ZA200402842B - Interleukin-12 as a veterinary vaccine adjuvant - Google Patents

Description

INTER2LEUKIN-12 AS A VETERINA_RY VACCINE ADJUVANT

CRO)SS-REFERENCE TO RELATESD U.S. APPLICATION=S

This application claims the benefit under 35 U.S.C. § 119 (e) of U.S. Provisional

Application No. 60/322,840, filed September 17, 2001. The prior application is incorporated hereizn by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED

RESEARCH OR DEVELOPMENT

Not Applicabole

REFERENCE TO A "Secguence Listing"

Not Applicabwle

BACKGROUND OF THES INVENTION

Field of the Inve-ntion

The preseent invention concerns a novel combination ceomprising an immunomodulator in conjunction with immmunoadjuvants that enhances the immunogenicity or physiological efficacy of veterirnary vaccines containing =an antigen and the new use of the combination to significantly improve the immunological response of an animal to the antigen when administered concurzrently or in admixture v=vith a vaccine composition.

WO= 03/024354 PCT/US02/29229 ¢ :

Description of the Related Art

All patents and publications cited in this specificatior are hereby incorporated by reference in their entirety. :

The etiology of man-y debilitating or fatal diseases has been established. For

S example, Bovine Respiratorsy Syncytial Virus (hereinafter referred to as "BRSV") is recomenized as a significant factor in Bovine Respiratory Disease Complex. The disease is characterized by rapid breathing, coughing, loss of appetite, cular and nasal discharge as well as elevated temperatures in cattle. Death can occur wakthin 48 hours after onset of

Symp=toms in an acute outbreamk. BRSYV is considered the mosst common viral pathogen in enzoeotic pneumonia in calvess, and has also been associated with pulmonary emphysema amorag newly weaned calves.

Another disease of lamrge animals, Strangles, is causead by a bacterial infection of

Strepmstococcus equi. Also k=nown as distemper or bam fTever, Strangles is a highly contagious disease of a horse's upper respiratory tract charamcterized by the presence of local and disseminated abscessses,

A variety of etiologic agents are known to cause disea.se in small animals. Disease in dowgs, for instance, is fourad to be associated with the presence of Ehrlichia canis, canin e parvovirus (CPV), canine parainfluenza virus (CPI), canine adenovirus type II (CANV/-2), canine adenovirus (CDV), canine coronawirus (CCV), Leptospira icterohemorrhagiae (LD, Legtospira canicola (LC), Leptozspira grippotyphosa (LG),

Leptospira pomona (LP) and the like. Similarly, disease in cazts is caused by transmittable viruses such as feline immun-odeficiency virus and feline lemkemia virus among others, bactezria such as feline Chlamyadia psittaci, etc.

There is a real need for= effective prophylaxis against theese types of etiologic agents that produce highly contagious, debilitating and deadly disezases in animals. However, veterinary vaccines often suffer from poor immunogenicity responses due to weak antigenic activities of certain etiologic agents or due to bioMogical variations from one animaml species to another. Reduced physiological efficacy is also problematic in any attempt to obtain proper humeoral immune responses in animals. Producing an adequate

. ® WO 03/024354 PCT/US02/29229 : level of serum antibodies. which reflect true protection agamnst the disease through concomitant cell-mediated immunity, is difficult to achieve. Moreover, physical and ’ chemical compatibilities of” the antigenic substances with each aclditive or combination of additives must be resolved through significant testing to preclude rendering sensitive antigens inactive. Troublesome side effects or potential toxicity from a narrow margin of safety provide yet another challenge to the development of a use=ful veterinary vaccination program. Establishing protective immunity is not a simple mamtter. Thus, research has focused on finding a reliable, nontoxic adjuvant that is compat ible with the antigen and able to improve the immmunogenicity and efficacy of animal vaccines without raising toxicity concerns.

A number of immunoadjuvants has been examined amnd many hold promisingz abilities to augment cell-mediated and humoral immune responsses to a variety of antigenss suffering from weak immunogenicity (see discussion in FR. Rabinovich, "Vaccines

Technologies: View to the Future,” Science 265:1401-1404 (S-eptember 2, 1994) and F_

Audibert, "Adjuvants: current status, clinical perspectives and future prospects,™

Immunology Today 14(6)=281-284 (1993)). Alum (aluminum potassium sulfate), found ix diphtheria, tetanus and he patitis B vaccines, stimulates the humsoral immune response bust not the cell-mediated irmmunity. As a result, the salt is not efficacious with all immunogens. The aluminum salts also have the disadvantage of" not lending themselves ox the vaccines to lyophilization or freezing. Due to the limitations of the aluminum saltss, research has turned to many alternative immunoadjuvants such as saponins, non-ioniec block polymer surfactants, monophosphoryl lipid A, muramyl dlipeptides (squalene oil) o-r tripeptides and cytokines. However, the selection of a suitable immunoadjuvant system i_s not an easy matter and requires substantial experimentation to discover if the system will enhance cell-mediated ancl humoral immune responses in a particular species of animal teo different immunogens. Maintaining the stability and the efficacy of the immunogens ar—e other important factors that can influence the selection procsess in finding whether thme immunoadjuvant system wwill function as desired in the animal.

Interlesukin-1 (IL-1) was the first cytokine to be found useful as an adjuvant in . amplifying thee secondary antibody response to bovine serum albumin by a cell-mediated immunity via dncreasing production of interleukin-2 (IL-2). Previous studies have shown : that recombirmant bovine IL-1B is useful as an ammunomodulator of bovine immune responses to viral infections (see Reddy et al, “Adjuvanicity of recombinant bovine interleukin-18= influence on immunity, infection and latency in bovine herpes virus—1 infection," Lyrmphokine Res. 2:295-300 (1990)). Ime these studies, r-BolL-1 B-treatment of calves increase=d antibody production against bovine herpes virus-1 (BHV-1), bovine vinms diarrhea (BVID) and parainfluenza-3 (PI-3) viruses, enhanced cytotoxic responses te virally infected MDBK cells, decreased viral sheddi ng of BHV-1 after challenge and hae lower recrudescence of BHV-1 following dexarnethasone injections. The reports suggested that recombinant bovine interleukin-18 can potentiate the activity of antigens when administe=red subcutaneously in an aqueous solution.

Clinical trials have been performed to assess the ability of cytokines such ass interferon a (IF"N-a) and interferon y (IFN-y) to improve the immunogenicity of hepatitis

B vaccines in non-responsive subjects.

Subsequent research in immunology has exarmined the importance and activity of other cytokines such as, for example, interleukin-12 (see, for example, Y.-W. Tang ef al., "Interleukin-12 Treatment during Immunization Elicits a T Helper Cell Type 1-like

Immune Resporase in Mice Challenged with Respiratory Syncytial Virus and Improves

Vaccine Immuneogenicity,” J. Infectious Diseases 172:734-738 (1995); S. Morris ef al, “Effects of IL-1 2 on in Vivo Cytokine Gene Expression and Ig Isotype Selection,” J.

Immunology, pp=. 1047-1056 (1994); J. Orange et al. , "Effects of IL-12 on the Response and Susceptibility to Experimental Viral Infections,” J. Immunology, pp. 1253-1264 (1994); G. Trimchieri, “Interleukin-12 and its role in the generation of Tul cells,”

Immunology Toc3ay 14(7):335-338 (1993); R. Gazzinelli er al., "Interleukin-12 is required for the T-lympho--cyte-independent induction of interfex-on y by an intracellular parasite and induces resistanc e€ in T-cell-deficient hosts," Proc. Natl. Acad. Sci. USA 920:6115-6119 (July 1993); R. Locksley, "Commentary: Interleukin-E 2 in host defense against microbial i

1d pathogens," Proc. INatl. Acad. Sci. USA 90:5879-5 880 (July 1993); B. Graham e~/ al., "Priming Immunization Determines T Helper Cytokine mRNA Expression Patterns in

Lungs of Mice Challenged with Respiratory Syncytial Virus,” J. Immunology 151:2032- 2040 (August 15, 1993); J. Sypek el al, "Resolution of Cutaneous Leishmamiasis:

Interleukin 12 Initiamtes a Protective T Helper Type 1 Immune Response,” J. Exp. Med. 177:1797-1802 (Jure 1993); F. Heinzel ef al., "Receombinant Interleukin 12 Cures Mice

Infected with Leishrnania major," J. Exp. Med. 177: 1505-1509 (May 1993); C. Tripp ef al., "Interleukin 12 and tumor necrosis factor a are costimulators of interferson y production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antag onist," Proc. Natl. Acad. Sci. "USA 90:3725-3729 (Apral 1993); R. Manetti ef al, "Natural Killer Cell Stimulatory F=actor (Interleukin 12 [IL- 12]) Induces T Helper Type 1 (T"hl)-specific Immune Responses and

Inhibits the Developsment of Il-4-producing Th Cells," J. Exp. Med. 177:1199-1204 (April 1993); C.-S. Hsieh et al., "Development of Tul CD4" T Cells Through I-12 Produced by Listeria-Induced Macrophages," Science 260:547-54 (April 23, 1993); P. Scott, "IL_-12:

Initiation Cytokine #for Cell-Mediated Immunity," Science 260:496-497 (April 23, 19993);

M. Gately ef al., "Regulation of Human Cytolytic Lymphocyte Responses by Interleukin 12," Cellular Immuanology 143:127-142 (1992); A_. D'Andrea et al., "Productiosn of

Natural Killer Cell Stimulatory Factor (Interleukin 12) by Peripheral Blood Mononuclear

Cells,” J. Exp. Med_ 176:1387-1398 (November 19922); B. Naume er al, "A compar—ative study of IL-12 (Cytotoxic Lymphocyte Maturation Factor)-, IL-2-, and IL-7-indfuced effects on Immunomagnetically purified CD56 NK cells,” J. Immunology 148:2429-22436 (April 15, 1992); S. Chan et al, "Induction of Interferon y Production by Natural Killer

Cell Stimulatory Factor: Characterization of the Respwonder Cells and Synergy with Other 25 . Inducers,” J. Exp. Med. 173:869-879 (April 1991); and M. Kobayashi er al, "Identification and Purification of Natural Killer Cell Stimulatory Factor (NKSKF), a

Cytokine with Multi_ple Biologic Effects on Human Lwymphocytes,” J. Exp. Med. 170=827- 845 (September 198 9)).

Interleukin-12 (hereinafter referred to as "IL~12") has demonstrated adjuavant activity in eliciting a «cell-mediated immunity against lesishmaniasis in BALB/c micee (L.

Afonso et al., "The A_djuvant Effect of Interleukin-12 Sn a Vaccine Against Leishnaania : major," Science 263:235-237 (January 14, 1994)). The conferral of protection agaimst . major was based on thes activity of IL-12 to induce the deevelopment of leishmanial-specific

CD4" Tul (T helper) ceells. U.S. Patent No. 5,571,515 ( Scott ef al.) and related divisSions,

U.S. Patent Nos. 5,723,127 and 5,976,539, describe she use of IL-12 as an adjumvant against leishmaniasis by enhancing the cell-mediated immune response to an ankxigen comprising the protozoan parasite. Based on the descmription of the use of IL-12 a_s an adjuvant in the leishmamiasis model and with a cancer va ccine, U.S. Patent No. 5,723_,127 is directed to antigenic compositions of selected antigerms and IL-12, and the methoc3 for increasing the ability of the compositions to elicit tthe host's cell-mediated immune response to the selectecd antigens. U.S. Patent No. 5,9776,539 is drawn to a composition of an antigen selected firom cancer cells or cancer cells transfected with a selected antsgen and IL-12 and the metThod of use thereof A further related continuation in this semries,

U.S. Patent No. 6,168,923 B1 (Scott ef al), claims a cosmposition comprising an antigen consisting of a pathogeric microorganism and IL-12 whiach elicits a vaccinated host's cell- mediated immune respo-nse against the microorganism arad a method of administering IL- 12 to increase the ability of an immunogenic composition to elicit a vaccinated host's cell- mediated immune respomse.

U.S. Patent No. 5,665,347 (Metzger et al.) disclo ses that, in addition to activastion of Tul (T helper) cells, IL-12 inhibits the functional activ-ity of B1 cell activity but not B2 cells, and B1 cells possess an IL-12 receptor. Patentees suggest that IL-12 may find use in treatment of B1 cell disorders like chronic lymphocytic leukemia, lymphomas and infectious mononucleosis.

U.S. Patent No. 5,817,637 (Weiner ef al.) relate to a pharmaceutical immuniz=ing kit that uses genetic mat erial as the immunizing agent in t wo separate inoculants. A third inoculant contains bupiwacaine that may be combined ~with other response enhanc=ing agents like transfecting, replicating or inflammatory agemmts, for example, lectins, groxovth

® : factors, cytokines (such as a-interferon, y-interferon, IL-1, IL_-2, IL-4, IL-6, IL-8, 1L-10,

IL-12, etc.) and lymphowkines.

U.S. Patent No . 5,985,264 (Metzger ef al.) concern the method of enhancing an immune response to a gpathogen in a neonatal host comprising the administration of IL-12 and an antigen to induce memory for protective responses as an adult. The neonatal host is mammalian, for exampele, human, murine, feline, canine, bowine or porcine, and includes the fetus as well as newborn to about 2 years after birth. The antigen is described as bacteria (e.g., S. pnemmoniae, N. meningidilis, H. influer az), viruses (e.g., hepatitis, measles, poliovius, human immunodeficiency, influenza, parainfluenza, respiratory syncytial), parasites (e.g, Leishmania, Schistosomes) and fungi (e.g, Candida,

Aspergillus).

U.S. Patent NO. 5,744,132 (Warne et al.) describes compositions and methods for providing concentratec preparations of IL-12 in a frozen, liquid or lyophilized formulation of the IL-12 protein, polysorbate, a cryoprotectant, bulkings agents and buffering agents.

U.S. Patent No. 5,853 ,714 (Deetz et al.) provides a method for purification of IL-12 using a hydrophobic interaction chromatography resin to make IL.-12 free of contaminants such as host cell proteins amd viruses.

In addition to the above art, there are several pa tents and publications in this crowded field that describe the use of IL-12 with certaim antigens, for example, as an adjuvant in paramyxowviridae vaccines (U.S. Patent No. 6_071,893, Graham ef al.), for enhancing oral tolerance and treating autoimmune diseases (WO 98/16248), for treating inflammation (U.S. “Patent No. 5,674,483, Tu ef al), was an adjuvant in Bordetella pertussis vaccines (WO 97/45139) or as a co-adjuvant with IL-13 in vaccines containing antigens such as influenza A, HIV, tetanus toxoid, ec. (WO 98/31384) and the like.

Further research has provided a variety of animal cytokines and the methods to produce them, for example, feline IL-12 (C. Leutenegger er al., Immunization of Cats against

Feline Immunodeficiency Virus (FIV) Infection by Using Minimalistic Immunogenic

Defined Gene Expression Vector Vaccines Expressing FIV gp140 Alone or with Feline

Interleukin-12 (IL-122), IL-16, or a CpG Motif," J. Virology 74(22):10447-10457 (Nov.

2000) and WO» 01/04155 A2?), avian IL-15 (WO 97/14433), ovine IL-5 or IL-12 (W/O : 97/00321), to n.ame just a few.

Other research, including some of the publi cations described hereinabove, heas focused on particular vaccine formulations and the mesthods of making them. U.S. Pate-nt

S No. 5,242,686 Chu ef al), for instance, is directed to a process for preparing a felire vaccine composition useful against chlamydia infections. The inactivated mammaliaan chlamydial cells or antigens may be combined with an immunogenically suitable adjuvarat and a physiologically acceptable carrier. The patent Bists the adjuvant, for example, as surfactants, polyaanions, polycations, peptides, tufisin, oil emulsions, immunomodulator—s such as interleukin-1, interleukin-2 and interferons, acrylic acid copolymers such a=s ethylene/maleic amhydride copolymer, copolymers of sty/rene with a mixture of acrylic acici and methacrylic acid or a combination thereof.

U.S. Patent No. 5,733,555 (Chu) and its contineaation, U.S. Patent No. 5,958,423 concern a vaccine composition for immunizing an armimal against infection caused by

Bovine Respiratory Syncytial Virus ("BRSV") which comtains a modified live BRSV alone or in combination with a Bovine Rhinotracheitis Virus I'V, a Bovine Viral Diarrhea Virus and a Parainfluenza 3 Virus, an adjuvant and a pharmaceutically acceptable carrier. The composition elicits protective immunity after a single administration via cell-mediated immunity, secretomry immunoglobulin A immunity andl a combination thereof. The adjuvant may further comprise a surfactant such as polyoxyethylene sorbitan monooleate.

The patents list other adjuvants such as squalane, squalene, block copolymers, saponin, detergents, Quil A, mineral oils, vegetable oils, interleukins such as interleukin-1, interleukin-2 and in terleukin-12, tumor necrosis factor, iraterferons, combinations such as saponin and aluminaum hydroxide or Quil A and alumimam hydroxide, liposomes, iscom adjuvant, synthet®c glycopeptides such as muramyl dipeptides, dextran, carboxypolymethyleme, EMA®, acrylic copolymer emulsieons such as Neocryl® A640 or mixtures thereof.

However, it Tas not been described or exemplifiecd in the art that IL-12 or other immunomodulators can effectively and markedly enhance the immunogenicity of weak,

® immunosupparessive or potentially toxic anti gens when specifically co-a-dministered with immunoadju-vants.

Tt is therefore an important object Of the present invention to provide a highly unique vaccsine possessing significantly impr-oved immunogenicity in mammals and birds that is comprised of weak or immunosupp-ressive antigens, or antigerns with a narrow margin of safety, in conjunction with the novel combination comprising the immunomodiulators and the immunoadjuvantss of this invention.

Anosther object is to provide a new method of using the combimnation comprising the immuneomodulators and the immunoadjuvants or the vaccine hat contains the combinatiomn to substantially improve the immmunogenicity of the vaccine by inducing a stronger sti mulation on cell-mediated immunity including T memory cell sand to provide a longer duration of immunity thereby requirimng smaller or less frequent d-osages of antigens over time a_nd lessening side effects or potemdtial for toxicity.

A fEurther object is to provide a mew method of potentiatin_g, accelerating or extending the immunological activity of an antigen in an avian or mammaalian species.

Further purposes and objects of the present invention will appear as the specification proceeds.

Thee foregoing objects are accomplished by providing a combination of immunomodulators and immunoadjuvants, and a vaccine in which an inmmunomodulator is co-formulaated with an immunoadjuvant an d a viral, bacterial, parasitic or fungal antigen.

The produmct of this invention produces a highly improved immunological response to the antigen as- compared to classical vaccines and other combinations cormprising a cytokine by itself. The background of the inventiom and its departure from the art will be further described hereinbelow.

BRIEF SUMMARW OF THE INVENTION

The present invention involves an Lmproved vaccine formulation that comprises an effective immunizing amount of an antigen, an immunomodulator— and one or more immunoaadjuvants in which the immunogeicity or physiological efficacy of the vaccine is

J signifficantly enhanced. The imvention includes the novel combination composition comprising the immunomodulato-rs and the immunoadjuvants thas markedly improves the immunological response of a vacscinated host to the antigen. Alsso, the present invention conce=rns a novel method for pote-ntiating, accelerating or extendin 8 the immunogenicity of 5S weak, immunosuppressive or marginally safe antigens which comparises administering to an avian or mammalian species a pharmacologically effective ammount of the aforesaid combi_nation composition or an effective vaccinating amount of the aforedescribed vaccine composition.

BRIEF DESCRIPTION OF THE DRAWING-S

Not Applicable

DETAILED DE SCRIPTION OF THE INVENTEON

In accordance with the pres ent invention, the novel vaccine composition comprises an effe ctive immunizing amount of an antigen, an immunomodlulator, one or more immunasadjuvants and a phamnaceutically acceptable carrier. Surprisingly, the incorpomration of the immunomoadulator and the immunoadjuv—ant(s) into vaccines significa ntly potentiates the immumogenicity and physiological effiecacy of the antigenic substance. The unique combinatior of the immunomodulator and Ammunoadjuvants has beneficial application for increasing the biological activity of numeroums antigens.

The antigen encompasses a wide variety of infectious age=nts contemplated by those of ordinary skill in the pharmaceutical or veterinary arts. The infectious agent, for example, may be viral, bacterial or fungal in nature. Other infectioues agents include, but are not limited to, parasites, tumor zantigens and antigens of other pathological diseases.

The particular antigen or combination of antigens to be emplosyed in the vaccine compositmon will depend upon the spescies to be vaccinated and the dessired results.

Tie antigen is incorporated with the immunomodulator and thes immunoadjuvant in varying a-mounts and usually ranges from about 0.0001% to about 1.0% by weight.

Examples of typical viral antigens imclude, but are not limited to, Bovine Respiratory

® Syncy=tial Virus, herpes simplex virus type 1 (HSV), bovine virus diarrhea (BVD), paraimfluenza-3 virus (PI), canine paxvovirus (CPV), canine parainfluenza virus (CPD), canines adenovirus type II (CAV-2), canine adenovirus (CDV), canine coronavirus (CCV), rabies. virus (particularly for, but not limited to, canine rambies vaccines), feline immumodeficiency virus (FIV), feline Leukemia virus (FeLV), felinee coronavirus (etiologic agent of feline infectious peritonitis (FIP)), Porcine Reprodiactive and Respiratory

Syndr-ome (PRRS) Virus, chicken herpes virus (etiologic agent o—f Marek's Disease), elc.

Typic.al bacterial antigens include, but are not limited to, Chlamydia, Ehrlichia,

Pasteaurella, Haemophilus, Salmoramella, Staphylococcus, Streptococcus, Borrelia, Mycogolasma (for example, swine disesase of Mycoplasma hyopne umoniae), etc. Typical parasitic antigens include, but are nowt limited to, Leptospira, Caoccidia, Hemosporidia,

Amoe bida, Trypanosoma, Leishmaniar, Giardia, Histomonas, etc. Typical fungal antigens include, but are not limited to, Coc=cidioides, Histoplasma, Bleastomyces, Aspergillus,

Cryptococcus, elc. “The immunomodulator is pressent in the improved vaccmne of the invention in varyirmg amounts and usually ranges from about 0.00001% to a bout 0.01% by weight.

Examples of suitable immunomodulators include, but are not limit. ed to, cytokines such as

IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, eefc., interferons such as a-in_terferon or y-interferon, tumomr necrosis factor, transforming garowth factor, colony stimulating factor and the like, or a combination thereof. Desirably. the immunomodulator comprises a cytokine. In a preferred embodiment, the immunomeodulator is interleukin-12 a nd most preferably, the homomlogous animal interleukin-12 svach as, for example, canine IL-12 is employed in caninee vaccines; feline IL-12 is empl oyed in cat vaccines; equin_e IL-12 is employed in horse vaccines and so forth. Human IL-12 or murine IL-12, such as recombinant human

IL-12= (commercially available froma Genetics Institute, Inc, Cambridge, MA) or reconmbinant murine IL-12 (commercially available from various suppliers, for example,

Reseanrch Diagnostics, Inc., Flanders, INJ and Cambridge Bioscience, Cambridge, England, see al_so D. Schoenhaut ef al., "Clonirag and Expression of Murine= IL-12," J. Immunology 24R8(1_):3433-3440 (June 1, 1992)), mmay suitably be employed for a variety of animal -_ 11 -

species altthough the immunopotentiati on effect may not be as great as the homologous animal IL-1 2 in some animal vaccines.

One= or more immunoadjuvants are present in the improve=d vaccine of the invention im varying amounts and ustaally range from about 0.05% to about 50% by weight. Erecamples of suitable immsnoadjuvants include, but are not limited to, metabolizabwle oils of plant or animal ori £in such as squalene (2,6,10,15, 19,23-hexamethyl- 2,6,10,14,18,22-tetracosahexaene) or preferably, squalane (2,6,10,15, 1 9,23-hexamethyl- tetracosane)®; block copolymers, for example, polyoxypropylene-polyoxyethylene block copolymers such as Pluronic® (commercially available from BASF Corporation, Mount

Olive, NJ); saponin such as Quil A (commercial name of a purified form of Quillaja saponaria, amvailable from Iscotec AB, Sweden and Superfos Biosect-or a/s, Vedbaek,

Denmark); ethylene/maleic copolymers such as EMA-31® (a linear ethylene/maleic anhydride copolymer having approximately equal amounts of ethy~lene and maleic anhydride, hmaving an estimated averages molecular weight of about 75 ,000 to 100,000, commercially available from Monsanto Co., St. Louis, MO), acrylic -acid copolymers, acrylic acid copolymer emulsions such ass Neocryl® (an uncoalesced aquaeous acrylic acid copolymer of acrylic acid and methacryli« acid mixed with styrene, commnercially available from Polyvimy! Chemicals, Inc, Wilminggton, MA); mineral oil emulsions such as MVP® (an oil-in-water emulsion of light mimeral oil, commercially available from Modern

Veterinary Products, Omaha, NE) or mixtures thereof.

The pereferred polyoxypropylene-polyoxyethylene block copolyme=rs of the present invention include varying amounts of polyoxypropylene and polyoxyethy~lene. Desirably, the block cogpolymer comprises polyoxyethylene in the amount of about 10-20% of the total molecul e and polyoxypropylene in an average molecular weight o-f about 3250 to 4000.

The emthylene/maleic copolymers of the invention are typically watesr soluble, white, free-flowing poowders having the followingg properties: a true density of a bout 1.54 g/mL, a softening pooint of about 170°C, a meching point of about 235°C, aa decomposition temperature omf about 274°C, a bulk density of about 20 Ibs/f? and a pH of about 2.3 (1%

® soluation).

A preferred acrylic acid copolymer emulsion of the invention is Neoc ryl® A640 whiich comprises an aqueous acrylic acid copolymer having a pH of 7.5, visco~sity of 100 eps (Brookfield, 25°C), a weight per gallon of 8.6 pounds as supplied containing 40% solids by weight, 38% solids by volume and an acid number of 48. Specifically_, Neocryl®

A640 is a latex emulsion of an uncoalesced aqueous acrylic acid copolymer of acrylic acid andl methacrylic acid mixed with styrene. Other useful products include, tout are not limAted to, Neocryl® A520 and A625, and thelike.

Preferred combinations of immunonnodulators and immunoadjuvants -comprise a mincture of the homologous animal IL-12, squalane and a polyoxyw/propylene- pol yoxyethylene block copolymer; a mixture of the homologous animal IL-12 and saponin; ancl a mixture of the homologous animal IL.-12, EMA-31® and Neocryl® AG40 with or without a mineral oil emulsion. Recombinant human or murine IL-12 may be substituted for the homologous animal IL-12, though a partial immunopotentiation effeect may be elicited. Under those certain circumstances, the efficacy or potency can be readily determined through routine tests and then the dosage of the active ingredment can be appropriately titrated in the patient or animal as needed.

A pharmaceutically acceptable carrier is also present in the vaccine composition of thiss invention in varying amounts. The amount of the nontoxic, inert carrier_, of course, willl be dependent upon the amounts selected for the other ingredients, the desired comcentration of the active antigenic substamce, the selection of the vial, syrirmge or other comventional vehicle size, efc. The carrier can be added to the vaccine at any= convenient time. In the case of a lyophilized vaccine, the carrier can, for example=, be added immediately prior to administration. Alternatively, the final product can be mmanufactured with the carrier. Examples of appropriate carriers include, but are not limite=d to, sterile waster, saline, buffers, phosphate-buffered saline, buffered sodium chloride, ve getable oils,

Minimum Essential Medium (MEM), MEM with HEPES buffer, etc.

Optionally, the composition may contain conventional, secondary adjuvants in varying amounts depending on the adjuvant and the desired result. The custormary amount ranges from about 0.02% to about 20% by weight or provides from about= 1 Hg to about 50 pg per dose, depending upon the other ingredients and desired effec. Examples of suitable secondary adjuvants include, but are not limited to, stabilizears; emulsifiers; : aluminum hydroxide; aluminum phosphate; pH adjusters such as sodiwum hydroxide,

Zhydrochloric acid, elc.; surfactants such as Tween® 80 (polysorbate 80_ commercially available from Sigma Chemical Co., St. Louis, MO); liposomes; iscom adjie vant; synthetic &lycopeptides such as muramyl dipeptides; extenders such as dextramn or dextran combinations, for example, with aluminum phosphate; carboxypolymethylene; bacterial cell walls such as mycobacterial cell wall extract; their derivati ves such as

Corynebacterium parvum; Propiomibacterium acne; Mycobacterium bovis, for example,

Bovine Calmede Guern (BCG); vaccinia or animal poxvirus proteins; su bviral particle adjuvants such as orbivirus; cholera toxin; N,N-dioctadecyl-N',N'-bis(2-Faydroxyethyl)-

Propanediamine (avridine); monophosphoryl lipid A; dimethyldioctadescylammonium bromide (DDA, commercially available from Kodak, Rochester, NY); synthetics and mixtures thereof. Desirably, alwminum hydroxide is admixed with othaer secondary adjuvants or an immunoadjuvant sach as Quil A. Examples of suitable stabilizers include, beut are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-

Amine or NZ-Amine AS. Examples of emulsifiers include, but are not limited to, mineral omil, vegetable oil, peanut oil and other standard, metabolizable, nontoxic Oils useful for imjectables or intranasal vaccines.

For purposes of this invention, these adjuvants are identified herein a.s “secondary” merely to contrast with the above-described immunoadjuvant that is an essential ingredient ira the vaccine for its effect in cornbination with the immunomodulator to significantly iracrease the humoral immune response of the mammal or the bird to the antigenic substance. The secondary adjuvants are primarily included in the vaccine formulation as pmrocessing aids although certain adjuvants do possess immunologically enhancing pmroperties to some extent and have a dual purpose.

As needed, conventional preservatives can be added to the vaccine in effective armounts ranging from about 0.0001% to about 0.1% by weight. Depemding on the ag preservative employed in the formulation, amounts Wbelow or above this rangze may be useful. Typical pmreservatives include, for example, potassium sorbate, sodium metabisulfite, pheno 1, methyl paraben, propyl paraben, thimerosal, elc.

The choice of inactivated, modified or otheer type of vaccine and rnethod of preparation of the ®mproved vaccine formulation of the present invention are known of readily determined by those of ordinary skill in mhe art. As an illustrati on of the preparation of inactivated vaccines, for example, thme immunomodulator, prefiferably the homologous animaal IL-12, is mixed with one or more antigens, one Or more immunoadjuvants a_nd, optionally, one or more secon dary adjuvants. The antigeens may be the inactivated FIV, FeLV, E. canis, CCV, Lepaospira species, etc. Ass a further illustration, the immnunomodutator, preferably the hormnologous animal IL-12, is mixed with antigens in the presence or absence of the immuncadjuvants or secondary adjuvants to prepare modified waccines. The antigens in this case= may be BRSV, S. equi, CPV, CAV- 2, CDV, CP, etc. It is appreciated, however, that the vaccines of the presemt invention may be made by a_ variety of standard techniques well known to those in the £¢ ormulations art and are not lim ited by the illustrations described terein.

The comb-ination comprising the immunonmodulators and the immu_noadjuvants may be prepared and administered as a separate p roduct. A pharmacologicamlly effective amount of this Emmunogenicity enhancing compmosition may be given, feor example, parenterally, oral3y or otherwise, to a mammal or a bird before, concucTently with, sequentially to oer shortly after the administratio n of a weak, immunosugppressive or marginally safe aratigen in order to potentiate, acce lerate or extend the immumnogenicity of the antigen. Typically, the immunogenicity enharmcing composition will be administered within 24 hours before the start of the vaccination process and, preferably within four hours before or concurrently with the vaccination _ If vaccination requires mmore than one dose of the antigmenic substance, then the immunogenicity enhancing compossition may be administered in sequential fashion with the admin. istration of the vaccine. Although less effective, the immunogenicity enhancing composi tion may be given after t’he vaccine to boost the immunity against the antigen, but rarely beyond 24 hours.

When given sepamrately from the vaccine, the combeination may further comprise- a pharmaceutically acceptzable carrier and optionally, secondary adjuvants which are described herein. Both the immunomodulator and the immmunoadjuvant may be present &n varying amounts, typically in a unit dosage container. While the dosage of the combinatiosn «depends upon the antigen, species, body weight of the host waccinated or to be vaccinatecd, ezic., the dosage of a phasrmacologically effective amount of the immunomodulator willl

Lasually range from about @.1 pg to about 100 Hg per dose and, preferably, about 5 pg to asbout 50 pg per dose. Thee immunoadjuvant will typically range from about I pg to abouzt 25 pg per dose. Although the presence and the amount of t_he particular inmunoadjuvanst ir the combination will influence the amount of the imrmunomodulator necessary toms irmprove the immune respomnse, it is contemplated that the pramctitioner can easily adjust the= effective dosage amount Of the immunomodulator through. routine testing to meet the particular circumstances.

When the homologous animal IL-12 is employed, the amount of the immmunomodulator in the v—accine may be significantly redueced due to its potency. For small animals like dogs, cass, erc., a range of about 0.02 pe= to about 2 pg per dose of homologous animal IL-12 iss typically used, about 0.1 pg to about 1 ug per dose of the anmmal IL-12 is preferably u_sed and about 0.5 pg per dose is more preferably used in the cormbination composition of the invention. For large animal s like horses, cattle, swine, erc_, a range of about 0.1 pgs to about 5 pg per dose of animal IL-12 is typically used and abomut 0.5 pg to about 2.5 ug per dose is preferably used. It sis appreciated that amounts below and above these given ranges may find their respective Lases in the smaller birds and extremely large animals. To retain biological activity, it is also recommended that the aninmal IL-12 be added to the -vaccine or unit dosage form imme=diately prior to use.

As a non-limiting example, a suitable canine vaccine may comprise the Ebony strai_n of E. canis at a conce=ntration/dose of 1 x 10° TCIDsc; B. burgdorferi IPS at a conc=entration/dose of 5 X E7Z B. burgdorferi B-31 at a concent-ration/dose of 5 X E8; 5% viv «of emulsigen SA; 1% w/v of EMA-31®; 3% v/v of MNleocryl® A640; 1:20,000 conceentration of thimerosal (5%); a suitable amount of 1X MEN diluent and canine IL-12

. ® WO 03/024354 PCT/US02/29229 : at a concentration per dose Of approximately 0.5 pug or human IL—12 at a concentration of approximately 10 pg per dose.

The present invention further embraces the novel m_ethod for potentiating, accelerating or extending the immunogenicity of weak, immunosmuppressive or marginally safe antigens which comprises administering to an avian or~ mammalian species a pharmacologically effective amount of the immunogenicity enharcing composition or an effective vaccinating or immaunizing amount of the vaccine formmulation described herein.

Potentiating the immunogemicity of the weak, immunosuppresssive or marginally safe antigens involves improving the potency of the antigens. Accelerating the immunogenicity 1 0 refers to speeding up the onset of action. Extending the immunogenicity means lengthening the duration of activity.

As a general rule, the vaccine of the present invention is c-onveniently administered parenterally (subcutaneou sly, intramuscularly, intravenoussly, intradermally or intraperitoneally), intrabuccally, intranasally, transdermally or- orally. The route of 1.5 administration contemplated by the present invention will depwend upon the antigenic substance and the co-formuslants. For instance, if the vaccine contains saponins, while non-toxic orally or intranasally, care must be taken not to inject —the sapogenin glycosides into the blood stream as they function as strong hemolytics. Also, many antigens will not be effective if taken orally. Preferably, the vaccine is admimistered subcutaneously, intramuscularly or, in the case of S. equi and others, intranasally.

The dosage of the vaccine will be dependent upon the selected antigen, the route of administration, species, body weight and other standard factors. Itis contemplated that a person of ordinary skill in the art can easily and readily titrate the appropriate dosage for an immunogenic response for each antigen to achieve the effective immunizing amount and method of administration.

Advantageously, by ‘using the antigen and an immunomod ulator such as a cytokine, preferably the homologous animal IL-12, in combination withh immunoadjuvants in a vaccine formulation, the Improved vaccine is highly antigemnic, eliciting a stronger stimulation of T memory cells than had been achievable in the pwast. The serum antibody titers to ammtigenic substances after vaccination with the formulation of the present invention aree much higher than the titers induced by the same formulation in the absence of the immuknomodulator. For instance, a previoms study showed that the serum antibody titers to BRESV at 14 days after vaccination with BRSV adjuvanted with a mixture of squalane aned a polyoxypropylene-polyoxyethylene block copolymer were about 1:125.

Surprisingly, the serum antibody titers to BRSV at 14 days after vaccination with. BRSV mixed with. squalane, a polyoxypropylene-p olyoxyethylene block copolymesr and recombinant human IL-12 are distinctly higher at about 1:395, and remarkably still higher at about 1:3&6 after 28 days. The significantly emhanced immunogenicity, the acce=lerated onset of actieon and the extended duration of immaunity are evidenced by heightenecll serum antibody titezrs (i.e., humoral immune response) and stronger stimulation of T mnemory cells. The sumbstantial improvement in the efficacy” of the vaccine of this selective inwention gives a profosund departure from the state of the axt.

As ussed herein, the "CFU" stands for colo ny forming units. An "infectious unit” of BRSYV, for example, is defined as the TCIDss. "T<«CIDso" or 50% Tissue Culture Infaectious

Dose is defimed as the dose which infects 50% of the tissue culture. For example, when a solution cont—aining an antigen is diluted 1:100, 1 infectious unit equals the amount: which affects 50% of the tissue culture. In the case of BRSV, the TCIDso is the amount cf virus which is requmired to infect or kill 50% of the tissume culture cells. The term “cell-mediated immunity* iracludes the stimulation of T-Helper Cells, T-Killer Cells and T-Dwelayed

Hypersensitiwity Cells as well as the stimulation of macrophage, monocyte and other lymphokine aand interferon production. The presence of cell-mediated immunity acan be determined by conventional in vivo and in vitro a ssays. Local immunity such as secretory

IgA can be determined by conventional ELISA or IFA assays showing a serum neutralizing antibody titer of 1 to 2 or greater. The cell-mediated or local imrmunity elicited accor-ding to the present invention is specific to or associated directly with the antigen. The= term “mammal” refers to humans, cattle, cows, sheep, deer, horses, swine, goats, dogs, cats and the like. The term "avian ™ refers to poultry such as chickeens or turkey and other types of domesticated or wild birds. Although veterinary use in anicnals is

° WO 03/024354 PC~T/US02/29229 preferred, it is comtemplated that the immunogenicity enhancing and vaccin e compositions described herein ray find beneficial medical use.

A further understanding of the presemt invention can be obta ined from the following non-linmiting examples. However, fhe examples are set fortdh only for the illustration of certain aspects of the invention znd are not to be construeed as limitations thereon. It is to Bbe understood that the example=s do not purport to be wholly definitive as to conditions anad scope of this invention. It should be further appreci-ated that when typical reaction conditions (e.g., temperature, mreaction times, efc.) have “been given, the conditions both sabove and below the specified ranges can also be used, thhough generally less conveniently=. The following experimental studies employ recombinamnt human IL-12 that is obtained From Genetics Institute, Inc, Cambridge, MA, a wholly-o~wned subsidiary of Wyeth, Madison, NJ. Unless otherwise expressed, the examples are conducted at room temperature (ab-out 23°C to about 28°C) and at atmospheric pressure, all parts and percents referrecd to herein are by weight, and all temperatures are expressed in degrees centigrade.

EXAMPIE1

Effect of Adj 1 city of Hi Vacci

A study» is performed to determine the effect of certain acljuvants on the immunogenicity of an inactivated vaccine «wf Streptococcus equi. To prepare the adjuvants, stoc-k solutions of recombinant human IL-12 (4.45 mg/mml), saponin, a stabilizer for meodified live vaccines (SGGK-3 , 25% viv) and a bacterialk growth medium (Modified Todd Hewitt Broth, MTHB) are -used. Three adjuvant ble nds are made to approximate 10s pg of IL-12 per dose, 50 ug of 1L-12 per dose and 10 p g of IL-12 plus 5 mg of saponin oer dose.

To prepare the adjuvant blend comprising about 10 pg of II -12 per dose, a rehydration dilLaent is made by adding about 0.056 mL of IL-12 to aboust 49.719 mL of a sufficient quantity of water to total 50 mL. Aan adjustment diluent is then made by adding

W/O 03/024354 PCT/US02/292229 LC) ) about 0.056 mL of IL-12 to about 12.5 mL of SGGK-3 (25% v/v) mixed with about 377.444 mL of MTHB.

To prepare the adjusvant blend comprising about 50 ug of IL-12 per dos=e, a re hydration diluent is made by adding about 0.281 mL of L1-12 to about 49.719 mL of a

S su ficient quantity of water teo total 50 mL. An adjustment diluent is then made by adding ab out 0.281 mL of IL-12 te about 12.5 mL of SGGK-3 (25% v/v) mixed with abmout 37.219 mL of MTHB.

To prepare the adjuvrant blend comprising about 1 0 pg of IL-12 plus 5 mg of saponin per dose, a rehydrat ion diluent is made by adding about 0.056 mL of IL-12 to about 0.625 mL of saponin ard the mixture to about 49.319 mL of a sufficient quantity~ of waster to total 50 mL. An adjustment diluent is then made By adding about 0.056 mL. of

IL—12 to about 0.625 mL of saponin and the mixture to abowit 12.5 mL of SGGK-3 (25% v/vD) mixed with about 37.819 mL of MTHB.

For the preparation of each vaccine, one vial of Pinnacle® IN. (an intranas=sal equine Strangles vaccine, cormmercially available from Fort Dodge Animal Health, Inc. _ a veterinary division of Wyeth, Madison, NJ) is rehydrated with about 2.5 mL of rehywdrating diluent. Ten dose=s of vaccine are prepared for e-ach group (approximately 20 mL of vaccine). After rehydraating the vaccine, about 0.467 mL of rehydrated vaccine is added to about 19.533 mL of 2xdjustment diluent to obtain an . amount of approximately 1 x 10’ «CFU per dose.

All horses subjected to -the test vaccines are vaccinatecd two times with three weelecs betwseen vaccinations. The vaaccine is administered intranasa_1ly with a syringe connecte=d to a catheter of about 5.5 inchees in length. The first vaccina tion is administered into thme left mostril and the second vaccination is administered into the right nostril. All of th_e horses in the control group aree vaccinated with a commercially available Streptococcirs equi vaccine (Stepguard® with Haviogen®, an adjuvant ~consisting of carboxypoly=- methsylene, manufactured by Bayer Animal Health, Inc., an agricultural division of Baye-r

Corpeoration) and receive two v-accinations three weeks apart. The commercial vaccine i=s admiristered intramuscularly according to the manufacturer's icastruction.

®

Five horses are not vaccinated and, instead, are inocculated with 1 mL (apeproximately 5 x 10° CFU/mL) of thee S. equi CF-32 strain inte each nostril 5 days before the contact challenge. A syringe with a catheter of about 58.5 inches in length is usexd to inoculate the horses. The five morses are observed daily fro m two days before to fivee days post challenge for clinical signs and rectal temperatur e. Nasal swabs are collected daily after challenge to monitor S. equi shedding. Twenty—one days post second vaeccination, all the vaccinated horses are commingled with the five direct challenged ho ses. The animals are observed daily from -2 days to 0 days posst challenge (DPC) to establish a baseline and 1 to 28 days DPC for various clinical signs. Animals are observed ad ditionally on 30, 33 and 36 DPC.

The below Table 1 shows that a djuvants IL-12 (approximat=ely 50 ug IL-12/dose) amd a combination of IL-12 with saponin are relatively bettesr immunostimulators cosmpared to the rest of the adjuvants used in the study as demonstraated by average clinical sc-ore, incidence of local lymph node abscess, S. equi shedding and disseminated abscess.

H-orses in these two groups show about 35% to about 40% reduction in the incidence of di sseminated abscess and about 23% to about 40% reduction of thes average clinical score ass compared to the group receiving the commercial vaccine without IL-12 or the combination of IL-12 and saponin.

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Eff Adi I . f Cattle Vacci

A study is performed to determine the effect of a certain adjuvant on the immunogenicity of a modRtified live vaccine of BRSV (Bo-vine Respiratory Syncytial Virus).

To prepare the adjuvant, stock solutions of SP oil adjuvant (5% v/v) and re=combinant human IL-12 (about 1,26-0 ug per mL) are used.

SP oil is preparesd by mixing 20 mL of Pluromic® L121 (a polyoxy propylene- polyoxyethylene block copolymer, commercially avadlable from BASF Corporation,

Mount Olive, NJ), 40 rmlL of squalane, 3.2 mL of po lysorbate 80 and 936.8 mL of a buffered salt solution arad homogenizing the mixture umntil a stable mass or emulsion is formed. Prior to homog enation, the ingredients or mixtmire is autoclaved. The —emulsion is further sterilized by filtration. Formalin and thimerosal are added to a final concentration of 0.2% and dilution of MW :10,000, respectively.

The adjuvant ble-nd, which comprises about 5% v/v of SP oil plus about 10 pg of

IL-12 per dose, is made= by adding about 0.278 mL of IL-12 to about 69.722. mL of 5% viv SP oil to make about 70 mL of about 5% v/v SP oil plus about 10 pg/do se of IL-12 adjuvant.

For preparation of the vaccine, BRSV are grown in MDBK cells (VE adin-Darby

Bovine Kidney cells; the MDBK cell line is derived fromm a kidney of a normal adult steer) and are harvested 6 day-s after inoculation. The vaccin_e cake is blended at BERSV titer of about 10°” TCIDso per dose with MEM and then is ly ophilized. The lyophili=zed vaccine cake is then rehydratecd with the above-described IL—12 containing adjuvanat diluent to make the final vaccine pereparation.

Nine calves, abwout 6 months of age, are vamccinated with the BR_SV vaccine subcutaneously, leavings seven calves as the control group. Serum antibody response is measured by detecting the specific antibody to BRS. The efficacy of the= vaccines is demonstrated by challemging the vaccinates and the comntrols with virulent BRRSV 28 days post vaccination.

The modified live BRSV vaccines adjuvante=d with SP oil + 1-12 induced a very high titer antibody re=sponse (about 1:366 at 28th d ay post vaccination) to BRSY. _Afier the virulent BRSV cheallenge, the severity of the disease is reduced in the vaccinated gsroup compared with the coantrol group (about 53% reduction). This indicates that the SP oil +

IL-12 adjuvant used imn this study is compatible with the BRSV modified virus vaccine= and can significantly enharace the efficacy of the BRSV meodified virus vaccine.

The below Table 2 shows the antibody respponse to BRSV of calves vaccin_ated with modified live BRSV and 11-12 containing adjuvasnt.

Table 2

Antibody Response to BRSV [ Group [Nemberof| 0DPY Tine DPV |7DPC [14DPC el ll a el

AE I EC I ER EN

The below Table= 3 shows the disease reduction of calves vaccinated with modifieed

Imve BRSV and IL-12 comtaining adjuvant after virulent BRSV challenge.

Table 3

Disease Reduction

Gow | Bamberor | Average | Disease oo]

I PS EN a = Disease reduction is the percentage of calves which do not show the disease after challenge as compared to econtrols, 2 "N/A" means not applicable.

: ° WO 03/0243354 PCT/US02/29229 : EXAMPILE3

Eff F Adi Eff Doe Vaceci

A study is performed to determine the effect of a certain adjuvant on the immunogenicity of a monovalent vaccine, killed bacterin, of Ehrlichia canis. To prepare the adjuvant, stock solutions of” recombinant human IL-12 (4.45 mg/mL), EMA-31® (1% v/v, a linear ethylene/maleic ankhydride copolymer having appro—ximately equal amounts of” ethylene and maleic anhydride. having an estimated average rmnolecular weight of about 75,000 to 100,000, commercially available from Monsanto &Co., St. Louis, MO) andd

Neocryl® A640 (3% viv, a latex emulsion of an uncoalessced aqueous acrylic acid copolymer of acrylic acid and methacrylic acid mixed with stywrene, having a pH of 7.55 viscosity of 100 eps (Brookfield, 25°C), a weight per gallon of 8.6 pounds as supplied containing 40% solids by wesight, 38% solids by volume ard an acid number of 48 , commercially available from Polyvinyl Chemicals, Inc., Wilm_ington, MA) are used. A working solution of IL-12 is prepared in a dilution buffer comprising phosphate buffered saline without magnesium and calcium. Forty-five pL of the IL -12 stock solution is added to 9,955 pL of the dilution buffer. The final concentration of the diluted IL-12 workinzg solution is 20 pg/mL.

For preparation of thes vaccine, approximately 1 x 10-* or 1 x 10° TCIDso of an inactivated Ebony strain of E. canis is blended with 1% v/v o-f EMA-31® and 3% viv of 208 Neocryl®. Two percent of thimerosal is added to the vaccine at a level of about 1:20,0080 as preservative. The diluted XL-12 working solution in the ammount of 500 pL per dose is mixed with the vaccine prior to injection. The vaccine for group 4 as shown in Table 4 below is blended with 100 pg/dose of Bovine Calmede Guern (BCG) bacterin.

Thirty-five dogs are randomized into six groups including four vaccination groups and two control groups. The vaccinates are vaccinated with ax monovalent Ebony strain of

E. canis vaccine at two antigen levels. As shown in Table 4 Sbelow, group 2 is vaccinateed with the antigen level of approximately 1 x 10° TCIDso and the rest are vaccinated with the antigen level of approximately 1 x 10° TCIDso. Groups 2_, 3 and § are vaccinated with a vaccine blended with 10 jg of IL-12 per dose. Group 4 is vaccinated with a vaccmne containing BCG as adjuvant. Two doses of each vaccine are given at 20 weekss of age and 23 weeks of age, respectively. To demonstrate the possibility of cross-protect ion, groups and 6 are Ineterogeneously challenged with a Broadford strain of E. canis ancd others are ’ homogeneously challenged with an Ebony sirain of E. canis. 5S Shown in the below Table 4, two out of 5 dogs (40%) in group 3 and two out of 6 dogs (33%) in group 4 are free of thrombocytopenia when the vacceinates are homogeneoussly challenged with the Ebony strain of £. canis. One hundred percent of the controls (groump 1) and the dogs vaccinated with lower dose vaccine (group 2) have severe thrombocytop enia until the study ends. In terms of mortality, five out of 6 (83%) controls are dead or eumthanized during the period of observation. The dogs vaccinated vavith IL-12 adjuvanted lover dose vaccine (group 2) and dogs vaccinated with vaccine a-djuvanted with BCG (group 4) have 33% mortality rate. Based on the morbidity and mont ality data, the IL-12 adj svanted E. canis vaccine has significantly enhanced protective immunity against homoge=neous £. camis challenge.

As commpared with the controls, the addition of IL-12 in combination wiath EMA- 31® and Neocsryl® greatly increases the efficacy of E. canis monovalent vaccine and significantly reclluces the mortality. The protection induced by the IL-12 combimation as shown in groupes 2 and 3 is antigen dose-dependent. Further, as compared to BBCG, the adjuvant respon_ses induced by the IL-12 combination play a critical role in the reeduction ofthe vaccinated dogs from lethal challenge of E=. canis.

Table 4 below shows the results of the pre-immunogenicity study of monov~alent E. canis vaccine adguvanted with recombinant human I-12. :

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EXAMPLE 4

Evaluati FH 11 R Dog Vacei

A study is performed to determine the effect of a certain adjuvant on the immunogenicity of a modified live and killed viruses and killed bacterin of Canine 5S Duramune@® 10 Vaccine (composed of lyophilized live, attenuated canine par-vovirus (CPV), canine parainfluenza virus (CPI), canine adenovirus type II (CAV-2), canine adenovirus (CDV) and a diluent containing canine coronavirus (CCV), Lepaospira icterohemowrhagiae (LY), Leptospira canicola (LC), Leptospira grippotyphosa (L&G) and

Leptospira gpomona (LP), killed virus and bacterin fractions, commercially availabl=e from

Fort Dodges Animal Health, Inc., a veterinary division of Wyeth, Madison, NJ). To prepare the= adjuvant, stock solutions of recombinant human IL-12 (4.45 mg/mL),

Duramune®» 10 immunogenicity vaccine, EMA-3 1®, Neocryl® A640 and thimerosal 2% v/v) are usec.

For ypreparation of the test vaccine, the initial adjuvant is prepared by bleending

Neocryl® and EMA-31® to a final concentration of about 3% and about 1%, respectively. Thimerosal is added at the concentration of about 1:20,000 as preservative.

To prepare the IL-12 adjuvanted diluent, the diluent portion of the Duramune® 10 vaccine is firsst blended with the above initia! adjuvant at a ratio of about 1:10, one part of

Duramune® 10 diluent and 9 parts of the initial adjuvant comprising Neocryl® and EMA- 31®. The recombinant human IL-12 is then added at a final concentration of about 1.0 pg or 40 ug per adose.

Prior #0 use, one part of the lyophilized portion of the Duramune® 10 Vaccine is rehydrated in 9 parts of the IL-12 adjuvanted diluent. Therefore, the fractions o=f the

Duramune® 10 vaccine used in this study is about 10-fold less than the conventional immunogenicity vaccine. In other words, the waccine tested in this study contain s an insufficient amount of antigen (subpotent) as cornpared to the regular vaccines designed for commercial sale.

A total of 15 dogs are randomized into three groups of 5 dogs each and vaccinated twice subcutameously at 10 weeks of age and 13 weeks of age. The first grougp is )

: ® vaccinated with m vaccine containing about 10 pg of IL-12. The se-cond group is vaccinated with a ~vaccine containing about 40 pg of IL-12. The third groump is vaccinated ) with a 1:10 diluted? Duramune® 10 placebo without IL-12.

The dogs zare bled for serum at 0 day posst vaccination one (0 DP-V1) and 0 day post vaccination two (0 DPV2), 7, 14, 21 and 28 DPV2. The antibod=y titers for the leptospiras are det-ermined by microagglutination teest (MAT).

The results are detailed in the below Tab-les 5-8. Significant diffesrence between the principal grougp and the placebo is observed ir LP and LG fractions. For LP fraction listed in Table 5, significant difference is observred at 0. DPV2 (group vaccinated with about 10 pg of IL -12) and 21 DPV2 (same groupe). For the fraction of LG listed in Table 6, the significant difference is observed at 0 DPV2 (group vaccinated with about 40 pg of

IL-12), 7, 14 and 21 DPV2 (both 10 and 40 pg groups). No significamnt difference is observed in the other two fractions (Tables 7 and 8). Therefore, IL-12 _addition to the mixture of EMA-331® and Neocryl® is shown to enhance the humoral imrune responses to the two leptosp iras, LP and LG.

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EXAMPLES

Eff F Adj I icity of Cat Wace;

To determine whesther recombinant human IL-12 can entmance the immunogenicity of an FIV-FelLV vaccine, IL-12 is blended with inactivated feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) at 5 pg per dose after EMEA-3 1®, Neocryl® A640 and MVP® (an oil-in-water emulsion of light mineral oil, commercially available from

Modem Veterinary Products, Omaha, NE) are added to the vaccine. The challenge route of administration for the vaccine is intraperitoneally. One group oa 20 kittens, eight weeks of age, are vaccinated twice with the FIV-FeLV vaccine, ancether group of 20 age- matched kittens are vaccimated with the same vaccine supplemerated with IL-12. Three weeks following the completion of vaccination, all vaccinates are «challenged with virulent

FeLV along with nine age—matched controls. The challenged cats are monitored weekly for viremia for 15 weeks. To monitor the challenged cats for FeLV viremia, the serum samples are tested weekly for the presence of FeLV p27 antigen using IDEXX FeLV antigen test kit. A cat is considered persistently infected with FellLV when FeLV p27 is «detected on three consecutive sampling points during weeks 3 thraaugh 12 after challenge exposure. All nine controls are found to become persistently infeacted with FeLV. Five ©ut of 20 vaccinates which receive the FIV-FeLV vaccine are found to become

Poersistently infected with FeeL'V while only one out of 20 vaccinates which receive the FIV-FelLV vaccine supplemented with IL-12 are found to becomee persistently infected vavith FeLV. This result indi cates that IL-12 in combination with EMA-31®, Neocryl® and MVP® may be used to emhance the immunogenicity of FeLV vaccines, oo

Claims (8)

1. oN PCT/US02/29229 CLAIMS:

   l. A composition for enhancing the immunogenicit=y of a veterinary vaccine, characterized by comprising a pharmacologically effe=ctive amount of an immunomodulator and an immunoadjuvant, wherein the tlhe immunomodulator is selected from the group corisisting of a cytokine, an interfero n, tumor necrosis factor, transforming growth factor, «colony stimulating factor and a cormbination thereof, and the immunoadjuvant is selected from the group consisting of a nmctabolizable oil, a block copolymer, an ethylene/mal eic copolymer, an acrylic acid cospolymer, an acrylic acid copolymer emulsion, a mineral oil emulsion and a mixture there of.
2. 2. The immunogenicity enhancing composition according to Claim 1, characterized in that the imrmunomodulator is a cytokine.
3. 3. The immunogenicity enhancing composition according to Claim 1, characterized in that the immunomodulator is interleukin-12.
4. 4. The immunogenicity enhancing composition according to Claim |, 15+ characterized in that the imrnunomodulator is a homologous araimal, recombinant human or recombinant murine inter leukin-12,
5. 5. The immunogenicity enhancing composition according to any one of Claims I to 4, characterized in that the metabolizable oil is squalene or squalane.
6. 6. The immuncogenicity enhancing composition according to any one of 2 Claims | to 4, characterized. in that the metabolizable oil is squaalane.
7. 7. The immuneogenicity enhancing composition according to any one of Claims 1 to 4, charactermzed in that the block copolymer is a polyoxypropylene- polyoxyethylene block copolymer.
8. 8. The immunogenicity enhancing composition according to any one of 2% Claims 1 to 4, characterized in that the ethylene/malei ¢ copolymer is a linear ethylene/maleic anhydride copolymer having equal amount_s of ethylene and maleic anhydride and an estimated. average molecular weight of about 75,000 to 100,000.

   9. The immumeogenicity enhancing composition according to any one of Claims 1 to 4, characterize«d in that the acrylic acid copolymer is a mixture of styrene and -34- AMENDED SHEET

   A PCT/US02/29229® an uncoalesced aqueous acrylic acicl copolymer of acrylic acid &and methacrylic acid.

   10. The immunogenicity enhancing composition according to any one of

   Cl.aims | 10 9, characterized in that the mixture is emulsified.

   11. The immunogenicity enhancing composition according to any one o f Cl aims 1 to 10, characterized in that the mineral oil emulsion Ms an oil-in-water emulsioe1 of” light mineral oil.

   12. The immunogeniciky enhancing composition according to any one o=f CHaims 1 to 11, characterized in that the immunoadju-vant is a mixture of a pO lyoxypropylene-polyoxyethylene block copolymer and squaBane.

   13. The immunogenicity enhancing composition acecording to any one of Claims 1 to 11, characterized in thaat the immunoadjuvant is a rmixture of a linear et-hylene/maleic anhydride copolyrmer, having equal amounts o fethylene and maleic armhydride and an estimated averag e molecular weight of about 75,000 to 100,000, and ar aczrylic acid copolymer emulsion ceomprising an emulsified mixture of styrene and an umcoalesced aqueous acrylic acid copolymer of acrylic acid an=d methacrylic acid.

   14. The immunogenicity enhancing composition ac=cording to any one of Claims 1 to 11, characterized in th at the immunoadjuvant is a mixture of a linear ethylene/maleic anhydride copolyemner, having equal amounts of ethylene and maleic a-nhydride and an estimated average molecular weight of about 75,000 to 100,000, an a_crylic acid copolymer emulsion ccomprising an emulsified mixture of styrene and an wncoalesced aqueous acrylic acid scopolymer of acrylic acid armd methacrylic acid, and a mineral oil emulsion.

   15. An improved veter-inary vaccine composition «characterized by comprising aan effective immunizing am ount of an antigen, amn immunomodulator, an i.mmunoadjuvant and a ph. armaceutically acceptablee carrier, wherein the i_mmunomodulator is selected freom the group consisting o f a cytokine, an interferon, tumor necrosis factor, transforrming growth factor, colon=y stimulating factor and. a «combination thereof, and the imrmunoadjuvant is selected frcom the group consisting o fa mmetabolizable oil, a block copolymer, an ethylene/maleic copolymer, an acrylic aid «copolymer, an acrylic acid copowlymer emulsion, a mineral oil emulsion and a mixtwre -35- AMENDED SHEET tL) PCT /US02/29229 thereof.

   16. T he vaccine composition accordirmg to Claim 15, characterize=d in that the immunomodulat or is a cytokine.

   17. T he vaccine composition accordirg to Claim 15, characterized in that the immunomodulat-or is interleukin-12.

   18. T he vaccine composition accordirg to Claim 15, characteriz ed in that the immunomodulawtor is a homologous animal, reccombinant human or recombinant murine interleukin-12.

   19. The vaccine composition accorclding to any one of Clairmns 15 to 18, characterized in that the metabolizable oil is squaalene or squalane.

   20. "The vaccine composition accor-ding to any one of Clairms 15 to IS, characterized ims that the metabolizable oil is squaalane.

   21. "The vaccine composition accor—ding to any one of Clairms 15 to 18, characterized ir that the block copolymer is a gpolyoxypropylene-polyoxyethylene block copolymer.

   22. “The vaccine composition accomding to any one of Clai-ms 15 to 18, characterized im that the ethylene/maleic copolymer is a linear ethylene/maleic anhydride copolymer having equal amounts of ethylene and maleic anhydride ancl an estimated average molecular weight of about 75,000 to 10m0,000.

   23. The vaccine composition acco rding to any one of Clamms 15 to 18, characterized in that the acrylic acid copo lymer is a mixture of styrene and an uncoalesced acqueous acrylic acid copolymer of ~ acrylic acid and methacrylic acid.

   24. The vaccine composition according to any one of Cla ims 15 to 18, characterized an that the mixture is emulsified.

   25. The vaccine composition according to any one of Clawims 15 to 18, characterized in that the mineral oil emulsion &s an oil-in-water emulsion of light mineral oil.

   26. The vaccine composition acceording to any one of Claims 15 to 18, characterized in that the immunoadjuvant is a mixture of the pol.yoxypropylene- polyoxyethyle=ne block copolymer and a metat>olizable oil. -36 - AMENDED SHEET

   A PCT/1802/29229

   217. The vaccine composition accorading to any one of Claims 15 to 18, charactesrized in that the metabolizable oil is squalane.

   28. The vaccine composition according to any onc of Claims 15 to 18, characterized in that the immunoadjuvant is a mixture of the ethylene/maleic anhydride copolymer and an acrylic acid copolymer emulsion.

   29. The vaccine composition accordirg to Claim 28, characterized i n that the immuno adjuvant further comprises a mineral oil emulsion.

   30. The vaccine composition accord ing to any one of Claims 1 5 to 29, characterized in that the antigen is selected from the group consisting o=f Bovine Respiratory Syncytial Virus, herpes simplex virus type 1, bovine virus diarrhea, parainflu enza-3 virus, canine parvovirus, canine parainfluenza virus, canine adenovirus type II, canine adenovirus, canine coronavirus, rabies virus, feline immunode=ficiency virus, fel ine leukemia virus, feline coronavirus, Porcine Reproductive and Resspiratory Syndrome (PRRS) Virus, chicken herpes virus, Chlamydia, Ehrlichia, Pas tewrella, Haemophilus, Salmoneila, Staphylococcus, Streptococcus, Mycoplasma, Borrelia, Leptospirea, Coccidia, Hemosporidia, Amoebida, Trypanosoma, Leishmania, Giardia, Histomoneuas, Coccidioides, Histoplasma, Blastomy ces, Aspergillus, Cryptococciees and a combination thereof,

   31. The vaccine composition according to Claim 30, characterized in that the antigen is Bovine Respiratory Syncytial Virus and the immunoadjuvant is a mix<ture of the block copolymer and the metabolizable oil.

   32. The vaccine composition according to Claim 30, characterized in €hat the antigen is _Ehrlichia canis and the immunoadjuvan_t is a mixture of the ethylene~maleic copolymer and the acrylic acid copolymer emulsion.

   33. The vaccine composttion according to Claim 30, characterized in t"hat the antigen is a combination of canine parvovirus, canine parainfluenza virus, canine adenovirus type II, canine adenovirus, canine coronavirus, Lept ospira icterohemor-rhagiae, Leptospira canicola, Leptospoira grippotyphosa and Leptospira pomona, and the immunoadjuvant is a mixture of thee ethylene/maleic copolymer a-nd the acrylic acid copolymer emulsion. -37- AMENDED SHEE™T

   . PCT/US02/29229

   Z=4. The vaccine composition according to Claim 30, characteriz ed in that the antigen Ms a combination of feline immunodeficiency virus and feline leukermia virus, and the immaunoadjuvant is a mixture of the cthylene/malcic copolymer, the acrylic acid copolymmer emulsion and the mineral oil ermulsion.

   3.5. The vaccine composition according to any one of Claims 15 to 34, charactemrized by further comprising a preservative. 36 The vaccine composition according to any one of Claims 15 to 34, charactemized by further comprising a seco mndary adjuvant. 37 The vaccine composition a<ccording to Claim 36, characterized in that the secondary adjuvant is selected from the group consisting of a stabilizer, aan emulsifier, aluminurm hydroxide, aluminum phosphawe, a pH adjuster, a surfactant, a liposome, an iscom acdjuvant, a synthetic glycopeptide, an extender, carboxypolymethylene, bacterial cell wall, a derivative of a bacterial cell wall, vaccinia, an animal poxvirus protein, a subviral particle adjuvant, chol era toxin, N,N-dioctadecy 1-N',N'-bis(2- hydroxyesthyl)propanediamine, monophosphoryl lipid A, dimethyldioctadecy-l-ammonium bromide and a mixture thereof.

   3.8. An improved veterinary vaccine composition characterized b=y comprising an effecztive immunizing amount of Streptococcus equi, in combination with a pharmacezutically acceptable carrier, sapo min and an homologous animal, recombinant human om recombinant murine [L-12. 3, Use of an immunomodula tor selected from the group corsisting of a cytokine, an interferon, tumor necrosis factor, transforming growth fa ctor, colony stimulatimg factor or a combination there of and an immunoadjuvant selected from the group consisting of a metabolisable oil, a block copolymer, an eth ylene/maleic copolymer, an acrylic acid copolymer, an acrylic acid copolymer emulsion, za mineral oil emulsion or amixture thereof in the preparation of a vaccine according to» any one of claims 1 “to 38.

   40. Use as claimed in claim 39, wherein the vaccine is administered concurreritly with, sequentially with or after the administration of a weak, immunos uppressive or marginally safe antx gen and potentiates, accelerates ox extends the - 38- AMEND»ED SHEET

   )) PCT/WS02/29229 immunity of the ar tigen.

   41. Uses as claimed in Claim 39 or 4 0, characterized by adminmstering the immunogenicity enhancing or vaccine composi tion subcutaneously, intrarmuscularly, intradermally, intr-aperitoneally, intranasally, intratouccally, transdermally or orally.

   42. Usee claimed in any one of claims 3 9 to 41, wherein the antiger is selected from the group coonsisting of Bovine Respiratory Syncytial Virus, herpes simmplex virus type 1, bovine virus diarrhea, parainfluenza—3 virus, canine parvovizrus, canine parainfluenza virwus, canine adenovirus type Il, canine adenovirus, canine coronavirus, rabies virus, felirme immunodeficiency virus, feline leukemia virus, feline scoronavirus, Porcine Reprodusctive and Respiratory Syndromee (PRRS) Virus, chicken merpes virus, Chlamydia, Ehrlichia, Pasteurella, Haemophilus, Salmonella, Staphylococcus, Streptococcus, Mycoplasma, Borrelia, Leptospira, Coccidia, Hemosporidiaz, Amoebida, Trypanosoma, Leishmania, Giardia, Histo monas, Coccidioides, Ilistoplasma, Blastomyces, Aspergillus, Cryptococcus and a cormbination thereof.

   43. A composition according to Clairm 1 or Claim 38, substantia=lly as herein described with reference to and as illustrated in amy of the examples.

   44. U se according to Claim 39 or Claim 43 or Claim 44 owr Claim 45, substantially as herein described with reference to and as illustrated iro any of the examples.

   -39. AMENDED SHEET