AU2008202282A1 - Immunogenic Compositions - Google Patents

Description

23-MAY-2008 12:29 FROM iH P -RR TO 061262M79999 P.044 00 0C Regulation 3.2

AUSTRALIA

PATENTS ACT, 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Actual Iaventors: Address for service in Australia: Invention Title: OTAGO INNOVATION LIMITED, ANIMAL HEALTH BOARD, INC. and AGRESEARCH LIMITED Frank Ernest ALDWELL, Bryce Malcolm BUDDLE, and lan George TUCKER A J PARK, Level 11, 60 Marcus Clarke Street, Canberra ACT 2601, Australia Antigenic compositions The following statement is a full description of this invention, including the best method of perfouning it known to us.

-1- (followed by page la) COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:29 FROM AJ PARK TO 00612628:3999 00 O ANTIGENIC COMPOSITIONS

O

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

00

BACKGROUND

oto 0 00 Most human and animal pathogens including those that cause tuberculosis initiate o 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 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 alterate 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. 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 antigenic 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 freezedrying procedures result in 30 to 50% loss of viability of BCG and impaired recovery of remaining live bacteria A composition which retains greater viability of organisms prior to use would contribute greatly to the 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 use of Freund's complete adjuvant (FCA) in human and veterinary vaccines is contraindicated la COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 2J-PHY-001 12: 29 FIROM A3 PIRI TO 0061262837999 P. 1 00 because Of toxic rections that have tenreported. Tior these reasons, Fftud's adjovza may also be unsujreblc for oral administration.

I" other oil-water emuliWi eMfActants 'have been required becaue of the high oil contear Detergent propertes of stictants render them unsuitable for parenteral or oral admiistatiun.

Furter, 'o"dc reacton eveft fcr appoved sorfactats have been xepfl A further drawvback with 00 emulsions are: that they are heceogeu syss Of one iromisalbe liquid disered in another. This ci ~~is unstable and resualn in separtion of the aqueous ph&se ovrte, This poses di iltie for o vaii accnea in stabLe "esumMreover, antigefts h-Aped in thie aqueous phase of waterin-oi emulsions am re nlkey to be protected from dqgadation in the stomack 0 0 ~~Uposoxnes =4d lpId vtsicles have also been explored for mse wth vaodner, panictlay with small antigenic components tha my be readily eacq%etL Generally, Irposomez and vesicles are not usefl(4 for encapsulation of larg andien such as live mileor$ansni.,Moreover, liposomes and 1$vesiclws are costly cnd time consming to _produoe,, end the extrction procedume used to a~ pteparation may result in alteration of the chemical structur or tWit of vacins prepaxations and hence their inmunogeoidty. For examuple, hteat and solvents may altef the biologcalnterty of antigenic compoents such as piomins.

It is terefr an object of t Present &inntion to provide an antigeric compositio Sad~er divetly system which addresses these desideraia or which at Icag provids the. publc with a useWb ohoic&- SUMMARY OF THMEf VENTION Accordingy, IA a ffis Aspec the present inventioa provides an anigenic composition comprising a pbsxmaoewieally acceptable lipd Oixmulation and at least one aatigcic component comprising an "kgucally offective awovnt Of live organisms, tam compositon WASn fw~tled for orA Preferably, the lipid formulation is in solid fo In a fulrthaer aspect the preset invention provkie an anktigehic composition comrising a pb=Armcedcaly acceptable lipid %bumxlatoa Which is in bolid form aod undergoes solid to fluid, trnsition tetween1 about $00C to 4M~ and At lcea one catigenic componet present in an anridgenically effioctime amholnL 2 COMS ID Na:ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-:-MnY-2008 1 P2:29 FROM A3 PARKTO0628399P7 TO 0061262837999 P.1-17 00 Prefexe lipid fommiations for use in the compositionts of the iivein contain long chain, Xt6cds.

Ci I terms of fatty acid composition, a preferred lipid formulation contains 40% to. IOOO/t 6M.% to I00OW6 mar preferably Soo% to 100%/, and evcon mor prefeAbly 90% to ci~ 1Go% Cie, aridor Cis fatty acids.

00 A fuxther preferred composition has a lipid formulation which contains less than 35016 00preferably less Than 256/, and mmr preferably less than 109 /0 C 14 fatty acids or Shorter.

o00 0 ~~In one embodiment the lpid foxmulato contains 206% to 60% satuated fatty acids;, to 60% monounsaturated fatty acids; and to 05% polynsauted fatty acids.

In a particrilarly preferred composition, the lipid formulation contein to 50%/ satuate fatty acids; to 55% monounsaturated fatty acids; amd The curret prefcred lipid formulaton for use in the invention ba= the tbnnnbe. 3% myzisfic a1cid; 26% palmnitic acid; 15% stearic acid; 40%/ esie acid; antd 6% linoleic acid TIhe antigenic component may be a protein, glynoproitei peptide or factor with a protein or peptide. component JAi one eanbodiment the zfnigenic cbmponent comprises live organisms. Pxeferablyr as a biolcoally pure cltre Prefe-rably, the live Organi== in the compositions of t invention are bacteria, particularly non-pathogenic becteW6a and mome pretmably bacteria belonging to the genu Mycobacewn.m A particularly prefered mycobacteam -for use in the invention is Mycjobacze~fum tort BCG.

3 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MIAY-20-E 12:30 FR'OM Hr PRRK TD 061262837999 P.a 0 0 In one embodiment, the composition comprises at least two antigenic components. The first 0 is preferably a live organism and the second antigenic component is preferably derived from an infectious agent, or is a weakly immunogenic protein or peptide.

In a further aspect, the invention provides a method for preparing an antigenic composition of the invention, the method comprising mixing the antigenic component(s) with the lipid formulation.

00 C' In a still further aspect, the invention also provides a method for immunising an animal, the O 10 method comprising administering to said animal an antigenic composition of the invention.

00 o In a further aspect, the invention provides a method for stimulating a mucosal immune Ci response in an animal, the method comprising administering to said animal an antigenic 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 antigenic compositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Aspects of the invention will now be described in relation to the accompanying drawings in which: 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 (10-250C) BCG formulations were wanned to 370C and emulsified in 7H9 broth. Numbers of viable organisms were determined by inoculating serial 10 fold dilutions of each emulsion onto 7HlI agar plates. The number of CFU/ul of formulation media was 4 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:30 FROM AJ PARK TO 1:16126,""37999 P. 09 0 0 determined after 2-3 weeks of culture. Results are representative of duplicate experiments and Sare 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 C with 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.

C Supematants were then collected and analysed using a sandwich ELISA. Each treatment 00 C group contained 6 mice. Spleens were individually processed. Results are expressed in pg/ml S 10 and are presented as means of triplicate determinations. Bar indicates standard error.

00 o 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 analysed using a sandwich ELISA. Each treatment group contained 5-6 mice. Spleens were individually processed. Results are expressed in pg/mi 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 bovis at an MOI of 2 bacilli per macrophage. Non-adherent autologous NPEC were added at a ratio of 10 NPEC per macrophage. 3 HJuracil incorporation was then assessed at 72h post infection. The mean 3 lH]uracil uptake by cell cultures which did not contaifi M. bovis was 460 cpm. Growth of 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 nonformulated non-vaccinated control Results are expressed as mean COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:30 FROM AJ PARK TO 0061262837999 1 0 stimulation index *Represents a mean which is significantly different from the mean of 0 non-vaccinated control 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 c- challenge to necropsy. The mean body weight of the possums immediately prior to challenge was 3.0 0.07 SE) kg. Bar indicates SE.

00 0 Figure 8. Effect of oral vaccination with formulated BCG on lung weight of possums S 10 challenged with MA 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 animal was compared with the body weight and expressed as a ratio. *Represents a mean ci 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 mycobacteria isolated from lungs following challenge with M bovis. Results are expressed as the geometric mean number of CFU (loglo)/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 mycobacteria isolated from spleen following challenge with M. bov:. Results are expressed as the geometric mean number of CPU (logloYg of tissue. *Represents a mean which is significantly different from the mean of non-vaccinated control group; bar indicates SE.

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 Results are expressed as mean stimulation index (Si).

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

6 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 2-M1 123 FROM. A3 PARKToU 125355 P1 TO 0061262837999 P.11 00 DETAILED DESCPflQN Accodingly, in a first aspect the invention provides an antigeajo compositiwO compising a.

phwanncutically accptble lipid formulation and at least one antigenic componeBut coniplisig ci an antigenicay effective amount of liv; organisms, the composition being formulate-d for oral adrrdnisfration.

00 Preferably, the lipd is in Solid form. Conveniently, the lpd is in solid form at 10 0 -C or above.

c Ini a I inther aspect the presalt invention provides an antigenizcomposition comprising a o 10 pharmacutically acceptable lipid formulation which is in solid form and undergoIes solid to o ~fluid transition between about 306C to 40 0 C, and *t least one atigeni; cOMPOnen present lB an anigenically e-ffective amount The lipids empLoyed wn the frulation~s above ar preferably siable for atnAl or human admiuistraioincludin consumpto and may be selected fr-om a broad range of' naturzil (vegetable or animal derived), or synthetic lipid produt including oils fats =nd waxes.

Most uwually, the lipid mateial will be iquid at teprtrsabove about 30 0 C. That is, the liid should be selected to aedeve meltinig poinat at physiologia tunperature ia the animal to wbhchhis admnstered mostuually hy tbeora route. Desirably, the lipid will be !a the forn of a solid at 104000 at atmospheric prcssurej and preferably is uti0 solid at from 2(Y'C to at atmospheric pressure. However the melting temperature of lipid is tot tsdumve and may include oil, fats and wares with a rangeo of melting temperatures.

Preferred lipids for ms herein vwkiego transition from the soWi phase to the liquid phase between about 300C md physiolgicaI, temperature of about 400C, commonly 37C.

Summaries of lipid phase behaviour are available in the amt see for example Accordingly, a skiled reader on select a liid having the desired properies and melt point based on information in the art and simple experimnt Snitable lipid formulations =a triglycerde Including glyceryl esas of oarboxylic acid, compounds consisting of an aliphatie chain and a -00011 ead, amd saturted a=d non-saturted fatty acids axdxmixtures thmeo 7 COMS ID No: ARCS-i 91 648 Received by IP Australia: Time (I-tm) 09:42 Date 2008-05-23 23-MrY-238 12:31 FROM AJ PARK TOI 0061262837999 P.12 00 o Currently preferred lipids are triglycerides containing primarily Cs to C20 acyl groups, for Sexample myristic, palmitic, stearic, oleic, linoleic, parinic, lauric, linolenic, arachidonic, and eicosapentaenoic acids, or mixtures thereof.

ec CI It has also been determined that for lipid formulations useful in the invention longer chain fatty acids, for example, C 16

-C

18 are preferred. Long chain fatty acids have been found to be l more effective in protecting organisms such as BCG in vaccines given to mice and possums.

00 Cl Viewed in this way, lipid formulations preferred for use in the invention contain: 40% to Cl o 10 100% preferably 60% to 100%, preferably 80% to 100%, and more preferably 90% to 100% 00 C1 and/or Cis fatty acids.

0 Cl Generally, C 1 6 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 Cis fatty acids represent from 40% to 90%, preferably from 50% to 80%, and more preferably from 60% to 70% Ci8 of the total fatty acid content.

Preferred lipid fomnulations also contain less than 35% C1 4 fatty acids or shorter, preferably less than 25%, and more preferably less than In terms of chain length, the preferred lipid formulation contains less than 5% fatty acids with Ci4 chains or shorter, 25% to 32% Ci6 fatty acids, and from 60% to 70% C18 fatty acid chains.

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 preferably 30%/ to 60%, and more preferably 40% to 55%; and polyunsaturated fatty acids in 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 saturated fatty acids, 40% to 50% monounsaturated fatty acid, and 5% to 9% polymusaturated fatty acid.

s COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-NY-2UIV FROM PJ PARK O0Zli2879 .1 FD 0061 2 28-17999 P. 13 00 The currently prefaed lipid formulation for use in- the invention has the formula. 3 mytistic Ctacid, 26% palinitic adid, 15% stcari; acid 40% oleic acid end 60/ lixtoeic acid as determined byHIPLC analysis.

Currently preferred lipids formulations also indlude animal drved fractionated lipid.

complexes, one or more hydrogenaed vegetable oils, especially olive oil or coconut oiL.

00 conmial suppositoiy bases and other lipid formulations or mixture thereof.

0 The lipid fonnulation is useffhl in the pzcpaton of *aigenic composious, and in protctig 00 10 antigeswithin the composition frxom degradatio. The lipid fbunulation is especially, useful in o mintdaining viability of ive organisms, particuArly bacteria. The lipid fonnulation acts to maintmin the organsm in a live, but dorm=u stae. This is particularly imnpoxtat for vaccines compsingUvcorganismns formulatd for oral adninisrh~on. The lipids also maintain axlligens int a uniforn saspension. Tha is, in the compositons of the invention t antigenic.

Qofpoflonets, and live orgaims in particular are uniformaly &diszWte thruou t. a solid or paste li, lipid mattix Te lipids also protect the. antiges from destraction by gastrointestinal seeions when orally administeed. Protecton fro macophaga attack is also likely when administered by othe routes such as subcutaneouWy- This allows for upttb. of the antigens ad pariculary live orgatitw tsroigh the gastrointestinal mucosa, and subsequent relieation of organisms in the hostL Rqcgaaion of the live orgams within the host sriwaates a protective inlme response as deenned by a reduction in severity of &iesag Wowing Formualations for a wide range of delivery routes may also juebAde additives sueb as fillem, exteders, binders, wetting agents emulifiers bufn agents, surfacltants, suspen agents, presrvatives, colourats salts, mntioxidants including mono sodium glutamate (MSG), vitamin such as ViA=i= X butylated hydroxansole (RIT), ialbwui detrose-cataas (ADC), protective* coatings, atnetants and odouauts, and agents to aid survival of organism 0aciaed in the lipid but are naot limited thereto.

Protective coatings or enterooatip mray be selected for exampi; from gcls, pam&tns, and plastics includig gelatin The coatings tintr aid in the preventio of exposr to Zas-6io acids and enzymes, when the oral adwinisttioa route is selected.

COMS ID No: ARCS-i 91 648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAtY-2008 12:32 FROMI HU PmRI O0622393P1 TO 0061--62837999 P.14 00 When used for oral adminisnariou, the formlation may also ine~ic additives Which, for examnple, imxprove palattility, such as flvouring agents. (including anise oil, choolWt and pcppecciat), and sweetencis (inoluding glucose, fructose, or any othe auMa or artfiia sweetener).

The antigenic, component may be a protein, glcoprotein, peptkk&. or facto with a protein or pepide component preferbly isolated and/or puriied or mixture thereof The component may 00 be derived from an agent which may be used to genat an immune response in an animalo ~Mos usually, the antigen will bear at least one epitope which ir. present on an organ wbiZ is 00 10 pathogtdc; in the animal species to be treated, Other anligenic sltnres such as a= zmo"z in the o ~art may also be used. For exazupi; polyssvowides, glycolii4 ond haptens conjugated to a Preferably, the anrigenkc component is a living oianimna. preforably a biologcally p=n culture The living organism in the coMositio my be selected from the gonp consistng of, fWWi, protozoas, bacteria and viruse. For eample, MV, SWV, Brucrla and, Anthvx. Preferably ft organism is a bactia Orgauismercaldy sMooted from ncn-pathogenie bactria awe Preferred for use in ompositions fonlaaed for oral or subvvtatrnn delivery. A preferred bacterium is a iinpa&hgtnc stran selected from the genus Afrobacaflwn including Ut rzdras complex (coMpriin U tubercUlosis AL bovis M. ofriCanu and Mt Microtit) M. aVk0AIntuceIIulce complex (comprising M. fnracdlulae ad M. aflw), Me pariatxbaradis. MW. vacc= X ammnats, Mt chelonais Mfoizwn tm ALamsai, M. hep=e M. mPavuan M ulcavns, YAt simia, Af hoemowphihm, Mt mbnoense M. Ahmoidei. Mt gaar, AL tenie complex and Mt nonchromogeniwn. LA a peiliculaty preferrd embodiment the agent is MOtile Claiete Guerin an altexwatd str=i of it bovis inldxing die t1OtT4Ugn strain 83/6235, Pasteur 1173P2, Glaxo 1077, hpmAeae 172, Prague, Russian, Brazliwi, Danish 1331, Copenhagen Cozmanbt and including funtionally equivalent variants and other attennated strains of AL bovtt clones, ntants and rWoflilumts of these stran either natural recvriinaats or those produced by any of a wide Of genetic: engieering techniqueg, and atigeic comnponents thereof.

It Will be appreciated from the foregoing that the antigenie component may be a qomplex of proteins Or peptides, or the like.

COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:32 FROM AJ PARK TO 00612628---7999 P.11 00 0 O In one embodiment, the composition includes at least two antigenic components selected from any of those identified above, and may include multiple combinations of subunit antigens.

Three or more antigenic components are feasible.

Ci The concentration of the antigenic 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 Cl immune response on administration to an animal. In particular, an immune response in the gut 00 c, associated lymphoid tissue of the small intestine. In the case ofmycobacteria a range of from S 10 1 x 10 5 to 1 x 10 t0 colony forming units (CFUy)ml is appropriate. Preferably, the 00 concentration is from 1 x 10 7 to 1 x 10 9 CFU/mI. For protein and peptide type antigens a 0 range of from 10-100,g per gram of formulation is appropriate. For virus-type antigens a C1 range of 1 x 10i to 1 x 10 1 0 preferably 1 x 10 5 to 1 x 10 8 Plaque Forming Units (PFU)/ml is appropriate. The immune response may be humoral, or cell mediated including a mucosal immune response.

Accordingly, in a further aspect the invention relates to a method for stimulating a mucosal immune response in an animal by administering an antigenic 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 antigenic component(s) and other ingredients (when used) as described above are added. Dispersal of the antigenic composition may be achieved by mixing, shaking or other techniques that do not adversely affect the viability of the antigenic component.

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 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 antigenic composition is a vaccine.

11 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:32 FROM AJ PARK TO 0061262837999 P.16 0 In an alternate embodiment, the antigenic composition is an adjuvant useful for administration o with a vaccine to increase efficacy of same. Mycobacterium-containing, and BCG-containing antigenic compositions in particular are preferred for use as adjuvants.

The antigenic composition of the invention can also be useful for generating a response to a (n second or further antigenic molecule of a type as indicated above for the antigenic component, particularly those that are weakly immunogenic. This may be achieved by co-delivery of the c second or further antigenic molecule in an antigenic composition of the invention by Sconjugating the antigenic molecule to the antigenic component of the composition 10 Conjugation may.be achieved using standard art techniques In particular, an antigen of 00 interest may be conjugated to an antigenic carrier or adjuvant by a linker group which does not interfere with antibody production in vive. The antigenic carrier or adjuvant may be any 0 of the antigenic 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 those that bind to Fc receptors. The second or further antigenic 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 an animal, the method comprising administering to said animal an antigenic composition of the invention.

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 12 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:33 FROM AJ DARK TO 0061262837999 P.17 00 practical method for vaccinating wildlife. In one embodiment the applicants have therefore o 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 Ci 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 Ci depot allows for sustained release of the antigenic component in a process which more closely 00 Ci 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 0 these forms of administration.

0 Ci 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 antigen component in combination with its antigenicity. Usual considerations such as weight, age, sex of the animal, concurrent treatments (if any), and nature of the antigen 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 10 5 to 1 x 1010, preferably 1 x 101 to 1 x 10 9 CFU/kilogram per dose. For peptide and protein type antigens the dose range will be from 1-10,000g, preferably 1000l g. For virus-type antigens the dose range will be from 1 x 10 3 to 1 x 1010, preferably 1 x 10 to 1 x 10 s PFU/ml. Whichever method of delivery is used, when live 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 rultidose 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 At 4°C the lipid formulation facilitates storage and maintenance of organisms in a dormant 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.

13 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:33 FROM AJ PARK TO 0061262837999

P.

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

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

I EXAMPLES 00 C 10 MATERIALS AND METHODS 00 0 Bacteria. M. bovs BCG Pasteur 1173P2 (Paster Institute, Paris) was used as the vaccine 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 For BCG formulation and macrophage infection, bacteria were grown to mid log phase in 175 ml flasks (Falcon) containing Middlebrook 7H9 medium (Difco, Detroit, Mich.) supplemented with albumin-dextrosecatalase (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% 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 fonning units (CFU) of BCG or M. bovi was determined as described previously 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 selected for testing in oral vaccine trials in mice and possums: Formupinn -an animal derived fractionated complex lipid ormu l K consisting of tryglycerides of purified hydrogenated coconut oil; Formlation N-Novarta B, a commercially 14 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-200B 12:33 FROM 4 r PARK TO 0061262-37999 19 00 available suppository base. The three formulations were analysed by gas chromatography to O 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 10 7 CFU/ml for C, vaccination of mice or 1 x 108 CFUhnl for vaccination of possums. To increase attractiveness and palatability for possums, 10 rag of glucose and 10 pl of anise oil (Pharmacare, Auckland ,1 NZ) were added per ml of formulation. For oral vaccination of mice, 10 mg of glucose, 1 mg

OO

00 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 N affect viability of BCG. BCG formulations were transferred to 15 ml tubes (falcon) and 00 o allowed to solidify with gentle mixing at 4OC. Formulations were removed from the tubes and 0 aseptically cut into Ig 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 0 C or at room temperature (10-25C) was determined as described previously Samples for culture were collected by warming 100 mg aliquots of the three BCG formulations to 37 0 C for 15 min and performing serial 10-fold dilutions in 7H9 broth. Numbers of viable organisms were determined by inoculating 100 pal of each emulsion onto Middlebrook 7H11 agar plates (Difco) supplemented with oleic acid-ADC (OADC; Becton Dickinson) and 0.5 glycerol.

Emulsions were dispersed using a glass spreader. Plates were sealed with parafilm and incubated in 5% C02 at 37 0 C. The number of colonies was counted after 2-3 weeks of culture. Results are expressed as CFU/g 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 ofM.

bovir 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 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:34 FROM AJ PARK T 0061262837999 0o consisted of lipid formulation alone. For dose response and time course experiments mice Swere given vaccine in two separate doses at 24 h intervals. For aerosol challenge experiments, 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 CN sacrificed by CO 2 inhalation and their spleens were removed aseptically.

C Spleen cell proliferation assay. Spleen cell suspensions were prepared by filtering cells 00 Cl through a cell strainer (70-pm mesh; Beckton Dickinson). Erythrocytes were lysed in 0.83% S 1t NH4Cl (pH Cells were washed twice in PBS and resuspended to 1 x 10 6 /ml in 00 Dulbeccos's modified Eagles medium (DMEM) containingl0% foetal calf serum (FCS), o mM HEPES penicillin at 100U/ml, streptomycin at 100g/ml, 5.5 x 10 5 M 2mercaptoethanol (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 10 per well) were plated out in triplicate wells in 96-well plates (Nune). Cells were cultured purified protein derivative from a culture of M. bovis (bovine PPD; CSL, Melboure, Australia), 60pg/ml final concentration or with medium alone. Cells were harvested 4 days later, after an 18-h pulse with 1 pCi of 3 H] thymidine (Amersham, Buckinghamshire, England)), and the incorporated thymidine was measured as previously described 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 epm for splenocytes cultured with medium only.

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 pl of either PBS or bovine PPD final concentration) was added to the wells. Cultures were incubated for 72 h in 5% CO2 at 37°C after which time 200 Pl of culture supernatant was collected and frozen at 70°C for cytoldne analysis. Interleukin-2 (IL-2) and interferon-gamma (TPN-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 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.

16 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 2:-MAY-200& 12:34 FROM AJ PARK TO 00612f-283?799P2 00 M. bovis inhibition assay. Peritoneal-derived macrophages were tested for inhibition of 0 O intracellular growth of M. bovis following co-culture with or without autologous lymphocytes. Experiments were performed according to a modification of previously CO 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 100U/ml penicillin (supplemented DMEM) at 2 x 106 /ml. 100 gl of cell suspension was dispensed into a 96 flat well plate (Nunc). After incubation for 2 h in 5% CO 2 at 37C the 00 0 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 iN population by incubation in 25 ml flasks (falcon). Nonadherent PEC (NPEC) were determined 00 O to comprise >90% lymphocytes following May-Gnmwald/Giemsa staining. Warm 0 supplemented DMEM was added to the adherent monolayer which was estimated to contain x 10 4 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 Non phagocytosed bacteria were removed by gentle washing. One hundred pl (containing 5 x 10' cells) of autologous NPEC was added to each well containing infected macrophages and cultures were further incubated in 5% CO 2 at 37 0

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 ptCi 3 H]uracil for 18 h. The cells were lysed with 0.1% saponin and the bacteria heat killed at 80-90 0 C for 20 minutes prior to harvesting onto glass fibre filters (Whatman Ic, Finland) using an automated cell harvester (Cambridge Teclmology, USA). The amount of 3 H]uracil incorporated was determined using a liquid P-scintillation counter (Wallac, Country).

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 ofM. bovis 83/6235 was prepared using a modification of a method described by Grover et al., 1967 and stored at -70 C. For preparing these suspensions, the bacterial cells were dispersed by sonication for 30 seconds and filtered through an 8 mn membrane filter. Mice were infected via the respiratory route using an aerosol chamber which produces droplet nuclei of the size 17 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 '"-MAY-2008 12:35 FROM AJ PARK T.3 061262837999 P.22 00 appropriate for entry into alveolar spaces. The concentration of viable M. bovis in the O nebuliser fluid was empirically adjusted to result in the inhalation and retention of 5-20 viable organisms per mouse lungs Buddle 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 i were performed under strict isolation conditions in a biohazard facility.

C Isolation of M- bovis. Mice were euthanased between 37 and 40 days after aerosol challenge.

oo The lungs and spleen from each mouse were processed individually for mycobacterial 10t isolation. The organs were homogenized in a Ten-Broeck grinder and samples centrifuged at 00 0 dilutions were made in TAB and a 0.1 ml volume of a diluted or undiluted sample was C0 inoculated onto a modified mycobacteria 7H11 agar 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 bacterial counts from the lung and spleen were loglo 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.

Vaccination and challenge of possums. Possums were trapped and housed as previously described BCG was fed to two groups of possums (5 animals/group). A 1 g pellet of formulated BCG (1 x 10 S CPU) was given to each possum in one group. A second group was given BCG (I x 10' 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 nonvaccinated 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 10 8 CPU/possum). All of the possums were challenged by the aerosol route 41 days after vaccination.

18 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:35 FROM AJ PARK TO 0061262e37999 P.23 00 In a second experiment, four oral lipid BCG formulations were compared with subcutaneous

O

Svaccination. Six possums per vaccine group were challenged by aerosol with virulent M.

bovis 8 weeks after vaccination. Lipid CK.N and F (a modification of K containing Sfoetal calf serum) Cf Aerosol challenge of possums with iM. bovis. The possums were challenged with M. bovis 83/6235, which was originally isolated from a lymph node of a possum from Taumaranui, New Zealand Single cell suspensions of the isolate were prepared using a modification of 00 a method described by Grover et al., 1967 and stored at -70 C. For preparing these Cl 10 suspensions, the bacterial cells were dispersed by sonication for 30 seconds and filtered C through an 8 M membrane filter. Possums anaesthetised with an intramuscular injection of 00 0 ketamine HCI (30 mg/kg; Parnell Laboratories, Auckland, New Zealand) were infected via o 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 (Buddle and de Lisle, unpublished). This challenge dose had previously been estimated from the number of primary tubercles observed grossly in the lungs 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.

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 hmg 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 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 19 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:35 FROM AJ PRRK TO 0061262837999 P.24 0 0 prepared for each dilution. Culture conditions and methods for identification of isolates were 0 carried out as previously described Possum peripheral blood lymphocyte proliferation assay. Proliferative responses to PPD- B and PPD-A (CSL Limited, Parkville, Australia) were measured using whole blood depleted C" 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 NH4

C

1, pH 7.2 at 37 0 C for 10 rain, washed twice C in PBS at 200C and made up to 3 ml in DMEM tissue culture medium supplemented with 00 ci 2mM glutamine and 2% normal possum serum. The cells (200 Ipl) were plated into flat bottom 96 well plates containing 50 pz PPD-B, PPD-A or Con A in PBS or PBS alone to give final 0 concentrations of 60 jig/m PPD or 5 pg/ml Con A. Plates were placed in a 5% CO 2 in air Sincubator for 72 hr, pulsed with lp Ciwell 3 H-tritiated thymidine (Amersham, UK), ci harvested after a further 18 h and 3 H 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 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 transformed prior to 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.

COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:36 FROM RJ PARK TO 0061262837999P.25 00 0 O 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: M, Formulation C.

89% total lipid (48.5% neutral, 40.5% polar -comprising 3% myristic acid, 26% palmitic acid, IC 15% steario acid, 40% oleic acid and 6% linoleic acid), 00

IN

Formulation K CN 47% lauric acid, 20% myristic acid, 12% palmitic acid, 12% stearic acid and 3% oleic acid.

00 0 Formulation N. Novarta B, a commercially available suppository base consisting of a mixture of esterified, hydrogenated, fractionated vegetable oils with synthetic triglyceride mixtures.

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, fomulations C and K maintained high levels of BCG viability with formulation C showing higher retention of viability compared to formulation K 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 0 C compared to formulation N.

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 formulation K (mean logio CFU/ug =10) at 22 days In contrast, formulation N showed a rapid loss of BCG viability (mean logj0 CFT/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.

21 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:36 FROM AJ PARrT TO 0061262837999 P.26 00 0 0 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 Ci 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- 0 formulated M. bovis BCG or M. bovis BCG in jam (non-formulated M. bovis BCG). Table 1 00 Cl shows that while both the LTA and IFN-y assays showed significant differences between the Cil O 10 formulated and non-fonurlated oral M. bovis BCG groups, the differences for the IL-2 assay 00 were not significant. The IFN-y assay was used in further experiments to monitor systemic o 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 or non-formulated M. bovis BCG at 8 weeks post vaccination. Fig 2 shows that a low level of IFN-y (<200pg/ml) was detected in the formulated group following oral immunization with CFU of M. bovis BCG, but there were no significant differences between the vaccine groups. When the dose was increased to 10 7 CFU, IFN-Y responses in the non-formulated group remained low whereas responses to formulated M. bovis BCG increased significantly (P 0.05). Similar differences were seen with 10' 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 107-10 9 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 IFNy 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 subcutaneous vaccination with M. bovis BCG. Fig. 3 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 nonformulated M. bovis BCG or formulation material alone remained low between 2 and 8 22 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:36 FREM AJ F'iK TO 0061262837999 P. 2 O weeks. These results show that immune responses following oral vaccination with formulated 0 AfL 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 Sinhibit growth of M. bovis in autologous macrophages. The addition of NPEC to M. bovisinfected macrophages from mice vaccinated with oral M. bovis BCG formulations was performed in order to determine whether lymphocyte-mediated effector mechanisms could Sinhibit intracellular growth ofM. bovis. Growth of M. bovis in macrophages was determined by 3 H]uracil uptake. The growth ofM. bovis within macrophages alone or when co-cultured I with NPEC from orally vaccinated mice is illustrated in Fig. 4 Macrophages prepared from o mice orally vaccinated with formulated or non-formulated M. bovis BCG or mice given o 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. bois-infected macrophages, the 3 Huracil counts were significantly reduced compared to co-culture of NPEC from mice vaccinated with non-formulated M. bovis BCG or formulation material alone 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 ofM. bovis in the host Oral vaccination with formulated M. bois 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 subcutaneously vaccinated with 1 x 106 CPU M. bovis BCG. Non-vaccinated mice served as 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 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 MI bovis BCG induced significant protection against aerosol challenge with virulent M. bois, although the protective efficacy of subcutaneous M. bovis BCG in the lung was greater than that for oral formulated M. bovis BCG group.

23 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12:37 FROM AJ PPRK TO 0061262837999 P.288 00 0 0 D. Immune responses and pathology in possums.

0 Lymphocyte blastogenic responses. The effect of oral vaccination with formulated BCG on the whole blood lymphocyte blastogenic responses to bovine PPD is shown in Fig.5 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- C' vaccinated control groups At 4 weeks following challenge with M. bovis all groups 00 showed a mean SI for PPD-B of >20. These results show that oral delivery of formulated Cl 10 BCG elicits strong immune responses to PPD-B in possums compared to non-formulated

O

Ci| BCG.

00 0 0 A further experiment compared immune responses to four oral lipid BCG formulations with subcutaneous vaccination (Fig 11), Subcutaneously vaccinated possums showed strong LTA 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=1-7) at 4 weeks post-vaccination but increased progressively and were sustained through to 8 weeks post-vaccination (SI=15-22). These results show that 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) indication that the type of lipid used to fdrmulated oral BCG is important for protection against tuberculosis.

Clinical findings. The mean body weight changes between challenge and necropsy for the different groups are shown in Figure 6. 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 groups decreased by 0.35 kg and 0.23 kg respecively during this period. However these differences were not statistically significant.

In a further experiment (table which compared four oral lipid BCG formulations with subcutaneous vaccination, the mean body weight changes between challenge and necropsy were significantly reduced for the subcutaneous vaccination group (mean weight loss 0.012 24 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 12: 37 FROMrl AJ PAR TO 0061-62837999 P.219 00 kg) and one of the oral lipid BCG groups (group F) (0.035 kg) compared to the nono vaccinated group (0.147 kg). By comparison, the mean weight loss for the remaining oral BCG groups were 0,060 kg (lipid 0.067 kg (lipid K) and 0.122 kg (lipid Possums c which did not show an immune response to vaccination (ie non-vaccinated and lipid N groups) showed greater body weight loss compared with those that responded.

ci 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. High 00 lung weight is associated with extensive tuberculous pneumonia In order to C lo standardise differences in lung weight with variation in body weight, the lung weight of each C, animal was compared with the body weight and expressed as a ratio. The ratio of mean lung 00 Sweight to body weight of the animals vaccinated with formulated BCG was 1.62. By 0 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 weight ratio of the possums vaccinated with formulated BCG was significantly different from the non-formulated BCG and non-vaccinated control groups 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 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 8 and 9. The mean lung bacterial counts for the non-formulated and formulated BCG groups were significantly lower than those for the non-vaccinated control group The mean spleen bacterial counts for the formulated BCG group were approximately 10-fold less than the non-formulated BCG group and approximately less than the non-vaccinated control group. The mean spleen bacterial counts for the COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23 -r1AY-2008 12:38 FROM AJ PARK T[I 00612628:37999 00 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 spleen bacterial counts for three of the orally vaccinated groups and the subcutaneously vaccinated group were significantly lower compared to the non-vaccinated group The remaining oral lipid BCG group (lipid N) did not show significantly reduce bacterial spleen counts. No significant differences were seen between the groups when bacterial lung counts were compared. In general, possums which did not show 10 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.

TABLE 1. Bovine PPD-stimulated spleen cell responses in mice 8 weeks after oral vaccination with differexit lipid formulations" Immunization IL-2 (pg/ml) .IFN-y (pg/ml) LTA (SI) Formulation only 110.62 51.44 1.67 (+1-14.38) Non formulated BCG 153.51 65.10 1.83 Formulation C BCG 430.43* 2160.95* 16.26* Formulation K BCG 230.23 1268.30* 7.26* Formulation NBCG 130.23 75.50 2.76 Is *Represents a mean which is significantly different from the mean of non-vaccinated (Formulation only) control group. P value 0.05 (Student t test) 26 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MPY-2Ul0:t 12:38 FROM AJ PARK T iF22399P3 T,'i 001'--11261-28:37999 P. 31 00 0 0 ci en ci TABLE 7. Effect of vaccination on protection of mice against aerosol clhalienige with Mycobacteriuna bovis Lugctelt resitac Spencteri resistance Vaccine group L unatera LeiaSpleen cterial Logea# Non-vaccinated 5-837- (±0O.362) NA 4.565- NA Formulated Oral 4 7 7 4 b 1.06 3 0 84 b (*0.176) 1.48 BCG Subcutaneous 3.498c (±0.237) 2.34 2.

6 6 0 P 1.90 BCG Values are log 10 numbers of CPU standard errorfM. bends fromn the lungs and spleen of 6 anals per group 37-40 days post-challage.

Data are expressed as levels of logl 0 resistance calculated by subtracting the loglo mean number of bacilli it the organs of vaccinated animnals from the logja 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 differcnt (P>0.05).

TABLE 3. Number of possunu responding to bovine PPI) in the lymphocyte proliferation assay following oral vaccination.

Weeks after vaccination BCG formulation Group sie 0 4 6 Formulated BCG 5 2 Non-formulated BCG 5 0 0 1 LControl (no BCG) 6 0 0 0 *No. of animals with stimulation index COMS ID No: ARCS-191 648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-Nmr-200812:138 FROM FtJ PA1RK TO 1E,2 -~.7999 F.2 00 00 00 TABLE 4. Pathological. and Microbiological findings for vaccinated possums challenged with M. bovis Vaccine Chae i bod weight/challenge Group we ht! Lipid C -0.060 Cbange in bOdy weight fchalenge body weight. Lipid lF BCG Non-vacciuated (P<0,0O5).

Spleen b&CteriaI count Lipid C, Lipid K, Lipid F, 'Boo Non-vaccinatd Lipid F Lipid N .Change 'a body weight between Post Mort=~ and challengc body weight at challenge (kg b Thin weight body weight at post mortem (Ig).

B~acterial colut, CRJ Ioglw g of tissue.

Significantly different to non-vaceinaled group.

IMbUSTRIAJ.

APPLICATION

The antigenic composition includes a lipid formulation which maintains antigens in a stable matrix, through which they are uniformly dispersed. This facilitates administration Of conSiSttet doSes of antigen avoiding dose dumping and ineffective low dosing. The lipid formulation has also been shown by the applicants to improve storage and viability of live organisms contained therein. The lipid fornmulation also protect$ the antigens and live Organism 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 con~ditions canl also be achieved without deteriorationt because Of the hydrophobic properties of the fonnulation.

28 COMS ID No: ARCS-i 91 648 Received by IP Austraiia: Time 09:42 Date 2008-05-23 23-MAY-200 12:38 FROM A PARK TO 0061262837999 P.33 00 0 0 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 Sbe achieved using the compositions of the invention. The compositions are also simple to 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 0 subcutaneously, but are particularly amenable to oral delivery. The applicants have found C 10 that the lipid formulation in the composition can protect viability of organisms and their N constituent antigens against degradation in the stomach, which enables live organisms to be o taken up through the gastrointestinal mucosa for processing, replication and presentation to o 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 Vaccination of wildlife, such as possums requires antigens 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 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 antigens, or for the co-delivery or conjugated delivery of antigenic molecules, particularly those which for reasons of dose or antigenicity are poorly immunogenic. The compositions of the invention are also useful as vaccine adjuvants.

29 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-2008 1e2:39 FROM AJ PARK TO 061262837999 P.34 00 0 O 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.

eN COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MnY-100 12:39 FROM AJ PARK TO 0061262837999 00 REFElENCE LISTING 0 0 1. Adwell, F. D. L. Keen, V. C. Stent, A. Thomson, G. F. Yates, G. W. de Lisle, and B- M. Buddle. 1995. Route of BCG administration in possums affects protection against bovine tuberculosis. New Zealand Veterinary Journal. 43:356-359.

n 2- Aldwell, F. D. N. Wedlock, and B. M. Buddle. 1996. Bacterial metabolism, cytokine mRNA transcription and viability of bovine alveolar macrophages infected with Mycobacterium bovis BCG or virulent M. bovis. Immunol Cell Biotl. 74:45-51.

00 3. Aldwell, F. D. N. Wedlock, and B. N. Buddle. 1997. Sequential activation of alveolar macrophages by PIFN-y and LPS is required for enhanced growth inhibition of virulent Mycobaoterium bovis but not M.bovis BCG. Immunologiy and Cell Biology.

00 o 4. Buddle, B. F. E. Adwell, D. L. Keen, N. A. Parlane, G. Yates, and G. W. de Lisle. 1997. Intraduodenal vaccination of brushtail possums with bacille Calmette- Gueriu enhances immune responses and protection against Mycobacterium bovis infection. Int J Tubere Lung Dis 1:377-83.

Buddle, B. F. E. Aldwell, A. Pfeffer, and G. W. de Lisle. 1994. Expeimental Mycobactrium bovis infection in the brushtail possum (Trichosurus vulpecula): pathology, haematology and lymphocyte stimulation responses. Vet MicrobioL 38:241-54.

6. Daugelat, C. H. Ladel, and S. I. 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. Lagranderie, and A. M. Balaznc. 1996. Stabilisation of BCG vaccines. New Approaches to Stablisation of Vaccine Potency. Dev.BIotStand.Basel, Karger. 87:251-261.

8. Lagranderie, P. Chavarot, A. M. Balazue, and G. Marebal. 2000.

Immunogenicity and protective capacity of Mycobacterium bovis B3CG after oral or intragastric administration in mice. Vaccine. 18:1186-95.

9. Masarova, J.,Mialovicova, D.,Geminer, P and Michalkova, B. 2001 Stability enhancement of Escherichia coil penicillin G acylase by glycosylation with yeast mannan. Biotechnol Appl Biochem 34:127-33 Morrison, 1. 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.

31 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23

Claims (16)

1. An antigenic compositiont comprising a pharrmactically acceptable lipid fonUilatiOn Mand at least one antigenis. component compilising ana antigencally effectiv =mt Of live organims; the composition being formulated for oral admintraton. 00 2. A composition according to claim I wherein the lipid fonjailaton is in SOlW fofn. 0 3. A composition accorfin to claim 1 or claim 2 'wherein thie lipid fonnuMWio undergoes 00 10solid to fluid tranition bactea about W0C to 4000. AUi antigeni composition comprisig a pharinaceatlcally accptable lipid formulaon which ndczgoes solid to flid tation between. about 30P0 to 40*C, and at least one antigemec component present in an antigeically effective amount. A composirt acciording to claim 4 Whein the antigerdo, component iS isolatd ad/or purifiad A composition according to claim 4 or claim 5 wheiren. tbs antigenic 1;OmPoomit is a pwtei 7 glyoprotein peptide, or facto with a pwoteia or peptide component Or ixture tereot

7. A composition according to any one of claims 4 to 6 which is formulated for paxrotal administration.

8. A cowuposii& =ccording to claim 7 wbich is fotmulated for subquanaeous composition according to ;my one of claim 4 to 6 which is formulated for oral A composition. according to claim 4 or claim 5 wherein the antigenic oPmpoet co)mprses liVCogaat 32 COMS ID No: ARCS-191 648 Received by IP Australia: Time (I-tm) 09:42 Date 2008-05-23

23-MAY-2008 12:39 FROM Aj PA~k TO 0061262837999 P.37 00 0 1 -3 A composition according to ayoeo claim s 1 o32r1 wherein the liveisHI or ganism areb= an bloicly pe culre. 12- A Composition according to claim 10 wherein the liveorgacvims esetd from th MICi00 U.ou conistn 4&c~t cope pmrsn M ~rc~r n %Amositind cordbinzt t lam 12maa whren heisiMI orot tham 00 10 ~14. A composition etccrding to claim 12 wheren the yacr is M.ce fom bmc, 18. A composition according to claim 14 wi$~n theA bari is McBaceriwn.. Gmi 16. A composilion accrding to cay ,R 1f wheI te1 hichcofw i select frstomA anmic o.abnifagldncml (opiig t nrclllead 11. A composi according to clain 19 werei the Mycobaclmpo is a living A comsl acoar toO cam1whri eantigenic~ component is a livingo Pptde 33 COMS ID No: ARCS-i 91 648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MAY-20L8 12: 40 FRIIAJ PARK TO OCFi 126 37R3~9 P.38 00 21. A comnposition ~ccordng to olaim20v tterein the living orgapibm is a Mycbacterur CC) 22. A oomposition aceording to claim 21 whreiu teeycobaclgrium is M bovtq. 2-3, A composition according to claim 22 whordin th-, M. b6v&s is BCGf. 00 21. A composition according t any e; of rein livn 0or3anis the protein U an O i~~innnmocntraceptiveproteim 00 n 22. A composition according to any o21 of cai= 20 u 23 wwheein thproten or pcS bvt is Poorly imoamiogwic,

26. A composition according to ay me of claims I to 25 whBCG.n te lipid forultion undergoes solid to fluidtrawstion between abott 30C tD 3M 27 A composition accordkng to ay on* of chd= I to 26 whm-ein tbo Hpid farmUlatdo contains 40% to 100, preftm~ably 60% to 100% prefisbly 803,4 to 1001/o, and more prmferably 90% to 1000/9 C16 anwolO Cla faty =cids- 020

28. A oamposition according to any on; of claims to 2 wherein the lipid fs laflon o taiao 10% to protefily 20ro to 35% wd more preftrly 25-/ to 320 Cie. acids; and 40ao to 90ny1, prefcrably 50019 to 8002 e and more preiffaby 60pe to Cis fhlttj adids.

29. A composition according to any oe of claims 1 to 26 wherein the lipid fonnulation contains less ten 350%, preferably 60% to 25-/Nand more prefera bly less t3d more. C14 ferb acids or C 1 bai is 28. A composition according to :any oe of claims I to 26 whein the lipid formulaion codtaink 10% to 40, re14 feay tds or dreerery 25% to 32% C 16 tty ad4, and W to Cis fa ory acsho 34 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-MRY-208 12:40 FROM HJ Pp'qK TO 00612683799 P. 39 00 (N

31. A coirposition according to any one of claims I to 26 wherin Ie lipid fwmvlation *cctaint to 60% satutated fatty acids; to 60% monounsantrated fatty acids; and s 0.5% to 15% polyunsatated fatty acids

0032. A composition accotding to claim 31 wherein the lipid formulation contains to 55% satUrated fatty acids: to 600/ met uratpA fatty a -ia (N -10 3 to l% olymsud faty ad&. 0 A composition according to clin 32 waetr ft lipid fonnulatica contains: 40P/6 to 50% sauated fatty acids; to 55% nononnsaamatcd fatty acids d 5% to 9% polyasatrated fatty acids- j4. A composition accoiding to any one of claim I to 26 wherein the lipid formulation has the fbrmulL- 3% myxistic acid; 26%. palmitic acid; 15% staiiec acid; 40% oleic acid; and 6% linoleic acid. Acompositiomn ording to aiy one of claims Iot 34 whichbis essentially fre of atweos component&

36. A composition according to any one of claims Ito 35 wheprein the composition consists esscfsiallyoflipa

37.A composition accocllng to any one of clama Ito 36 which further comptises in-detxtnacatalasc (ADC).

38. A composition according to aty os of claims ito 37 which is a vaccine.

39. A composifionaccording to any one of claims ito 38 which is a vaccine adjuvant COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23 23-NfY-ZWH133 12:40 FROM RJ PARK T71 06126283-9999 P. 00 A nietbod for imimising an animal, th, mthod compising administering to said Mirna a composition aocordinugto any one ofdaims 1 to 39-

41.A method for stimulating a iucosal inmune resonse in an animal, the method comprising administering to said animal a compositido according to any one of claims I to 39. 00

42. A method according to claim 40 or claim 41 wherein the administraton is oral 0 administration, and the composition is lbnnLated for oral adu trtion. 00

43- A method according to claim 40 or claim 41 wberein the administration is subutanecmS Cl administration, and the composition is tbrmlated for subcutaneous adndnistraionL 36 COMS ID No: ARCS-191648 Received by IP Australia: Time 09:42 Date 2008-05-23