NZ562036A - Immunogenic compositions wherein the immunogenic components are dispersed throughout a lipid matirx - Google Patents

Abstract

Disclosed are immunogenic compositions comprising either a pharmaceutically acceptable lipid formulation and at least one immunogenic component comprising an immunogenically effective amount of live organisms, the composition being formulated for oral administration, or comprising a pharmaceutically acceptable lipid formulation which undergoes solid to fluid transition between about 10 degrees C to 40 degrees C, and at least one immunogenic component present in an immunogenically effective amount, wherein in each composition the lipid formulation is a lipid matrix in paste or solid form, and the immunogenic components are dispersed throughout the lipid matrix.

Description

New Zealand Paient Spedficaiion for Paient Number 562036 562036 NEW ZEALAND PATENTS ACT, 1953 No: Divided out of NZ 546141 Date: Dated 26 July 2001 COMPLETE SPECIFICATION IMMUNOGENIC COMPOSITIONS We, OTAGO INNOVATION LIMITED, c/- T D Scott & Co, Chartered Accountants, Level 6 Otago House, 481 Moray Place, Dunedin, New Zealand; ANIMAL HEALTH BOARD, INC., a New Zealand company of Level 9, Guardian Trust House, Willeston Street, Wellington, New Zealand; and AGRESEARCH LIMITED, a New Zealand company and 15 Crown Research Institute under the Crown Research Institutes Act 1992, of 5th Floor, Tower Block, Ruakura Research Centre, East Street, Hamilton, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 1148875-1 INTELLECTUAL PROP" OFFICE OF ' 2 7 NOV r ec f 562036 IMMUNOGENIC COMPOSITIONS The reader's attention is directed to our related divisional application NZ 577542.

TECHNICAL FIELD The present invention broadly relates to the use of lipids to formulate immunogenic compositions, particularly live bacterial vaccines, and to methods for immunising animals using the compositions.

BACKGROUND Most human and animal pathogens including those that cause tuberculosis (TB), initiate infection via the mucosal surfaces. Accordingly, protective immunity against such pathogens may require induction of strong mucosal immune responses. However, mucosal immune responses are generally 15 weak following parenteral immunisation. Despite the obvious need for vaccines, particularly TB vaccines, to protect against mucosal sites, the vaccines in use today are given by intradermal or subcutaneous injection. The development of more effective compositions, and/or delivery systems for vaccines by alternate routes is therefore desirable. Oral administration of vaccines in particular has a number of advantages including ease of administration and targeting of the mucosal immune response. 20 Despite this, oral vaccination of animals and man to provide mucosal and/or systemic immunity has to date been largely ineffective. Efficacy of such vaccines has been hampered by degradation of the vaccine as it passes through the gut. In particular, most immunogenic compounds possess peptide bonds that are readily broken down by gastric and proteolytic enzymes in the gut.

A number of vaccines rely on the use of freeze-dried preparations of organisms. For example, the current vaccine for human TB is based on freeze-dried preparations of a live attenuated bacterium called Bacille Calmette Guerin (BCG). However, it has been shown that freeze-drying procedures result in 30 to 50% loss of viability of BCG and impaired recovery of remaining live bacteria (7). A composition which retains greater viability of organisms prior to use would contribute greatly to the 30 effectiveness of such vaccines.

To improve immune responses, antigens have been mixed with a number of adjuvant substances to stimulate immunogenicity. These adjuvants are primarily alum and oil-in-water emulsions. The latter group is typified by the Freund's mineral oil adjuvants. However, the 35 use of Freund's complete adjuvant (FCA) in human and veterinary vaccines is contraindicated intellectual property office of NrZ. t 6 JUN 2009 RECEIVED 562036 because of toxic reactions that have been reported. For these reasons, Freund's adjuvant may also be unsuitable for oral administration.

In other oil-in-water emulsions surfactants have been required because of the high oil content. 5 Detergent properties of surfactants render them unsuitable for parenteral or oral administration. Further, toxic reactions even for approved surfactants have been reported. A further drawback with emulsions are that they are heterogeneous systems of one immiscible liquid dispersed in another. This is unstable and results in separation of the aqueous phase over time. This poses difficulties for maintaining vaccines in stable suspension. Moreover, 10 antigens trapped in the aqueous phase of water-in-oil emulsions are unlikely to be protected from degradation in the stomach.

Liposomes and lipid vesicles have also been explored for use with vaccines, particularly with small immunogenic components that may be readily encapsulated. Generally, liposomes and 15 vesicles are not useful for encapsulation of large antigens such as live microorganisms. Moreover, liposomes and vesicles are costly and time consuming to produce, and the extraction procedures used in their preparation may result in alteration of the chemical structure or viability of vaccine preparations and hence their immunogenicity. For example, heat and solvents may alter the biological integrity of immunogenic components such as 20 proteins.

It is therefore an object of the present invention to provide an immunogenic composition and/or delivery system which addresses these desiderata or which at least provides the public with a useful choice.

SUMMARY OF THE INVENTION Accordingly, in a first aspect the present invention provides an immunogenic composition comprising a pharmaceutically acceptable lipid formulation and at least one immnugenic component comprising an immunogenically effective amount of live organisms, the 30 composition being formulated for oral administration, and wherein the lipid formulation is a lipid matrixin paste or solid form, and the live organisms are dispersed throughout the lipid matrix.

In a further aspect the present invention provides an immunogenic composition comprising a 35 pharmaceutically acceptable lipid formulation which undergoes solid to fluid transition 1148875-1 3 562036 between about 10X to 40°C, and at least one immunogenic component present in an immunogenically effective amount, wherein the lipid formulation is a lipid matrix in a paste or solid form and the immunogenic component is dispersed throughout the lipid matrix.

Preferably, the lipid formulation undergoes solid to fluid transition between about 30°C to 40°C.

Preferred lipid formulations for use in the compositions of the invention contain long chain fatty acids.

In terms of fatty acid composition, a preferred lipid formulation contains 40% to 100%, preferably 60% to 100%, more preferably 80% to 100%, and even more preferably 90% to 100% Ci6 and/or Cig fatty acids.

A further preferred composition has a lipid formulation which contains less than 35%, preferably less than 25%, and more preferably less than 10%> C14 fatty acids or shorter.

In one embodiment, the lipid formulation contains: % to 60% saturated fatty acids; 25% to 60% monounsaturated fatty acids; and 0.5 to 15% polyunsaturated fatty acids.

In a particularly preferred composition, the lipid formulation contains: % to 50% saturated fatty acids; 40% to 55% monounsaturated fatty acids; and % to 9% polyunsaturated fatty acids.

A current preferred lipid formulation for use in the invention has the formula: 3% myristic acid; 26% palmitic acid; 15% stearic acid; 40% oleic acid; and 6% linoleic acid.

A further preferred lipid formulation for use in the invention is a coconut oil.

The immunogenic component may be a protein, glycoprotein, peptide or factor with a protein or peptide component. 1148875-1 4 562036 In one embodiment, the immunogenic component comprises live organisms.

Preferably, the live organisms in the compositions of the invention are bacteria, particularly non-pathogenic bacteria, and more preferably bacteria belonging to the genus Mycobacterium. 5 A particularly preferred mycobacterium for use in the invention is Mycobacterium bovis BCG.

In one embodiment, the composition comprises at least two immunogenic components. The first is preferably a live organism and the second immunogenic component is preferably 10 derived from an infectious agent, or is a weakly immunogenic protein or peptide.

Described is a method for preparing an immunogenic composition of the invention, the method comprising mixing the immunogenic component(s) with the lipid formulation.

In a still further aspect, the invention also provides a method for immunising a non-human animal, the method comprising administering to said animal an immunogenic composition of the invention.

In a further aspect, the invention provides a method for stimulating a mucosal immune 20 response in a non-human animal, the method comprising administering to said animal an immunogenic composition of the invention.

Administration of the composition in these methods is preferably by the oral route The invention also relates to the use of lipid formulations in the preparation of the immunogenic compositions of the invention, and use of the composition of the invention for preparing medicaments useful in the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Aspects of the invention will now be described in relation to the accompanying drawings in which: 1148875-1 562036 Figure 1. Fatty acid composition of lipid formulations. Lipids were analysed by gas chromatography according to standard protocols. The fatty acid composition of each lipid is expressed as a percentage of the total fatty acid composition.

Figure 2. BCG viability following formulation and storage in lipids at 4°C (2a) or room temperature (10-25°C) (2b). BCG formulations were warmed to 37°C and emulsified in 7H9 broth. Numbers of viable organisms were determined by inoculating serial 10 fold dilutions of each emulsion onto 7H11 agar plates. The number of CFU/ul of formulation media was determined after 2-3 weeks of culture. Results are representative of duplicate experiments and 10 are expressed as means.

Figure 3. Bovine PPD induced IFN-y responses following oral vaccination with varying doses of formulated M. bovis BCG. Mice were sacrificed at 8 weeks after oral immunization with formulated M. bovis BCG (circles), non-formulated M. bovis BCG (triangles) or 15 formulation material only (diamonds). Splenocytes were incubated with bovine PPD for 72 h. Supernatants were then collected and analysed using a sandwich ELISA. Each treatment group contained 6 mice. Spleens were individually processed. Results are expressed in pg/ml and are presented as means of triplicate determinations. Bar indicates standard error.

Figure 4. Antigen-induced splenic IFN-y responses to M bovis BCG vaccination in BALB/c mice. Mice were euthanased at 2, 4, 6 and 8 weeks after vaccination with 106 CFU subcutaneous M. bovis BCG (squares), oral delivery of 107 CFU of formulated M. bovis BCG (circles), non-formulated M bovis BCG (triangles), or formulation material only (diamonds). Splenocytes were incubated with bovine PPD for 72 h. Supernatants were then collected and 25 analysed using a sandwich ELISA. Each treatment group contained 5-6 mice. Spleens were individually processed. Results are expressed in pg/ml and are presented as means of triplicate determinations. Results at 8 weeks are from 2 separate experiments. P value < 0.05 (Student t test). Bar indicates standard error.

Figure 5. Growth inhibition of M. bovis by macrophages co-cultured with nonadherent peritoneal exudate cells (NPEC). Macrophages were infected with M. bovis at an MOI of 2 bacilli per macrophage. Non-adherent autologous NPEC were added at a ratio of 10 NPEC per macrophage. [3H]uracil incorporation was then assessed at 72h post infection. The mean [3H]uracil uptake by cell cultures which did not contain M. bovis was 460 cpm. Growth of 1148875-J 6 562036 intracellular bacilli from co-cultured macrophages and NPEC was expressed as means of triplicates. The results are representative of two experiments. *Represents a mean which is significantly different from the mean of Formulation only control group; bar indicates standard error.

Figure 6. Effect of oral vaccination of possums with formulated BCG on in vitro peripheral blood lymphocyte blastogenic responses to PPD-B. Formulated BCG non-formulated BCG.-b-; non-vaccinated control -X-; Results are expressed as mean stimulation index (SI). *Represents a mean which is significantly different from the mean of non-vaccinated control 10 group. Bar indicates SE.

Figure 7. Effect of oral vaccination with formulated BCG on body weight of possums challenged with M. bovis. Mean body weight change was determined over the period from challenge to necropsy. The mean body weight of the possums immediately prior to challenge 15 was 3.0 + 0.07 (+ SE) kg. Bar indicates SE.

Figure 8. Effect of oral vaccination with formulated BCG on lung weight of possums challenged with M. bovis. Mean lung weight was determined at necropsy. In order to standardise differences in lung weight with variation in body weight, the lung weight of each 20 animal was compared with the body weight and expressed as a ratio. * Represents a mean which is significantly different from the mean of non-vaccinated control group; bar indicates SE.

Figure 9. Effect of oral vaccination of possums with formulated BCG on mean numbers of 25 mycobacteria isolated from lungs following challenge with M. bovis. Results are expressed as the geometric mean number of CFU (logio)/g of tissue. ^Represents a mean which is significantly different from the mean of non-vaccinated control group; bar indicates SE.

Figure 10. Effect of oral vaccination of possums with formulated BCG on mean numbers of 30 mycobacteria isolated from spleen following challenge with M. bovis. Results are expressed as the geometric mean number of CFU (logio)/g of tissue. *Represents a mean which is significantly different from the mean of non-vaccinated control group; bar indicates SE. 1148875-1 7 562036 Figure 11. Comparison of immune responses to four oral lipid BCG formulations or to subcutaneous vaccination. The figure shows the effect on in vitro peripheral blood lymphocyte blastogenic responses to PPD-B in possums following vaccination (week 0) and challenge (week 8). Results are expressed as mean stimulation index (SI).

Figure 12. is a diagram of a generic vaccine delivery system according to the invention.

DETAILED DESCRIPTION Accordingly, in a first aspect, the invention provides an immunogenic composition comprising a pharmaceutically acceptable lipid formulation and at least one immunogenic component comprising an immunogenically effective amount of live organisms, the composition being formulated for oral administration, and wherein the lipid formulation is a 15 lipid matrix in paste or solid form, and the live organisms are dispersed throughout the lipid.

Preferably, the lipid is in solid form. Conveniently, the lipid formulation undergoes solid to fluid transition between about 10°C to 40°C.

In a further aspect the present invention provides an immunogenic composition comprising a pharmaceutically acceptable lipid formulation which undergoes solid to fluid transition between about 10°C to 40°C, preferably 30°C to 40°C, and at least one immunogenic component present in an immunogenically effective amount, wherein the lipid formulation is a lipid matrix in paste or solid form and the immunogenic component is dispersed throughout 25 the lipid matrix.

The lipids employed in the formulations above are preferably suitable for animal or human consumption and may be selected from a broad range of natural (vegetable or animal derived), or synthetic lipid products including oils, fats and waxes.

Most usually, the lipid material will be liquid at temperatures above about 30°C. That is, the lipid should be selected to achieve melting point at physiological temperature in the animal to which it is administered, most usually by the oral route. Desirably, the lipid will be in the form of a solid or semi-solid phase at 10-30°C at atmospheric pressure, and preferably is still 35 solid or semi-solid at from 20°C to 30°C at atmospheric pressure. However the melting 1148875-1 8 562036 temperature of lipid is not exclusive and may include oils, fats and waxes with a range of melting temperatures.

Preferred lipids for use herein will undergo transition from the solid phase to the liquid phase 5 between about 30°C and physiological temperature of about 37°C. Summaries of lipid phase behaviour are available in the art, see for example (10). Accordingly, a skilled reader can select a lipid having the desired properties and melt point based on information in the art and simple experiment.

Suitable lipid formulations are triglycerides including glyceryl esters of carboxylic acids, compounds consisting of an aliphatic chain and a -COOH end, and saturated and non-saturated fatty acids and mixtures thereof.

Currently preferred lipids are triglycerides containing primarily Cs to C20 acyl groups, for 15 example myristic, palmitic, stearic, oleic, linoleic, parinic, lauric, linolenic, arachidonic, and eicosapentaenoic acids, or mixtures thereof.

It has also been determined that for lipid formulations useful in the invention longer chain fatty acids, for example, C16-C18, are preferred. Long chain fatty acids have been found to be 20 more effective in protecting organisms such as BCG in vaccines given to mice and possums. Viewed in this way, lipid formulations preferred for use in the invention contain: 40% to 100% , preferably 60% to 100%, preferably 80% to 100%, and more preferably 90% to 100% Ci6 and/or Cig fatty acids.

Generally, Cj6 fatty acids represent from 10% to 40%, more preferably 20% to 35%, and even more preferably 25% to 32% of the total fatty acid content, and Cig fatty acids represent from 40% to 90%, preferably from 50% to 80%, and more preferably from 60% to 70% C53 of the total fatty acid content.

Preferred lipid formulations also contain less than 35% C14 fatty acids or shorter, preferably less than 25%, and more preferably less than 10%.

In terms of chain length, the preferred lipid formulation contains less than 5% fatty acids with C]4 chains or shorter, 25% to 32% C16 fatty acids, and from 60% to 70% Cig fatty acid chains. 1148875-1 9 562036 In terms of their fatty acid contents, lipid formulations for use in the invention may contain: saturated fatty acids in an amount from 20% to 60%, preferably 30% to 55%, and even more preferably 40% to 50%; monounsaturated fatty acids in an amount from 25% to 60%, preferably 30% to 60%, and more preferably 40% to 55%; and polyunsaturated fatty acids in 5 an amount of from 0.5% to 15%, preferably 3% to 11%, and more preferably 5% to 9%.

A particularly preferred lipid formulation for use in the invention comprises 40% to 50% saturated fatty acids, 40% to 50% monounsaturated fatty acid, and 5% to 9% polyunsaturated fatty acid.

A currently preferred lipid formulation for use in the invention has the formula 3% myristic acid, 26% palmitic acid, 15% stearic acid, 40% oleic acid, and 6% linoleic acid as determined by HPLC analysis.

Currently preferred lipids formulations also include animal derived fractionated lipid complexes, one or more hydrogenated vegetable oils, especially olive oil or coconut oil, commercial suppository bases and other lipid formulations or mixtures thereof.

The lipid formulation is useful in the preparation of immunogenic compositions, and in 20 protecting immunogens within the composition from degradation. The lipid formulation is especially useful in maintaining viability of live organisms, particularly bacteria. The lipid formulation acts to maintain the organisms in a live, but dormant state. This is particularly important for vaccines comprising live organisms formulated for oral administration. The lipids also maintain immunogens in a uniform suspension. That is, in the compositions of the 25 invention the immunogenic components, and live organisms in particular, are uniformly distributed throughout a solid or paste like lipid matrix. The lipids also protect the immunogens from destruction by gastrointestinal secretions when orally administered. Protection from macrophage attack is also likely when administered by other routes such as subcutaneously. This allows for uptake of the immunogens and particularly live organisms 30 through the gastrointestinal mucosa, and subsequent replication of organisms in the host. Replication of the live organisms within the host stimulates a protective immune response as determined by a reduction in severity of disease following challenge with virulent bacteria.

Formulations for a wide range of delivery routes may also include additives such as fillers, 35 extenders, binders, wetting agents, emulsifiers, buffing agents, surfactants, suspension agents, 1148875-1 562036 preservatives, colourants, salts, antioxidants including mono sodium glutamate (MSG), vitamins such as vitamin E, butylated hydroxanisole (BHA), albumin dextrose-catalase (ADC), protective coatings, attractants and odourants, and agents to aid survival of organisms contained in the lipid but are not limited thereto.

Protective coatings or enterocoatings may be selected, for example, from gels, paraffins, and plastics including gelatin. The coatings further aid in the prevention of exposure to gastric acids and enzymes when the oral administration route is selected.

When used for oral administration, the formulation may also include additives which, for example, improve palatability, such as flavouring agents (including anise oil, chocolate and peppermint), and sweeteners (including glucose, fructose, or any other sugar or artificial sweetener).

The immunogenic component may be a protein, glycoprotein, peptide, or factor with a protein or peptide component or mixtures thereof. The component may be derived from an agent which may be used to generate an immune response in an animal.

Most usually, the immunogen will bear at least one epitope which is present on an organism 20 which is pathogenic in the animal species to be treated. Other immunogenic structures such as are known in the art may also be used. For example, polysaccharides, glycolipids, and haptens conjugated to a carrier.

Preferably, the immunogenic component is a living organism, preferably a biologically pure 25 culture.

The living organism in the composition may be selected from the group consisting of: fungi, protozoans, bacteria and viruses. For example, HIV, SIV, Brucella and Anthrax. Preferably the organism is a bacterium. Organisms currently selected from non-pathogenic bacteria are 30 preferred for use in compositions formulated for oral or subcutaneous delivery. A preferred bacterium is a non-pathogenic strain selected from the genus Mycobacterium including M, tuberculosis complex (comprising M. tuberculosis, M. bovis, M. africanum and M. microtii), M. avium-intracellulare complex (comprising M intracellulars and M. avium), M, paratuberculosis, M. vaccae, M. smegmatis, M. chelonae, M. fortuitum, M. kansasii, M. i, M. ulcerans, M. simiae, M. haemophilum, M. malmoense, M. shimoidei, 11 OFFICE OF N.Z. lb DEMW8 RECEIVED 562036 M gastri, M. terrae complex, M. bovis and M. nonchromogenicum. In a particularly preferred embodiment the agent is Bacille Calmette Guerin (BCG), an attenuated strain of M. bovis including the following strains: 83/6235, Pasteur 1173P2, Glaxo 1077, Japanese 172, Prague, Russian, Brazilian, Danish 1331, Copenhagen, Connaught and including functionally 5 equivalent variants and other attenuated strains of M. bovis, clones, mutants and recombinants of these strains either natural recombinants or those produced by any of a wide range of genetic engineering techniques, and immunogenic components thereof.

It will be appreciated from the foregoing that the immunogenic component may be a complex 10 of proteins or peptides, or the like.

In one embodiment, the composition includes at least two immunogenic components selected from any of those identified above, and may include multiple combinations of subunit immunogens. Three or more immunogenic components are feasible.

The concentration of the immunogenic component(s) in the composition may vary according to known art protocols provided it is present in an amount which is effective to stimulate an immune response on administration to an animal. In particular, an immune response in the gut associated lymphoid tissue of the small intestine. In the case of mycobacteria a range of from 20 1 x 105 to 1 x 1010 colony forming units (CFU)/ml is appropriate. Preferably, the concentration is from 1 x 107 to 1 x 109 CFU/ml. For protein and peptide type immunogens a range of from 10-1000|_tg per gram of formulation is appropriate. For virus-type immunogens a range of 1 x 103 to 1 x 1010, preferably 1 x 105 to 1 x 108 Plaque Forming Units (PFU)/ml is appropriate. The immune response may be humoral, or cell mediated including a mucosal 25 immune response.

Accordingly, in a further aspect the invention relates to a method for stimulating a mucosal immune response in a non-human animal by administering an immunogenic composition of the invention to the animal.

The composition may be prepared using techniques known in the art. Conveniently, the lipid formulation is heated to liquefy if required, and the immunogenic component(s) and other ingredients (when used) as described above are added. Dispersal of the immunogenic composition may be achieved by mixing, shaking or other techniques that do not adversely ty of the immunogenic component. 12 DECEIVED 562036 Further preferred compositions for use in the invention are also essentially free of aqueous components including water. The term "essentially free" as used herein means that the composition contains less than 10% aqueous components, and preferably less than 5% 5 aqueous components. As indicated above, the presence of components, particularly aqueous solvents, reduces the protective effect of the lipid formulation especially in the gut.

In one embodiment, the immunogenic composition is a vaccine.

In an alternate embodiment, the immunogenic composition is an adjuvant useful for administration with a vaccine to increase efficacy of same. Myco bacterium-containing, and BCG-containing immunogenic compositions in particular are preferred for use as adjuvants.

The immunogenic composition of the invention can also be useful for generating a response 15 to a second or further immunogenic molecule of a type as indicated above for the immunogenic component, particularly those that are weakly immunogenic. This may be achieved by co-delivery of the second or further immunogenic molecule in an immunogenic composition of the invention by conjugating the immunogenic molecule to the immunogenic component of the composition. Conjugation may be achieved using standard art techniques 20 (9). In particular, an immunogen of interest may be conjugated to an immunogenic carrier or adjuvant by a linker group which does not interfere with antibody production in vivo. The immunogenic carrier or adjuvant may be any of the immunogenic components including the organisms identified above but are preferably Mycobacterium, and more preferably BCG. Suitable linker groups include mannose receptor binding proteins such as ovalbumin and 25 those that bind to Fc receptors. The second or further immunogenic molecule is preferably a protein or peptide. A particularly preferred protein is an immunocontraceptive protein. The lipid again acts as the delivery matrix. An example of this vaccine delivery system is given in Figure 12. When the composition is administered an enhanced immune response to the conjugated molecule or co-delivered molecule results.

In a further aspect the invention also provides a method for immunising a non-human animal, the method comprising administering to said animal an immunogenic composition of the invention.

TmilECmL PROPERTV OFFICE OF N.Z. 16 DEC 2008 1148875-1 13 RECEIVED] 562036 The term "animal" as used herein refers to a warm-blooded animal, and particularly mammals. Humans, dogs, cats, birds, cattle, sheep, deer, goats, rats, mice, rabbits, possums, badgers, guinea pigs, ferrets, pigs and buffalo are examples of animals within the scope of the meaning of the term. Monogastric and ruminant animals in particular are contemplated within this term.

The compositions of the invention may be administered by a variety of routes including parenteral (subcutaneous, intradermal, intramuscular), mucosal, aerosol and oral administration, but are not limited thereto. In one embodiment, oral administration is preferred. The compositions may be orally administered in the form of pellets, tablets, capsules, lozenges, or other suitable formulations. Oral administration enjoys wide consumer acceptance where the use of needles and syringes can be avoided and is an economical and practical method for vaccinating wildlife. In one embodiment the applicants have therefore provided a novel live vaccine formulated for oral administration.

In an alternate embodiment, the compositions may be formulated for parenteral administration by injection. This form of administration may also include injectable and subcutaneous depot formulations compatible with body tissues. Time release absorption from the depot may be achieved using the lipid formulation alone or with additional biodegradable polymers. The depot allows for sustained release of the immunogenic component in a process which more closely approximates the infection process, facilitating the mounting of an immune response in the animal to which the composition is administered. A lipid protective effect also occurs with these forms of administration.

The composition can be administered as a single dose, particularly for parenteral administration, or in repeated doses over time. For example, an initial dose and booster doses at spaced intervals. The dosage for administration is determined by the release rate of the immunogen component in combination with its immunogenicity. Usual considerations such as weight, age, sex of the animal, concurrent treatments (if any), and nature of the immunogen to be treated may also be taken into account. Generally the dose range for oral vaccination will be as given above, i.e. 1 x 103 to 1 x 1010, preferably 1 x 107 to 1 x 109 CFU/kilogram per dose. For peptide and protein type immunogens the dose range will be from l-lQ,000|^g, preferably lO-lOOOpg. For virus-type immunogens the dose range will be from 1 x 103 to 1 x 1010, preferably 1 x 105 to 1 x 10s PFU/ml, Whichever method of delivery is used, when live — "erty 1 b DEC 2008 i 1148875-1 14 RECEiyjED 562036 organisms are used in the vaccine formulation they are expected to multiply within the host to facilitate the immune response.

The composition may also be formulated as a single dose preparation or as a multidose 5 preparation for mass vaccination programmes.

Until required for use, the compositions of the invention may be stored for limited periods at room temperature, or preferably under normal refrigeration conditions at approximately 4°C. At 4°C the lipid formulation facilitates storage and maintenance of organisms in a dormant 10 but viable state without deterioration. For parenteral delivery, the composition is then warmed to 30 to 40°C to liquefy prior to administration. For oral administration the composition is a solid or a paste.

It will be appreciated that the above description is provided by way of example only and 15 variations in both the materials and techniques used which are known to those persons skilled in the art are contemplated.

Non-limiting examples illustrating the invention will now be provided.

EXAMPLES MATERIALS AND METHODS Bacteria. M. bovis BCG Pasteur 1173P2 (Pasteur Institute, Paris) was used as the vaccine 25 strain. The M. bovis strain used for macrophage infection studies and for possum challenge was M. bovis 83/6235 (AgResearch, Wallaceville, New Zealand) which was originally isolated from a tuberculous lesion in a brushtail possum and has been used in previous macrophage and possum inoculation studies (1, 4). For BCG formulation and macrophage infection, bacteria were grown to mid log phase in 175 ml flasks (Falcon) containing 30 Middlebrook 7H9 medium (Difco, Detroit, Mich.) supplemented with albumin-dextrose-catalase (ADC; BBL, Becton Dickinson, Maryland, USA). Bacilli were harvested by centrifugation and washed twice in phosphate buffered saline (PBS) prior to storage at -70° C. For possum challenge, M. bovis was grown to mid-log phase in tween albumin broth (TAB) containing Dubos broth base (Difco Laboratories, Detroit, USA) supplemented with 0.006% 1148875-1 562036 v/v alkalinized oleic acid, 0.5% w/v albumin fraction V and 0.25% w/v glucose and the numbers of bacteria were estimated by the degree of turbidity. Dilutions for inoculating the possums were made in TAB. The number of colony forming units (CFU) of BCG or M. bovis was determined as described previously (5).

Formulation composition. Three lipid products were selected on the basis of melting temperature and the ability to maintain BCG in uniform suspension for formulating with BCG. Lipids which were liquid at 37°C but became solid below 30°C were chosen for testing in BCG viability studies. Following viability testing, the following three formulations were 10 selected for testing in oral vaccine trials in mice and possums: Formulation C -an animal derived fractionated complex lipid; Formulation K - consisting of tryglycerides of purified hydrogenated coconut oil; Formulation N-Novarta B, a commercially available suppository base. The three formulations were analysed by gas chromatography to determine the percentage of fatty acid groups.

Formulation of BCG. Pelleted BCG was resuspended in formulation medium which had been warmed to 37°C. BCG was resuspended at a concentration of 1 x 107 CFU/ml for vaccination of mice or 1 x 108 CFU/ml for vaccination of possums. To increase attractiveness and palatability for possums, 10 mg of glucose and 10 j.il of anise oil (Pharmacare, Auckland 20 NZ) were added per ml of formulation. For oral vaccination of mice, 10 mg of glucose, 1 mg of monosodium glutamate (Sigma), and 10% v/v ADC was added per ml of formulation. These additives were dispersed with the formulation lipids and were previously shown not to affect viability of BCG. BCG formulations were transferred to 15 ml tubes (falcon) and allowed to solidify with gentle mixing at 4°C. Formulations were removed from the tubes and 25 aseptically cut into lg pellets as required for viability testing and vaccination studies. Pellets were tested for dispersal of BCG by culturing on 7H11 agar plates and counting CFU as described below.

BCG viability. The number of CFU in the formulations following storage at 4°C or at room 30 temperature (10-25°C) was determined as described previously (4). Samples for culture were collected by warming 100 mg aliquots of the three BCG formulations to 37°C for 15 min and performing serial 10-fold dilutions in 7H9 broth. Numbers of viable organisms were determined by inoculating 100 |il of each emulsion onto Middlebrook 7H11 agar plates (Difco) supplemented with oleic acid-ADC (OADC; Becton Dickinson) and 0.5 % glycerol. 1148875-1 16 562036 Emulsions were dispersed using a glass spreader. Plates were sealed with parafilm and incubated in 5% CO2 at 37°C. The number of colonies was counted after 2-3 weeks of culture. Results are expressed as CFU/fig of BCG formulation.

Vaccination of mice. Specific pathogen free female BALB/c mice (6-8 weeks old) were obtained from the University of Otago Department of Animal Laboratory Sciences, Dunedin. Mouse experiments were conducted under ethics approval from the University of Otago Animal Ethics Committee (Approval No: 51/2000). Mice were separated into individual cages and taken off food for 12h prior to oral vaccination. Non-formulated controls consisted of M 10 bovis BCG in Craig's preservative-free strawberry jam (Heinz-Watties Ltd., Hastings, New Zealand). A previous study had shown that M. bovis BCG viability over a 24 h interval was not affected by mixing M. bovis BCG in the jam (data not shown). Non-vaccinated controls consisted of lipid formulation alone. For dose response and time course experiments mice were given vaccine in two separate doses at 24 h intervals. For aerosol challenge experiments, 15 mice were given a single oral dose (5 x 107 CFU) or vaccinated subcutaneously with 1 x 106 CFU. The mice were observed at various intervals during consumption of pellets and jam to ensure the full dose was eaten. At various time-points post vaccination, the mice were sacrificed by CO2 inhalation and their spleens were removed aseptically.

Spleen cell proliferation assay. Spleen cell suspensions were prepared by filtering cells through a cell strainer (70-jj.m mesh; Beckton Dickinson). Erythrocytes were lysed in 0.83% NH4CI (pH 7.2). Cells were washed twice in PBS and resuspended to 1 x 106/ml in Dulbeccos's modified Eagles medium (DMEM) containingl0% foetal calf serum (FCS), 20 mM HEPES penicillin at lOOU/ml, streptomycin at 100fig/ml, 5.5 x 10"5 M 2-25 mercaptoethanol (DMEM-10%FCS; all from Gibco-BRL, USA). Cells were resuspended to a concentration of 107 per ml in RPMI plus 10% foetal calf serum (Gibco). Splenocytes (5 x 105 per well) were plated out in triplicate wells in 96-well plates (Nunc). Cells were cultured with purified protein derivative from a culture 'of M bovis (bovine PPD; CSL, Melbourne, Australia), 60|ng/ml final concentration or with medium alone. Cells were harvested 4 days 30 later, after an 18-h pulse with 1 pCi of [3H] thymidine (Amersham, Buckinghamshire, England)), and the incorporated thymidine was measured as previously described (5). A stimulation index (SI) was obtained by dividing the mean counts per minute (cpm) for the triplicate cultures incubated with bovine PPD by the mean cpm for splenocytes cultured with medium only. 1148875-1 17 562036 In vitro assay for cytokine production by spleen cells. Spleen cell suspensions were prepared as described above for the spleen cell proliferation assay. One ml of cell suspension was dispensed into 24 well plates (Costar) and 100 of either PBS or bovine PPD (60)j,g/ml 5 final concentration) was added to the wells. Cultures were incubated for 72 h in 5% CO2 at 37°C after which time 200 jil of culture supernatant was collected and frozen at 70°C for cytokine analysis. Interleukin-2 (IL-2) and interferon-gamma (IFN-y) capture ELISAs were performed according to the manufacturers instructions using a commercial kit (R&D Systems, Duoset, City, Country) Cytokine levels in culture supernatants were quantified by 10 extrapolation from standard curves. The minimum sensitivities of the two ELISAs were determined to be 50 pg/ml for IFN-y and 35 pg/ml for IL-2.

M. bovis inhibition assay. Peritoneal-derived macrophages were tested for inhibition of intracellular growth of M. bovis following co-culture with or without autologous 15 lymphocytes. Experiments were performed according to a modification of previously described protocols. Peritoneal exudate cells (PEC) were obtained by lavage from female BALB/c mice. Cells were collected in PBS supplemented with 1% BSA and 20 U/ml of heparin, washed once and resuspended in DMEM medium containing 10% foetal calf serum and lOOU/ml penicillin (supplemented DMEM) at 2 x 106 /ml. 100 (il of cell suspension was 20 dispensed into a 96 flat well plate (Nunc). After incubation for 2 h in 5% CO2 at 37°C the nonadherent cells were removed, washed and resuspended at a density 5 x 106/ml in supplemented DMEM. Nonadherent cells were selectively depleted of the remaining adherent population by incubation in 25 ml flasks (Falcon). Nonadherent PEC (NPEC) were determined to comprise >90% lymphocytes following May-Grunwald/Giemsa staining. Warm 25 supplemented DMEM was added to the adherent monolayer which was estimated to contain 5 x 104 cells/well. This population was found to be 98% positive with a non-specific esterase staining kit (catalogue no. 181-B; Sigma, St. Louis, Mo, USA) and is henceforth referred to as macrophages. Macrophages were infected with M. bovis at an MOI of 2 bacilli per macrophage as described previously (2). Non phagocytosed bacteria were removed by gentle 30 washing. One hundred |nl (containing 5 x 105 cells) of autologous NPEC was added to each well containing infected macrophages and cultures were further incubated in 5% CO2 at 37°C. The resulting 10:1 NPEC-to-macrophage ratio was selected to approximate that of the ratio found in peripheral blood mononuclear cells. Control wells consisted of M. bovis-infected macrophages alone or uninfected NPEC and macrophages. After 72 h, cells were pulsed with 1148875-1 18 562036 1.0 juCi [3H]uracil for 18 h. The cells were lysed with 0.1% saponin and the bacteria heat killed at 80-90°C for 20 minutes prior to harvesting onto glass fibre filters (Whatman Inc, Finland) using an automated cell harvester (Cambridge Technology, USA). The amount of [3H]uracil incorporated was determined using a liquid P-scintillation counter (Wallac, 1205 5 betaplate scintillation counter (Pharmacia, UK)).

Aerosol challenge of mice with M. bovis. Six mice per vaccine group were challenged by aerosol with virulent M. bovis 8 weeks after vaccination. A single cell suspension of M. bovis 83/6235 was prepared using a modification of a method described by Grover et al., 1967 and 10 stored at -70 C. For preparing these suspensions, the bacterial cells were dispersed by sonication for 30 seconds and filtered through an 8 ]um membrane filter. Mice were infected via the respiratory route using an aerosol chamber which produces droplet nuclei of the size appropriate for entry into alveolar spaces. The concentration of viable M. bovis in the nebuliser fluid was empirically adjusted to result in the inhalation and retention of 5-20 viable 15 organisms per mouse lungs (B. Buddie and G. de Lisle, unpublished data). A similar procedure has been shown to result in reproducible, uniform infection of the lungs of guinea pigs. The aerosol infection and subsequent maintenance and manipulation of infected mice were performed under strict isolation conditions in a biohazard facility.

Isolation of M. bovis. Mice were euthanased between 37 and 40 days after aerosol challenge. The lungs and spleen from each mouse were processed individually for mycobacterial isolation. The organs were homogenized in a Ten-Broeck grinder and samples centrifuged at 3500 g for 20 min. The deposits were resuspended in 1 ml of distilled water. Appropriate dilutions were made in TAB and a 0.1 ml volume of a diluted or undiluted sample was 25 inoculated onto a modified mycobacteria 7H11 agar (1). Two replicates were prepared for each dilution. Culture conditions and methods for identification of isolates were carried out as previously described (1) Analysis of data Statistically analyses of differences in the mean cytokine levels and logio transformed spleen cell proliferation responses for the vaccine groups were determined using the Student t test. The bacteria] counts from the lung and spleen were logio transformed and analysed using analysis of variance. For statistical purposes, when no bacteria were cultured from tissues, half the lowest detectable count (% CFU/organ) was used. 1148875-1 19 562036 Vaccination and challenge of possums. Possums were trapped and housed as previously described (4). BCG was fed to two groups of possums (5 animals/group). A 1 g pellet of formulated BCG (1 x 108 CFU) was given to each possum in one group. A second group was 5 given BCG (1x10 CFU) in jam to control for the formulation procedure. The jam had previously been shown not to inhibit BCG viability (data not shown). A third group (6 animals/group) was given pellets containing formulation medium only and served as non-vaccinated controls. Possums were observed during consumption of pellets to ensure the full pellet was eaten. The following day the vaccinations were repeated (total BCG dose 2 x 108 10 CFU/possum). All of the possums were challenged by the aerosol route 41 days after vaccination.

In a second experiment, four oral lipid BCG formulations were compared with subcutaneous vaccination. Six possums per vaccine group were challenged by aerosol with virulent M 15 bovis 8 weeks after vaccination using lipids C, K, N and F (a modification of K containing 10% foetal calf serum) Aerosol challenge of possums with M. bovis. The possums were challenged with M. bovis 83/6235, which was originally isolated from a lymph node of a possum from Taumaranui, 20 New Zealand (5). Single cell suspensions of the isolate were prepared using a modification of a method described by Grover et aL 1967 and stored at -70EC. For preparing these suspensions, the bacterial cells were dispersed by sonication for 30 seconds and filtered through an 8|i membrane filter. Possums anaesthetised with an intramuscular injection of ketamine HC1 (30 mg/kg; Parnell Laboratories, Auckland, New Zealand) were infected via 25 the respiratory route by using an aerosol chamber which produces droplet nuclei of the size appropriate for entry into alveolar spaces. The concentration of viable M. bovis in the nebuliser fluid was empirically adjusted to result in the inhalation and retention of 10-20 viable organisms per possum (Buddie and de Lisle, unpublished). This challenge dose had previously been estimated from the number of primary tubercles observed grossly in the lungs 30 of non-vaccinated possums at 4 weeks post-infection. A similar procedure has been shown to result in reproducible, uniform infection of the lungs of guinea pigs (Wiegeshaus et al., 1970; Smith et al., 1970). The aerosol infection and subsequent maintenance and manipulation of infected possums were performed under strict isolation conditions in a biohazard facility. 1148875-1 562036 Necropsy of possums. All possums were killed between 56 and 57 days after challenge and subjected to extensive gross post-mortem examination. The lungs were separated from surrounding tissues and weighed.

Isolation of M. bovis from possum tissues. From each animal, a sample of lung and spleen each weighing approximately 1 g, was taken from a macroscopic lesion, or, if no lesion was present, a sample was taken from a pre-determined part of the organ and processed individually for mycobacterial isolation. Samples were weighed, homogenized in a Ten-Broeck grinder and decontaminated in 0.75% cetyl-pyridinium chloride for 1 h. Samples were 10 centrifuged at 3500 g for 20 min and deposits resuspended in 1 ml of distilled water. Appropriate dilutions were made in TAB and a 0.1 ml volume of a diluted or undiluted sample was inoculated onto a modified mycobacteria 7H11 agar plate. Two replicates were prepared for each dilution. Culture conditions and methods for identification of isolates were carried out as previously described (1).

Possum peripheral blood lymphocyte proliferation assay. Proliferative responses to PPD-B and PPD-A (CSL Limited, Parkville, Australia) were measured using whole blood depleted of red blood cells. Responses to Con A were also tested. Briefly, 1 ml of heparinised blood was mixed with 50 ml 0.17 M Tris- 0.16 M NH4CI, pH 7.2 at 37°C for 10 min, washed twice 20 in PBS at 20°C and made up to 3 ml in DMEM tissue culture medium supplemented with 2mM glutamine and 2% normal possum serum. The cells (200 ul) were plated into flat bottom 96 well plates containing 50 tul PPD-B, PPD-A or Con A in PBS or PBS alone to give final concentrations of 60 .ug/ml PPD or 5 |ng/ml Con A. Plates were placed in a 5% CO2 in air incubator for 72 hr, pulsed with lja Ci/well 3H-tritiated thymidine (Amersham, UK), 25 harvested after a further 18 h and 3H counted in a Micro Beta Trilux (Wallac, Finland). The stimulation index (SI) was calculated by dividing counts per minute (cpm) from triplicate cultures stimulated with PPD by cpm from triplicate cultures with medium and PBS.

Analysis of data Statistically significant differences of mouse cytokine secretion were determined using the Student t test (GraphPad, San Diego, Calif.). These studies were performed twice with similar results. For possum lymphocyte proliferation responses, stimulation indices of >3.5 were scored as a positive response as this represents a response at least three standard deviations above the mean of the background (mean SI for PPD-B prior to vaccination). The possum 1148875-1 21 562036 body weight changes, lung weights, lymphocyte blastogenic responses and bacterial counts for the different treatment groups were initially compared by one-way analysis of variance. Duncan's multiple range test was then used to compare the means for individual groups. Lymphocyte proliferation responses and bacterial counts from the lung and spleen were log 10 transformed prior to 5 analysis. For statistical purposes, when no bacteria were cultured from tissues, half the lowest detectable count (5 CFU/g tissue) was used.

RESULTS A. Fatty acid composition of formulation lipids. Lipids selected for use in formulating oral BCG were analysed by gas chromatography. Fig 1 shows the fatty acid composition of the 3 lipids used in mice and possum vaccination trials.

The relative percentage of fatty acids in the three lipid formulations are shown in Fig 1.

Chemical analysis of lipids by HPLC showed that the 3 formulations comprised the following mixtures of fatty acids: Formulation C, 89% total lipid (48.5% neutral, 40.5% polar -comprising 3% myristic acid, 26% palmitic acid, 15% 20 stearic acid, 40% oleic acid and 6% linoleic acid), Formulation K 47% lauric acid, 20% myristic acid, 12% palmitic acid, 12% stearic acid and 3% oleic acid.

Formulation F Formulation K containing 10% foetal calf serum.

Formulation N. Novarta B, a commercially available suppository base consisting of a mixture of esterified, hydrogenated, fractionated vegetable oils with synthetic triglyceride mixtures. 30 comprising: 44% lauric acid, 20% myristic acid, 16% palmitic acid, 19% stearic acid.

B. BCG viability following formulation.

The viability of formulated BCG following storage at 4°C is shown in Fig 2a. Over a period of 16 weeks, formulations C and K maintained high levels of BCG viability with formulation C showing 35 higher retention of viability (98%) compared to formulation K (52%). In contrast, formulation N showed a progressive loss of BCG viability resulting in greater than 97% loss of viable organisms by 16 weeks. These results suggest that formulations C and K are more suited to maintaining BCG viability at 4°C compared to formulati<^^[^^[j^L PROPERTY j " OFFiCE OF N.Z. j ' 6 flEC 2008 deceived 562036 The viability of formulated BCG following storage at room temperature (10-25°C) is shown in Fig 2b. Formulations C and K maintained high levels of BCG viability with formulation C showing prolonged retention of viability (mean logio CFU/ug =10) at 40 days compared to 5 formulation K (mean logio CFU/ug =10) at 22 days In contrast, formulation N showed a rapid loss of BCG viability (mean logio CFU/ug =10) at 12 days. These results suggest that formulations C and K are more suited to maintaining BCG viability at room temperature compared to formulation N.

C. Immunogenicity of formulated BCG in mice.

Oral delivery of formulated M. bovis BCG induces immune responses in mice. To determine a suitable method of measuring systemic immune responses following oral delivery 15 of M bovis BCG, we compared bovine PPD-induced splenocyte proliferation (LTA), and splenic IL-2 and IFN-y responses at 8 weeks following oral delivery of 107 CFU of lipid-formulated M. bovis BCG or M. bovis BCG in jam (non-formulated M. bovis BCG). Table 1 shows that while both the LTA and IFN-y assays showed significant differences between the formulated and non-formulated oral M. bovis BCG groups, the differences for the IL-2 assay 20 were not significant. The IFN-y assay was used in further experiments to monitor systemic immune responses due to importance of IFN-y in protection against tuberculosis.

To determine the effect of dose of M. bovis BCG following oral delivery, we compared splenic IFN-y responses to bovine PPD in mice vaccinated with varying doses of formulated 25 or non-formulated M. bovis BCG at 8 weeks post vaccination. Fig 3 shows that a low level of IFN-y (<200pg/ml) was detected in the formulated group following oral immunization with 106 CFU of M. bovis BCG, but there were no significant differences between the vaccine groups. When the dose was increased to 107 CFU, IFN-y responses in the non-formulated group remained low whereas responses to formulated M. bovis BCG increased significantly 30 (P < 0.05). Similar differences were seen with 108 CFU of M, bovis BCG. When the vaccine dose was increased to 109 CFU of BCG, an increase in the levels of IFN-y was seen in the non-formulated group while the formulated group remained high. At doses of M. bovis BCG ranging from 107-109 CFU, IFN-y responses in the formulated M. bovis BCG group were significantly greater that those of non-formulated M. bovis BCG. The increase in IFN- 1148875-1 23 562036 y responses seen at the high dose in the non-formulated group shows that considerably higher doses of oral M. bovis BCG are required for induction of immune responses compared to formulated M. bovis BCG. To determine the time course of immune responses to oral M. bovis BCG, we compared splenic IFN-y responses at 2 weekly intervals following oral or 5 subcutaneous vaccination with M. bovis BCG. Fig. 4 shows that IFN-y responses following subcutaneous vaccination peaked at 4 weeks and gradually declined at weeks 6 and 8. By comparison, IFN-y responses following oral vaccination with formulated M. bovis BCG first increased at 6 weeks and remained high at 8 weeks post vaccination. IFN-y responses to non-formulated M. bovis BCG or formulation material alone remained low between 2 and 8 10 weeks. These results show that immune responses following oral vaccination with formulated M. bovis BCG are delayed compared to subcutaneous vaccination but appear to persist at least to 8 weeks.

Peritoneal-derived lymphocytes from mice orally vaccinated with formulated M bovis 15 inhibit growth of M. bovis in autologous macrophages. The addition of NPEC to M. bovis-infected macrophages from mice vaccinated with oral M. bovis BCG formulations was performed in order to determine whether lymphocyte-mediated effector mechanisms could inhibit intracellular growth of M. bovis. Growth of M. bovis in macrophages was determined by [3H]uracil uptake. The growth of M. bovis within macrophages alone or when co-cultured 20 with NPEC from orally vaccinated mice is illustrated in Fig. 5 Macrophages prepared from mice orally vaccinated with formulated or non-formulated M. bovis BCG or mice given formulation material alone showed no differences in their ability to control M. bovis growth. When NPEC from mice vaccinated with formulated M. bovis BCG were co-cultured with autologous M. /wra-infected macrophages, the [3H]uracil counts were significantly reduced 25 compared to co-culture of NPEC from mice vaccinated with non-formulated M. bovis BCG or formulation material alone (/*<().05). These results demonstrate that lymphocytes from mice orally vaccinated with formulated M. bovis BCG activate macrophages to inhibit intracellular growth of M. bovis. Control of intracellular growth of M. bovis in vitro may reflect growth inhibition in vivo leading to reduced dissemination of M. bovis in the host.

Oral vaccination with formulated M. bovis BCG protects against aerosol challenge with virulent M. bovis. In order to determine the protective efficacy of formulated oral M. bovis BCG, mice were orally vaccinated with 5 x 107 CFU formulated M. bovis BCG or subcutaneouslv vaccinated with 1 x 106 CFU M. bovis BCG. Non-vaccinated mice served as 1148875-1 24 562036 controls. Mice were challenged with virulent M. bovis by the aerosol route 8 weeks after vaccination and euthanased 37-40 days after challenge. Table 2 shows that subcutaneous M. bovis BCG vaccination reduced the bacterial lung count by approximately 2.34 logs and the bacterial spleen count by 1.90 logs. By comparison, formulated oral M. bovis BCG reduced 5 the bacterial lung count by approximately 1.0 log and the bacterial spleen count by 1.48 logs. The results in Table 2 showed that oral formulated M. bovis BCG and subcutaneous M. bovis BCG induced significant protection against aerosol challenge with virulent M bovis, although the protective efficacy of subcutaneous M. bovis BCG in the lung was greater than that for oral formulated M. bovis BCG group.

D. Immune responses and pathology in possums.

Lymphocyte blastogenic responses. The effect of oral vaccination with formulated BCG on 15 the whole blood lymphocyte blastogenic responses to bovine PPD is shown in Fig.6 and Table 3. At 6 weeks after vaccination, the mean stimulation indices (Sis) to PPD-B for the formulated BCG group were significantly higher than for non-formulated BCG and non-vaccinated control groups (P<0.05). At 4 weeks following challenge with M. bovis all groups showed a mean SI for PPD-B of >20. These results show that oral delivery of formulated 20 BCG elicits strong immune responses to PPD-B in possums compared to non-formulated BCG.

A further experiment compared immune responses to four oral lipid BCG formulations with subcutaneous vaccination (Fig 11), Subcutaneously vaccinated possums showed strong LTA 25 responses which peaked at 4 weeks post vaccination (Mean SI 42.5) and gradually dropped to SI = 30 by 8 weeks In contrast, Lipid N formulated oral BCG failed to elicit an LTA response during the 8 week vaccination period. Oral BCG formulated in lipids C,K and F induced LTA responses which were weak (SI=l-7) at 4 weeks post-vaccination but increased progressively and were sustained through to 8 weeks post-vaccination (SI=15-22). These results show that 30 systemic immune response to oral vaccination is delayed compared with subcutaneous vaccination but that they may persist longer. Formulation N did not induce LTA responses above those seen with the non-vaccinated possums nor did it protect against aerosol challenge with M. bovis (see Table 4) an indication that the type of lipid used to formulated oral BCG is important for protection against tuberculosis. 1148875-1 562036 Clinical findings. The mean body weight changes between challenge and necropsy for the different groups are shown in Figure 7. The mean body weight of possums vaccinated with formulated BCG increased by 0.02 kg between the time of challenge and necropsy. In contrast, the mean body weights for the non-formulated BCG and non-vaccinated control 5 groups decreased by 0.35 kg and 0.23 kg respectively during this period. However these differences were not statistically significant.

In a further experiment (Table 4), which compared four oral lipid BCG formulations with subcutaneous vaccination, the mean body weight changes between challenge and necropsy 10 were significantly reduced for the subcutaneous vaccination group (mean weight loss 0.012 kg) and one of the oral lipid BCG groups (group F) (0.035 kg) compared to the non-vaccinated group (0.147 kg). By comparison, the mean weight loss for the remaining oral BCG groups were 0.060 kg (lipid C), 0.067 kg (lipid K) and 0.122 kg (lipid N). Possums which did not show an immune response to vaccination (ie non-vaccinated and lipid N 15 groups) showed greater body weight loss compared with those that responded.

Pathology. Macroscopic lesions were observed in the lungs of all of the challenged animals. The extent of tuberculous pneumonia can be estimated from the lung weights (Fig. 8). High lung weight is associated with extensive tuberculous pneumonia (3) (4). In order to 20 standardise differences in lung weight with variation in body weight, the lung weight of each animal was compared with the body weight and expressed as a ratio. The ratio of mean lung weight to body weight of the animals vaccinated with formulated BCG was 1.62. By comparison the ratio of mean lung weight to body weight of the non-formulated BCG and non-vaccinated control groups were 2.86 and 3.0 respectively. The lung weight to body 25 weight ratio of the possums vaccinated with formulated BCG was significantly different from the non-formulated BCG and non-vaccinated control groups (P<0.05). Typically, the lung lesions were small consolidated areas or lobar consolidation with a yellow necrotic area in the centre of the lesion. Swollen bronchial lymph nodes were observed in animals with the most extensive lung lesions.

In the second experiment (Table 4) which compared four oral lipid BCG formulations with subcutaneous vaccination, there were no significant differences in the ratio of mean lung weight to body weight between the vaccination groups. However possums which did not 1148875-1 26 562036 show an immune response to vaccination (ie non-vaccinated and lipid N groups) had higher mean lung weights compared with those that responded.

Bacteriology Mycobacterium bovis was isolated from the lung and spleens of the M. bovis challenged possums. The mean numbers of M. bovis isolated from the lungs and spleen for the different groups are shown in Figs 9 and 10. The mean lung bacterial counts for the non-formulated and formulated BCG groups were significantly lower than those for the non-vaccinated control group (P<0.05). The mean spleen bacterial counts for the formulated BCG group were 10 approximately 10-fold less than the non-formulated BCG group and approximately 40-fold less than the non-vaccinated control group. The mean spleen bacterial counts for the formulated BCG group were significantly lower than those for non-formulated BCG and the non-vaccinated control groups (P<0.05).

In the second experiment which compared four oral lipid BCG formulations with subcutaneous vaccination (Table 4), spleen bacterial counts for three of the orally vaccinated groups and the subcutaneously vaccinated group were significantly lower compared to the non-vaccinated group (P<0.05). The remaining oral lipid BCG group (lipid N) did not show significantly reduced bacterial spleen counts. No significant differences were seen between 20 the groups when bacterial lung counts were compared. In general, possums which did not show an immune response to vaccination (ie non-vaccinated and lipid N groups) had higher mean bacterial spleen and lung counts compared with possums that had responded to vaccination. 1148875-1 27 562036 TABLE 1. Bovine PPD-stimuIated spleen cell responses in mice 8 weeks after oral vaccination with different lipid formulations" Immunization IL-2 (pg/ml) IFN-y (pg/ml) LTA (SI) Formulation only 110.62 (+/-14.06) 51.44 (+/-14.38) 1.67 (+/-0.49) Non formulated BCG 153.51 (+/-25.22) 65.10 (+/-20.05 1.83 (+/-0.51) Formulation C BCG 430.43* ( +/-66.44) 2160.95* (+7-273.40) 16.26* (+/-1.20) Formulation K BCG 230.23 ( +/-54.13) 1268.30* (+/-76.80) 7.26* (+/-0.83) Formulation N BCG 130.23 ( +/-54.70) 75.50 (+/-16.80) 2.76 (+/-0.33) *Represents a mean which is significantly different from the mean of non-vaccinated (Formulation only) control 5 group. P value < 0.05 (Student t test) 1148875-1 28 562036 TABLE 2. Effect of vaccination on protection of mice against aerosol challenge with Mycobacterium bovis Vaccine Group Lung bacterial count1 Logio resistance Spleen bacterial count Logio resistance# Non-vaccinated .837a (±0.362) NA 4.565a(±0.189) NA Formulated Oral BCG 4.774b (±0.270) 1.06 3.084b (±0.176) 1.48 Subcutaneous BCG 3.498° (+0.237) 2.34 2.660b (±0.181) 1.90 1 Values are logi0 numbers of CFU ± standard error of M. bovis from the lungs and spleen of 6 animals per group 37-40 days post-challenge.

# Data are expressed as levels of logio resistance calculated by subtracting the Iog10 mean number of bacilli in the organs of vaccinated animals from the log10 mean number of bacilli in the organs of non-vaccinated animals, NA-not applicable.

Figures in columns with the same superscript letter are not significantly different (P>0.05).

TABLE 3. Number of possums responding to bovine PPD in the lymphocyte proliferation assay following oral vaccination.

Weeks after vaccination BCG formulation Group size 0 4 6 Formulated BCG 0* 2 Non-formulated BCG 0 0 1 Control (no BCG) 6 0 0 0 *No. of animals with stimulation index >3.5 1148875-1 29 562036 TABLE 4. Pathological and microbiological findings for vaccinated possums challenged with M. bovis Vaccine Group Change in body weight/challenge weight8 Lung weight/PM body weightb Lung bacterial count0 Spleen bacterial countc Lipid C -0.060 22.54 .634 1.301 * (±0.033) (±2.63) (±0.385) (±0.373) Lipid K -0.067 .49 .481 1.321* (±0.030) (±2.12) (±0.428) (±0.289) Lipid N -0.122 23.47 6.038 2.200 (±0.037) (±2.85) (±0.273) (±0.498) Lipid F -0.035 * 16.28 .342 0.934 * (±0.031) (±4.11) (±0.290) (±0.230) sc BCG -0.012 * .07 .384 1.270 * (±0.055) (±3.86) (±0.427) (±0.309) Non- -0.147 24.10 6.048 2.553 vaccinated (±0.048) (±3.50) (±0.166) (±0.465) Change in body weight /challenge body weight: Lipid F, BCG < Non-vaccinated (P<0.05).

Spleen bacterial count: Lipid C, Lipid K, Lipid F, BCG < Non-vaccinated; Lipid F < Lipid N (P<0.05), a Change in body weight between post mortem and challenge (kg)/ body weight at challenge (kg) b Lung weight (g) / body weight at post mortem (kg). c Bacterial count, CFU logio / g of tissue.

* Significantly different to non-vaccinated group.

INDUSTRIAL APPLICATION The immunogenic composition includes a lipid formulation which maintains immunogens in a stable matrix, through which they are uniformly dispersed. This facilitates administration of consistent doses of immunogen, avoiding dose dumping and ineffective low dosing. The lipid formulation has also been shown by the applicants to improve storage and viability of live 20 organisms contained therein. The lipid formulation also protects the immunogens and live organisms from degradation by stomach acids and enzymes. Losses in viability of organisms in lipid based formulations are also significantly lower than those reported for freeze-dried products. Storage under humid or moist conditions can also be achieved without deterioration because of the hydrophobic properties of the formulation. 1148875-1 562036 It has been demonstrated that the viability of organisms, particularly bacteria in vaccine preparations is important for inducing strong and long lasting protective immunity. This may be achieved using the compositions of the invention. The compositions are also simple to 5 prepare, more affordable to produce, and find increased consumer acceptance and safety where the use of needles and syringes can be avoided.

The inventive compositions may be administered in a variety of ways including subcutaneously, but are particularly amenable to oral delivery. The applicants have found 10 that the lipid formulation in the composition can protect viability of organisms and their constituent immunogens against degradation in the stomach, which enables live organisms to be taken up through the gastrointestinal mucosa for processing, replication and presentation to the immune system. Moreover, the applicants have determined that the doses to be administered can be effective at doses lower than previously anticipated for oral delivery (8).

Vaccination of wildlife, such as possums requires immunogens to be delivered by the mucosal route. Oral bait vaccines therefore represent a practical and cost effective delivery option. Oral vaccination of humans is also a more cost effective method of vaccination and likely to find favour with users.

When administered in other ways such as subcutaneously, the lipid formulation still provides protection from attack, for example, by macrophages. With subcutaneous administration, or administration by injection, the formulation of a lipid depot also allows sustained release to mimic the infection process, and facilitate the mounting of an immune response.

It will be appreciated that the compositions of the invention also provide substantial advantages over many higher cost, injectable vaccine formulations.

The compositions are effective to induce immune responses to a wide range of infectious 30 organisms, including gastrointestinal and respiratory pathogens, and preferably tuberculosis.

The compositions of the invention may also be used as a vaccine delivery system for a wide range of immunogens, or for the co-delivery or conjugated delivery of immunogenic molecules, particularly those which for reasons of dose or immunogenicity are poorly 35 immunogenic. The compositions of the invention are also useful as vaccine adjuvants.

INTELLECTUAL PROPERTY OFFICE OF N.Z. 1 6 DEC4® d c r F IV F D 31 562036 It will further be appreciated by those persons skilled in the art that the present description is provided by way of example only and that the scope of the invention is not limited thereto.

The term "comprising" as used in this specification means "consisting at least in part of'. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of 15 the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention 20 as defined in the claims of this application. 1148875-1 32 562036 REFERENCE LISTING 1. Aldwell, F. E., D. L. Keen, V. C. Stent, A. Thomson, G. F. Yates, G. W. de Lisle, and B. M. Buddie. 1995. Route of BCG administration in possums affects protection against bovine tuberculosis. New Zealand Veterinary Journal. 43:356-359. 2. Aldwell, F. E., D. N. Wedlock, and B. M. Buddie. 1996. Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis. Immunol Cell Biol. 74:45-51. 3. Aldwell, F. E., D. N. Wedlock, and B. N. Buddie. 1997. Sequential activation of 10 alveolar macrophages by IFN-y and LPS is required for enhanced growth inhibition of virulent Mycobacterium bovis but not M.bovis BCG. Immunologiy and Cell Biology. 75:. 4. Buddie, B. M., F. E. Aldwell, D. L. Keen, N. A. Parlane, G. Yates, and G. W. de Lisle. 1997. Intraduodenal vaccination of brushtail possums with bacille Calmette- Guerin enhances immune responses and protection against Mycobacterium bovis infection. Int J Tuberc Lung Dis. 1:377-83.

. Buddie, B. M., F. E. Aldwell, A. Pfeffer, and G. W. de Lisle. 1994. Experimental Mycobacterium bovis infection in the brushtail possum (Trichosurus vulpecula): pathology, haematology and lymphocyte stimulation responses. Vet Microbiol. 38:241-54. 6. Daugelat, S,, C. H. Ladel, and S. H. Kaufmann. 1995. Influence of mouse strain and vaccine viability on T-cell responses induced by Mycobacterium bovis bacillus Calmette-Guerin. Infect Immun. 63:2033-40. 7. Gheorghiu, M., M. Lagranderie, and A, M. Balazuc. 1996. Stabilisation of BCG 25 vaccines. New Approaches to Stablisation of Vaccine Potency. Dev.BIol.Stand.Basel, Karger. 87:251-261. 8. Lagranderie, M., P. Chavarot, A. M. Balazuc, and G. Marchal. 2000. Immunogenicity and protective capacity of Mycobacterium bovis BCG after oral or intragastric administration in mice. Vaccine. 18:1186-95. 9. Masarova, J.,Mislovicova, D.,Gemeiner, P and Michalkova, E. 2001 Stability enhancement of Escherichia coli penicillin G acylase by glycosylation with yeast mannan. Biotechnol Appl Biochem 34:127-33 10. Morrison, I. M. and Hawke, J. C 1979, Influence of elevated levels of linoleic acid on the thermal properties of bovine milk fat Lipids 14: 391-4. 1148875-1 33 562036

Claims (92)

WHAT WE CLAIM IS:

1. An immunogenic composition comprising a pharmaceutically acceptable lipid formulation and at least one immunogenic component comprising an 5 immunogenically effective amount of live organisms, the composition being formulated for oral administration, and wherein the lipid formulation is a lipid matrix in paste or solid form, and the live organisms are dispersed throughout the lipid matrix. 10

2. A composition according to claim 1 wherein the live organisms are uniformly dispersed throughout the lipid matrix.

3. A composition according to claim 1 or claim 2 wherein the lipid formulation undergoes solid to fluid transition between about 10°C to 40°C. 15

4. A composition according to any one of claims 1 to 3 wherein the lipid formulation undergoes solid to fluid transition between about 30°C to 40°C.

5. A composition according to any one of claims 1 to 4 wherein the lipid formulation 20 undergoes solid to fluid transition between about 30°C to 37°C.

6. A composition according to any one of claims 1 to 3 wherein the lipid formulation has a melt temperature between about 20°C to 30°C. 25

7. An immunogenic composition comprising a pharmaceutically acceptable lipid formulation which undergoes solid to fluid transition between about 10°C to 40°C, and at least one immunogenic component present in an immunogenically effective amount, wherein the lipid formulation is a lipid matrix in a paste or solid form and the immunogenic component is dispersed throughout the lipid matrix. 30

8. A composition according to claim 7 wherein the immunogenic component is uniformly dispersed throughout the matrix.

9. A composition according to claim 7 or claim 8 wherein the lipid formulation 35 undergoes solid to fluid transition between about 30°C to 40°C. 1148875-1 34 — 1 INTELLECTUAL PKOetW ! office of n.z. ■ , 23 APR 2009 562036

10. A composition according to any one of claims 7 to 9 wherein the lipid formulation undergoes solid to fluid transition between about 30°C to 37°C. 5

11. A composition according to claim 7 or claim 8 wherein the lipid formulation has a melt temperature between about 20°C to 30°C.

12. A composition according to any one of claims 7 to 11 wherein the immunogenic component is isolated or purified. 10

13. A composition according to any one of claims 7 to 12 wherein the immunogenic component is a protein, glycoprotein, or peptide.

14. A composition according to claim 13wherein the protein, glycoprotein or peptide is 15 present in a range of from 10-1000|j,g per gram of lipid formulation.

15. A composition according to any one of claims 7 to 14 which is formulated for parenteral administration. 20

16. A composition according to claim 15 which is formulated for subcutaneous administration.

17. A composition according to any one of claims 7 to 14 which is formulated for oral administration. 25

18. A composition according to any one of claims 7 to 12 wherein the immunogenic component comprises live organisms.

19. A composition according to any one of claims 1 to 18 which comprises at least two 30 immunogenic components.

20. A composition according to claim 19 wherein one immunogenic component is live organisms and one immunogenic component is a protein or peptide. 1148875-1 35 intellectual property office of n.z. 23 APR 2009 received 562036 10 15 20 25 30

21. A composition according to any one of claims 1 to 6, and 18 to 20 wherein the live organisms are a biologically pure culture.

22. A composition according to claim 21 wherein the live organisms are selected from the group consisting of fungi, protozoa, bacteria, and viruses.

23. A composition according to claim 22 wherein the virus is HIV or SIV.

24. A composition according to claim 22 wherein the bacteria is selected from Brucella, Anthrax, and Mycobacterium.

25. A composition according to claim 24 wherein the bacteria is Mycobacterium.

26. A composition according to claim 24 or claim 25 wherein the Mycobacterium is selected from M. tuberculosis complex (comprising M. tuberculosis, M. bovis, M. africanum and M. microtii), M. avium-intracellulare complex (comprising M. intracellulare and M. avium), M. paratuberculosis, M. vaccae, M. smegmatis, M. chelonae, M. fortuitum, M. kansasii, M. leprae, M. marinum, M. ulcerans, M. simiae, M. haemophilum, M. malmoense, M. shimoidei, M. gastri, M. terrae complex, M. bovis and M. nonchromogenicum, including immunogenic components thereof.

27. A composition according to claim 26 wherein the Mycobacterium are functionally equivalent variants, natural or genetically engineered clones, mutants, and recombinants of the strains in claim 26.

28. A composition according to any one of claims 24 to 27 wherein said Mycobacterium is selected from M. tuberculosis, M. microtii, M. intracellulare, and M. avium, including immunogenic components thereof.

29. A composition according to any one of claims 24 to 27 wherein the Mycobacterium is M. bovis.

30. A composition according to claim 29 wherein the M. bovis is Bacille Calmette Geurin 1148875-1 (BCG). 36 562036

31. A composition according to claim 30 wherein the M. bovis is the BCG Danish strain 1331.

32. A composition according to claim 30 wherein the M bovis is the BCG Brazilian 5 strain.

33. A composition according to claim 30 wherein the M bovis is a BCG strain selected from Pasteur 1173P2, 83/6235, Glaxo 1077, Japanese 172, Prague, Russian, Copenhagen and Connaught strains. 10

34. A composition according to claim 30 or claim 33 wherein the M. bovis is the BCG Japanese 172 strain.

35. A composition according to any one of claims 24 to 34 wherein the Mycobacterium is 15 present in a concentration of 1 x 105 to 1 x 10to colony forming units (CFU)/ml.

36. A composition according to claim 35 wherein the Mycobacterium concentration is lxlO7 to IxlO9 CFU/ml. 20

37. A composition according to claim 22 or claim 23 wherein the live organism is a virus, and the virus is present in a concentration of lxlO3 to lxlO10 Plaque Forming Units (PFU)/ml.

38. A composition according to claim 37 wherein the virus is present in a concentration of 25 1x10s to lxl0s PFU/ml.

39. A composition according to claim 20 wherein the protein is an immunocontraceptive protein. 30

40. A composition according to claim 20 wherein the protein or peptide is poorly immunogenic.

41. A composition according to any one of claims 1 to 40 wherein the lipid formulation 35 comprises 40% to 100% of: /7MTr."~~—-— 1148875 1 37 I 2 3 APR 2009 i

MMce i \/ & pii 562036 (a) Ci6 fatty acids* or (b) Cig fatty acids; or (c) C16 and C \ g fatty acids. 5 42. A composition according to claim 41 wherein the lipid formulation comprises 60% to 100% of: (a) C]6 fatty acids; or (b) Ci8 fatty acids; or (c) Ci6 and Cig fatty acids. 10

43. A composition according to claim 42 wherein the lipid formulation comprises 80% to 100% of: (a) C]6 fatty acids; or (b) Cis fatty acids; or 15 (c) Cjg and Cis fatty acids.

44. A composition according to claim 43 wherein the lipid formulation comprises 90% to 100% of: (a) Ci6 fatty acids; or 20 (b) Cis fatty acids; or (c) Cig and C^ fatty acids.

A composition according to any one of claims 1 to 40 wherein the lipid formulation comprises 10% to 40% Ci6 fatty acids; and 40% to 90% Cis fatty acids.

A composition according to claim 45 wherein the lipid formulation comprises 20% to 35% Cig fatty acids, and 50% to 80% Cig fatty acids.

47. A composition according to claim 46 wherein the lipid formulation comprises 25% to 30 32% Ci6 fatty acids, and 60% to 70% Cis fatty acids.

48. A composition according to any one of claims 1 to 40 wherein the lipid formulation comprises less than 35% C14 fatty acids or shorter. 45. 25 46. 562036

49. A composition according to claim 48 wherein the lipid formulation comprises less than 20% Ch fatty acids or shorter.

50. A composition according to claim 48 wherein the lipid formulation comprises less 5 than 10% C]4 fatty acids or shorter. 10 30

51. A composition according to any one of claims 1 to 40 wherein the lipid formulation comprises less than 5% C]4 fatty acids or shorter; 25% to 32% C]6 fatty acids; and 60% to 70% Cig fatty acids.

52. A composition according to any one of claims 1 to 40 wherein the lipid formulation comprises: 20% to 60% saturated fatty acids; 25% to 60% monounsaturated fatty acids; and 15 0.5% to 15% polyunsaturated fatty acids.

53. A composition according to claim 52 wherein the lipid formulation comprises: 30% to 55% saturated fatty acids; 30% to 60% monounsaturated fatty acids; and 20 3% to 11% polyunsaturated fatty acids.

54. A composition according to claim 53 wherein the lipid formulation comprises: 40% to 50% saturated fatty acids; 40% to 55% monounsaturated fatty acids; and 25 5% to 9% polyunsaturated fatty acids.

55. A composition according to any one of claims 1 to 40 wherein the lipid formulation comprises about 3% myristic acid; about 26% palmitic acid; about 15% stearic acid; about 40% oleic acid; and about 6% linoleic acid.

56. A composition according to any one of claims 1 to 40 wherein the lipid formulation comprises about 47% lauric acid, about 20% myrisitc acid, about 12% palmitic acid, about 12% stearic acid, and about 3% oleic acid. 1148875-1 39 intellectual propgrtv office OP 'V z 2 3 APR 2uuy received 562036

57. A composition according to any one of claims 1 to 40 wherein the lipid formulation comprises a coconut oil.

58. A composition according to claim 57 wherein the coconut oil comprises a 5 hydrogenated coconut oil.

59. A composition according to any one of claims 1 to 40, 57 or 58 wherein the lipid formulation comprises an olive oil. 10

60. A composition according to claim 59 wherein the olive oil is hydrogenated olive oil.

61. A composition according to any one of claims 1 to 60 which is essentially free of aqueous components. 15

62. A composition according to any one of claims 1 to 61 wherein the composition consists essentially of immunogenic components and lipid.

63. A composition according to any one of claims 1 to 62 which further comprises albumin-dextrose-catalase (ADC). 20

64. A composition according to any one of claims 1 to 14 and 17 to 63 which further comprises one or more flavouring agents, attractants or odourants.

65. A composition according to any one of claims 1 to 14, and 17 to 64, wherein said 25 composition is protectively coated.

66. A composition according to claim 65 wherein the protective coating is gelatin.

67. A composition according to any one of claims 1 to 65 which is a vaccine. 30

68. A composition according to any one of claims 1 to 65 which is a vaccine adjuvant suitable for use with another vaccine.

69. A composition according to any one of claims 1 to 68 for use in immunising an 35 animal. 1148875-1 40 intellectual property OFRCE OF w.z. 1 6 JUN 2009 D ET A P i w r— nj 562036

70. A composition according to any one of claims 1 to 68 for use in stimulating a mucosal immune response in an animal. 5

71. A method for immunising a non-human animal, the method comprising administering to said animal a composition according to any one of claims 1 to 68.

72. A method for stimulating a mucosal immune response in a non-human animal, the method comprising administering to said animal a composition according to any one 10 of claims 1 to 68.

73. A method according to claim 71 or claim 72 wherein the administration is oral administration, and the composition is formulated for oral administration. 15

74. A method according to claim 71 or claim 72 wherein the administration is subcutaneous administration, and the composition is formulated for subcutaneous administration.

75. A method according to any one of claims 71 to 74 wherein the animal is a cattle, deer, 20 sheep, possum or badger.

76. A use of a composition according to any one of claims 1 to 68 for the manufacture of a medicament for immunizing an animal. 25

77. A use of a composition according to any one of claims 1 to 68 for the manufacture of a medicament for use in stimulating a mucosal immune response in an animal.

78. A use of a lipid formulation as described in any one of claims 1 to 11 and 41 to 62 for the manufacture of a medicament for immunizing an animal. 30

79. A use of a lipid formulation as described in any one of claims 1 to 11 and 41 to 62 for the manufacture of a medicament for stimulating a mucosal immune response in an animal. 1148875-1 41 562036

80. A use of claim 78 or claim 79 wherein the medicament further comprises one or more immunogenic components as described in any one of claims I, 7, 8, 12 to 14 and 18 to 40.

81. A use of claim 80 wherein one immunogenic component is live organisms.

82. A use of any one of claims 76 to 81 wherein the medicament is formulated for oral administration.

83. A use of any one of claims 76 to 81 wherein the medicament when administered is administered orally.

84. A use of any one of claims 76 to 81 wherein the medicament is formulated for subcutaneous administration.

85. A use of any one of claims 76 to 81 wherein the medicament when administered is administered subcutaneously.

86. A use according to any one of claims 76 to 85 wherein the medicament is a vaccine.

87. A use according to any one of claims 76 to 85 wherein the medicament is a vaccine adjuvant suitable for use with another vaccine.

88. A use according to any one of claims 76 to 87 wherein the animal is a human, cattle, deer, sheep, possum or badger.

89. An immunogenic composition as defined in claim 1 or claim 7 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.

90. A method as defined in claim 71 for immunising an animal substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 1148875-1 42 property OFFICE OF N.Z. 1 6 JUN 2009 DC Ac i i 562036

91. A method as defined in claim 72 for stimulating a mucosal immune response in an animal substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 5

92. A use as claimed in any one of claims 76 to 88 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 1148875-1 43 i 'ntelofrcea8fps0zperty ' 16 JUN 2009 I B-ELO. Er x