NZ254695A - Vaccine with ovine cytokine polypeptide as adjuvant - Google Patents

Description

New Zealand No. 254695 International No. PCT/AU93/00425 Priority Dato(s): j Compete Specification Filed: .2©.l§.l5i3..

Class: Pubflcetion D«tt:..w2.f!...D.CI..1396.. i P.O. Journal No: 1*±£.?L.

NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Cytokine applications Name, address and nationality of applicant(s) as in international application form: THE UNIVERSITY OF MELBOURNE, an Australian body corporate of Grattan Street, Parkville, Victoria 3052, Australia; AUSTRALIAN WOOL RESEARCH AND PROMOTION ORGANISATION, an Australian body corporate of 369 Royal Parade, Parkville, Victoria 3052, Australia - |— (FOLLOWED BY PAGE 1A) 254695 WO 94/04174 *■ w I W V %f PCT/AU93/00425 - lA- CYTOKINE APPLICATIONS The present invention relates to vaccine compositions including ovine cytokines/ and to methods of use of such vaccines.

Cytokines are important polypeptides which display significant immuno-regulatory and inflammatory activities in animals. Whilst the ovine immune system has been extensively utilised in lymphocyte recirculation studies and as a large animal model for the study of immune 10 responses to infectious diseases, little is known about either the production of cytokines by ovine leukocytes or the regulation of cellular function by cytokines.

An important cytokine produced by macrophages is Interleukin-1 (IL-1). The bioactivities ascribed to IL-1 15 are produced by two molecules (IL-la and IL-1S) encoded for by two distinct genes.

Other important cytokines include Interleukin-6 (IL-6) and the Interleukin-2 (IL-2).

The production of interleukin-2 and expression of 20 its high affinity receptor is essential for T cell proliferation and differentiation in the development of an immune response.

Although the importance of IL-2 and its Receptor in humans and various other species has been demonstrated, 25 its involvement in the ovine immune response to infection and disease is as yet uncharacterised. This is of particular importance due' to the potential application of cytokines as adjuvants in, e.g. vaccine compositions.

In international application PCT/AU91/00358, to 30 applicants, the entire disclosure of which is incorporated herein by reference there is disclosed a method for the production of synthetic ovine cytokines including interleukin-lB and interleukin-2. However,. the effect, if any, of such synthetic ovine cytokines on the ovine immune 35 response to infection and disease has not been established. For example, in vaccine compositions known generally in the prior art, it is often desirable to enhance the immunogenic effect of an antigen in order to obtain a stronger immune response by utilising such an 4 6 9 5 pct/a u93/00425 adjuvant. However certain adjuvants have been found to lack stability and may even generate unwanted side effects such as chronic inf 1 animation.

A particular problem is also encountered in the 5 prior art where multi-component vaccines are used. Such multi-component vaccines often suffer from antigenic competition which may significantly reduce their potential effectiveness.

Accordingly, it is an object of the present 10 invention to overcome/ or at least alleviate/ one or more of the difficulties relating to the prior art.

Accordingly, in a first aspect of the present invention there is provided a vaccine composition including an antigen against a disease of interest; 15 a non-tozic adjuvant including a recombinant polypeptide having ovine cytokine or ovine cytokine receptor activity, or mimotopes/ derivatives or fragments thereof; and a non-tozic coadjuvant selected to stabilise 20 and/or enhance the immune response to the recombinant polypeptide adjuvant.

It has surprisingly been found that use of the non-toxic adjuvants according to the present invention may enhance immune response to the vaccine composition by for 25 example 4 to 10 fold or more. Thus the time of effect and/or degree of biological effect may be extended greatly.

The antigen may be derived from any source including viral, fungal, bacterial or parasite antigens, auto-immunity related antigens or tumor-associated 30 antigens. The antigen may also be derived from plants, for example allergens and plant toxins.

The vaccine composition is particularly suitable for treatment of parasitic diseases.

These parasites include, for example, Haemonchus 35 contortus, Trichostrongylus colubriformus and Ostertagia circumcincta (gastrointestinal nematodes), Bacteroides Rodosus (foot rot), Lucilia cuprina (blowfly sti.

Staph, aureus (mastitis) and C.ovis (cheesy glanjt^* ** The^ cytokines may function as adjuvants in combination with t*..

\ N \v_ PCT/A U93/O0425 existing antigens or new recombinant antigens that are being developed.

The recombinant polypeptide having ovine cytokine or cytokine receptor activity, or mimotope thereof, or 5 fragment thereof may preferably be selected from the group consisting of IL-la, II-IB, IL-1R, IL-2, IL-2R, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-10R, IL-12, IFN-y/ IFN-yR, TNF-a, TNF-aR, GMCSF, GMCSFR, TGF-fi and 10 TGF-BR, TNP, more preferably IL-la, IL-1B, IL-2 or IL-6, or mixtures thereof.

The antigen . may be present in the vaccine composition in any suitable amounts. The antigen may be present in amounts of from approximately 1% to 99% by 15 weight based on the total weight of the vaccine composition, preferably approximately 10% to 50% by weight.

The vaccine composition may further include a carrier or excipient therefor for veterinary use.

Whilst the vaccine composition may be utilised in 20 the treatment of ruminant animals, especially sheep, goats and cattle, it may also be utilised in the treatment of other animal species, in particular pigs, cats, dogs and horses.

The cytokines may be used to induce, enhance or 25 modulate an immune response against an antigen.

The vaccine composition containing the recombinant polypeptide and antigen'may be incorporated in any pharmaceutical^ acceptable vehicle with or without added co-adjuvants or immunostimulatory molecules. 30 The co-adjuvant may be of any suitable type. The co-adjuvant may be selected from vegetable oils or emulsions thereof, surface active substances, e.g., hexa-decylamine, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecyl-ammonium bromide, 35 N,H-dicoctadecyl-H'-N'bis(2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; immune stimulating complexes (ISCOMS); oil emulsions; and mineral PCT/AL'93/00425 gels and suspensions. A mineral suspension such as alum, i.e. aluminium hydroxide (Al(OH>3), aluminum phosphate or aluminium sulphate is preferred.

The antigen may preferably be absorbed onto the 5 mineral suspension to further modulate the protective immune response to the cytokine antigen. A vector-soluble polymeric adjuvant such as Pluronic gel has been found to be suitable.

The co-adjuvant may be present in any suitable 10 amounts. The co-adjuvant may be present in amounts of from approximately 5 to 75% by weight, preferably approximately 20% to 50% by weight, based on the total weight of the non-toxic adjuvant.

Utilisation of the recombinant polypeptide is 15 particularly suitable where a site-specific immune response is sought, for example in the mucosa, mammary gland, gut, mouth, skin or the like.

For different diseases, different recombinant cytokines or cytokine receptors, or combinations thereof 20 may be more suitable. Similarly, the route of delivery of the cytokine may vary for both different cytokines and different vaccine preparations.

The recombinant polypeptide may also be utilised as a non-toxic adjuvant in combination with a viral vector. 25 Utilisation of the recombinant polypeptide is particularly suitable where a plurality of antigens are to be used in a multi-component vaccine. It has been surprisingly found that the adjuvants according to the present invention function to reduce or eliminate 30 competition between the various antigens in a multi-component vaccine.

Accordingly, in a preferred aspect, there is provided a vaccine composition including an effective amount of a plurality of antigens against diseases of interest; a non-toxic adjuvant including a recombinant polypeptide having ovine cytokine or ovine cytokine rec activity, or mimotopes, derivatives or fragments th^freo 4 6 9 5 WO 94/04174 PCT/AL'93/00425 and a non-toxic co-adjuvant.

The antigens may be present in any suitable combination. A combination of 3, 4, or 5 antigens may be 5 used.

The non-toxic adjuvant may be an ovine IL-la, IL-1B or IL-2, preferably in combination with an aluminium hydroxide as co-adjuvant.

The non-toxic adjuvant may be present in amounts 10 effective to reduce or eliminate competition between the various antigens. The non-toxic adjuvant may be present in amounts of from approximately 1 to 75% by weight, based on the total weight of the vaccine composition, preferably approximately 7.5% to 50% by weight. 15 The compositions according to the invention may take any form suitable for administration including forms suitable for oral or parenteral (including implant) use. For oral administration the compositions may take the form of, for example solutions, syrups or suspensions e.g. in 20 agueous buffer, or solid compositions such as tablets or capsules, prepared by conventional means. For parenteral use, the compositions may for example take a form suitable for injection, such as a suspension, solution or emulsion in an agueous or oily vehicle optionally containing 25 formulatory agents such as suspending, stabilising, solubilising and/or dispersing agents. The agueous or oily vehicle may include any other adjuvant known per se.

In a further aspect of the present invention there is provided a method for the treatment of disease in 3 0 non-human animals which method includes administering to a non-human animal a therapeutically or prophylactically effective amount of a vaccine composition including an antigen against a disease of interest; a non-toxic adjuvant including a recombinant 3 5 polypeptide having ovine cytokine or ovine cytokine receptor activity, or mimotopes, derivatives or fragments thereof; and a non-toxic coadjuvant selected to stab)i'£i*s*e0< and/or enhance the immune response to the recoqjalnan \i PCT/A U93/00425 polypeptide adjuvant.

Preferably the vaccine composition is a multi-component vaccine composition as described above.

The vaccine composition may be administered via 5 any suitable route. The vaccine composition may be administered to an animal in a variety of ways. These include intradermal, transdermal (such as by slow release polymers), intramuscular, intraabomasomal, intraperitoneal, intravenous, subcutaneous, oral, intranasal, intramaromary, 10 intrarectal and intravaginal routes of administration.

Preferably the animal may be subjected to a plurality of vaccinations. For example a primary and secondary vaccination may be administered. Desirably the non-toxic adjuvant is included in both the primary and 15 secondary vaccines. However where the adjuvant is included in only one of the vaccines, the secondary vaccine is preferred.

It has surprisingly been found that the effectiveness of the vaccine may be affected by the route 20 of vaccination. An intramuscular, subcutaneous or intradermal route of administration is preferred. An intramuscular route is particularly preferred.

The present invention will now be more fully described with reference to the following examples. It 25 should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the figures: Figure 1 is a graph showing secondary antibody titres for various combinations of the model antigen, avidin with ovine IL-1B.

Figure 2 is a graph showing secondary antibody titres for various combinations of the model antigen, 35 avidin with IL-1B, together with the co-adjuvant Al(OH)3.

Figure 3 is a graph showing secondary antibody titres for various combinations of the model antigen, avidin, with and without IL-lfl in a Pluronic gel.

Figures 4a and 4B are graphs showing 14 day post 4 69 5 primary and 14 day post secondary antibody titres for various combinations of the model antigen, avidin, with and without Al(OH)3 and IL-lfi.

Figures 5a and 5b are graphs showing primary and secondary antibody titres for various combinations of the model antigen, avidin with various adjuvant combinations.

Figure 6 is a graph of serum assay versus time for a combination of an experimental vaccine antigen isolated from the nematode Haemonchus contortus with IL-16 or IL-2 and optionally the co-adjuvant Al(OH)3.

Figure 7 is a graph showing 21 day post secondary antibody titres for Avidin plus IL-1B in A1(0H)3 introduced via differing routes.

Figure 8 is a graph showing 21 day post secondary antibody titres for Avidin plus IL-1B in A1(0H)3 where the IL-1B is present in either the primary and/or secondary immunisations.

Figure 9 is a graph showing 28 day post primary and 14 day post secondary antibody titres for Avidin plus IL-la in A1(0H)3 introduced via differing routes.

Figure 10a shows the primary anti-avidin antibody response in the presence and absence of alum when injected intra-muscularly to test the adjuvant ability of IL-la- Figure 10b shows the secondary anti-avidin antibody response in the presence and absence of alum when injected intramuscularly to test the adjuvant ability of IL-la.

MATERIALS AMD METHODS Animals For trials using avidin as a model Ag, merino cross-bred sheep between 12 and 3 6 months of age were used. Five month old merino lambs from H-contortus free pastures were used in the trial involving the purified experimental H. contortus antigen. All animals were housed in the School of Veterinary Science, The University of Melbourne and fed oats and lucerne chaff ad libitum.

Recombinant PNA techniques Standard recombinant DNA techniques were used and oligonucleotide-directed in vitro mutagenesis was performed using the in vitro Mutagenesis System develoEJP^TT3^55^-. ^ 1 ° f A by Amersham Ltd. (U.K.). Oligonucleotides ^»Vre ' v synthesised on an Applied Biosystems 391 DNA-Synthesisfer.

Growth and induction of bacterial cultures ^ ^ E.coli strain IB392 [rel-1 tonA22 HfrC laci^® ^ ;r P02A T2r traD(M13r y2r)] was transformed w&sj' the Celltech expression vector containing the modified ovine complementary DNA (cDNA) sequences (see Results 'section) for production of recombinant proteins. Cultures were grown overnight in M9 medium at 30°C and then diluted 1 in 20 in fresh medium and grown at 34°C until an OD^qq 5 of 1 was reached. Plasmid amplification was induced by 11 incubating cells at 42°C for 20 minutes followed by a 5 hour incubation at 37°C to allow expression of recombinant protein. Cells were harvested by centrifugation (5000 rpm, 30 rain) and pellets stored at 20°C until required. io Qvine cytokine bioassavs The NOB—1/CTLL assay for measuring IL-1 activity and the WEHI-164 assay for determination of TNFa activity were performed as previously described. Proliferation of Concanavalin A (Con A) stimulated ovine 15 lymph node cells was used to assay for ovine IL-2 activity. Briefly, ovine lymph node cells were dispersed using a fine stainless steel mesh, washed and resuspended at 1 x 106/ml in RPMI-1640 supplemented with 10% v/v fetal calf serum, 2 raM glut amine, 100 U/ml penicillin and 20 0.1 mg/ml streptomycin (RF10). Cells were cultured for 72 hours at 37°C with 5% C02 in the presence of 10 jig/ml of Con A then harvested and washed 3 times. Cells were resuspended at 4 x 10 /ml in RF10 and 100 jil of this suspension added to flat bottom microtitre plates 25 containing 100 yl of appropriately diluted test rovIL-2. After incubation for 48 hours at 37°C with 5% CO,, wells were pulsed with 1 yCi/well of H-thymidine (Amersham, United Kingdom) and harvested 24 hours later for counting. All test samples were assayed in duplicate. 30 Immunisations and infections For induction of anti-avidin responses, sheep were injected intradermally (id) or intramuscularly (im) with 100 fig of egg white avidin (Calbiochem, San Diego, California) in a phosphate buffered saline (PBS) based 35 formulation containing aluminium hydroxide (alum, Commonwealth Serum Laboratories, Melbourne, Australia) prepared as a 6 mg/ml solution and used at a final concentration of 1 mg/ml. Recombinant ovIL-la or ovILIB were incorporated into this mixture at the concentrations PCT/ A U93/00425 described in the results. For id immunisations a total ~ volume of 200 >il was administered into the inner thigh in 2 x 100 fil injections and for im immunisations a total volume of 2 ml was injected into the biceps femoris 5 muscle. Primary immunisations were given at day 0 and secondary immunisations on day 28. Sheep were bled prior to immunisation and then at 14 days post primary and post secondary immunisations.

For induction of anti-H. contortus responses, 10 sheep were injected id with 10 fig of a unique glycoprotein present on the surface of third and fourth stage H. contortus larvae. Immunisation with this Ag has been demonstrated to confer significant protection against subsequent challenge with H. contortus larvae (H. Jacobs, 15 unpublished results). The Ag was formulated in alum as described above and where indicated 10 pg of rovlL-lfi was included in the formulation. Sheep were immunised on days 0, 32 and 53 (identical preparations on each occasion) and sera collected weekly throughout the 20 course of the trial. Twenty one days after the final immunisation all sheep in the trial were orally infected with 10,000 viable third stage H. contortus larvae. Fecal samples were collected starting at 20 days post infection and fecal egg counts performed every second or third day 25 thereafter.

Measurement of anti-a-viflin an<3 anti-K, contortus antigen responses Sera were assayed by ELISA to determine avidin and H. contortus Ag specific antibody (Ab) titres. For 30 the avidin ELISA, plates were coated for 1 hour at 37°C with avidin (50 pl/well of a 10 pg/ml solution) in carbonate coating buffer (pH 9.6). Following 3 washes withe PBS + 0.05% Tween 20, plates were blocked for 1 hour at 37°C with skim milk powder (2% w/v in PBS). After 3 35 further wash steps, serial twofold serum dilutions were prepared in duplicate and added to the plates and incubated for 90 minutes at 37°C. Plates were then washed 3 times prior to the addition of 50 }il per well of rabbit anti-sheep Ig conjugated to horseradish peroxidase (HRPO, DAKO, Denmark). Conjugate was diluted at 1:5000 in ' blocking solution and incubated at 37°C for 1 hour prior to 3 further washes. Plates were developed with 100 pl/well of TMB substrate (3,3*.5,5'-tetramethyl-benzidine 5 dihydrochloride hydrate 97%, Aldrich Chem. Co., Milwaukee, Wisconsin). After 10 minutes, the reaction was stopped by the addition of 100 til/well of 2 M H^SO^. Absorbance was determined using a Titertek multiscan MCC plate reader in dual wavelength mode (450-690nm) . The assay of H. 10 contortus specific Ab was essentially identical to that described for avidin specific antibodies except that plates were coated with the experimental H. contortus Ag.

Avidin specific Ab levels were determined for serum of individual animals and group mean titres were 15 then calculated. Only pooled sera from each group were assayed for H. contortus specific Ab. Any minor variation in the background Ab levels was accounted for by subtraction of the prebleed Ab level. Results were examined statistically by the Students t test using the 20 log of the reciprocal serum dilution that produced half the maximal absorbance.

Isotvpino of the H. contortus specific Ab response For determination of the effects of rovIL-lB on the isotype of the H. contortus specific Ab response 25 pooled sera from each group were assayed basically as described above but with the inclusion of isotype specific second antibodies and *where necessary appropriate HRPO conjugates. H. contortus specific Ab of the IgM isotype was determined using a HRPO conjugated rabbit anti-sheep 30 igM reagent (KPL Inc., Gaithersberg, U.S.A.) at a final dilution of 1:100. H. contortus specific IgG^ antibodies were determined using an ovine igG^ specific monoclonal Ab (ascites used at a concentration of 1:100,00, gift from Dr. K. Beh, CSIRO Animal Health, Sydney) and a HRPO 35 conjugated rabbit anti-mouse Ig reagent (Dako, Denmark). IgG2 antibodies specific for the H. contortus Ag were detected using rabbit anti-sheep IgG2 antisera (used at a concentration of 1:1000, gift from Dr. K. Beh, CSIRO Animal Health, Sydney) and a HRPO conjufated sheep PCT/A U93/00425 anti-rabbit Ig reagent (Silenus, Hawthorn, Australia). All assays were blocked with a 2% w/v solution of bovine serum albumin. Sera were titrated and assayed for the specific isotypes in triplicate with results expressed as 5 the ratio (IgG1:IgG2, IgG^IgM, lgG2:IgM) of the log of the mid-point titre for each isotype.

Production and purification of rovIL-lB Ovine IL-1B was cloned from a cDNA library produced with RHA isolated from LPS stimulated ovine 10 alveolar macrophages. A number of cDNA isolates were sequenced and the predicted amino acid sequence determined. To confirm that the cloned cDNA did code for a protein product with the expected biological activity, small scale mammalian expression studies were performed. 15 Transfection supernatants, following transfection of COS cells with the IL-18 cDNA in an appropriate expression vector, were shown to contain IL-1 activity using the EJOB1/CTLL IL-1 bioassay.

To allow expression of this cDNA in a bacterial 20 system the DNA was modified in vitro to remove the eukaryotic leader sequence and to place an ATG initiation codon and an appropriate restriction endonuclease site immediately upstream of the amino terminal amino acid of the mature form of IL-1B. This modified form of ovine 25 IL-lfi cDNA was then cloned into a suitable bacterial expression vector and the resultant plasmid used to transform E.coli strain IB392. Temperature induction of this transformed strain resulted in the production of a protein band with a size of approximately 18kDa, the size 30 expected for recombinant ovine IL-1B. This protein was expressed at approximately 30% of total cellular protein. The protein was found in the soluble fraction of total cell extracts and was released from harvested cells by gentle freeze thaw extractions. The protein was further 35 purified by anion exchange chromatography resulting in a recombinant product of greater than 95% purity. This product was shown to possess IL-1 activity with a specific activity • of 2x10 units per mg protein in the NOB1/CTLL assay.

WO 94/04174 PCT/AU93/00425 This purified recombinant ovine IL-1B has been ► used in a number of vaccine trials to determine its potential as a vaccine adjuvant. These trials have used the model protein avidin, existing commercial vaccines and 5 experimental parasite and bacterial vaccines. The results of these trials are set out below.

Production and purification of rovIL-la Ovine IL-la cDNA encodes a polypeptide of 268 amino acids that, based on analysis of human IL-la, is 10 processed to a mature protein of 150 amino acids with a predicted molecular weight of 17,230. The sequence coding for the cleaved portion of the protein was removed and a Bglll site and ATG codon placed upstream of the glutamine residue (amino acid 119) corresponding to the 15 predicted amino terminal amino acid of mature ovine IL-la by two rounds of oligonucleotide directed in vitro mutagenesis.

Induced cells were found to produce a protein corresponding in size to that expected for rovIL-la 20 (17,000 MW) at about 15% of total cellular protein. Similar to rovTNFa, rovIL-la was found to purify in both the soluble and insoluble fractions (50% soluble, 50% insoluble), however we were able to obtain active protein from the insoluble fraction using 6M urea as the 25 denaturation agent (results not shown). In addition we purified rovIL-la from the soluble fraction which was « obtained by subjecting cells resuspended in 50 mM Tris pH8 to 3 freeze thaw cycles (dry ice, 37°C) followed by centrifugation (15,000 g, 20 minutes). This soluble 30 material was then passed down a DE52 anion exchange column (0-250 mM KC1 in 50 mM Tris pH8) and the rovIL-la containing fraction further purified by gel filtration (Superose 12, 3 M urea, 1 mM DTT, 100 mM KC1 in 50 mM Tris pH8) and a second anion exchange column (Mono Q, 0-250 mM 35 KC1 in 40 niM Tris pH 8). Purified rovIL-la was then tested using the N0B1/CTLL bioassay and shown to have a specific activity of 1 x 10 units/mg protein.

PCT/ A U93/00425 EXAMPLE 1 MOUSE TRIAL 1 AVIPIN PLUS Ifr-lfi IN PPg EXPERIMENTAL PROTOCOL: 5 v»g avidin administered subcutaneous ly in PBS Group Number Primary Number of Mice Treatment 1 4 Avidin 2 4 Avidin + 10 iig IL-lfi 3 4 Avidin + 1 |ig IL-1B 4 4 Avidin + 0.1 jig IL-1B Time Procedure Day 0 Primary immunisations Day 14 Bleed : Test serum by anti-avidin ELISA Secondary immunisations: 5 jig avidin in PBS Day 28 Bleed : Test serum by anti-avidin ELISA 20 RESULTS; See Figure 1.

CONORS I PES; No enhancement of antibody titre was observed in either the primary or the secondary responses where IL-1B 25 was administered in a soluble form.

EXAMPLE 2 MOUSE TRIAL 2 AVIDIN PLUS IL-1B IN ALfOH^ EXPERIMENTAL PROTOCOL: 5 i*g avidin administered subcutaneous ly in Al(OH)3 Group Number Number of Mice Prima ry Treatment 1 2 3 4 4 4 Avidin + Al(OH)3 Avidin + Al(OH)3 + 5 yg IL-1B Avidin + Al(OH)3 + 0.5 yig IL-1B PCT/A U93/00425 Time Prcceflmre Day 0 Primary immunisations, Day 14 Bleed ; Test serum by anti-avidin ELISA Secondary immunisations: 5 >ig avidin in PBS 5 Day 28 Bleed : Test serum by anti-avidin ELISA RESULTS: See Figure 2.

Primary Response: No differences between the three groups were 10 observed after the primary immunisations.

Secondary Response: Avidin + Al(OH)3 resulted in an 8 fold increase in antibody titre between the primary and the secondary immunisations. When IL-lfi is also used the increase is 15 more than 16 fold. No difference between the two doses of IL-1 was observed.

The addition of IL-lfi resulted in a 2 to 4 fold increase in antibody titre compared to avidin plus 20 A1(OH)g following secondary immunisation. No differences were seen after the primary immunisations. No differences between the doses of IL-lfi tested were observed.

EXAMPLE 3 MOUSE TRIAL 3 AVIDIN PLUS IL-lfi IN PLURONIC GEL RIMENTAL PROTOCOL: ' jig avidin administered subcutaneous ly in 25% Pluronic Gel.

Group Number Primary Number of Mice Treatment 1 4 Avidin in Pluronic 2 4 Avidin + 10 pg IL-lfi in Pluronic 3 4 Avidin + 1 IL-1B in Pluronic 4 4 Avidin + 0.1 jig IL-lfi in Pluronic Tj.me Prpcefluse Day 0 Primary iramuni sat ions Day 14 Bleed : Test serum by anti-avidin ELISA Secondary immunisations: 5 yg avidin in PBS 5 Day 28 Bleed : Test serum by anti-avidin ELISA RESULTS: See Figure 3.

CONCLUSIONS: When avidin was administered in pluronic gel the 10 addition of IL-lfi resulted in increases in antibody titre after secondary immunisations. No differences between groups were seen after the primary immunisations. No differences between the doses of IL-lfi tested were observed.

EXAMPLE 4 SHEEP IL-lfi DOSE RESPONSE TRIAL EXPERIMENTAL PROTOCOL: 100 yg of avidin administered intradermally in aluminium hydroxide [Al(OH>3] with doses of IL-lfi 20 ranging from 0.01 to 100 yg.

Group Number Number of Sheep Treatment 1 2 Avidin in PBS 2 2 Avidin in Al(OH)3 3 4 Avidin in Al(OH)3 + 0.01 yg IL-IB 4 4 Avidin in Al(OH)3 + 0.1 yg IL-lfi 4 Avidin in Al(OH)3 + 1 yg IL-lfi 6 4 Avidin in Al(OH)3 + yg IL-IB 7 4 Avidin in Al(OH)3 + 100 yg IL-lfi lime Procedure Day 0 Prebleed + Primary Immunisation Day 14 Primary bleed : Test serum by anti-avidin ELISA Day 28 Secondary immunisation Day 42 Secondary bleed : Test serum by anti-avidin ELISA RESULTS: See Figures 4a and 4b.

Primary Response: Both the 100 and 10 yg doses gave responses 8 fold higher than the 0.01, 0.1 and 1 yg doses which showed antibody titres similar to that of avidin + 5 AL(OH)3 alone.

Secondary Response: In general the antibody titres are 4 fold higher after the secondary iromuni sat ions. The same pattern of responses as in the primary are observed, with the 100 and 10 10 yg doses giving 8 to 10 fold increases in antibody levels compared to the lower doses and the avidin plus Al(OE')3 control. conclusions: The 0.01, 0.1 and 1 yg doses showed no 15 improvement in antibody titre over the avidin plus AlCOH)^ control. The 100 and 10 yg doses gave 8 to 10 fold increases with no significant difference in response seen between these two doses.

EXAMPLE 5 SHEEP ADJUVANT TRIAL aim: To compare anti avidin responses following intradermal injection of avidin with a number of different adjuvants including ovine IL-IB.

EXPERIMENTAL PROTOCOL: 100 yg of avidin administered intradermally with various adjuvants.

Group Number 30 (3 Sheep per Group) Treatment 1 Avidin in PBS 2 Avidin in Al Primary Response: Using the response to avidin in PBS (group 1) as a baseline control, MDP as an adjuvant had no effect. IL-1 administered in a soluble form (group 5) produced only a slight improvement in antibody titre. Avidin in 20 Al(OH>2 resulted in an 8 fold increase while avidin in Al(OH)3 plus saponin or IL-IB resulted in 16 and 32 fold increases respectively.

Secondary Response: In general the antibody titres are 4 fold higher 25 after the secondary immunisations. The same pattern of responses as in the primary are observed with avidin in Al(OH)3 plus IL-IB giving the best antibody response. DTH Response: See Table 1.

To give some idea of the relative stimulations of cell mediated immunity, DTH respoonses to a 1 yg intradermal injection of soluble avidin were measured. The response was assessed on a scale of 0 to 3 with respect to induration and erythema.

PCT/ A U93/00425 - 18 -TABLE 1 DTH RESPONSES - SHEEP ADJUVANT TRIAL 24 and 48 hours post a 1 yg intradermal injection of avidin in PBS. 24 hours 48 hours Sheep Group Redness Lump Redness Lump 76 Avidin + 82 Avidin 89 Avidin 68 Avidin + Al(OH>3 + 69 Avidin + ai(oh>3 + ++ 93 Avidin + Al(OH)3 ++ 80 Avidin + il-1 87 Avidin + il-ib 90 Avidin + il-ib 74 Avidin + il-ib + Al(OH)3 +++ +/- ++ 79 Avidin + il-ib + Al(OH)3 +++ +++ + +++ 86 Avidin + il-ib + ai3 produced the best response. The results of DTH testing showed that immunisation with avidin and IL-IB in Al(OH)3 stimulated a greater cellular response compared to that of the other 5 groups. example s TRIAL OF IL-IB AS AN ADJUVANT FOR THE SHEEP RESPONSE TO AN EXPERIMENTAL NEMATODE PARASITE VACCINE AIM: To test the ability of IL-IB to act as an adjuvant, in conjunction with Al(OH)3, for the sheep response to an experimental vaccine antigen isolated from the nematode Haemonchus contortus.

EXPERIMENTAL PROTOCOL: 10 yg of experimental antigen administered intradermally in Al(OH)3 containing, where indicated, 10 yg of IL-IB.

Group Number (10 Sheep per Group) Treatment 1 Antigen in Al(OH)3 2 Antigen in Al(OH)3 plus IL-IB 3 PBS in Al(OH)3 plus IL-IB Time Procedure Day 0 Prebleed Primary immunisation : 10 yg vaccine intradermal route Day 14 Primary bleed : Test serum by anti-experimental antigen ELISA Day 32 Secondary immunisation : 10 yg vaccine intradermal route Day 46 Secondary bleed : Test serum by anti-experimental 35 antigen ELISA Day 53 Tertiary immunisation : 10 yg vaccine intradermal route Day 67 Tertiary bleed : Test serum by anti-experimental antigen ELISA RESULTS: Antiboflv Response: See Figure 6.

For the control group receiving Ag in alum only, 5 H. contortus specific Ab was detectable following the primary vaccination with the titre increasing following each of the subsequent vaccinations. Incorporation of rovIL-lB into the formulation significantly increased the H. contortus specific Ab titre following each 10 vaccination. Following primary vaccination, pooled sera from the group receiving Ag and rovIL-lB ift alum contained H. contortus specific Ab 3 fold higher than that contained in sera pooled from sheep receiving Ag in alum only. This difference increased to 4 fold following the secondary 15 immunisation and was 7 fold following the tertiary immunisation.

CONCLUSIONS: The adjuvant formulation of IL-lfi in Al(OH)3 is superior to Al(OH)3 for enhancing the sheep response to 20 a nematode parasite vaccine antigen.

Influence Of rovIL-lB on the isotype profile of the anti-H. contortus response The influence of rovIL-lB on the isotype profile of the H. contortus specific humoral response was 25 determined. Pooled sera collected after each vaccination from the Ag in alum group and the Ag and rovIL-lB in alum group were titrated using reagents specific for the ovine IgM, IgG1 and IqG2 isotypes. Results are expressed as isotype ratios, and ratios in the presence or absence of 30 rovIL-IB compared (Table 2). As expected both the ratio of IgG^sIgM and IgG2:lgM increased following secondary and tertiary vaccinations. Results demonstrate, however, that rovIL-lfi exerted little influence over the Ag specific isotype profile. While the presence of rovIL-lB 35 resulted iin a marginal increase in IgG1 over IgG2 anc3 IgM after primary immunisation, this effect was not observed after either the secondary or tertiary immunisations and was not found to be significant.

TAQhE Z Ratio of isotvpes of H. contortus Aa specific antibody IL-1Q Isotype ratios* IgGl:IgG2 IgGlJlgM IgG2:IgM Response Primary Secondary Tertiary 1.00*0.0,5 1.10±0.04 1.12*0.03 1.08*0.01 1.14*0.02 1.09*0.03 1.09±0.05 1.27*0.11 1.60±C.05 1.19±0.05 1.36*0.07 1.58±0.04 1.10*0.08 1.15*0.08 1.44*0.03 1.11*0.03 1.20*0.08 1.45*0.03 * the value shown represents the ratio of the log of the mid-point titre for each isotype in an Ag specific ELISA EXAMPLE 7 SHEEP 16 ROUTE TRIAL EXPERIMENT All PROTOCOL: fig of avidin in aluminium hydroxide [Al(OH)j] with either 10 or 100 fig IL-lfi administered via either the intradermal (id, 200 fil final volume), intramuscular (im, 2 ml final volume) or subcutaneous (sc, 2 ml final volume) routes.

Group No.

No. of Sheep Treatment Route 1 4 Avidin in Al(OH)3 + 100 fig IL-lfi sc 2 4 Avidin in Al(OH)3 + fig IL-lfi sc 3 4 Avidin in Al(OH)3 sc 4 4 Avidin in Al(OH)3 + 100 fig IL-lfi im 4 Avidin in Al(OH)3 + fig IL-lfi im 6 4 Avidin in Al(OH)3 im 7 4 Avidin in Al(OH)3 + 100 fig IL-lfi id Time Day 0 Day 14 Day 28 Day 49 RESULTS Procedure Prebleed + Primary immunisation Primary bleed: Test serum by anti-avidin ELISA Secondary immunisation (identical to primary) Secondary bleed: Test serum by anti-avidin ELISA See Figure 7 CONCLUSIONS: Recombinant ovine IL-lfi was able to enhance the anti-avidin antibody response in all reoutes of administration with the best response seen following intramuscular administration. A dose of 100 fig of IL-lfi produced a slightly higher antibody tirrc than a dose of 10 fig. The response following intradermal vaccination was greater than the response following subcutaneous vaccination but IL-lfi was able to increase the antibody levels in all cases.

PCT/AL'93/00425 - 23 -EXAMPLE 8 ADMINISTRATION OF IL-IB IN EITHER PRIMARY OR SECONDARY VACCINATION EXPERIMENTAL PROTOCOL: 100 yg of avidin in aluminium hydroxide [Al(OH)3] was administered to groups of 3 sheep. 10 yg of IL-IB was included in both or either the primary or secondary vaccinations and the resultant antibody titres compared.

Group NO. Of Primary Secondary No.

Sheep Immunisation Immunisation 1 3 Avidin in Al(OH)3 Avidin in Al(OH)3 2 3 Avidin in Al(OH)3 Avidin in Al(OH)3 + 10 yg IL-IB + 10 yg IL-IB 3 3 Avidin in Al(OH).

Avidin in Al(OH)3 + 10 yg IL-IB 4 3 Avidin in Al(OH)3 Avidin in Al(OH)3 + 10 yg IL-IB Time Procedure Day 0 Prebleed + Primary immunisation Day 14 Primary bleed: Test serum by anti-avidin ELISA Day 28 Secondary immunisation (identical to primary) 25 Day 42 Secondary bleed: Test serum by anti-avidin ELISA RESULTS: See Figure 8 CONCLUSIONS: The addition of recombinant ovine IL-IB in both 30 the primary and secondary vaccinations resulted in the highest anti-avidin antibody titres. Sheep given rovIL-lB in the second immunisation only were found to respond better than both the control group (avidin in Al(OH)_ both 1° and 2° p<0.01) and than the group which received 35 rovIL-lB in only the primary immunisation (p<0.01).

EXAMPLE 9 SHEEP IL-ltt DOSE RESPONSE TRIAL EXPERIMENTAL PROTOCOL: 100 yg of avidin administered intradermally (id) or intramuscularly (im) in aluminium hydroxide [A1(0H)3] with 3 doses o£ IL-la, 1, 10 and 100 yg.

Group No.

No. of Sheep Treatment Route 1 4 Avidin in ai(oh)3 id 2 4 Avidin in Al(oh)3 + 1 yg IL-la id 3 4 Avidin in Al(oh)3 + yg IL-la id 4 4 Avidin in Al(oh)3 + 100 yg IL-la id 4 Avidin in Al(oh)3 im 6 4 Avidin in Al(oh)3 + 100 yg IL-IB im Time Procedure 15 Day 0 Prebleed + Primary immunisation Day 14 Primary bleed: Test serum by anti-avidin ELISA Day 28 Secondary immunisation Day 42 Secondary bleed: Test serum by anti-avidin ELISA RESULTS; See Figure 9 Primary Response; After id immunisation the 1, 10 and 100 yg doses gave responses 2-4 fold higher than the control avidin + Al(OH)3 group. After im immunisation, a 100 25 yg dose of IL-la resulted in a 4 fold increase in antibody levels. t Secondary Response: The same pattern of responses as in the primary are observed with both id and im administration of 30 rovIL-la giving 8 to 10 fold increases in anti-avidin antibody levels. im administration gave a slightly higher response than id administration.

CONCLUSIONS: Recombinant ovine IL-la administered either 35 intradermally or intramuscularly in alum gave an 8 fold increase in the anti-avidin antibody response. No significant difference was seen between the three doses of IL-la tested, 1, 10 or 100 yg. The intramuscular route of administration resulted in slightly higher . antibody levels than intradermal administration.

EXAMPLE 19 SHEEP IL-lcc INTRAMUSCULAR DOSE RESPONSE TRIAL AIM; To test the adjuvant ability of recombinant ovine IL-la to enhance the specific antibody response to avidin when delivered intramuscularly either with or without aluminium hydroxide (alum[Al(OH)3]).

EXPERIMENTAL PRQCQTQL: 100 yg of avidin administered intramuscularly (im) with 5 doses of ILOla, 0.01/ 0.1,1, 10 and 100 yg in the presence or absence of aluminium hydroxide.

Group No. of No. Sheep Treatment Route 1 Avidin in Al(OH)3 im 2 Avidin in Al(OH)3 + 0.01 yg IL-la im 3 Avidin in Al(OH)3 + 0.1 yg IL-la im 4 Avidin in Al(OH)3 + 1 yg IL-la im Avidin in Al(OH)3 + 10 yg IL-la im 6 Avidin in Al(OH)3 + 100 yg IL-IB im 7 Avidin in PBS im 8 Avidin in PBS + 0.01 yg IL-la im 9 Avidin in PBS + 0.1 yg IL-la im Avidin in PBS + 1 yg IL-la im 11 Avidin in PBS + 10 yg IL-la im 12 Avidin in PBS + 100 yg IL-la im Time Procedure Day 0 Prebleed + Primary Immunisation Day 14 Primary bleed : Test serum by anti-avidin ELISA Day 21 Secondary immunisation Day 35 Secondary bleed : Test serum by anti-avidin ELISA 35 RESULTS; See Figures 10(a) and 10(b).

Primary Response: "After im immunisation, the groups receiving avidin in alum plus either 10 or 100 yg of rovIL-la . gave responses 2-4 fold higher than the control avidin + alum group. Groups receiving either 1, 0.1 or 0.01 pg rovIL-la showed no increase in antibody levels over the control group. When avidin was administered with 5 rovIL-la in PBS (no alum) no increase in response was observed.

Secondary Response: The same pattern of responses seen following primary immunisation was observed when avidin was 10 administered in alum plus the various doses of rovIL-la. The group receiving 100 pg of rovIL-la showed an 8 fold increase in anti-avidin antibody levels while the group receiving 10 pg showed a 4 fold increase. After a second immunisation, rovIL-la administered intramuscuarly in PBS 15 (no alum) also resulted in a dose related increase in anti-avidin antibody. The increase in antibody levels occured over the whole range of rovIL-la tested with the 100 pg dose giving antibody levels higher than, and the 10 pg dose similar to that seen after immunisation with 20 avidin in alum alone.

CONCLUSIONS: 100 pg of recombinant ovine IL-la administered intramuscularly in alum resulted in an 8 fold increase in the anti-avidin antibody response following secondary 25 vaccination. A dose of 10 pg gave a 4 fold increase. Doses of 1, 0.1 and 0.01 pg showed no enhancement over the avidin in alum control group. When rovIL-la was administered in a soluble manner (no alum) a dose related increase in response was observed with the antibody levels 30 obtained using 10 pg rovIL-la similar to that seen with alum alone while a further increase was observed in the 100 pg group.

Finally, it is to be understood that various other modifications and/or alterations may be made without 35 departing from the spirit of the present invention as outlined herein. 4 69 5 WO 94/04174 PCT/AL'93/00425

Claims (13)

Claims

1. A vaccine composition including an antigen against a disease of interest; a non-toxic adjuvant including a recombinant 5 polypeptide having ovine cytokine or ovine cytokine receptor activity, or mimotopes, derivatives or fragments thereof; and a non-toxic coadjuvant selected to stabilise and/or enhance the immune response to the recombinant 10 polypeptide adjuvant.

2. A vaccine composition according to claim 1, wherein the antigen is an antigen against a parasitic disease selected from Haemonchus contortus, Trichostrongyius colubriformus and Ostertagia 15 circumcincta, Bacteroides rodosus, Lucilia cuprina, Staph, aureus and C.ovis.

3. A vaccine composition according to claim 2, wherein the recombinant polypeptide is selected from the group consisting of IL-la, IL-IB, IL-1R, IL-2, IL-2R, 20 IL-31 IL-3R, IL—4, IL-4R, IL-£ , IL-5R, IL-6, IL-6R, IL-7, IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL^IO, IL-10R, IFN-y, IFN-yR, TNF-a, TNF-aR, GMCSF, GMCSFR, TGF-B and TGF-BR, or mimotopes, derivatives or fragments thereof.

4. A vaccine composition according to claim 3, 25 wherein the recombinant polypeptide is selected from the group consisting of IL-la, IL-IB, IL-2 and IL-6, or mixtures thereof.

5. A vaccine composition including substantially 1 to 95% by weight based on the 30 total weight of the vaccine composition of an antigen against a parasitic disease selected from the group consisting of Haemonchus contortus, Trichostrongylus colubriformus and Ostertagia circumcincta, Bacteroides rodosus, Lucilia cuprina, Staph, aureus and C.ovis; 35 substantially 1 to 75% by weight of a non-tozic adjuvant including a recombinant polypeptide having ovine cytokine or ovine cytokine receptor activity selected f] group consisting of IL-la, IL-IB, IL-1R, IL-2, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-| fi rs* C\;WO 94/04174;2 rj A r> 3;pct/au93/00425;- 28 -;IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-10R, IFN-y, IFN-yR, TNF-a, TNF-oR, GMCSF, GMCSFR, TGF-13 and TGF-BR, or mimotopes, derivatives or fragments thereof; and substantially 5 to 75% by weight of a non-toxic 5 co-adjuvant selected from the group consisting of vegetable oils or emulsions thereof, surface active substances, octadecyl amino acid esters, methoxyhexadecylglycerol, pluronic polyols; polyamines, dextransulfate, poly IC, carbopol; peptides, dimethylglycine, tuftsin; immune 10 stimulating complexes (ISCOMS); oil emulsions; mineral gels, mineral suspensions; and mixtures thereof.;

6. A vaccine composition according to claim 5, wherein the co-adjuvant is a mineral suspension selected from the group consisting of aluminium hydroxide,;15 aluminium phosphate and aluminium sulphate.;

7. A vaccine composition according to claim 6, including a plurality of antigens against diseases of interest.;

8. A method for the treatment of disease in non-human 20 animals, which method includes administering to a non-human animal a therapeutically or prophylactically effective amount of a vaccine composition including an antigen against a disease of interest;;a non-toxic adjuvant including a recombinant 25 polypeptide having ovine cytokine or ovine cytokine receptor activity, or mimotopes, derivatives or fragments thereof;;and a non-toxic coadjuvant selected to stabilise and/or enhance the immune response to the recombinant 3 0 polypeptide adjuvant.;

9. A method according to claim 8, wherein the vaccine composition includes substantially 1 to 95% by weight based on the total weight of the vaccine composition of an antigen 35 against a parasitic disease selected from the group consisting of Haemonchus contortus, Trichostrongyius colubrif ormus and Ostertagia circumcincta, Bacteroyi^t ® Rodosus, Lucilia cuprina, Staph, aureus and C.ovis;;substantially l to 75% by weight of a rson-rfcpxic;NX*1 WO 94/04174 25 4 69 5 PCT/AL'93/00425 - 29 - adjuvant including a recombinant polypeptide having ovine cytokine or ovine cytokine receptor activity selected from the group consisting of IL-la, IL-IB, IL-1R, IL-2, IL-2R, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-7, 5 IL-7R, IL-8, IL-8R, IL-9, IL-9R, IL-10, IL-10R, IFN-y IFN-yR, TNF-a, TNF-aR, GMCSF, GMCSFR, TGF-B and TGF-BR, or mimotopes, derivatives or fragments thereof; and substantially 5 to 75% by weight of a co-adjuvant selected from the group consisting of vegetable oils or 10 emulsions thereof, surface active substances, octadecyl amino acid esters, methoxyhexadecylglycerol, pluronic polyols; polyamines, dextransulfate, poly IC, carbopol; peptides, dimethylglycine, tuftsin; immune stimulating complexes (ISCOMS); oil emulsions; mineral gels; mineral 15 suspensions; and mixtures thereof.

10. A method according to claim 9, wherein the vaccine composition is administered to the non-human animal intraderinally, intramuscularly or subcutaneously.

11. A method according to claim 10, wherein the 2 0 non-human animal is subjected to at least a primary and secondary vaccination.

12. A vaccine composition according to claim 1, substantially as hereinbefore described with reference to any one of the examples. 25

13. A method according to claim 8, substantially hereinbefore described wth reference to the accompanying examples. 35