Macrophage Colony-Stimulating Factor Recombinant Ovine IL-1p Recombinant Ovine TNFa Recombinant Ovine GM-CSF Aluminium hydroxide Glutathione-S-transferase An antigen from Taenia ovis Recombinant 45W fused to GST Phosphate buffered saline Incomplete Freund's adjuvant Intramuscularly Enzyme Immunoassay Optical Density PBS containing Tween Tetra-methyl benzidine Phytohemagglutinin

A

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WO 95/30436 PCT/AU95/00261 17- In the Figures.

Figure 1 is a schematic representation of the construction of pGEX-2T.1L- 1 expression vector. PCR amplification of plasmid DNA harbouring ovine IL-1P was carried out in a total volume of 100 1 containing 20ng plasmid DNA, 100ng of each primer, 200uM of each dNTPs, 1 x Taq polymerase buffer, 1.6mM MgCI 2 and 2.5U Taq DNA polymerase. The sample was overlaid with 100gl of mineral oil and subjected to cycles of denaturation at 94 0 C for 1 min, annealing at 50 0 C for 2min and extension at 72 0 C for 2min using a DNA thermal cycler. After the final cycle, incu ation at 72 0

C

was continued for 10min. The PCR product was polished with T4 DNA polymerase I and applied to a 1% w/v agarose gel and purified from the gel slice using a Geneclean kit (B10 101, USA), ligated to the Smal site of pUC119 and transformed into JM109.

The clones with inserts were sequenced to confirm the IL-1 cDNA sequence encoding the mature protein. The IL-I 3 DNA insert obtained from the BamHI and Hincll digest was ligated into the BamHI and Smal site of the expression vector, pGEX-2T. This would be in frame with the thrombin cleavage site behind the carboxy terminus of Sj26, a 26kDa glutathione-S-transferase of the parasite helminth Schistosoma japonicum.

Figure 2 is a photographic representation of an SDS-PAGE analysis of recombinant ovine IL-l purified by glutathione Sepharose affinity purification. Lane 1: molecular weight markers (Biorad); lane 2: crude lysate after sonication and loaded onto affinity column; lane 3: insoluble pellet after sonication; lane 4: proteins that did not bind to the column; lane 5: ovine GST-IL- 1 fusion protein after glutathione elution; lane 6: ovine IL-1 following thrombin cleavage on the affinity column.

Figure 3 shows the biological activity of recombinant OvlL-1 preparations in an ovine thymocyte proliferation assay and in the murine NOB-1/CTLL assay. The results shown are mean values from triplicate wells. Thymocyte assay: A, NOB-1/CTLL assay: 0. IL-1 is shown as solid symbols and IL-1-GST as open symbols. Background CPM for unstimulated wells were 1511 and 4396 for the NOB-1/CTLL and thymocyte assays, respectively.

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cI~I I i: i r t i WO 95/30436 PCT/AU95/00261 -18- Figure 4 is a schematic representation of the construction of pGEX-2T.TNFa expression vector.

Figure 5 is an SDS-PAGE analysis of proteins expressed in E. coli strain JM109. Lane i: proteins in the insoluble fraction from growth at 37 0 C. Lane 2: Soluble fraction from growth at 37 0 C. Total accumulated proteins at 30 0 C and the corresponding insoluble.

fraction (lane 3) and soluble fraction (lane Lane 5: Molecular weight markers (BioRad). Lane 6: Fusion protein cleaved with thrombin to yield GST and rOvTNF.

Lane 7: purified rOvTNF with carrier protein BSA w/v) added.

Figure 6 is a graphical representation showing the cytotoxic effect of recombinant ovine and human TNF-a on murine L929 cells.

Figure 7 is a graphical representation showing the adjuvant effect of rOvIL-lP in.

aqueous 45W vaccine formulations as compared to AlOH. Sera collected 2 weeks post second vaccination were titrated in an EIA against purified 45W. The results shown are the mean ODs obtained from the 5 sheep per group at each serum dilution.

PBS PBS IOug IL-1 PBS +O10ug IL- -1 0- AIOH -U AIOH AIOH 10ug IL-1 AIOH 100ug IL-1 Quil A Figure 9 is a graphical representation showing the adjuvant effect of rOvIL-10 in Quil A and IFA vaccine formulations. Sera collected 2 weeks post second vaccination were titrated in an EIA against purified 45W. The results shown are the mean ODs obtained th enOsotie rmte5see e ru tec eu iuin !9 .V 0gIL1--PS H~gI-1--AO

-A

WO 95/30436 PCT/AU95/00261 19from the 5 sheep per group at each serum dilution.

IFA -W IFA +10 ug IL-1 IFA 100ug IL-1 Quil A Quil A 10ug IL-1 -W Qui A 10Oug IL-1 Figure 10 is a graphical representation showing the adjuvant effect of rOvTNF-a in aqueous 45W vaccine formulations as compared to AlOH and non-vaccinated control sheep. Sera collected 2 weeks post second vaccination were titrated in an EIA against The results shown are the mean ODs obtained from the 5 sheep per group at each serum dilution.

PBS PBS 10ug TNF PBS 100ug TNFI -w AIOH Controls Figure 11 is a graphical representation showing the adjuvant effect of rOvTNF-a in A1OH 45W vaccine formulations as compared to Quil A and non-vaccinated control sheep. Sera collected 2 weeks post second vaccination were titrated in an EIA against The results shown are the mean ODs obtained from the 5 sheep per group at each serum dilution.

AlOH -V AJOH 10ug TNF AIOH 100ug TNF Quil A Controls Figure 12 is a graphical representation showing the adjuvant effect of rOvTNF-a in Quil A 45W vaccine formulations. Sera collected 2 weeks post second vaccination were titrated in an EIA against GST-45W. The results shown are the mean ODs obtained from the 5 sheep per group at each serum dilution.

W- Quil A Quil A 10ug TNF Quil A 100ug TNF Controls -1 /4 0 WO 95/30436 PCT/AU95/00261 Figure 13 is a graphical representation showing the adjuvant effect of rOvTNF-a and rOvIL-13 both alone and in combination when incorporated in A1OH 45W vaccine formulations. Antibody responses of non-vaccinated control sheep and those vaccinated with 45W in AlOH alone or Quil A are shown for comparison. Sera collected 2 weeks post second vaccination were titrated in an EIA against GST-45W. The results shown are the mean ODs obtained from the 5 sheep per group at each serum dilution.

AIOH

AIOH 10ug IL-1 Quil A AIOH 10ug TNF AIOH 10ug TNF 10ug IL-1 Controls i Figure 14 is a graphical representation showing the antibody titres from sheep vaccinated with the recombinant Haemonchus contortus tropomyosin antigen with aluminium hydroxide alone and in combination with either 1 gg/dose or 10 pg/dose of rGM-CSF.

NIL

ALUM

-ALUM +GM(lug) 0 Figure 15 is a graphical representation showing the titres of antibody over a ten week period.

NIL

ALUM

ALUM+IL-1+GM(1 ug) ALUM+IL- +GM(1 Oug) -~QUIL A ALUM+IL-1 j~.

SUBSTITUTE SHEET (Rule 26) 1~ I 1 1, WO 95/30436 PCT/AU95/00261 -21 EXAMPLE 1

ANIMALS

Merino wethers, 12-18 months old, were used in subsequent examples to test the efficacy of the vaccine adjuvant.

EXAMPLE 2

CYTOKINES

Recombinant ovine interleukin-1 P (rOvIL-1 P) was prepared as described in International Patent Application No. PCT/AU91/00419. Briefly, rOvIL-lp and tumour necrosis factor-a (rOvTNF-a) were prepared as described as follows.

1. Construction of ovine interleukin-1 expression vector The ovine IL-1 p cDNA encoding the mature IL-1 P protein was obtained by polymerase chain reaction (PCR) using the following primers:- 5' GGATCC GCA GCC GTG CAG TCA 3' (SEQ ID NO. 1) and 5' CCGGTCGAC TAG GGA GAG AGG GTT TCC ATT C 3' (SEQ ID NO. The primers were synthesized with a cohesive BamHI 5' end and a blunt HincII 3' end. The amplified fragment was treated with the Klenow fragment of DNA polymerase I and ligated into the SmaI site of pUC119 and dideoxy sequencing using the T7 polymerase sequencing kit (Pharmacia) was performed to confirm the DNA sequence. The plasmid pUC 19 harboring the DNA sequence encoding the mature IL-1p protein was digested with BamHI and Hincll. The insert was ligated into the BamHI and Smai site of the expression vector pGEX-2T (Smith aio Johnson, 1988). Transformants of E.coli strain JM109 were produced. The recombinant plasmid was designated pGEX-2T.IL-1.

2. Expression and affinity purification of recombinant interleukin-1 Overnight cultures of pGEX-2T plasmids were diluted 1:50 in 250ml of Luria Broth bacto-tryptone, 5g/L yeast extract, 10g/L NaCI) or Terrific Broth (16.43g/L

K

2

HPO

4 .3H 2 0, 2.31g/L KHzPO 4 tryptone 12g/L, yeast 24g/L, glycerol 4ml/L) with 100/gg/ml ampicillin. The cultures were grown for 2h at 37 0 C before adding IPTG (isopropyl--thiogalactopyranoside) to 0.2mM (or as indicated in the legend). After 4h, II L 11

I

WO 95/30436 PCT/AU95/00261 22the cultures were harvested and centrifuged. The pellets were weighed and resuspended in the appropriate volume of buffer (50mM Tris.CI, pH7.5; 10ml/g of wet weight of pellet). The cells were lysed on ice by sonication and then centrifuged. The supernatant was loaded onto a 5ml glutathione Sepharose column (sulphur-linkage,Sigma). The flow through was kept and the column was washed thoroughly with at least 5 bed volumes of 50mM Tris.CI, pH7.5. The recombinant protein was eluted either as a fusion product with 5mM glutathione or as free form by cleavage with human thrombin (lOU/ml; ICN) at room temperature for lh. The eluted proteins were analysed on a 15% w/v SDS-PAGE (Laemmli, 1970) stained with 0.05% w/v Coomassie Brilliant Blue R.

3. Protein assay Protein concentrations were estimated by the Bradford dye assay (Biorad) using bovine serum albumin as standard.

4. Assay of ovine IL-1 IL-1 was assayed in two separate bio-assays, 1) thymocyte proliferation in the presence of sub-mitogenic concentrations of lectin and 2) by the ability to stimulate the murine cell line NOB-1 to secrete IL-2 as measured by proliferation of the IL-2 dependent cell line CTLL. Recombinant human IL-1p (National Institute for Biological Standards and Control, Hertforshire, England) was used in both assays as a positive control.

For the thymocyte assay, the thymus was aseptically collected from a euthanised lamb (2-8 weeks old). The thymus was then cut into small pieces before being passed through a stainless steel sieve to produce a single cell suspension. The thymocytes were suspended in Dulbecco's modification of Eagle's medium (DMEM; Flow Laboratories, Australia) supplemented with 20 mM HEPES, 9 mM sodium bicarbonate, 2 mM L-glutamine, 100 IU/ml penicillin, 100 tg/ml streptomycin and 10% v/v heat-inactivated foetal bovine serum (FBS; Flow Laboratories). After three washes in DMEM, thymocytes were resuspended in DMEM and viable cells counted by trypan blue exclusion. The assay was performed in triplicate in 96-well tissue culture plates with 7 x 105 thymocytes per well in a total volume of 200 jl per well containing 2 ig/ml of the lectin phytohemagglutinin (PHA) and serial dilutions of IL-13. Cells were incubated WO 95/30436 PCT/AU95/00261 -23 at 37 0 C, in a humidified atmosphere of 5% C02 in air, for 72 h before the addition of 3H thymidine (0.5 pCi/well). Cells were harvested 16 h later, using an automated cell harvester, and the amount of 3 H thymidine incorporation determined by counting in a Gas proportional p radiation counter.

The NOB-1/CTLL assay was performed as previously described (Gearing et al., 1987) with minor modifications. Briefly, 2 x 105 NOB-1 cells/well were added in triplicate to flat-bottomed 96 well tissue culture plates with various dilutions of rOvIL-1 in a total volume of 200 il/well of DMEM. NOB-1 cells were incubated for 24 h at 37 0 C before 50tl of culture supernatant were transferred to a second 96 well plate along with 100gl of CTLL cells at a concentration of 4 x 104 per ml. The CTLL cells were incubated for h at 37C before the addition Cf 3 H thymidine (0.5 pCi/well). After a further 3 h incubation, cells were harvested and 3H thymidine incorporation measured as above.

5. Expression and purification of reco'mbinant ovine IL-If The gene encoding the mature form of ovine IL-l was cloned into the BamHI and Smai site of the expression plasmid pGEX-2T behind the C-terminus of Sj26, glutathione-S-transferase. Figure 1 is a schematic illustration of the expression plasmid showing the site of thiombin cleavage of the fusion protein and the restriction sites used to insert the IL-1 gene. Induction of the tac promoter of the expression plasmid pGEX-2T.lL- 1 resulted in high level expression of a fusion protein. The recombinant protein was recovered in the soluble fraction and represented approximately 25% of total accumulated proteins as estimated by densitometric scanning of SDS polyacrylamide gels.

Affinity chromatography of the soluble fraction (Fig.2, lane 2) on a glutathione-Sepharose column, followed by cleavage with thrombin, yielded the free form of mature rOvlL- 1 with a molecular weight of ap~roximately 18 Ja (Fig.2, lane Alternatively, elution from the solumn with glutathione yielded a fusion protein with a molecular weight of 44kDa consisting of GST linked to IL-1 (Fig.2, lane Overall, the amount of rOvlL-1 purified from Luria broth and Terrific broth cultures was 2-3mg/L and 8-10mg/L, respectively.

T~ I WO 95/30436 PCT/AU95/00261 -24- 6. Bio-Assay Results Pure and GST fused rOvlL-1p were active in both the thymocyte and the NOB-1/CTLL assays. Figure 3 shows the activity of both preparations in the thymocyte co-stimulation assay. Specific activities of 5.4 x 103 U/mg and 5.6 X 107 U/mg for GST-IL-lp and IL-13, respectively, were calculated from the amount of cytokine required to give half-maximal activity in the thymocyte assay. The large increase in specific activity of IL-1~ over the GST-IL-1p fusion was also demonstrated in the NOB-1/CTLL assay although higher specific activities were obtained; 6.2 x 104 and 9.1 x 107 U/mg for GST-IL-1p and IL-1p respectively (Fig. 4).

7. Construction of TNF expression vector The ovine TNF-a cDNA encoding the mature TNF-a protein was obtained by PCR using the pUC118-TNF-ca DNA as template and the following set of primers: CGCGGATCC CTC AGG TCA TCT TCT CAA GCC 3' (SEQ ID NO. 3) BamHI site incorporated) and 5' TCA CAG GGC AAT GAT CCC AAA GTA 3' (SEQ ID NO.

PCR amplification of plasmid DNA harbouring ovine TNF-a was carried out. The purified PCR product was ligated into the BamHI and Smal site of the expression vector, pGEX-2T. This was in frame with the thrombin cleavage site behind the carboxy terminus of Sj26, a 26kDa glutathione-S-transferase of the parasite helminth Schistosoma japonicum (Smith and Johnson, 1988). The ligated DNA was electroporated into Escherichia coli JM109 and the recombinant plasmid designated pGEX2T.TNF-a.

8. Expression and eurification of recombinant ovine TNF The method used is as described for preparation of recombinant cvine interleukin-1.

9. Assay of recombinant ovine TNF-a Recombinant ovine TNF-a was assayed by 1) cytotoxicity on L929 cells and 2) ovine thymocyte proliferation in the presence of submitogenic concentrations of lectin.

Recombinant human TNF-a (2.3 X 106 units/ml) was used in both assays as a positive control.

WO 95/30436 PCT/AU95/00261 25 For the cytotoxicity assay, L929 murine fibroblast cells were added to 96 well culture plates (2.5 x 10 5 /well) in Dulbecco's modification of Eagle's medium (DMEM; Flow Laboratories, Australia) supplemented with 20 mM HEPES, 9 mM sodium bicarbonate, 2 mM L-glutamine, 100 lU/ml penicillin, 100 gg/ml streptomycin and 10% v/v heat-inactivated foetal bovine serum (FBS; Flow Laboratories). Aft.r 24 hours culture at 37°C in 5% v/v CO 2 the culture media was aspirated and 100L of serial dilutions of TNF-cl preparations in DMEM containing 1% v/v FBS and 10gg/ml cycloheximide added to triplicate wells. After a further 24 hours incubation, culture media was aspirated, plates washed twice with PBS and remaining viable cells stained for minutes with 100ijL/well of 0.5% w/v crystal violet in 20% v/v methanol. Plates were then washed extensively with PBS before the addition of 100 L/well of 33% v/v acetic acid. The solubilized crystal violet was then quantified by measuring the optical density (OD) of each well at 550nm. The percentage cytolysis was calculated by the formula: Cytolysis (1 (OD test well OD untreated well)) x 100 Expression and affinity purification of recombinant ovine TNF Figure 4 is a schematic representation of the expression construct showing the site of thrombin cleavage of the fusion protein and restriction enzyme sites used to clone the OvTNF-ca gene encoding the mature protein. Induction of the tac promoter resulted in expression of a fusion protein of approximately 43 kDa as shown by SDS-PAGE A analysis (Fig. The amount of recombinant protein in the soluble fraction was only 5.3% of total proteins as estimated by densitometric scanning of the COmassie-blue stained gel (Fig.5, lane When the growth temperature was lowered to 30 0 C. The level of expression of soluble recombinant product was increased to 8.6% (Fig. 5, lane 4).

Elution of the bound protein with glutathione yielded a fusion protein of 43 kDa.

Thrombin digestion of the fusion protein was performed following elution of the fusion protein with glutathione. This gave rise to GST of 26kDa and the rOvTNF-c of 17kDa lane The GST moiety was rebound on the glutathione affinity column leaving the rOvTNF-ac in the flowthrough. BSA was added to the purified rOvTNF-co as carrier C 4 WO 95/30436 PCT/AU95100261 -26lane 7).

11. Bio-assay Recombinant ovine and human TNF-a demonstrated similar levels of activity in the L929 assay (Fig. In repeated assays, both TNF-a preparations routinely generated cytotoxicity for L929 cells at concentrations of approximatelty Ing/ml. This equatesto a biological activity of 1 x 106 units per ml which is within assay variablility of that quoted for the human preparation. The rOvTNF-a GST fusion protein exhibited the same activity in the L929 assay as the purified protein.

Before use in vivo, the purified cytokines were passed through a Detoxi column (Pierce) to remove the possibility of endotoxin contamination.

Recombinant ovine IL-Ip was expressed and purified as previously described (Seow et al, 1994). rGM-CSF was obtained from a Chinese haitster ovary cell expression system and its specific.activity was determined from a liquid proliferation assay using bone marrow cells.

EXAMPLE 3 VACCINE PREPARATIONS The recombinant Taenia ovis fusion protein GST-45W (Johnson et al., 1989) was used as the model antigen in all studies. Vaccines were formulated with 50g/dose of GSTand 0, 10 or 100g/dose of IL-1p and/or TNF-a in either phosphate buffered saline (PBS) or the conventional adjuvants Quil A (1 or 5mg/ml), incomplete Freund's adjuvant (IFA; 1:1, oil:water) and aluminium hydroxide (6 mg/ml). Sheep were injected intra muscularly (1ml) into the left hind leg for the primary inoculation and 4 weeks later boosted with an i/m injection of the same vaccine preparation into the right hind leg.

i WO 95/30436 PCT/AU95/00261 -27- EXAMPLE 4

SEROLOGY

Serum examples were collected from all animals before the primary inoculations and then at weekly intervals until 4 weeks post secondary inoculation. Sera was stored at 0 C until assayed for antibodies to 45W using the enzyme immunoassay (EIA).

described below. Pre-bleed sera from all sheep was screened for antibodies to prior to the commencement of experiments and any animals demonstrating significant antibody levels to 45W (EIA OD 0.2 at 1/300 serum dilution) were excluded. For the EIA, either recombinant 45W, thrombin cleaved and purified from the GST moiety or (as indicated), was bound to 96-well microtitre plates (Nunc Maxisorb) by incubating 0.2tg per well in 100l of 50mM carbonate buffer (pH 9.6) for 20hrs at The plates were then post-coated (Ihr at 20 0 C) with 100gl per well of phosphate buffered saline (PBS: 0.9% w/v, pH 7.2) containing 1% w/v sodium casein. After 4 washes with phosphate buffered saline containing 0.05% v/v Tween 20 (PBST), 100l of serial dilutions of serum samples were added to the wells for lhr at 20 0 C. The plates were then washed 4 times with PBST before the addition of 100pl per well of a 1/1000 dilution of horseradish peroxidase conjugated anti-ovine IgG monoclonal antibody Silenus, Australia) in PBST for 1 hr at 20 0 C. Plates were washed 5 times with PBST and 1001l of tetra-methyl benzidine (TMB) substrate (Bos et al, 1981) added to each well for 30min at 20 0 C before the reaction was stopped by the addition of 50tl of

H

2

SO

4 per well and the absorbance read at 450nm.

EXAMPLE ADJUVANT ACTIVITY OF rOvIL-1B Sheep were randomly allocated into 12 groups of 5 as detailed in Table 2. Serum samples were collected prior to first vaccination and then at weekly intervals until 4 weeks post secondary vaccination. Serum antibody levels to purified 45W were quantified by EIA. All vaccine formulations contained 50ug of GST-45W per dose.

The addition of either 10 or 100g of rOvIL-lp to the aqueous (PBS; Fig. 7) and A1OH (Fig. 8) vaccine formulations resulted in significant increases in serum IgG Both concentrations of rOvIL-lp tested stimulated antibody production to the same i i WO 95/30436 PCT/AU95/00261 -28extent. The incorporation of rOvIL- 1 in the Quil A (5mg/dose) and IFA formulations did not further enhance the antibody levels elicited (Fig. 9).

EXAMPLE 6 ADJUVANT ACTIVITY OF rOvTNF-a An experiment similar to that described in Example 5 for rOvIL-P was conducted with rOvTNF-a to ascertain its adjuvant potential. The vaccine preparations for this experiment are lister: 'able 3. Serum antibody levels were quantified by EIA using antigen. As in Example 5, adjuvant effects were seen in the PBS (Fig. and AlOH (Fig. 11) vaccine groups when either 10 or 100g of rOvTNF-a were incorporated. Recombinant OvTNF-a alone as adjuvant was sufficient to induce antibody production with the 100tg dose being more effective than 10g. There was no enhancement of antibody titre in the Quil A groups when rOvTNF-a was added (Fig.

12).

EXAMPLE 7 ADJUVANT ACTIVITY OF rOvIL-1/ AND rOvTNF-a IN COMBINATION The combination of 10tg of both IL-1P and TNF-a was studied in AIOH vaccine formulations. Table 4 shows the vaccine groups, and the antibody titres to at two weeks post second vaccination, are shown in Figure 13. The two cytokines exerted synergistic co-adjuvant effects when administered with A1OH which resulted in a 15 fold increase in antibody titre compared to that obtained when A1OH alone was used as adjuvant (Fig. 13). The level of antibody obtained with the AOH-cytokine combination was commensurate with that obtained with Quil A.

w IL r i i-- WO 95/30436 PCT/AU95/00261 29 TABLE 2 rOvIL-f Vaccine Preparations VACCINE FORMULATION ig of rOvIL-1 P 1/PBS 0 2/PBS 3/PBS 100 4/ QUIL A (5 mg/dose) 0 QUIL A (5 mg/dose) 6/ QUIL A (5 mg/dose) 100 7/IFA (Oil:H 2 0, 1:1) 0 8/ IFA (Oil:H 2 0, 1:1) 9/ IFA (Oil:H 2 0, 1:1) 100 A1OH (6 mg/dose) 0 11/ AOH (6 mg/dose) 12/ AlOH (6 mg/dose) 100 All vaccine preparations were formulated to a volume of lml per dose containing of GST-45W fusion protein. There were 5 sheep per vaccine group. Animals were injected i/m in the left rear leg on day 0 and received a second i/m injection in the right leg on day 28.

WO 95/30436 PCT/AU95/00261 TABLE 3 rOvTNF-a vaccine preparations VACCINE FORMULATION jlg of rOvTNF-a 1/ PBS 0 2/ PBS 3/ PBS 100 4/ QUIL A (1mg/dose) 0 QUIL A (1mg/dose) 6/ QUIL A (Img/dose) 100 7/ AlOH (6mg/dose) 0 8/ AlOH (6mg/dose) 9/ AlOH (6mg/dose) 100 Controls (no vaccine) All vaccine preparations were formulated to a volume of lml per dose containing of GST-45W fusion protein. There were 5 sheep per group. Animals were injected i/m in the left rear leg on day 0 and received a second i/m injection in the right rear leg on day 28.

I__

il. I1 I WO 95/30436 PCT/AU95/00261 -31 TABLE 4 Combined rOvTNF-a and rOvlL3 vaccine preparations VACCINE FORMULATION jpg of rOvTNF-a I.g of rOvIL-1 1/ A1OH (6mg/dose) 0 0 2/ AlOH (6mg/dose) 10 0 3/ AlOH (6mg/dose) 0 4/ A1OH (6mg/dose) 10 QUIL A (1mg/dose) 0 0 6/Controls (no vaccine) All vaccine preparations were formulated to a volume of 1ml per dose containing of GST-45W fusion protein. There were 5 sheep per group. Animals were injected i/m in the left rear leg on day 0 and received a second i/m injection in the right rear leg on day 28.

EXAMPLE 8 ADJUVANT ACTIVITY OF rOvIL-1# AND rOvGM-CSF This example shows the ability of ovine IL-l and ovine GM-CSF to boost antibody levels to the protective H.C. antigen tropomyosin in the presence of the conventional adjuvant A1OH 3 The results are shown in Table 5 and Figures 14 and WO 95/30436 PCT/AU95/00261 -32- TABLE Vaccine formulation pg of rOvIL-13 pg of rOvGM-CSF 1. Nil 0 0 2. Al(OH) 3 (6mg/dose) 0 0.

3. Al(OH) 3 10 0 4. Al(OH) 3 0 1 Al(OH) 3 0 6. AI(OH)3 10 1 7. Al(OH) 3 10 8. QuilA(lmg/dose) 0 0 Table 5 shows the various formulations used. Each group consisted of 5 sheep.

Vaccines were formulated with 50 pg/dose of recombinant Haemonchus contortus tropomyosin antigen and aluminium hydroxide with 10 pg/dose of IL-1P and/or .Lg/dose of GM-CSF. Another group of 5 sheep were vaccinated with the same antigen with either Quil A or aluminium hydroxide. Sheep were injected subcutaneously into the right hind leg for primary inoculation and 4 weeks later boosted with another subcutaneous injection of the same vaccine preparation. Blood was collected from all animals just before the primary inoculation and then fortnightly until week 10. The sera were stored at -20 0 C until all sera was collected. The titre of antibodies to tropomyosin (41 kDa) was measured using standard enzyme immunoassay (EIA).

Figure 14 is a graphical representation showing the antibody titres from sheep vaccinated with the recombinant Haemonchus contortus tropomyosin antigen with aluminium hydroxide alone and in combination with either 1 Jtg/dose or 10 p.g/dose of rGM-CSF.

j WO 95/30436 PCT/AU95/00261 33 Figure 15 is a graphical representation showing the mean titres of antibody over the ten week period. The highest amount of antibody was detected at week 6 post primary inoculation in all the groups. Figure 15 was generated using data from only 4 animals in the aluminium hydroxide +IL-13 GM-CSF (10ig) vaccination group as one animal failed to respond to the booster vaccination. However, statistical analysis was done on data from all 5 animals and therefore the results of the analysis are conservative. The difference in mean titres at this time were significantly difference (F( 5 24 43.5, p<0.0001) and the treatments formed three distinct clusters that are significantly different from each other (Tukey HSD); cluster 1 the nil treatment; cluster 2 aluminium hydroxide, aluminium hydroxide IL- 1 and aluminium hydroxide IL- GM-CSF (I1g), and cluster 3 aluminium hydroxide IL-1p GM-CSF (10tg) and Quil A.

The addition of the IL- 1 to aluminium hydroxide enhanced mean antibody titre by 1.7 fold compared to aluminium hydroxide alone. The combination of IL-1 and GM-CSF (1jg) added to aluminium hydroxide increased the mean titre 1.3 fold over aluminium hydroxide alone. At this time the combination of IL-1p and GM-CSF (0Og) added to aluminium hydroxide increased the mean titre 2.3 fold over aluminium hydroxide alone. Quil A was the most effective adjuvant with the mean titre 4.9 fold higher than aluminium hydroxide. It has been previously shown with other antigens that IL-1 can act as an adjuvant. This is the first demonstration that the combination of IL-1 and GM-CSF with aluminium hydroxide was a better adjuvant than aluminium hydroxide alone. The antibody titre with the combination of IL- 1 and GM-CSF was lower than with Quil A.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

U_ I WO 95/30436 PCT/AU95/00261 34

BIBLIOGRAPHY

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WO 95/30436 PCT/AU95/00261 37 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT (other than US): COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH

ORGANISATION

APPLICANT (US only): CORNER, L A; ROTHEL, J S; SEOW, H-F; and WOOD, P R; and McWATERS P (ii) TITLE OF INVENTION: AN ADJUVANT (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT INTERNATIONAL FILING DATE: 04-MAY-1995 (vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: PM5438/94 (AU) FILING DATE: 04-MAY-1994 (viii) ATTANEY/AGENT INFORMATION: L'VAE: HUGHES, E J L (0 EFERENCE/DOCKET NUMBER: EJH/EK (ix) FELECOMMUNICATION INFORMATION: TELEPHONE: (613) 254 2777 TELEFAX: (613) 254 2770 y N- 4C II WO 95/30436 -38- INm'ORi'tf.'XON FOR SEQ ID NO:1: iQ SGENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: PCT/AU95/00261 GGATCCGCAG CCGTGCAGTC A INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEO ID NO:2: CCGGTCGACt AGGGAGAGAG GGTTTCCATT C INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGCGGATCCC TCAGGTCATC TTCTCAAGCC INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TCACAGGGCA ATGATCCCAA AGTA

A

I

WO 95/30436 PCT/AU95/00261 -39 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID CTG GTT CCG CGT GGA TCC GCA GCC GTC 29 Leu Val Pro Arg Gly Ser Ala Ala Val 1 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Leu Val Pro Arg Gly Ser Ala Ala Val 1 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTC TCT CCC TAGTCGGGAA TTCAT Lea Ser Pro INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 3 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Ser Pro

Claims (22)

1. An adjuvant composition, said composition comprising at least two cytokine activities acting in synergy to enhance an immune response to an antigen when administered to an animal or bird, wherein said composition comprises either the combination of IL-1P and TNF-a or the combination of IL-1P and GM-CSF a functional derivative of one or more cytokines thereof.

2. The adjuvant composition according to claim 1 wherein the cytokines are fused 10 together or one or both of each cytokine are fused with another molecule.

3. The adjuvant composition according to claim 2 wherein one or both of the cytokines are fused to glutathione-S-transferase (GST). 15 4. The adjuvant composition according to any one of claims 1 to 3 wherein the animal is a human or livestock animal. C 5. The adjuvant composition according to any one of the previous claims further comprising an antigen.

6. The adjuvant composition according to claim 5 wherein the antigen is derived from a bacterium, virus, insect, yeast, fungus, nematode or cestode.

7. The adjuvant composition according to claim 6 wherein the antigen is a nematode antigen.

8. The adjuvant composition according to claim 7 wherein the nematode antigen is from Haemonchus, Trichostrongylus or Ostertagia.

9. The adjuvant composition according to claim 8 wherein the antigen is H.contortus P:\OPER\MRO\23411-95.CLA 26/98 -41- tropomyosin. The adjuvant composition according to claim 6 wherein the cestode antigen is from Taenia ovis.

11. A multi-compartment pharmaceutical pack comprising a first compartment adapted to contain a first cytokine comprising IL-1p and a second compartment adapted to contain a second cytokine, comprising TNF-a or GM-CSF, wherein said first and second cytokines when mixed act in synergy to enhance an immune response to an antigen and the first and o second compartments are combined with a particular antigen prior to use. c

12. A method of enhancing, increasing, upwardly modulating or otherwise facilitating an immune response in an animal or a bird to an antigen, said method comprising administering S' to said animal or bird an immune-enhancing effective amount of an adjuvant composition which comprises two cytokines which act in synergy to enhance an immune response to an 15 antigen, wherein said adjuvant composition comprises the combination of IL-1 and TNF-a or the combination of IL-1 and GM-CSF or a functional derivative of one or more cytokines thereof.

13. The method according to claim 12 wherein the animal is selected from a human, livestock animal, laboratory test animal, a domestic animal and a captive wild animal.

14. The method according to claim 12 wherein the bird is a chicken or other poultry bird. The method according to any one of claims 12 to 14 wherein the cytokines are administered sequentially.

16. The method according to any one of claims 12 to 14 wherein the cytokines are administered simultaneously. S 17. The method according to any one of claims 12 to 16 further comprising administration I i tr iwttffWF c C CC P:\OPER\MRO\23411-95.CLA -2/6/98 -42- of the cytokines with an antigen against which an immune response is desired.

18. The method according to claim 17 wherein the antigen is derived from a bacterium, virus, insect, yeast, fungus, nematode or cestode.

19. The method according to claim 18 wherein the antigen is a nematode or cestode antigen. The method according to claim 19 wherein the nematode antigen is derived from Haemonchus, Trichostrongylus or Ostertagia.

21. The method according to claim 20 wherein the antigen is H. contortus tropomyosin.

22. The method according to claim 19 wherein the cestode antigen is 45W from Taenia 15 ovis.

23. A vaccine composition comprising an antigen and a synergistic combination which comprises at least IL-13 and TNF-a; or (ii) IL-1P and GM-CSF or a functional derivative or one or more cytokines thereof, wherein said synergistic combination is capable of enhancing an immune response to said antigen when administered to an animal or bird, said vaccine composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.

24. A genetic adjuvant comprising one or more nucleic acid molecules encoding at least two polypeptides having cytokine activities which are capable of acting in synergy to enhance an immune response to an antigen, wherein said two polypeptides having cytokine activities are selected from the combination of IL-1l and TNF-a or the combination of IL-1 and GM- CSF, and wherein said genetic adjuvant enhances an immune response to an antigen when administered to an animal or bird. *r S C V T 0 ',Nr a3 1 7 PMOPIR\MRO2341 l-95,CLA -26198 -43- The genetic adjuvant according to claim 24 further comprising one or more nucleic acid molecules which encode the antigen.

26. The genetic adjuvant according to claims 24 or 25 wherein the antigen is derived from a bacterium, virus, insect, yeast, fungus, nematode or cestode.

27. The genetic adjuvant according to claim 26 wherein the antigen is a nematode or cestode antigen. 10 28. The genetic adjuvant according to claim 27 wherein the nematode antigen is from Haemonchus, Trichostrongylus or Ostertagia. Ie

29. The genetic adjuvant according to claim 28 wherein the Haemonchus antigen is H. contortus tropomyosin.

30. The genetic adjuvant according to claim 27 wherein the cestode antigen is the antigen derived from Taenia ovis. I I DATED this SECOND day of JUNE, 1998. Commonwealth Scientific and Industrial Research Organisation by DAVIES COLLISON CAVE Patent Attorneys for the Applicants 51AT QA<