NZ713360B2 - Novel vaccine compositions comprising immunostimulatory oligonucleotides - Google Patents

Abstract

The invention provides a vaccine composition comprising an antigen component and an adjuvant component, wherein the adjuvant component comprises a P-class immunostimulatory oligonucleotide and either a combination of a saponin and a sterol; or an oily phase comprising an oil and, optionally, one or more emulsifiers, said oily phase comprising 2-20% v/v of the vaccine composition, wherein said antigen component is an EHV antigen or a Leishmania antigen. more emulsifiers, said oily phase comprising 2-20% v/v of the vaccine composition, wherein said antigen component is an EHV antigen or a Leishmania antigen.

Description

NOVEL VACCINE COMPOSITIONS COMPRISING STIMULATORY OLIGONUCLEOTIDES FIELD OF THE INVENTION This invention relates to novel adjuvants for enhancing immune response to Leishmania and Equine Horse Virus vaccines.

BACKGROUND Equine herpes virus is a major equine pathogen responsible for viral-induced abortion ogical disease such as paresis, infections of the upper respiratory tract, and neonatal foal disease (NFD). NFD results from close to term transplacental ion of fetuses, which are born weak with severe respiratory disease and some with jaundice due to liver infection by EHV-1. These s usually die within a few days after birth. Equine rhinopneumonitis virus (EHV-4) is the major cause of acute atory tract disease ("rhinopneumonitis") and infects most horses during their first two years of life. Rhinopneumonitis is characterized by fever, anorexia, and e serous nasal discharge that later becomes mucopurulent. On rare occasions EHV4 infection causes abortion in pregnant mares. Furthermore EHV1 and EHV4 establish persistent, lifelong latent infections. Upon reactivation the viruses cause recurrent disease, accompanied by virus shedding and transmission to other animals.

Control of equine herpes virus infection and their diseases remain inadequate, in particular against EHV1 mediated abortions, paresis and neonatal foal disease resulting from close to term transplacental infection of foetus. Although inactivated as well as modified live vaccines are available, neither vaccine appears to block infection sufficiently, nor do they prevent the establishment of latency by wild-type virus. Hence there is a great need for safe vaccines with ed protection against field infections of these viruses, particularly against infections caused by EHV1.

Leishmaniasis is a major and severe parasitic disease of humans, canids (dogs, wolves, foxes, coyotes, jackals), and felids (lions, tigers, domestic cats, wild cats, other big cats, and other s ing hs and lynx). The agent of leishmaniasis is a protozoan parasite and belongs to the leishmania donovani complex. This parasite is widely distributed in temperate and subtropical countries of Southern Europe, Africa, Asia, South America and Central America (Desjeux P., Trans. R. Soc. Trop. Med. Hyg., 2001, 95: 239—43). Leishmania donovani infantum (L. infantum) is responsible for the feline and canine disease in rn Europe, Africa, and Asia. In South America and Central America, the agent is Leishmania donovani chagasi (L. chagasi), which is closely related to L. infantum. In humans, the agent is Leishmania donovani donovani (L. donovani), which is y related to L. infantum and L. chagasi.

These parasites cause visceral aniasis and/or cutaneous leishmaniasis.

Visceral leishmaniasis results in clinical ms like fever, cachexia, hepatosplenomegaly (enlargement of the liver and spleen), and blood cytopenia.

Cutaneous aniasis occurs in g presentations, from the imited and even self-healing ous forms to fatal systemic disease. Lesions of ous leishmaniasis may occur anywhere on the body but the most common sites are those which are exposed to the environment and are therefore more susceptible to bites from the sand flies. The initial papule rapidly gives rise to an ulcer. Systemic leishmaniasis is rare but is invariably fatal if not treated promptly. Systemic leishmaniasis affects the internal body organs, specifically the spleen and the liver.

In canines, the disease is associated with ous symptoms or with visceral symptoms or both cutaneous and visceral symptoms, and is lethal in the absence of therapy.

Numerous treatments have been described but none is fully satisfactory due to toxicity of the ent itself or a tendency for the animal to e.

Accordingly, new vaccine formulations are needed to protect domestic animals against EHV and Leishmania infections.

SUMMARY OF INVENTION The instant invention addresses drawbacks of the art by providing, in one aspect, a vaccine composition comprising an antigen component selected from the group consisting of an EHV antigen or a Leishmania antigen and an adjuvant component, wherein the adjuvant component ses a P-class immunostimulatory oligonucleotide and a combination of a saponin and a sterol; or an oily phase FRPSULVLQJ DQ RLO DQG RSWLRQDOO\ RQH RU PRUH HPXOVLILHUV VDLG RLO\ SKDVH FRPSULVLQJ Y Y RI WKH YDFFLQH FRPSRVLWLRQ > @ ,Q D VHW RI PHQWV WKH 3 FODVV VWLPXODWRU\ ROLJRQXFOHRWLGH ZKLFK PD\ EH PRGLILHG DQG RU VWDELOL]HG LV SUHVHQW LQ WKH DPRXQW RI —J SHU GRVH H J —J SHU GRVH > @ ,Q D VHW RI HPERGLPHQWV WKH DGMXYDQW FRPSRQHQW FRPSULVHV D VDSRQLQ VXFK DV 4XLO $ RU D SXULILHG IUDFWLRQ WKHUHRI DQG D VWHURO VXFK DV FKROHVWHURO HDFK LQ WKH DPRXQW RI EHWZHHQ DERXW —J PO ,Q D VSHFLILF HPERGLPHQW HDFK RI 4XLO $ DQG FKROHVWHURO LV SUHVHQW LQ WKH DPRXQW RI —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³$GMXYDQW PHDQV DQ\ VXEVWDQFH WKDW VHV WKH KXPRUDO RU FHOOXODU LPPXQH UHVSRQVH WR DQ DQWLJHQ $GMXYDQWV DUH JHQHUDOO\ XVHG WR DFFRPSOLVK WZR REMHFWLYHV 7KH VORZ WKH UHOHDVH RI DQWLJHQV IURP WKH LQMHFWLRQ VLWH DQG WKH VWLPXODWLRQ RI WKH LPPXQH V\VWHP > @ $QWLERG\ UHIHUV WR DQ LPPXQRJOREXOLQ PROHFXOH WKDW FDQ ELQG WR D VSHFLILF DQWLJHQ DV WKH UHVXOW RI DQ LPPXQH UHVSRQVH WR WKDW DQWLJHQ JOREXOLQV DUH VHUXP SURWHLQV FRPSRVHG RI OLJKW DQG KHDY\ SRO\SHSWLGH FKDLQV KDYLQJ FRQVWDQW D QH[W LV SDJH and "variable" regions and are divided into classes (e.g., IgA, IgD, IgE, lgG, and IgM) based on the composition of the constant regions.

"Antigen" or "immunogen" refers to any substance that stimulates an immune response. The term includes , inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term n also includes polynucleotides, polypeptides, recombinant proteins, synthetic es, protein extract, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, individually or in any combination thereof. The term n also includes antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

"Bacterin" means a suspension of one or more killed bacteria which may be used as a component of a vaccine or immunogenic composition.

"Buffer" means a chemical system that prevents change in the concentration of another chemical substance, e.g., proton donor and acceptor systems serve as buffers preventing marked changes in en ion concentration (pH). A further example of a buffer is a solution containing a e of a weak acid and its salt (conjugate base) or a weak base and its salt gate acid).

"Cellular immune response" or "cell mediated immune response" is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of nes, chemokines and similar molecules produced by ted T-cells, white blood cells, or both.

“Consisting essentially” as applied to the adjuvant formulations refers to formulation which does not contain ted additional adjuvanting or modulating agents in the amounts at which said agent exert measurable adjuvanting or immunomodulating effects.

"Dose" refers to a vaccine or immunogenic composition given to a subject. A "first dose" or "priming vaccine" refers to the dose of such a composition given on Day 0. A "second dose" or a "third dose" or an "annual dose" refers to an amount of such composition given uent to the first dose, which may or may not be the same vaccine or immunogenic composition as the first dose.

"Humoral immune response" refers to one that is mediated by antibodies.

"Immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune se to an antigen. Immune responses can usually be determined using standard immunoassays and neutralization , which are known in the art. "lmmunologically tive amount" or "immunologically effective amount" or "effective amount to produce an immune response" of an antigen is an amount effective to induce an immunogenic response in the ent. The immunogenic response may be sufficient for diagnostic purposes or other testing, or may be adequate to prevent signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a e agent. Either humoral immunity or cell-mediated immunity or both may be induced. The genic response of an animal to an genic composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain, whereas the tive immunity conferred by a vaccine can be evaluated by measuring, e.g., ion in clinical signs such as mortality, morbidity, temperature number, l physical condition, and overall health and performance of the subject. The immune response may comprise, without limitation, induction of cellular and/or humoral immunity. "lmmunogenic" means g an immune or antigenic response. Thus an immunogenic ition would be any ition that induces an immune response. ostimulatory molecule" refers to a molecule that generates an immune response.

"Lipids" refers to any of a group of organic compounds, including the fats, oils, waxes, sterols, and triglycerides that are insoluble in water but soluble in nonpolar organic solvents, are oily to the touch, and together with carbohydrates and proteins constitute the principal structural material of living cells.

“Pharmaceutically acceptable" refers to substances, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, tion, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use.

"Reactogenicity" refers to the side effects elicited in a subject in response to the administration of an adjuvant, an immunogenic, or a vaccine composition. It can occur at the site of administration, and is usually assessed in terms of the pment of a number of symptoms. These symptoms can include inflammation, s, and abscess. It is also assessed in terms of occurrence, duration, and severity. A "low" reaction would, for example, involve swelling that is only detectable by palpitation and not by the eye, or would be of short duration. A more severe reaction would be, for example, one that is visible to the eye or is of longer duration.

"Room Temperature" means a temperature from 18 to 25°C.

"Saponin" refers to a group of surface-active glycosides of plant origin composed of a hydrophilic region (usually l sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure.

"Steroids" refers to any of a group of organic compounds belonging to biochemical class of lipids, which are easily soluble in organic solvents and slightly e in water. Steroids comprise a four-fused ring system of three fused cyclohexane (six-carbon) rings plus a fourth cyclopentane (five-carbon) ring.

"Sterols" refers to compounds in animals which are ically ed from terpenoid sors. They comprise a steroid ring structure, having a hydroxyl (OH) group, y attached to carbon-3. The arbon chain of the fatty-acid substituent varies in length, usually from 16 to 20 carbon atoms, and can be saturated or unsaturated. Sterols commonly contain one or more double bonds in the ring structure and also a variety of tuents attached to the rings. Sterols and their fatty-acid esters are essentially water insoluble.

"Subject" refers to any animal for which the administration of an adjuvant composition is d. It includes s and non-mammals, including primates, livestock, companion animals, laboratory test animals, captive wild animals, aves (including in ova), reptiles, and fish. Thus, this term includes but is not limited to monkeys, humans, swine; , sheep, goats, equines, mice, rats, guinea pigs, hamsters, rabbits, felines, canines, chickens, turkeys, ducks, other poultry, frogs, and lizards.

"Therapeutically effective amount" refers to an amount of an antigen or vaccine that would induce an immune response in a subject receiving the antigen or vaccine which is adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus or a bacterium. l immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced. The immunogenic response of an animal to a vaccine may be evaluated, e.g., ctly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and l health and performance of the subject. The amount of a vaccine that is therapeutically effective may vary depending on the particular nt used, the particular antigen used, or the condition of the subject, and can be determined by one skilled in the art.

"Treating" refers to preventing a disorder, condition, or disease to which such term applies, or to preventing or reducing one or more symptoms of such disorder, condition, or disease.

"Treatment" refers to the act of "treating" as defined above.

"Triterpeniods" refers to a large and diverse class of naturally ing organic molecules, derived from six arbon isoprene (2-methyI-1,3-butadiene) units, which can be led and ed in thousands of ways. Most are multicyclic structures which differ from one r in functional groups and in their basic carbon skeletons.

These molecules can be found in all s of living things.

"Vaccine" refers to a composition that includes an antigen, as defined herein.

Administration of the e to a subject results in an immune response, generally against one or more specific diseases. The amount of a vaccine that is therapeutically effective may vary depending on the particular antigen used, or the condition of the subject, and can be determined by one skilled in the art.

As noted above, the instant invention provides a vaccine composition comprising an antigen component ed from the group consisting of an EHV antigen or a Leishmania antigen and an adjuvant component, n the nt component ses (or, in different set of embodiments, ts essentially of, or in yet different set of embodiments, consists of) a P-class immunostimulatory oligonucleotide and a combination of a saponin and a sterol; or an oil, said oil sing 2-20% v/v of the vaccine composition.

In a set of embodiments the vaccine compositions of the instant invention are non-liposomal. In embodiments where the oil is present, the antigen would generally be dispersed or dissolved in the aqueous phase and not enveloped by liposomes or similar structures. In embodiments where the saponin and the sterol are present, said saponins and said s would form helical es and the antigen would be in admixture with, but not integrated within, said helical micelles.

Antigens Different ns derived from Leishmania or EHV are suitable for the instant invention. The antigens may include, without limitation, whole sms (inactivated, attenuated, and modified live), nucleotides, polynucleotides, peptides, polypeptides, recombinant proteins, synthetic peptides, protein t, polysaccharides, carbohydrates, fatty acids, teichioc acid, oglycans, lipids, or glycolipids, individually or in any combination thereof.

Live, modified-live, and attenuated viral strains that do not cause disease in a subject have been isolated in non-virulent form or have been attenuated using methods well known in the art, including serial passage in a suitable cell line or exposure to ultraviolet light or a chemical mutagen. lnactivated or killed viral strains are those which have been vated by s known to those skilled in the art, including treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), sterilizing radiation, heat, or other such methods.

The amount of antigen used to induce an immune response will vary considerably depending on the antigen used, the subject, and the level of response desired, and can be determined by one skilled in the art. For vaccines containing modified live or attenuated EHV, a therapeutically effective amount of the antigen generally ranges from about 102 Tissue e Infective Dose (TCID) so to about 1010 TCle, inclusive. For many such s, a therapeutically effective dose is generally in the range of about 102 TCID50 to about 108 TCID50, inclusive. In some embodiments, the ranges of therapeutically effective doses are about 103 TCIDso to about 106 TCleo, inclusive. In some other embodiments, the ranges of therapeutically effective doses are about 104 Toto50 to about 105 TC|D50, ive.

For vaccines containing inactivated EHV, a therapeutically effective amount of the n is generally at least about 100 relative units per dose, and often in the range from about 1,000 to about 4,500 relative units per dose, inclusive. In other embodiments, the therapeutically effective amount of the n is in a range from about 250 to about 4,000 relative units per dose, inclusive, from about 500 to about 3,000 relative units per dose, inclusive, from about 750 to about 2,000 relative units per dose, inclusive, or from about 1,000 to about 1,500 relative units per dose, inclusive.

The number of cells for a ania antigen administered in a vaccine ranges from about 1x102 to about 1x1010 per dose, inclusive. In other embodiments, the number of cells ranges from about 1x 103 to about 1x109 per dose, inclusive, or from about 1x104 to about 1x108 per dose, inclusive, or from about 1x105 to about 1x107 per dose, inclusive, or from about 1x 106 to about 1x108 per dose, inclusive.

A therapeutically effective amount of antigen in es containing inactivated viruses can also be measured in terms of Relative Potency (RP) per mL. A therapeutically effective amount is often in the range from about 0.1 to about 50 RP per mL, inclusive. In other embodiments, the therapeutically effective amount of the antigen is in a range from about 0.5 to about 30 RP per mL, inclusive, from about 1 to about 25 RP per mL, inclusive, from about 2 to about 20 RP per mL, inclusive, from about 3 to about 15 RP per mL, inclusive, or from about 5 to about 10 RP per mL, ive.

Suitable non-limiting examples of Leishmania ns include whole organisms, protein extracts, roteins, outer capsid proteins and extracts, and nucleic acid sequences which encode said outer capsid proteins. Specific antigens may e Gp63, PSA-2 and Fucose Mannose Ligand. In another set of embodiments, leishmanial excretory-secretory antigens (ESAs) may be used. ESAs for at least of seventeen ESAs of relative molecular weights 11, 13, 16, 18, 21, 23, 26, 29, 33, 35, 42, 51, 54, 58, 64, 70 and 80 kDa have been identified. Two fractions, F1 (11, '13 and 16 kDa} and F3 (26, 29 and 33 kDa), were found to be highly tmmunogenic as they significantiy induced NABPH oxidase and SOD activities as wail as NOX, TNF~G, iFN-v and 51.42 production in stimulated RAW 264.7 macrophages. Further, these ns also induced significant proliferation of human peripheral blood mononuclear cells along with increased production of $va and £1.42. See (Sour et al., Experiments} Parasitology, 2012; 132(3). 355-361.

The most common varieties of EHV are EHV-1 and EHV-4. EHV is composed of an icosahedral nucleocapsid containing the viral genome, surrounded by an amorphous envelope, which contains eleven glycoproteins (93, 9C, 9D, gE, gG, gH, gl, gK, gL, 9M and 9N). See Paillot et al., Open Vet Sc J, 2008; 2: 68-91.

Suitable non-limiting examples of EHV antigens include whole viruses (inactivated, attenuated, and modified live), n extracts, pe proteins and nucleic acid sequences which encode said proteins, as well as inant subunit antigens or vectored antigens.

Sagonins and CgGs Triterpenoid saponins suitable for use in the adjuvant compositions can come from many sources, either plant derived or synthetic equivalents, including but not limited to, Quillaja saponaria, tomatine, g extracts, oms, and an alkaloid glycoside urally r to dal saponins. Thus, triterpenoids suitable for use in the adjuvant compositions include saponins, squalene, and lanosterol. The amount of triterpenoids suitable for use in the nt compositions depends upon the nature of the triterpenoid used. However, they are generally used in an amount of about 1 pg to about 5,000 pg per dose. They also are used in an amount of about 1 pg to about 4,000 pg per dose, about 1 pg to about 3,000 pg per dose, about 1 pg to about 2,000 pg per dose, and about 1 pg to about 1,000 pg per dose. They are also used in an amount of about 5 pg to about 750 pg per dose, about 5 pg to about 500 pg per dose, about 5 pg to about 200 pg per dose, about 5 pg to about 100 pg per dose, about 15 pg to about 100 pg per dose, and in an amount of about 30 pg to about 75 pg per dose.

If a saponin is used, the adjuvant compositions generally contain an immunologically active saponin fraction from the bark of Quillaja saponaria. The saponin may be, for example, Quil A or another purified or partially ed saponin preparation, which can be obtained commercially. For example, Quill A is sold in the USA by EM Sergeant Company. 08-7, 08-17, 08-18, and 08-21 may be obtained from Antigenics Company, Massachusetts, USA. Thus, saponin extracts can be used as mixtures or ed individual ents such as QS-7, QS-17, 08-18, and 08-21. In one ment the Quil A is at least 85% pure. In other embodiments, the Quil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.

CpG oligonucleotides are a ly described class of pharmacotherapeutic agents that are characterized by the presence of an unmethylated CG dinucleotide in specific base-sequence contexts (CpG . (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. Prog Respir Res. Basel, Karger, 2001, vol 31, pp 229- 232, which is orated herein by reference). These CpG motifs are not seen in eukaryotic DNA, in which CG eotides are suppressed and, when present, usually methylated, but are present in bacterial DNA to which they confer immunostimulatory properties.

The adjuvants of the instant invention utilize a so-called P-class immunostimulatory oligonucleotide, more preferably, modified P-class immunostimulatory oligonucleotides. P-class immunostimulatory oligonucleotides are CpG oligonucleotides characterized by the presence of palindromes, generally 6-20 nucleotides long. The P-Class oligonucleotides have the ability to spontaneously self- assemble into concatamers either in vitro and/or in vivo. These oligonucleotides are, in a strict sense, single-stranded, but the presence of palindromes allows for formation of concatamers or possibly stem-and-loop structures. The overall length of P-class immunostimulatory oligonucleotides is between 19 and 100 nucleotides, e.g., 19-30 nucleotides, 30-40 tides, 40-50 tides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, 90-100 nucleotides.

In one aspect of the ion the immunostimulatory oligonucleotide ns a ' TLR activation domain and at least two palindromic regions, one palindromic region being a 5‘ palindromic region of at least 6 nucleotides in length and connected to a 3' palindromic region of at least 8 nucleotides in length either directly or through a spacer.

The P-class immunostimulatory oligonucleotides may be modified according to techniques known in the art. For example, J-modification refers to iodo-modified nucleotides. E-modification refers to ethyl-modified tide(s). Thus, E-modified P- class immunostimulatory oligonucleotides are P-class immunostimulatory oligonucleotides, wherein at least one nucleotide (preferably 5’ nucleotide) is ethylated.

Additional modifications include attachment of o-benzimidazol, O-Methylation, modification with proynyl-dU, e modification, 2-bromovinyl attachment (preferably to uridine).

The s immunostimulatory oligonucleotides may also contain a modified internucleotide linkage including, without limitations, phosphodiester linkages and phosphorothioate linkages. The oligonucleotides of the instant invention may be synthesized or obtained from commercial sources.

P-Class oligonucleotides and modified P-class oligonucleotides are further disclosed in published PCT ation no. W02008/068638, published on Jun. 12, 2008. Suitable non-limiting examples of modified P-class immunostimulatory oligonucleotides “*u are provided below ( refers to a phosphorothioate bond and “_” refers to a phosphodiester bond).

SEQ ID NO: 1 5' T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3' SEQ ID NO: 2 5' T*C-G*A"C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 3 5' T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' SEQ ID NO: 4 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 5 5' JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3' SEQ ID NO: 6 5' JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C* G*T 3' SEQ ID NO: 7 5' EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' SEQ ID NO: 8 5' JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' SEQ ID NO: 9 5' JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C* G*T 3' SEQ ID NO: 10 5' T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3'.

The amount of P-class stimulatory oligonucleotide for use in the nt compositions depends upon the nature of the P-class immunostimulatory oligonucleotide used and the intended s. However, they are generally used in an amount of about 20 pg to about 500 pg per ml. They also are used in an amount of about 25 pg to about 400 mg per ml, about 40 pg to about 250 pg per ml, about 50 pg to about 200 pg per ml, about 100 g per ml to about 200 pg per mi.

Sterols suitable for use in the nt compositions include sterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. These sterols are well known in the art and can be purchased commercially. For example cholesterol is disclosed in the Merck Index, 12th Ed., p. 369. The amount of sterols suitable for use in the adjuvant compositions depends upon the nature of the sterol used. However, they are generally used in an amount of about 1 pg to about 5,000 pg per ml. They also are used in an amount of about 1 pg to about 4,000 pg per ml, about 1 pg to about 3,000 pg per ml, about 1 pg to about 2,000 pg per ml, and about 1 pg to about 1,000 pg per ml. They are also used in an amount of about 5 pg to about 750 pg per ml, about 5 pg to about 500 pg per ml, about 5 pg to about 200 pg per ml, about 5 pg to about 100 pg per ml, about pg to about 100 pg per ml, and about 30 pg to about 75 pg per mi.

The preparation of the compositions containing the saponin and the sterol according to the instant invention is within the ordinary skill in the art. , an aqueous mixture is prepared, said mixture comprising the antigen, the P-class immunomodulatory oligonucleotide, and the n. The sterol is then lly (or dropwise) added to that mixture. 0in Phase In additional embodiments, the adjuvant component comprises an oily phase in the amount of about 2 to about 20% v/v of the vaccine composition, e.g., about 3 to about 15%, about 5 to about 15%, about 10 to about 15%, about 10 to about 20 % etc.

The oily phase generally comprises an oil and, optionally, one or more emulsifiers. In one embodiment, the oily phase comprises two emulsifiers, one of which is lipid-soluble and the other is water-soluble.

Multiple oils and combinations thereof are le for use of the instant invention. These oils include, without limitations, animal oils, vegetable oils, as well as non-metabolizable oils. miting examples of vegetable oils suitable in the instant invention are corn oil, peanut oil, soybean oil, coconut oil, and olive oil. Non-limiting example of animal oils is ne. Suitable non-limiting examples of non- metabolizable oils include light mineral oil, straight chained or branched saturated oils, squalane and the like.

In a set of ments, the oil used in the adjuvant formulations of the instant ion is a light l oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique. The term is synonymous with ”liquefied paraffin", "liquid petrolatum" and "white l oil." The term is also intended to e "light l oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, J. T. Baker (Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred mineral oil is light mineral oil commercially available under the name DRAKEOL®.

In another set of embodiments, the oil is SP oil, which ses a polyoxyethylene-polyoxypropylene block copolymer (available from BASF, Mt. Olive, NJ), squalane (available from Kodak, Rochester, NY), and polyoxyethylene sorbitan monooleate (TWEEN®80, available from Sigma Chemical, St. Louis, MO). Squalane is used at approximately 5% (w/v), yethylene-polyoxypropylene block copolymer is used at approximately 2.5% (w/v), polyoxyethylene an monooleate (TWEEN®-80), is used at approximately 0.2%(w/v) ed in a pharmaceutically acceptable t of either buffer, water, normal saline or cell culture growth media.

The oily phase includes oil and oil-soluble emulsifiers (e.g., SPAN 80), if any such emulsifiers are present. The volume of the oily phase is calculated as a sum of volumes of the oil and the oil soluble emulsifier. Thus, for example, if the volume of the oil is 10% and the volume of the oil-soluble emulsifier is 2% of a composition, then the oily phase would be present at 12% v/v of the composition. Similarly, if the oil is present in the amount of about 5% and the lipid-soluble emulsifier is present in the amount of about 6% of a composition, then the oily phase is t at about 11% v/v of the composition.

Emulsifiers suitable for use in the present emulsions include natural biologically compatible emulsifiers and non-natural synthetic surfactants. Biologically compatible emulsifiers e phospholipid compounds or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithin), such as soy or egg lecithin. Lecithin can be obtained as a mixture of atides and triglycerides by water-washing crude vegetable oils, and separating and drying the ing hydrated gums. A refined product can be obtained by fractionating the e for acetone insoluble phospholipids and glycolipids remaining after removal of the triglycerides and vegetable oil by acetone washing. Alternatively, lecithin can be obtained from various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylethanolamine. The olipids may be isolated from natural sources or conventionally synthesized.

In additional embodiments, the emulsifiers used herein do not include lecithin, or use in in an amount which is not logically effective.

Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention include sorbitan-based non-ionic surfactants, e.g. fatty-acid- substituted sorbitan surfactants (commercially available under the name SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name OL M- 53), polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL®), yethylene fatty l ethers (BR|J®); polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethylene isooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants are the tants available under the name SPAN® and TWEEN®, such as TWEEN-80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN-80 (sorbitan monooleate).

Generally speaking, the emulsifier(s) may be t in the vaccine composition in an amount of 0.01% to 10% by volume, preferably, 0.1% to 5%, more preferably 1% to 3%.

Preparation of the vaccine compositions comprising the oily phase is straightforward and entails mixing the aqueous phase comprising the antigen, the P- class immunostimulatory ucleotide, and, optionally, a soluble emulsifier, with an oily phase containing the oil and, optionally, a lipid-soluble emulsifier. The resulting mixture is emulsified.

Other ents of the compositions can include pharmaceutically acceptable excipients, such as carriers, ts, and diluents, isotonic agents, buffering agents, stabilizers, preservatives, vaso-constrictive agents, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents e water, saline, dextrose, ethanol, glycerol, oil, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, ol, lactose, and the like. Useful stabilizers include gelatin, albumin, and the like.

As used herein, "a pharmaceutically-acceptable carrier" includes any and all solvents, dispersion media, coatings, nts, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, ic agents, adsorption delaying agents, and the like. The carrier(s) must be "acceptable" in the sense of being compatible with the other components of the compositions and not deleterious to the subject. Typically, the carriers will be sterile and pyrogen-free, and selected based on the mode of administration to be used. It is well known by those skilled in the art that the preferred formulations for the ceutically acceptable carrier which comprise the compositions are those pharmaceutical carriers approved in the applicable regulations promulgated by the United States (US) Department of Agriculture or US Food and Drug Administration, or equivalent government agency in a non-US country. ore, the pharmaceutically accepted carrier for commercial production of the compositions is a carrier that is already approved or will be approved by the appropriate government agency in the US or foreign country.

The compositions optionally can include compatible pharmaceutically acceptable (i.e., sterile or non-toxic) , semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, ol, and e, among others. Stabilizers include albumin, among others.

The itions can also contain otics or preservatives, including, for example, gentamicin, merthiolate, or chlorocresol. The various s of antibiotics or preservatives from which to select are well known to the d artisan. stration and Use of the Compositions Dose sizes of the compositions typically range from about 0.5 mL to about 5 mL, inclusive, depending on the subject and the antigen. For example, for a canine or feline, a dose of about 0.5 to about 1 mL is typically used, while in larger animals a dose of about 1-5 mL is typically used. However, these adjuvants also can be ated in microdoses, wherein doses of about 100 to about 500 uL can be used.

The routes of administration for the adjuvant compositions include, without limitations, subcutaneous, intramuscular, parenteral, oral, oronasal, intranasal, intratracheal, topical, etc. Any suitable device may be used to administer the compositions, ing syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the n, and the subject, and such are well known to the skilled artisan.

The invention will be further described in the following non-limiting examples.

EXAMPLES Example 1: Leishmania antigen Fifty tory-reared male and female beagle dogs were obtained from ll BioResources (NY), and were ized 5 dogs per each of 10 groups (Table 1). The dogs were between 3 and 4 months of age at the time of the first injection. Three injections were administered at 21—day intervals; injection sites were observed, and blood samples were collected at prescribed intervals for assay. P-CpG of SEQ ID NO: 8 was used for this study.

Table 1: Experimental Design for Leishmania Antigen Study in Dogs <N=S>___-— --—— P-CpG(100ug) QC (scug/SOug) 3doses Days: P-CpG(200ug) g/50 ug) o,21&42 CpG - P class oligonucleotide Q - Quil A C - terol MDP - Muramyl Dipeptide FML - Fucose Mannose Ligand E-SA - Emulsigen®-SA PMBC were isolated on days -1, 7, 28, and 49. Isolated PBMC were cultured in ) and stimulated with FML (20 ug/ml), LICE (Leishmania lnfantum Crude Extract, 17.5 ug/ml) and ConA (Concanavalin A, 2.5 ug/ml). lFN-g was measured by ELlSpot using commercially available kits. The results of the experiments are summarized in the tables below.

Table 2: PBMC ELISPot Stimulation Index Results for Leishmania Antigen Study FML Ag - Stimulation Index on Day: LICE Ag stimulation Index on Day: Table 3: PBMC ELISPot Spot Forming Cells (SFC)/ 106 PBMCs Results for Leishmania Antigen Study LICE Ag sums/10‘ PBMCs on Day: HN U1 NN \lN RD H U1O U1 00 N00 U1 \I H\l U1 (TI N HO Taken together, these data demonstrate that CpG (100 pg) with QC appears to be the best adjuvant for FML, with t to lFN-g Stimulation Index. CpG (100 pg) with SP oil induced the highest lFN-g SFCs, but also had the t background stimulation. Surprisingly, the positive control, Leishmune vaccine was slightly better than the Neg. Control (PBS or FML alone), even though it was adjuvanted with 500 ug Quil A per dose. In other words, the addition of CpG allowed to reduce Quil A 10 times and still get superior results. The effects of previously known adjuvants MPD and E-SA were comparable or ly better than Leishmune®, but inferior to CpG with QC.

Example 2: EHV antigen Animals Cross-bred intact male and female equine, age 11-12 months, were divided into ent groups containing 5 horses per group. Horses were maintained in outdoor facilities during the ation phase. Since nearly all horses are exposed to EHV early in life, horses are not truly nai've. SN titers are used to select animals that have low titers however previous exposure may impact interpretation of test results.

Conventional killed EHV-1 was used as antigen in this study.

The mental design is provided in Table 4.

Table 4: Experimental Design for EHV Antigen Study in Horses Days VOL/Dose 2 doses . P-CpG (SEQ ID NO: QuilA (50ug) Days: 0 & 21 EHV Antigen 8; 100 pg /dose) Cholesterol (50ug) EHV Antigen.

P-CpG (SEQ ID NO: 56 SP Oil0 . 8; 100 pg /dose) The results are summarized in the tables below.

Table 5: PBMC ELISPot Stimulation Index Results for EHV Antigen Study in Horses EHV1 Ag — ation Index PBMCs on Group Day: II-n Table 6: PBMC ELISPot SFCs/106 PBMCs Results for EHV Antigen Study in Horses EHV1 Ag - SFCs/1o" PBMCs on Day: 292 299 Horses Table 8: Summary Results Table for vity Post-Second ation for EHV Antigen Study in Horses (Day 29) Assay T01 ..................... ................. .............

IFNgELISpOt x~~~~i~£:~2“.E.-;:;E.Rw.......................gs...............:-1...........

NlL4ELlSpotSFC“““““““ ........................ ................. .............

ILIOELISA .....................é RZBRTPCR .xt.................~.~.~~~~~s~.~';;:l:i.;e..;.s...........................gs.................:............. - no response + Below e Response ++ Average Response +++ Above Average Response ++++ Significantly Above Average Response These results trate that P-class oligonucleotides provide strong adjuvanting effects in combination with Quil A/Cholesterol and with SP Oil.

All publications cited in the specification, both patent publications and non-patent ations, are indicative of the level of skill of those skilled in the art to which this invention ns. All these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the ples and applications of the present invention. It is ore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims. &/$,06 $ YDFFLQH FRPSRVLWLRQ FRPSULVLQJ DQ DQWLJHQ FRPSRQHQW DQG DQ DGMXYDQW FRPSRQHQW ZKHUHLQ WKH DQWLJHQ FRPSRQHQW FRPSULVHV DQ HTXLQH UKLQRSQHXPRQLWLV YLUXV (+9 DQWLJHQ DQG WKH DGMXYDQW FRPSRQHQW FRPSULVHV D 3 FODVV VWLPXODWRU\ ROLJRQXFOHRWLGH DQG 63 RLO ZKHUHLQ VDLG 63 RLO FRPSULVHV Y Y RI WKH YDFFLQH FRPSRVLWLRQ 7KH H FRPSRVLWLRQ RI FODLP ZKHUHLQ WKH DGMXYDQW FRQVLVWV HVVHQWLDOO\ RI WKH 3 FODVV LPPXQRVWLPXODWRU\ ROLJRQXFOHRWLGH DQG 63 RLO 7KH YDFFLQH FRPSRVLWLRQ RI FODLP RU FODLP ZKLFK LV D QRQ OLSRVRPDO FRPSRVLWLRQ 7KH YDFFLQH FRPSRVLWLRQ RI DQ\ RQH RI FODLPV ZKHUHLQ 63 RLO LV SUHVHQW LQ WKH DPRXQW RI Y Y RI WKH YDFFLQH FRPSRVLWLRQ 7KH YDFFLQH FRPSRVLWLRQ RI DQ\ RQH RI FODLPV ZKHUHLQ 63 RLO LV SUHVHQW LQ WKH DPRXQW RI Y Y RI WKH LWLRQ 7KH YDFFLQH LWLRQ RI DQ\ RQH RI FODLPV ZKHUHLQ VDLG 3 FODVV LPPXQRVWLPXODWRU\ ROLJRQXFOHRWLGH LV W LQ WKH DPRXQW RI —J PO 7KH YDFFLQH FRPSRVLWLRQ RI FODLP ZKHUHLQ VDLG 3 FODVV LPPXQRVWLPXODWRU\ ROLJRQXFOHRWLGH LV SUHVHQW LQ WKH DPRXQW RI —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