HK1201445B - Novel adjuvant compositions - Google Patents

Abstract

This invention relates to novel adjuvant compositions. This invention relates to adjuvant formulations comprising various combinations of thterpenoids, sterols, immunomodulators, polymers, and Th2 stimulators; methods for making the adjuvant compositions; and the use of the adjuvant formulations in immunogenic and vaccine compositions with different antigens. This invention further relates to the use of the formulations in the treatment of animals.

Description

Novel adjuvant compositions

The application is a divisional application of an invention patent application 'novel adjuvant composition' with application number 200980124560.5(PCT/IB2009/052724) at the date of 2009, 6 and 24.

Background

Technical Field

The present invention relates generally to novel adjuvant formulations for enhancing immune responses to antigens, which are useful in immunogenic and vaccine compositions, but do not produce toxic or unwanted side effects in a subject. The invention also relates to methods for the preparation and use of said adjuvants, immunogenic compositions and vaccine compositions.

History and description of related Art

Bacterial, viral and parasitic infections are widely distributed in humans and animals. The diseases caused by these infectious agents are often resistant to antimicrobial drug therapy, making no effective treatment. Therefore, more and more people use vaccinology methods to control infectious diseases. The whole infectious pathogen can be made suitable for use in a vaccine formulation after chemical inactivation or suitable genetic engineering treatment. Alternatively, the protein subunits of the pathogen may be expressed in a recombinant expression system and purified for use in vaccine formulations. Vaccines can be made more effective by including suitable adjuvants in the compositions.

Interest in the use of vaccinology methods to treat cancer in animals and humans continues to increase. The therapeutic approach for treating cancer involves vaccinating a cancer patient with a vaccine comprising a tumor-specific antigen and an adjuvant. However, none of the many vaccines under development that have this property have been approved by the governing body. Vaccines have not been shown to shrink tumors-this is a standard measure of cancer drug efficacy.

The term "adjuvant" refers generally to any substance that increases a humoral or cellular immune response to an antigen. Adjuvants are used to achieve two purposes: it slows the release of antigen from the injection site and it stimulates the immune system. Conventional vaccines typically consist of a crude preparation of inactivated or killed or modified live pathogenic microorganisms. Impurities associated with cultures of these pathogenic microorganisms can act as adjuvants to enhance the immune response. However, the immunity elicited by vaccines made with homogeneous preparations of pathogenic microorganisms or purified protein subunits as antigens is often inadequate. Therefore, it becomes necessary to add certain foreign substances (such as adjuvants). Furthermore, synthetic and subunit vaccines are expensive to manufacture. The addition of an adjuvant may allow for the stimulation of a similar immune response using a smaller dose of antigen, thereby reducing the manufacturing cost of the vaccine. Thus, the efficacy of certain injectable agents can be significantly increased when the agent is combined with an adjuvant.

Many factors must be considered in selecting an adjuvant. Adjuvants should slow the release and absorption rates of the antigen in an effective manner, and minimize toxicity, allergenicity, irritation, and other undesirable effects on the host. To be satisfactory, the adjuvant should be non-virucidal, biodegradable, capable of sustaining a high level of immunity, capable of stimulating cross-protection, compatible with a variety of antigens, effective in a variety of species, non-toxic and safe for the host (e.g., no injection site reaction). Other desirable adjuvants are characterized by being microdosable, dose sparing, having good shelf stability, being able to withstand drying, being able to be made oil-free, being able to exist as a solid or liquid, being isotonic, being easy to manufacture and being inexpensive to manufacture. Finally, it is highly desirable to be able to configure adjuvants to induce either humoral or cellular immune responses or both, as required by the vaccination regimen. However, the number of adjuvants that can meet the above conditions is limited.

The choice of adjuvant depends on the requirements of the vaccine, whether it be to increase the magnitude or function of the antibody response, to increase the cell-mediated immune response, to induce mucosal immunity, or to reduce the antigen dose. A large number of adjuvants have been proposed, however, none of them have been shown to be ideally suited for all vaccines. The first Adjuvant reported in the literature is Freund's Complete Adjuvant (FCA) which contains a water-in-oil emulsion and an extract of Mycobacterium. Unfortunately, FCA is poorly tolerated and may cause uncontrolled inflammation. Since FCA was discovered more than 80 years ago, efforts have been made to reduce unwanted adjuvant side effects.

Some other substances that have been used as adjuvants include metal oxides (e.g., aluminum hydroxide), alum, inorganic chelates of salts, gelatin, various paraffin-type oils, synthetic resins, alginates, mucoid and polysaccharide compounds, caseinates, and blood-derived substances such as fibrin clots. While these agents are generally effective in stimulating the immune system, none are entirely satisfactory due to adverse effects in the host (e.g., the production of sterile abscesses, lesions, oncogenic or allergenic responses) or unsatisfactory pharmaceutical properties (e.g., rapid dispersal or poorly controlled dispersal of the agent from the site of injection, or swelling).

Synthetic oils and petroleum derivatives have been used as adjuvants because they have been shown to disperse relatively slowly in the body, but they may be unsatisfactory because they often break down into aromatic hydrocarbons, which may be carcinogenic. Furthermore, some of these substances have been found to produce sterile abscesses and may never be completely expelled from the body. When properly selected and formulated to the correct concentration, the oil is relatively safe and non-toxic.

Saponins extracted from the bark of Quillaja saponaria (Quillaja saponaria) have been used as adjuvants for some time. See, Lacaille-Dubois, M and Wagner H. (A review of the biological and pharmacological activities of saponin, A. many veterinary vaccines used today comprise Quil A, which is a saponin fraction from the bark of the Chinesia saponaria molina tree in south America.

The use of saponins as adjuvants has various disadvantages. Saponins are soluble and therefore stimulate non-specific immune responses. However, the goal of vaccinology is to stimulate a targeted response against one or more specific antigens. Saponins have a strong affinity for cholesterol. It forms a complex with cholesterol found in the cell membrane and hemolyzes the cell. It has also been shown to cause necrosis at the injection site and is difficult to formulate into a particulate structure. When used in vaccines containing modified live enveloped viruses, saponins can disrupt the viral envelope and thereby inactivate viral antigens.

In order to overcome the hemolytic and virucidal properties of Quil a, it has now been combined with cholesterol and phospholipids to form a special construct known as an immunostimulatory complex (ISCOM) or ISCOM matrix (ISCOMATRIX). See, Ozel m., et al; j.ultrastruc.and Molec.struc.Re102,240-248 (1989). ISCOMs typically induce a Th1 cytotoxic T cell response when combined with an antigen. However, while greatly diminishing the hemolytic properties of Quil a, Quil a in combination with cholesterol does not completely eliminate such properties. Another limitation of ISCOMs is that protein antigens must have a sufficiently large hydrophobic domain to interact with an ISCOM in order to be incorporated into the ISCOM. Highly hydrophilic proteins cannot be incorporated into ISCOMs. Finally, ISCOMs can stimulate an unwanted autoimmune response in a subject.

Immunomodulators have been used as adjuvants, and examples thereof include dimethyldioctadecylammonium bromide (hereinafter referred to as "DDA") and ravirine (avirdine). DDA is a lipophilic quaternary ammonium compound (amine) with two 18 carbon alkyl chains and two methyl groups bound to a positively charged quaternary ammonium molecule, molecular weight 631. Its use as an adjuvant was found by Gall (Immunol. V.11, p.369, 1966). DDA has been reported to stimulate a strong cell-mediated immune response, and has also been shown to induce a humoral immune response. A number of papers have been published demonstrating the efficacy of DDA as an adjuvant to protein antigens, haptens (hapten), tumours, viruses, protozoa and bacteria. (see, Korsholm, K S., et al., Immunology, vol.121, pp.216-226,2007). Most studies have been performed in laboratory animals, however only a few are performed in larger animals such as chickens (see, Katz, D., equivalent. FEMS Immunol Med. Microbiol. Vol7 (4): 303-313,1993), pigs and cattle. DDA can effectively induce delayed-type hypersensitivity (DTH) responses in laboratory animals as well as larger animals. However, it is not readily soluble in water.

Polymers have also been used as adjuvants, examples of which include diethyl-aminoethyl (DEAE) -dextran, polyethylene glycol, and polyacrylic acid (e.g.:the polysaccharide DEAE-dextran is known in the art as a very strong adjuvant. However, it is associated with unacceptable reactogenicity.The polymer is a polymer of acrylic acid crosslinked with polyalkenyl ether or diethylene glycol.Have been used in a variety of vaccines, but their use as adjuvants has not been demonstrated.

Some adjuvants have been shown to stimulate a Th2 response, examples of which include hydrated N- (2-deoxy-2-L-leucylamino-b-D-glucopyranosyl) -N-octadecyl dodecanoamide acetate (also known under the trade name Bay when it is in acetate form)And aluminum. Bay is a type ofWith purified virusThe vaccine or subunit vaccine combination can increase antibody production in virus-challenged mice. Preclinical trials in other animal species (pig, sheep, horse) gave comparable results in terms of antibody production. From BayThe increase in induced antibody synthesis is specifically dependent on the antigen and not the result of polyclonal stimulation.

No adjuvant formulation prior to the present invention possessed the broad desirable characteristics that an ideal adjuvant would have. Efforts are ongoing to find new adjuvants for use in vaccines that overcome the drawbacks of conventional adjuvants. In particular, adjuvant formulations that elicit potent cell-mediated and humoral immune responses against a wide range of antigens in humans and animals without the side effects and formulation difficulties of conventional adjuvants are highly desirable.

Disclosure of Invention

The present invention relates to novel adjuvants, immunogenic compositions and vaccine compositions. In particular, the invention relates to adjuvant formulations comprising a Th1 stimulant, an immunomodulator, a polymer and a Th2 stimulant. The invention also relates to immunogenic and vaccine compositions comprising such adjuvant formulations and one or more antigens, and methods of making the adjuvants and vaccine compositions.

In one embodiment, the adjuvant composition comprises a combination of a saponin, a sterol, and a quaternary ammonium compound. In one embodiment, the adjuvant composition comprises Quil a, cholesterol, and DDA.

In another embodiment, the adjuvant composition comprises a combination of a saponin, a sterol, a quaternary ammonium compound, and a polymer. In one embodiment, the adjuvant combination is Quil a, cholesterol, DDA, and polyacrylic acid.

In another embodiment, the adjuvant composition comprises a saponin, a sterol, a quaternary ammonium compound, a polyA combination of a compound and a glycolipid. In one embodiment, the adjuvant combination is Quil a, cholesterol, DDA, polyacrylic acid, and Bay

In one embodiment, an immunogenic composition comprising an adjuvant formulation and an immunologically effective amount of an antigen, wherein the adjuvant formulation comprises a saponin, a sterol, a quaternary ammonium compound, and a polymer, is prepared by a method comprising the steps of:

a) preparing a composition of antigens in a buffer;

b) adding saponin to the composition of step a;

c) adding a sterol to the composition of step b;

d) adding a quaternary ammonium compound to the composition of step c;

e) adding a polymer to the composition of step d.

In one embodiment of the method, the saponin is Quil a, the sterol is cholesterol, the quaternary ammonium compound is DDA and the polymer is polyacrylic acid.

In one embodiment, a vaccine comprising an adjuvant formulation and an immunologically effective amount of an antigen, wherein the adjuvant formulation comprises a saponin, a sterol, a quaternary ammonium compound, a polymer, and a glycolipid, is prepared by a method comprising the steps of:

a) preparing a composition of the antigenic component in a buffer;

b) adding saponin to the composition of step a;

c) adding a sterol to the composition of step b;

d) adding a quaternary ammonium compound to the composition of step c;

e) adding a polymer to the composition of step d; and

f) adding glycolipids to the composition of step e.

In one embodiment of the method, the saponin is Quil a, the sterol is cholesterol, the quaternary ammonium compound is DDA, the polymer is polyacrylic acid and the glycolipid is Bay

It has now been found that the adjuvant compositions reported herein have surprising and unexpected properties that exceed those expected for such combinations. Surprisingly, it has now been found that the virucidal properties of Quil a/cholesterol in these adjuvant compositions have been eliminated. It is suitable for use as a diluent for lyophilized modified live viral antigens. The adjuvant compositions described herein can be configured to elicit a very effective immune response against a cell-mediated immune response, a humoral immune response, or both. Furthermore, injection site reactions can be largely avoided by using these adjuvant formulations. The reactogenicity is lower than that of several individual components comprising the combination adjuvant. In addition, these adjuvant formulations provide the ability to be stored for long periods of time.

The applicants have found that these novel adjuvant compositions are highly immunogenic when combined with one or more of a large number of different antigens across a wide range of species. It can be used with one or more viral, bacterial, parasitic, recombinant protein and synthetic peptide antigens and combinations thereof. The novel vaccine adjuvant compositions are useful in therapeutic vaccines to treat cancer.

Accordingly, the present invention provides adjuvants, immunogenic compositions and vaccine compositions. Furthermore, the present invention provides a method for manufacturing the composition. The invention also provides the use of the composition in the treatment of disease. The invention also provides its use for the preparation of a medicament for treating a subject for a disease, in particular for a disease as described below. The invention also provides the use thereof for the manufacture of a medicament for preventing or alleviating a disease in a subject.

The invention also provides their use in the preparation of a medicament for: the treatment of cats against infections caused by feline leukemia virus, the treatment of avians against avian coccidiosis (avian coccidiosis), the treatment of cattle against disease caused by Escherichia coli, the treatment of cattle against disease caused by bovine viral diarrhea virus, the treatment of pigs against disease caused by Mycoplasma pneumoniae (Mycoplasma hyopneumaonia), the treatment of cats against disease caused by feline influenza virus, the treatment of subjects to cancer, the treatment of dogs against disease caused by canine coronavirus (canine coronavirus), the treatment of cattle against disease caused by bovine rotavirus (bovine rotavirus), and the treatment of dogs against disease caused by canine influenza virus. The invention also provides the use of an adjuvant as a marker vaccine to help confirm that an animal has received vaccination. The invention also provides the use of CpG to enhance the effect of adjuvants.

Detailed description of the drawings

FIG. 1 shows gel electrophoresis by radioimmunoprecipitation experiments, which shows the antibody pattern between the NS2/3 protein and the E2 protein of BVD virus. The group treated with PreZent A showed antibody responses to both NS2/3 protein and E2 protein, while the group treated with QCDC and QCDCR showed antibody responses to only E2 protein and no response to NS2/3 protein.

Detailed Description

Definition of

When used in connection with a measurable numerical variable, "about" or "approximately" refers to the indicated variable value and all variable values within experimental error of the indicated value (e.g., within 95% confidence interval of the mean) or within 10% of the indicated value (whichever is greater), unless "about" is used to indicate an interval in weeks wherein "about 3 weeks" is 17 to 25 days and about 2 to about 4 weeks is 10 to 40 days.

An "adjuvant" refers to any substance that increases the humoral or cellular immune response to an antigen. Adjuvants are commonly used to achieve two purposes: slowing the release of antigen from the injection site and stimulating the immune system.

"alkyl" refers to both straight and branched saturated hydrocarbon moieties.

"amine" refers to a chemical compound containing nitrogen. Amines are a class of compounds derived from ammonia by replacement of a hydrogen atom with a hydrocarbon group. "quaternary amine" refers to an ammonium-based compound having 4 hydrocarbon groups.

An "antibody" refers to an immunoglobulin molecule that binds to a specific antigen as a result of an immune response to the antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains with "constant" and "variable" regions, and are classified into several classes (e.g., IgA, IgD, IgE, IgG, and IgM) according to the composition of the constant regions.

"antigen" or "immunogen" refers to any substance that stimulates an immune response. The term includes killed, inactivated, attenuated or modified live bacteria, viruses or parasites. The term antigen also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, or lipids, or fragments thereof, either individually or in any combination thereof. The term antigen also includes antibodies, such as anti-subject genotypic antibodies or fragments thereof, as well as synthetic peptidomimetics (mimotopes) that mimic an antigen or antigenic determinant (epitope).

"bacterin" refers to a suspension of one or more killed bacteria that can be used as a component of a vaccine or immunogenic composition.

"buffer" refers to a chemical system that prevents a change in the concentration of another chemical, such as: the proton donor and acceptor system can act as a buffer to prevent significant changes in hydrogen ion concentration (pH). Another example of a buffer is a solution containing a weak acid and its salt (conjugate base) or a mixture of a weak base and its salt (conjugate acid).

A "cellular immune response" or "cell-mediated immune response" is an immune response mediated by T-lymphocytes or other leukocytes or both, which includes the production of cytokines, chemokines, and similar molecules made by activated T cells, leukocytes or both.

"Cholesterol" means a compound of formula C₂₇H₄₅White crystalline form of OH. It is a cyclic hydrocarbon alcohol classified as a lipid. It is insoluble in water, but soluble in a variety of organic solvents.

"delayed-type hypersensitivity (DTH)" refers to the inflammatory response that develops 24 to 72 hours after exposure to antigens recognized as foreign by the immune system. This type of immune response involves mainly T cells rather than antibodies (made by B cells).

"dose" refers to a vaccine or immunogenic composition administered to a subject. By "first dose" or "priming vaccine" is meant the dose of such composition administered on day 0. The "second dose" or "third dose" or "annual dose" refers to the amount of such composition administered after the first dose, which may or may not be the same vaccine or immunogenic composition as the first dose.

"emulsifier" refers to a substance used to make an emulsion more stable.

"emulsion" refers to a composition of two immiscible liquids in which droplets of one liquid are suspended in a continuous phase of the other liquid.

"esters" refers to any of the classes of organic compounds corresponding to inorganic salts, which are formed from the condensation reaction of organic acid units with alcohol molecules, wherein one water molecule is eliminated.

By "excipient" is meant any non-antigenic vaccine component.

"homogenization" refers to the process of mixing one or more similar or dissimilar components into a homogeneous mixture.

By "humoral immune response" is meant an immune response mediated by antibodies.

"hydrophobic" means insoluble in water, less prone to absorb moisture, or adversely affected by water; incompatible with water or low affinity for it.

An "immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or both a humoral and a cellular immune response in response to an antigen. Immune responses can generally be determined using standard immunoassays and neutralization assays known in the art.

An "immunoprotective amount" or "immunologically effective amount" or "effective amount to generate an immune response" of an antigen is an amount effective to induce an immunogenic response in a recipient. The immunogenic response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by a vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.

"immunogenic" refers to the elicitation of an immune or antigenic response. Thus, the immunogenic composition can be any composition that is capable of inducing an immune response.

"immunostimulatory complex" or ISCOM refers to a specific structure formed when Quil A is combined with cholesterol and phospholipids.

An "immunostimulatory molecule" refers to a molecule that generates an immune response.

"lipid" refers to any group of organic compounds that are insoluble in water but soluble in non-polar organic solvents, oily to the touch, and constitute the main building blocks of living cells together with carbohydrates and proteins, including fats, oils, waxes, sterols, and triglycerides.

By "lipophilic" is meant exhibiting significant affinity for or solubility in lipids.

"liposome" refers to a tiny spherical particle formed of a lipid bilayer enclosing an aqueous compartment, which is used medically to carry a drug, antigen, vaccine, enzyme, or another substance to a targeted cell in vivo.

"Medicinal agent" refers to any agent used for preventing, curing or ameliorating diseases, or preventing some physiological condition or disease.

By "parenteral administration" is meant the introduction of a substance (such as a vaccine) into a subject by or via a route not including the alimentary canal. Parenteral administration includes subcutaneous, intramuscular, transdermal, intradermal, intraperitoneal, intraocular and intravenous administration.

"pharmaceutically acceptable" refers to a substance that is suitable for contact with the tissues of a subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, and effective for its intended use, under the judicious medical judgment.

"reactogenicity" refers to the side effects elicited by a subject in response to administration of an adjuvant, immunogenic, or vaccine composition. It may occur at the site of administration and is usually assessed in terms of the number of symptoms developed. These symptoms may include inflammation, redness, and abscesses. It can also be evaluated with respect to occurrence, duration and severity. For example: a "low" response will involve swelling, or a short duration, that is detectable only by touch and not by eye. More severe reactions are, for example, reactions which can be detected visually or which last longer.

"ambient temperature" means a temperature of 18 to 25 ℃.

"saponin" refers to a group of plant-derived surface-active glycosides, which consist of a hydrophilic region (usually several sugar chains) bound to a hydrophobic region with steroid or triterpene structure.

"steroid" refers to any of a group of organic compounds belonging to the biochemical class of lipids that are readily soluble in organic solvents and sparingly soluble in water. Steroids contain a four-fused ring system with 3 fused cyclohexane (six carbon) rings plus a 4 th cyclopentane (five carbon) ring.

"sterol" refers to a compound biologically produced from a terpene precursor in an animal. Which contain a steroid ring structure with a hydroxyl (OH) group, typically attached to carbon-3. The hydrocarbon chain of the fatty acid substituents varies in length, typically from 16 to 20 carbon atoms, and may be saturated or unsaturated. Sterols typically contain one or more double bonds in the ring structure as well as a variety of different substituents attached to the ring. Sterols and their fatty acid esters are substantially insoluble in water.

By "subject" is meant any animal in need of administration of the adjuvant composition. It includes mammals and non-mammals, including primates, domestic animals, companion animals, laboratory test animals, wild animals in captivity, birds (including eggs), reptiles, and fish. Thus, the term includes, but is not limited to, monkeys, humans, pigs; cattle, sheep, goats, horses, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, chickens, turkeys, ducks, other birds, frogs and lizards.

“TCID₅₀"means" tissue culture infectious dose "and is defined as the amount of virus dilution required to infect 50% of a given batch of inoculated cell culture. TCID can be calculated using a variety of methods₅₀Including the Spearman-Karber method (Spearman-Karber method) as used throughout the present specification. The spearman-kappa method is described in: B.W.Mahy&H.O.Kangro,Virology Methods Manual,p.25-46(1996)。

By "therapeutically effective amount" is meant an amount of antigen or vaccine that induces an immune response in a subject receiving the antigen or vaccine sufficient to prevent or reduce the signs or symptoms of disease (including adverse health effects or complications thereof) caused by infection with a pathogen, such as a virus or bacterium. Humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced. The immune response of an animal to a vaccine can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain. The protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition, and overall health and efficacy of the subject. The therapeutically effective amount of the vaccine may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, and may be determined by one of skill in the art.

"treating" or "treatment" refers to preventing a disorder, condition, or disease to which the term applies, or preventing or alleviating one or more symptoms of such disorder, condition, or disease.

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

"triterpene" refers to a large and diverse number of naturally occurring organic molecules derived from six penta-carbon isoprene (2-methyl-1, 3-butadiene) units, which can be assembled and modified in thousands of ways. Most of them are of a multi-ring configuration, differing from each other in the functional group and its basic carbon skeleton. These molecules can be found in all kinds of living things.

By "vaccine" is meant a composition comprising an antigen as defined herein. Administration of a vaccine to a subject can generate an immune response that is substantially directed against one or more specific diseases. The amount of vaccine that is therapeutically effective may vary depending on the particular antigen used, or the condition of the subject, and may be determined by one of skill in the art.

Components of the composition

Triterpenes and CpGs

Triterpenes suitable for use in the adjuvant composition can be from a variety of sources (plant-derived or synthetic equivalents), including, but not limited to: quillaja saponaria, lycopersicin, ginseng extract, mushroom and alkaloid glycosides that are structurally similar to steroid saponins. Thus, triterpenes suitable for use in the adjuvant composition include saponins, squalene and lanosterol. The amount of triterpene suitable for use in the adjuvant composition depends on the nature of the triterpene used. However, it is typically used in amounts of about 1 microgram to about 5000 microgram per dose. The amount may also be from about 1 microgram to about 4000 microgram per dose, from about 1 microgram to about 3000 microgram per dose, from about 1 microgram to about 2000 microgram per dose, and from about 1 microgram to about 1000 microgram per dose. The amount may also be from about 5 micrograms to about 750 micrograms per dose, from about 5 micrograms to about 500 micrograms per dose, from about 5 micrograms to about 200 micrograms per dose, from about 5 micrograms to about 100 micrograms per dose, from about 15 micrograms to about 100 micrograms per dose, and from about 30 micrograms to about 75 micrograms per dose.

If saponins are used, the adjuvant composition typically contains an immunologically active saponin fraction from the bark of quillaja saponaria. The saponin may be, for example: quil a or other purified or partially purified saponin preparations (which are commercially available). Thus, saponin extracts may be used as a mixture or as individual components (such as QS-7, QS-17, QS-18 and QS-21) that are purified. In one embodiment, Quil a is at least 85% pure. In another embodiment, Quil a is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.

CpG ODN is a recently reported class of pharmacotherapeutic agents characterized by the presence of unmethylated CG dinucleotides in specific base-sequence contexts (CpG motifs). (Hansel TT, Barnes PJ (eds): New Drugs for Asthma, Allergy and COPD. prog Respir Res.Basel, Karger,2001, vol31, pp229-232, which is incorporated herein by reference). These CpG motifs are not present in eukaryotic DNA (in which CG dinucleotides are suppressed and are typically methylated when present), but are present in bacterial DNA to which immunostimulatory properties can be conferred by these CpG. These immunostimulatory properties include the induction of a Th1 type response and the release of large amounts of IFN-A, IL-12 and IL-18. CpG ODNs (18-24 bp in length) possess immunomodulatory properties similar to bacterial DNA. Cell surface proteins can absorb these molecules with different results. However, this immunomodulatory property and CpG uptake can be significantly enhanced by vectors such as QCDC, QCDCR and other combinations listed in this patent.

The amount of CpG suitable for use in the adjuvant composition depends on the CpG used and the nature of the species expected. However, it is generally used in an amount of about 1 microgram to about 20 milligrams per dose. The amount may also be from about 1 microgram to about 10 milligrams per dose, from about 1 microgram to about 5 milligrams per dose, from about 1 microgram to about 4 milligrams per dose, from about 1 microgram to about 3 milligrams per dose, from about 1 microgram to about 2 milligrams per dose, and from about 1 microgram to about 1 milligram per dose. The amount may also be from about 5 micrograms to about 750 micrograms per dose, from about 5 micrograms to about 500 micrograms per dose, from about 5 micrograms to about 200 micrograms per dose, from about 5 micrograms to about 100 micrograms per dose, from about 10 micrograms to about 100 micrograms per dose, from about 15 micrograms to about 100 micrograms per dose, and from about 30 micrograms to about 75 micrograms per dose.

Sterols

Sterols suitable for use in the adjuvant composition include beta-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. These sterols are well known in the art and are commercially available. For example: cholesterol is disclosed in Merck Index,12th ed., p.369. The amount of sterol suitable for use in the adjuvant composition depends on the nature of the sterol used. However, it is typically used in amounts of about 1 microgram to about 5000 microgram per dose. The amount may also be from about 1 microgram to about 4000 microgram per dose, from about 1 microgram to about 3000 microgram per dose, from about 1 microgram to about 2000 microgram per dose, and from about 1 microgram to about 1000 microgram per dose. The amount may also be from about 5 micrograms to about 750 micrograms per dose, from about 5 micrograms to about 500 micrograms per dose, from about 5 micrograms to about 200 micrograms per dose, from about 5 micrograms to about 100 micrograms per dose, from about 15 micrograms to about 100 micrograms per dose, and from about 30 micrograms to about 75 micrograms per dose.

Immunomodulator

The adjuvant composition may further comprise one or more immunomodulators, such as quaternary ammonium compounds (e.g., DDA) and interleukins, interferons or other cytokines. These materials are commercially available. The amount of immunomodulator suitable for use in the adjuvant composition depends on the nature of the immunomodulator used and the subject. However, it is typically used in amounts of about 1 microgram to about 5000 microgram per dose. The amount may also be from about 1 microgram to about 4000 microgram per dose, from about 1 microgram to about 3000 microgram per dose, from about 1 microgram to about 2000 microgram per dose, and from about 1 microgram to about 1000 microgram per dose. The amount may also be from about 5 micrograms to about 750 micrograms per dose, from about 5 micrograms to about 500 micrograms per dose, from about 5 micrograms to about 200 micrograms per dose, from about 5 micrograms to about 100 micrograms per dose, from about 15 micrograms to about 100 micrograms per dose, and from about 30 micrograms to about 75 micrograms per dose. In a particular example aspect, an adjuvant composition comprising DDA can be prepared by simply mixing an antigen solution with a freshly prepared DDA solution.

Polymer and method of making same

The adjuvant composition may further comprise one or more polymers such as, for example: DEAE dextran, polyethylene glycol and polyacrylic acid, and polymethacrylic acid (e.g.:). Such materials are commercially available. The amount of polymer suitable for use in the adjuvant composition depends on the nature of the polymer used. However, it is generally used in an amount of about 0.0001% volume to volume (v/v) to about 75% v/v. In other embodiments, it is used in an amount of about 0.001% v/v to about 50% v/v, about 0.005% v/v to about 25% v/v, about 0.01% v/v to about 10% v/v, about 0.05% v/v to about 2% v/v, and about 0.1% v/v to about 0.75% v/v. In another embodiment, it is used in an amount of about 0.02% v/v to about 0.4% v/v. The molecular size of the DEAE-dextran may be in the range of 50,000Da to 5,000,000Da, or it may be in the range of 500,000Da to 2,000,000 Da. These materials are commercially available or can be prepared from dextran.

Another particular example is polyacrylic acid (e.g.:polymer) having an average equivalent weight of 76. It is prepared from starting polymer particles having an average diameter of about 0.2 to 6.0 microns.The polymer can swell in water up to 1000 times its original volume and 10 times its original diameter to form a gel when exposed to a pH environment above the pKa of the carboxylate group. At higher pH than the pKa of the carboxylate group, the carboxylate group ionizes, resulting in repulsion between negatively charged groups, resulting in increased swelling of the polymer.

Th2 stimulant

The adjuvant composition may further include one or more Th2 stimulants such as, for example: bay is a type ofAnd aluminum. The amount of Th2 stimulant suitable for use in the adjuvant composition depends on the nature of the Th2 stimulant used. However, it is generally used in an amount of about 0.01 mg to about 10 mg per dose. In other embodiments, it is used in an amount of about 0.05 mg to about 7.5 mg per dose, about 0.1 mg to about 5 mg per dose, about 0.5 mg to about 2.5 mg per dose, and about 1 mg to about 2 mg per dose. One particular example is BayIt is a glycolipid with the chemical name "N- (2-deoxy-2-L-leucylamino- β -D-glucopyranosyl) -N-octadecyldodecanoamide acetate", which can be synthesized according to the procedure found in Lockhoff,0.(Angew. chem. int. Ed. Engl. 30: 1611-1620; 1991). it is proposed to store it in an airtight container at 2-8 ℃ which is slightly hygroscopic in chemical or physical properties, does not form polymorphs, is chemically stable in air and light at temperatures up to 50 ℃ and in aqueous solvents at ambient temperatures at pH2-12Are amphiphilic molecules that form micelles in aqueous solution.

Antigens and diseases

The adjuvant composition may contain one or more antigens. The antigen may be any of a variety of substances that are capable of producing a desired immune response in a subject. While Quil a alone is virucidal, Quil a plus cholesterol detoxifies Quil a when it forms a helical micelle (see, U.S. patent No.7,122,191). It has now been found that the adjuvant compositions described herein are non-virucidal, non-hemolytic or membrane-lytic. Thus, the antigen used with these adjuvant compositions can be one or more viruses (inactivated, attenuated, and modified live viruses), bacteria, parasites, nucleotides, polynucleotides, peptides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, carbohydrates, fatty acids, teichoic acids (teichooc acids), peptidoglycans, lipids, or glycolipids, individually or in any combination thereof.

Antigens for use with the adjuvants of the invention also include immunogenic fragments of nucleotides, polynucleotides, peptides, polypeptides, which may be isolated from the organisms mentioned herein.

Live, modified live and attenuated virus strains that do not cause disease in a subject have been isolated in a 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 chemical mutagens. Inactivated or killed viral strains are those that have been inactivated by methods known to those skilled in the art, including treatment with formalin, Beta Propiolactone (BPL), diethylene imine (BEI), sterilizing radiation, heat, or other such methods.

Two or more antigens may be combined together to produce a multivalent composition that can protect a subject against a variety of diseases caused by pathogens. Currently, commercial manufacturers of vaccines and end users prefer multivalent vaccine products. While conventional adjuvants are generally limited to different antigens (monovalent or multivalent) with which they can be effectively used, the adjuvants described herein can be effectively used in a wide range of antigens (monovalent as well as multivalent). Thus, the antigens described herein can be combined in a single composition comprising an adjuvant described herein.

Some examples of bacteria that can be used as antigens with adjuvant compositions include, but are not limited to: acinetobacter calcoaceticus (Acinetobacter calcoaceticus), Acinetobacter pasteurianus (Acetobacter pasanianus), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Aeromonas hydrophila (Aeromonas hydrophyllum), Alicyclobacillus acidocaldarius (Alicyclobacillus acidocaldarius), Archaeoglobus fulgidus (Arhaegenus fulgidus), Bacillus pumilus (Bacillus pumilus), Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus subtilis (Bacillus subtilis), Bacillus thermocatenulatus (Bacillus thermocatenulatus), Bordetella bronchiseptica (Bordetella), Chlamydia cepacia cepacicola (Bukkeri), Corynebacterium glutamicum (Corynebacterium glutamicum), Escherichia coli (Campylobacter), Campylobacter coli (Campylobacter coli), Campylobacter coli (Campylobacter) and Escherichia coli (Campylobacter coli), Campylobacter coli (Campylobacter) are strain, Campylobacter coli (Campylobacter) are strain, Campylobacter strain, listeria monocytogenes (Listeria monocytogenes), Escherichia canis (Ehrlichia canis), Escherichia coli (Escherichia coli), Haemophilus influenzae (Haemophilus influenzae), Haemophilus somnus, Helicobacter suis (Helicobacter suis), Lactobacillus intracellularis (Lawsonia intracellularis), Legionella pneumophila (Legionella pneumophila), Mycobacterium morganii (Moraxella sp), Mycobacterium bovis (Mycobacterium bovis), Mycoplasma hyopneumoniae (Mycoplasma pneumoniae), Mycoplasma filamentous (Mycoplasma urenum), Mycoplasma urenum (Mycoplasma urella), Mycoplasma urella vulgaris (Mycoplasma urella), Porphyromonas vulgaris (Porphyromonas), Porphyromonas (Porphyromonas) and Porphyromonas (Porphyromonas), Porphyromonas (Porphyromonas), Porphyromonas (Porphyromonas), pseudomonas aeruginosa (Pseudomonas wisconsis), Pseudomonas aeruginosa, Pseudomonas fluorescens C9(Pseudomonas fluorescens C9), Pseudomonas fluorescens SIKW1(Pseudomonas fluorescens SIKW1), Pseudomonas fragi (Pseudomonas fragrans), Pseudomonas shallownsis (Pseudomonas luteola), Pseudomonas oleovorans (Pseudomonas inovorans), Pseudomonas pseudomonads B11-1(Pseudomonas B11-1), Alcaligenes eutrophus (Alcaligeselutherophus), Pseudomonas immortalis (Pseudomonas imminobilis), Salmonella typhi (Salmonella typhimurium), Salmonella rickettsiamensis (Salmonella typhimurium), Salmonella typhimurium (Salmonella typhimurium), Salmonella typhi (Salmonella typhi), Salmonella typhi, Salmon, Staphylococcus suis (Staphylococcus hyicus), Streptomyces albus (Streptomyces albus), Streptomyces cinnamoneumoniae (Streptomyces cinnnamoneus), Streptococcus suis (Streptococcus suis), Streptomyces exfoliates (Streptomyces exfoliates), Streptomyces scabies (Streptomyces scabies), Sulfolobus acidocaldarius (Sulfolobus acidocaldarius), Synechocystis sp, Vibrio cholerae (Vibrio cholerae), Spirospira burgeri (Borrelia burgdorferi), Spirospira denticola (Treharynia denticola), Spirospira minicola (Treponema minuta), Spirospira trichotheca (Treponema pallidum), Spirospira curvata (Treponema pallidum), Leptospira curvata (Leptospira pallidum), Leptospira canicola (Leptospira pallida), Leptospira canicola (Leptospira grippospora), Leptospira canicola (Leptospira pallida), Leptospira canicola (Leptospira), Leptospira canicola (Leptospira), Leptospira (Leptospira), Leptospira canicola (Leptospira), Leptospira canicola (Leptospira), Leptospira (Leptospira, leptospira pratensis (Leptospira borgpetersenii Hardjo-prajitno), Leptospira interrogans (Leptospira interrogans), Leptospira icterohaemorrhagiae (Leptospira icheromorrhagiae), Leptospira pomona (Leptospira pomona), and Leptospira bratislava (Leptospira bratislava), and combinations thereof.

Inactivated viruses as well as live attenuated viruses may be used in adjuvant compositions. Some examples of viruses that can be used as antigens include, but are not limited to: avian herpes virus, bovine herpes virus, canine herpes virus, equine herpes virus, feline viral rhinotracheitis virus, Marek's Disease virus, ovine herpes virus, porcine herpes virus, pseudorabies virus, avian paramyxovirus, bovine respiratory syncytial virus, canine distemper virus, canine parainfluenza virus, canine adenovirus, canine parvovirus, bovine parainfluenza virus 3, ovine parainfluenza virus 3, bovine plague virus, Border Disease virus (Border Disease virus), Bovine Viral Diarrhea Virus (BVDV), BVDV type I, BVDV type II, Classical swine fever virus (classic virus), avian leukemia virus, bovine immunodeficiency virus, bovine leukemia virus, bovine tuberculosis virus, porcine infectious anemia virus, feline immunodeficiency virus, feline leukemia virus (FeLV), Newcastle Disease virus (Newcastle Disease virus), progressive ovine pneumonia virus, Ovine lung adenocarcinoma virus, Canine Coronavirus (CCV), pantropic CCV (pandropic CCV), canine respiratory coronavirus, bovine coronavirus, feline calicivirus, feline enteric coronavirus, feline infectious peritonitis virus, porcine epidemic diarrhea virus, porcine thromboencephalomyelitis virus, porcine parvovirus, porcine circovirus type I (PCV), PCV type II, Porcine Reproductive and Respiratory Syndrome (PRRS) virus, transmissible gastroenteritis virus, turkey coronavirus, bovine epidemic fever virus, rabies rotavirus, vesicular stomatitis virus, lentivirus, bird flu virus, rhinovirus, equine influenza virus, porcine influenza virus, canine influenza virus, feline influenza virus, human influenza virus, eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus, West Nile virus, Western equine encephalitis virus, human immunodeficiency virus, human papilloma virus, varicella zoster virus, herpes virus, bovine respiratory virus, porcine reproductive and respiratory syndrome virus, Hepatitis b virus, rhinovirus, and measles virus, and combinations thereof.

Examples of peptide antigens include Bordetella bronchiseptica (Bordetella bronchiaseptica) p68, GnRH, IgE peptides, Fel d1, and cancer antigens, and combinations thereof. Other examples of antigens include nucleotides, carbohydrates, lipids, glycolipids, peptides, fatty acids and teichoic acids and peptidoglycans, and combinations thereof.

Some examples of parasites that may be used as antigens with adjuvant compositions include, but are not limited to, Anaplasma (anaplasia), Fasciola hepatica (liver fluke), coccidia, Eimeria (Eimeria), Neospora caninum (Neospora caninum), Toxoplasma gondii (Toxoplasma gondii), Giardia lamblia (Giardia), Dirofilaria (Dirofilaria) (heartworm), Ancylostoma (crocortus), Trypanosoma (hookworm), Trypanosoma (Trypanosoma spp.), Leishmania spp. (Leishmania spp.), Trichomonas (Trichomonas spp.), Cryptosporidium parvum (Cryptosporidium parvum), Babesia (basalis), Schistosoma (Schistosoma), Taenia (xenocia), Strongyloides (strongylis), Trichomonas (Trichomonas), Trichomonas (sargastidea), Trichomonas (Trichomonas), Trichomonas (Trichomonas), and combinations thereof. Also contemplated are ectoparasites, including but not limited to: ticks (ticks) including hard ticks (Ixodes), Rhipicephalus (Rhipicephalus), dermanyssus (Dermacentor), Amblyomma (Amblyomma), Boophilus (Boophilus), ryocymus (Hyalomma), and haemanthus (Haemaphysalis), and combinations thereof.

The amount of antigen used to induce an immune response will vary considerably depending on the nature of the antigen used, the subject and the level of response desired, and can be determined by one skilled in the art. In the context of vaccines containing modified live or attenuated viruses, a therapeutically effective amount of antigen is generally from about 10²Tissue Culture Infectious Dose (TCID)₅₀(including) to about 10¹⁰TCID₅₀(iii) in the range of (inclusive). For many of these diseasesFor toxicity, a therapeutically effective dose is generally from about 10²TCID₅₀(including) to about 10⁸TCID₅₀(iii) in the range of (inclusive). In some embodiments, the therapeutically effective dose is generally at about 10³TCID₅₀(including) to about 10⁶TCID₅₀(iii) in the range of (inclusive). In some other embodiments, the therapeutically effective dosage system is about 10⁴TCID₅₀(including) to about 10⁵TCID₅₀(iii) in the range of (inclusive).

For vaccines containing inactivated virus, the therapeutically effective amount of antigen is typically at least about 100 relative units per dose, typically in the range of about 1,000 to about 4,500 relative units per dose. In other embodiments, a therapeutically effective amount of antigen is from about 250 (inclusive) to about 4,000 relative units (inclusive) per dose, from about 500 (inclusive) to about 3,000 relative units (inclusive) per dose, from about 750 (inclusive) to about 2,000 relative units (inclusive) per dose, or from about 1,000 (inclusive) to about 1,500 relative units (inclusive) per dose.

In vaccines containing inactivated virus, the therapeutically effective amount of antigen may also be measured in terms of Relative Potency (RP) per ml. The therapeutically effective amount will generally be in the range of about 0.1 (inclusive) to about 50PR (inclusive) per ml. In other embodiments, a therapeutically effective amount of antigen ranges from about 0.5 (inclusive) to about 30PR (inclusive) per ml, about 1 (inclusive) to about 25PR (inclusive) per ml, about 2 (inclusive) to about 20PR (inclusive) per ml, about 3 (inclusive) to about 15PR (inclusive) per ml, or about 5 (inclusive) to about 10PR (inclusive) per ml.

In one embodiment, the FeLV antigen was produced from FL74-UCD-1 cell line (ATCC accession number CRL-8012) persistently infected with the KT-FeLV-UCD-1 feline leukemia virus strain. The amount of FeLV antigen in the vaccine was measured as the amount of gp70 virus protein per ml. The therapeutically effective amount of FeLV antigen is typically in the range of about 100 (inclusive) to about 350,000 ng/ml (inclusive) as measured by the amount of gp70 viral protein per ml. In another embodiment, the range is from about 1,000 (inclusive) to about 300,000 ng/ml (inclusive), or from about 2,500 (inclusive) to about 250,000 ng/ml (inclusive), or from about 4,000 (inclusive) to about 220,000 ng/ml (inclusive), or from about 5,000 (inclusive) to about 150,000 ng/ml (inclusive), or from about 10,000 (inclusive) to about 100,000 ng/ml (inclusive).

The number of cells of bacterial antigen administered in the vaccine is about 1 × 10 per dose⁶To about 5 × 10¹⁰(including) a colony forming unit (CFU.) in other embodiments, the number of cells ranges from about 1 × 10⁷To about 5 × 10¹⁰CFU/dose, or about 1 × 10⁸To about 5 × 10¹⁰In yet other embodiments, the cell number ranges from about 1 × 10²To about 5 × 10¹⁰CFU/dose, or about 1 × 10⁴To about 5 × 10⁹CFU/dose, or about 1 × 10⁵To about 5 × 10⁹CFU/dose, or about 1 × 10⁶To about 5 × 10⁹CFU/dose, or about 1 × 10⁶To about 5 × 10⁸CFU/dose, or about 1 × 10⁷To about 5 × 10⁹CFU (inclusive) per dose.

The number of cells of parasite antigen administered in the vaccine is about 1 × 10 per dose²(inclusive) to about 1 × 10¹⁰In other embodiments, the cell number ranges from about 1 × 10³(inclusive) to about 1 × 10⁹(containing), or about 1 × 10⁴(inclusive) to about 1 × 10⁸(containing), or about 1 × 10⁵(inclusive) to about 1 × 10⁷(containing), or about 1 × 10⁶(inclusive) to about 1 × 10⁸(including).

It is well known in the art that significantly greater amounts of inactivated virus are required to stimulate comparable serological response levels when using conventional adjuvants compared to modified live or attenuated viruses. However, it has been surprisingly found that about the same amount of inactivated virus and modified live virus stimulates similar levels of serological response by the adjuvant compositions described herein. Furthermore, when compared to conventional adjuvants, smaller amounts of modified live, attenuated, and inactivated viruses are required to achieve the same level of serological response when using the adjuvants described herein. These unexpected findings demonstrate that resources are protected and costs are reduced during the preparation of immunogenic and vaccine compositions. In the context of vaccines with widespread use, millions of doses need to be made each year, and therefore these savings are important.

Excipient

Aqueous adjuvants offer certain advantages. They are generally easy to formulate and administer, and can induce few or less severe injection site reactions. However, aqueous adjuvants with antigens tend to diffuse from the injection site, are cleared by the liver of the subject and generate unwanted non-specific immune responses. It has been surprisingly found that the aqueous adjuvant compositions described herein can remain at the injection site until metabolized by the organism, which occurs over a long period of time, and provide a targeted immune response.

Oils typically provide a long and slow release profile when added as a component of an adjuvant. In the present invention, the oil may or may not be metabolized. The oil may be in the form of an oil-in-water, water-in-oil or water-in-oil-in-water emulsion.

Oils suitable for use in the present invention include alkanes, alkenes, alkynes, their corresponding acids and alcohols, and their ethers and esters, and mixtures thereof. Individual compounds of the oil are light hydrocarbon compounds, i.e. such components have 6 to 30 carbon atoms. The oil can be synthetically prepared or purified from petroleum products. This moiety may have a linear or branched configuration. It may be fully saturated or have one or more double or triple bonds. Some non-metabolizable oils for use in the present invention include, for example: mineral oil, paraffin oil and cycloparaffins.

The term oil is also intended to include "light mineral oils", i.e.: oil obtained by distillation of petroleum in a similar manner, but with a slightly lower specific gravity than white mineral oil.

Metabolizable oils include metabolizable, non-toxic oils. The oil can be any vegetable, fish, animal or synthetically prepared oil that can be metabolized by the body of the subject to which the adjuvant is administered and is not toxic to the subject. Sources of vegetable oils include nuts, seeds and grains.

The invention provides oil-in-water emulsions comprisingAnd (4) preparing a preparation. The formulation comprises an aqueous component, lecithin, mineral oil, and a surfactant. Patents describing the components of the formulation include U.S. patent No.5,084,269 and US6,572,861.

Typically, the oil component of the present invention is present in an amount of from 1% to 50% (by volume); or from 10% to 45%; or from 20% to 40%.

Other components of the composition may include pharmaceutically acceptable excipients such as carriers, solvents and diluents, isotonic agents, buffers, stabilizers, preservatives, vasoconstrictors, antibacterial agents, antifungal agents, and the like. Typical carriers, solvents, and diluents include water, saline, dextrose, ethanol, glycerol, oil, and the like. Representative isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, lactose and the like. Useful stabilizers include gelatin, albumin, and the like.

Surfactants are used to help stabilize emulsions selected as carriers for adjuvants and antigens. Surfactants suitable for use in the present invention include natural biocompatible surfactants and non-natural synthetic surfactants. The biocompatible surfactant comprises a phospholipid compound or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholines (lecithins), such as soya or egg lecithins. Lecithin, which is a mixture of phospholipids and triglycerides, can be obtained by washing the crude vegetable oil with water, separating and drying the resulting hydrated gum. A refined product can be obtained by fractionating a mixture of the remaining acetone-insoluble phospholipid and glycerolipid after acetone washing to remove triglyceride and vegetable oil. Alternatively, lecithin may be obtained from different commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylethanolamine. The phospholipids may be isolated from natural sources or synthesized in a conventional manner.

Is suitable forNon-natural synthetic surfactants useful in the present invention include sorbitan-based nonionic surfactants such as: fatty acid substituted sorbitan surfactants (which may be referred to by the nameOrCommercially available), fatty acid esters of polyethoxylated sorbitolPolyethylene glycol esters of fatty acids from sources such as castor oilPolyethoxylated fatty acids (e.g., those known under the name SIMULSOL)Stearic acid obtained), polyethoxylated isooctylphenol/formaldehyde polymerPolyoxyethylene fatty alcohol ethersPolyoxyethylene nonyl phenyl etherPolyoxyethylene isooctyl phenyl ether

Generally, if two or more surfactants are used, the surfactant or combination of surfactants is present in the emulsion in an amount of from 0.01% to 10%, preferably from 0.1% to 6.0%, more preferably from 0.2% to 5.0% by volume.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. The carrier must be "acceptable" in the sense of being compatible with the other components of the composition and not injurious to the subject. Typically, the carrier will be sterile and pathogen-free and will be selected according to the mode of administration to be used. Preferred formulations of pharmaceutically acceptable carriers comprising the compositions are known to those skilled in the art to be pharmaceutical carriers approved by applicable rules promulgated by the United States (US) department of agriculture or the united states food and drug administration, or equivalent governmental agencies in non-US countries. Thus, a pharmaceutically acceptable carrier for use in the commercial manufacture of the compositions is one that has been approved or will be approved by the appropriate governmental agency in the united states or foreign countries.

The compositions optionally may comprise compatible pharmaceutically acceptable (i.e., sterile or non-toxic) liquid, semi-solid, or solid diluents that function as pharmaceutical carriers, excipients, or media. Diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and others.

The compositions may also contain antibiotics or preservatives, including, for example: gentamicin, thimerosal, or chlorocresol. The skilled person is well aware of the different classes of antibiotics or antiseptics that may be used for selection.

Process for preparing a composition

Process for the preparation of an adjuvant formulation

ISCOMs can be prepared by combining a saponin, a sterol, and a phospholipid. For example: ISCOMs can contain 5% to 10% by weight Quil a, 1% to 5% cholesterol and phospholipids, and the remainder protein. The ratio of saponin to sterol in the adjuvant formulation is typically 1: 100 (weight to weight) (w/w) to 5: 1 (w/w). In some embodiments an excess of sterol is present, wherein the ratio of saponin to sterol is at least 1: 2(w/w), or 1: 5 (w/w). In other embodiments, the saponin is in excess relative to the sterol, and the saponin to sterol ratio used is about 5: 1 (w/w). ISCOM and ISCOMATRIX are available from Isconova AB (Sweden).

In some embodiments of the present invention, the substrate is,in combination with DDA, in an amount of at least 0.1 part by weight per part by weight of DDAIn other embodiments, at least 0.5 parts by weight of DDA is used per part by weight of DDAIn still other embodiments, at least 1 part by weight is used per part by weight of DDAIn combination with DDA to form a complex, whereby the tertiary amine functionality of DDA will immunofunctionalize the carboxylic acid side groups on the polymer. This allows a particular immune cell to target both the antigen and the adjuvant and deliver both the antigen and adjuvant to the cell at the optimum time and concentration.

Adjuvants described herein generally do not require any special carrier and will be formulated in aqueous or other pharmaceutically acceptable buffers. In some cases, the vaccine of the disclosed embodiments will be present in a suitable carrier, such as, for example: additional liposomes, microspheres or encapsulated antigen particles. The antigen may be contained within or outside the vesicle membrane. In general, the soluble antigen is outside the vesicle membrane, and the hydrophobic or lipidated antigen is contained within or outside the vesicle membrane.

Adjuvant compositions can be formulated in a variety of different forms depending on the route of administration, storage requirements, and the like. For example, it may be prepared in the form of a sterile aqueous solution or dispersion suitable for injection, or lyophilized using lyophilization, vacuum drying, or spray drying techniques. The lyophilized composition can be reconstituted in a stabilizing solution, such as saline or HEPES, prior to use. Thus, the adjuvant composition may be used as a solid, semi-solid or liquid dosage form.

Adjuvants may be prepared using techniques known in the art. For example: the saponin can be mixed with cholesterol in a suitable detergent and then formed into liposomes or ISCOMs by solvent extraction techniques. Saponins can also be combined with cholesterol to form helical micelles, as described in U.S. patent No.7,122,191.

Phosphate Buffered Saline (PBS) may be used as the aqueous buffer medium; the pH of the buffer may be neutral or slightly basic or slightly acidic. Thus, the pH may be in the range of pH6 to 8. The pH is generally from about 7.0 to about 7.3. The buffer may have an intensity of 10 to 50mM PO₄And 10 to 150mMPO₄In the meantime. In one example 0.063% PBS was used. The pH can be adjusted as desired using NaOH or HCl. Typical concentrations include 1N to 10N HCl and 1N to 10N NaOH.

The amount of adjuvant used will depend on the antigen used and the amount of antigen desired to be administered. It also depends on the intended species and the desired formulation. Generally, the amount of adjuvant is within the range of conventional adjuvant amounts. For example: adjuvants typically comprise about 1 microgram (inclusive) to about 1000 microgram (inclusive) in a 1 milliliter dose. Similarly, antibiotics typically comprise from about 1 microgram (inclusive) to about 60 microgram (inclusive) in a 1 milliliter dose.

The adjuvant formulation may be homogenized or microfluidized. The formulation is subjected to a primary mixing step, typically by passing the formulation through one or more homogenizers one or more times. Any commercially available homogenizer can be used for this purpose, such as: ross emulsifier (Hauppauge, new york), Gaulin homogenizer (Everett, MA) or Microfluidics (Newton, MA). In one embodiment, the formulation is homogenized at 10,000rpm for 3 minutes. Microfluidization homogenization can be achieved by using commercially available microfluidizers such as model number 110Y from Microfluidics (Newton, MA), model number Gaulin30CD (Gaulin corporation, Everett, MA), and model number Rainnie minilab8.30h (Miro Atomizer Food and Dairy corporation, Hudson, wisconsin). These microjet homogenizers operate by forcing a fluid under high pressure through small pores, so that the two fluid streams interact at high speed in an interaction chamber to form a composition having sub-micron sized droplets. In one embodiment, the formulation is homogenized by microfluidizing it through a 200 micron restricted size chamber at 10,000+/-500 psi.

The adjuvant compositions described herein can be homogenized and microfluidized. In one embodiment, the antigen is added to a suitable buffer. The solution was stirred and the saponin was slowly added to the antigen solution. Then, sterol is slowly added to the antigen/saponin solution, and the quaternary ammonium compound is slowly added to the antigen/saponin/sterol solution. The resulting composition was homogenized and then microfluidized. After microfluidization, the polymer is added to the microfluidized composition. Depending on the components used, the order of these steps may be varied to optimize the preparation of the composition.

Immunogenic compositions and methods of making vaccine compositions

The adjuvant compositions described herein are useful in the preparation of immunogenic and vaccine compositions. In the context of vaccines or immunogenic compositions, each dose contains a therapeutically effective amount of one or more antigens, which amount varies according to the age and general condition of the subject, the route of administration, the antigenic nature, and other factors. The amounts and concentrations of other components in the vaccine or immunogenic composition can be adjusted to modify the physical and chemical properties of the composition and can be readily determined by one skilled in the art. An advantageous property of the adjuvant composition is that it can be constructed entirely according to the desired characteristics of the composition. For example: if a stronger Th1 response is desired, the amount of Th1 stimulant may be increased. Similarly, if a stronger Th2 response is desired, the amount of Th2 stimulant may be increased. A balanced Th1/Th2 response was also achieved. The immunogenic and vaccine compositions may also be homogenized according to the methods described above or homogenized by microfluidization.

Application and use of compositions

Application of the composition

The dose size of the composition is typically in the range of about 1 milliliter (inclusive) to about 5 milliliters (inclusive), depending on the subject and the antigen. For example: a dose of about 1 ml is typically used on the canine or feline side, while about 2 to 5ml is typically used on the bovine side. However, these adjuvants may also be formulated in minute doses, where a dose of about 100 microliters may be used.

Routes of administration of the adjuvant compositions include parenteral, oral, oronasal, intranasal, intratracheal, topical and in ovo routes. Any suitable device may be used to administer the composition, including syringes, droppers, needleless injection devices, patches, and the like. The route and device chosen will depend on the adjuvant composition, antigen and subject, as is well known to those skilled in the art.

Use of a composition

One of the requirements of any vaccine adjuvant formulation for commercial use is to establish the stability of the adjuvant solution for long term storage. Adjuvant formulations are provided herein that are easy to manufacture and can remain stable for at least 18 months. In one embodiment, the formulation may remain stable for about 18 months. In another embodiment, the formulation may remain stable for about 18 to about 24 months. In another embodiment, the formulation may remain stable for about 24 months. Accelerated testing procedures also indicate that the formulations described herein are stable.

One advantageous property of the adjuvant composition of the invention is that it can be administered safely and effectively to a wide variety of subjects. It is expected in the art that combinations of adjuvants will exhibit greater reactogenicity than the individual components. However, the compositions described herein exhibit reduced reactogenicity when compared to compositions in which either or both components are used, while still maintaining the effect of the adjuvant. It has also been surprisingly found that the adjuvant compositions described herein show improved safety when compared to other adjuvant compositions.

The adjuvant compositions described herein can be used to generate a desired immune response in a subject. It is effective in a variety of species. Any animal to which administration of the adjuvant composition is desired is a suitable subject. It includes mammals and non-mammals, including primates, domestic animals, companion animals, laboratory test animals, captive wild animals, birds (including eggs), reptiles, and fish. Thus, the term includes, but is not limited to: monkey, human, pig; cattle, sheep, goats, horses, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, chickens, turkeys, ducks, other birds, frogs and lizards.

The adjuvants described herein can be used to show serological differences between infected and vaccinated animals. Thus, it can be used in marker vaccines, where the antigen in the vaccine can elicit a different antibody pattern in the vaccinated animal than the wild-type virus). Marker vaccines are typically used with companion diagnostic tests that measure differences in antibody patterns and demonstrate which animals have been vaccinated and which are infected with wild-type virus. Such techniques can be used to control and eradicate viruses from a population of subjects.

The following examples are presented as illustrative embodiments and should not be used to limit the scope of the present invention. Many alterations, variations, modifications and other uses and applications of the invention will become apparent to those skilled in the art.

Examples

Example 1 Quil A/Cholesterol (QC) solution

Quil A (Superfos) was dissolved in water to prepare a stock solution of 50 mg/ml. Cholesterol (fabrich Inc.) was dissolved in ethanol to make a stock solution of 18 mg/ml. The cholesterol stock solution was then filtered using a 0.2 micron filter.

The concentrations of Quil a and cholesterol in different formulations can range from Quil a to cholesterol as low as 1/1 (micrograms/ml) to as high as 1000/1000 (micrograms/ml). To prepare 50/50(μ g/ml) of the Quil a/cholesterol stock solution, the Quil a stock solution was diluted with water to a concentration of 50 μ g/ml. While stirring the solution, the cholesterol stock solution was slowly added to a final concentration of 50 μ g/ml.

Example 2 DDA (D) solution

Dimethyldioctadecylammonium bromide (DDA; Fluka Analytical) was dissolved in ethanol to prepare a stock solution of 18 mg/ml. The DDA stock solution was filtered using a 0.2 micron filter.

Example 3 Quil A/Cholesterol/DDA (QCD) solution

Stock solutions of Quil A/cholesterol were prepared as in example 1 at the desired concentrations. DDA stock solutions were prepared as in example 2 and slowly added to the Quil A/cholesterol stock solution. The solutions were mixed to achieve the desired final concentration. The pH of the solution is adjusted with NaOH or HCl as necessary to achieve the desired final pH (typically in the range of about 6.9 to about 7.5).

Example 4.(C) Solutions of

Will be provided with(Noveon, mexico) was dissolved in deionized water to make a 1.5% stock solution. In another embodiment, the compound will beDissolved in deionized water to make a 0.75% stock solution.

Example 5.(DC) solution

DDA stock solutions were prepared as in example 2. 0.75% of the preparation is carried out as in example 4Stock solutions. The solutions were mixed to achieve the desired final concentration.

Example 6 Quil A/cholesterol/DDA-(QCDC) solution

Stock solutions of Quil A/cholesterol/DDA were prepared as in example 3. 0.75% of the preparation is carried out as in example 4Stock solutions. Will be provided withThe stock solution was slowly added to the QuilA/cholesterol/DDA stock solution to achieve the desired final concentration. The pH of the solution is adjusted with NaOH or HCl to achieve the desired final pH (typically in the range of about 6.9 to about 7.5).

Example 7 Bay(R) solution

To prepare BayStock solution, the glycolipid N- (2-deoxy-2-L-leucylamino- β -D-glucopyranosyl) -N-octadecyldodecanoamide was dissolved in ethanol (60%, v/v.) then Tween 20 and glacial acetic acid were added, in one example 3.49gm of N- (2-deoxy-2-L-leucylamino- β -D-glucopyranosyl) -N-octadecyldodecanoamide was dissolved in 44.64 ml of ethanol/water (60%, v/v.) this solution was combined with 1.12 ml of Tween 20 and 0.68 ml of glacial acetic acid.

Example 8 Quil A/cholesterol/DDA-/Bay(QCDCR) solution

Quil A/cholesterol/DDA @, prepared as in example 6Stock solutions. Bay is prepared as in example 7Stock solutions. Bay is toThe solution is slowly added Quil A/cholesterol/DDA-In solution to achieve the desired final concentration. The pH of the solution is adjusted with NaOH or HCl as necessary to achieve the desired final pH (typically in the range of about 6.9 to about 7.5).

Example 9 DEAE dextran solution (X)

A stock solution of DEAE dextran (X) was prepared by dissolving 200 mg/ml of DEAE dextran in water. The solution may be autoclaved at 120 degrees celsius (C) for about 20 minutes.

Example 10 Quil A/Cholesterol/DDA/DEAE solution (QCDX)

Stock solutions of Quil A/cholesterol/DDA were prepared as in example 3. A DEAE stock solution was prepared as in example 9. The solution was added directly to the homogenizer to combine them. The mixing time is more than 1000 seconds^-1The shearing force of (2) is carried out by flash mixing (flash mixing method). The mixing was carried out as follows: the aqueous solution is fed directly into the oil phase containing the non-polar adjuvant and the antigen component and stirred until a homogeneous and stable mixture is obtained. Typically, this step may be at least several minutes or more depending on the desired particle size.

Example 11 oil composition (O)

Drakeol mineral oil was combined with Tween 85 and Span85 (Span85), heated to about 55 deg.C, cooled and sterile filtered to prepare an oil stock solution. Thus, this mixture comprises an oil phase matrix component for the oil-based carrier. If cholesterol and/or DDA are chosen as a co-acting immunomodulator in one of these compositions, they are also added to this mixture prior to filtration, as they are soluble in the oil phase.

Example 12 Quil A/Cholesterol/DDA/DEAE/oil composition (QCDXO)

Stock solutions of Quil A/cholesterol/DDA/DEAE were prepared as in example 10. An oil storage composition was prepared as in example 11. The solution was a combination of Quil-A, DEAE dextran and water to achieve the amount at the concentration. The reaction was stirred at room temperature or higher for several minutes or more to mix the aqueous phase, and then sterile filtered and stored for addition to the oil phase. The aqueous phase was slowly added to the continuously mixed oil phase.

Example 13 preparation of immunogenic compositions or vaccine compositions

To prepare an immunogenic composition or vaccine composition comprising an antigen and one of the adjuvants described above, the desired antigen is added to a suitable buffer. Then, the components of the desired adjuvant are added as described above. The resulting solution was brought to final volume with buffer.

Example 13a antigen, Quil A, Cholesterol, DDA,

For preparing a composition comprising antigen, Quil A, cholesterol, DDA andthe immunogenic composition or vaccine composition of (4), adding the desired antigen to a suitable buffer. Stock solutions of Quil a were prepared as in example 1 and slowly added to the antigen solution. Cholesterol stock solutions were prepared as in example 1 and slowly added to the antigen/Quil A solution. Stock solutions of DDA were prepared as in example 2 and slowly added to the antigen/Quil a/cholesterol solution. Homogenizing antigen/Quil a/cholesterol/DDA/solution and microfluidizing. Preparation 0.75% as in example 4And (3) solution. After homogenizing with a microfluid jet, subjecting the mixture toThe solution (0.05%, v/v) was added to the microfluidized composition and the pH adjusted to about 6.9 to about 7.5 with NaOH or HCl.

Example 13b. antiRaw material, Quil A, cholesterol, DDA,Bay

For preparing a vaccine comprising antigen, Quil A, cholesterol, DDA,And BayThe immunogenic composition or vaccine composition of (4), adding the desired antigen to a suitable buffer. Stock solutions of Quil a were prepared as in example 1 and slowly added to the antigen solution. Cholesterol stock solutions were prepared as in example 1 and slowly added to the antigen/Quil A solution. Stock solutions of DDA were prepared as in example 2 and slowly added to the antigen/Quil a/cholesterol solution. Homogenizing antigen/Quil a/cholesterol/DDA/solution and microfluidizing. Preparation 0.75% as in example 4And (3) solution. After homogenizing with a microfluid jet, subjecting the mixture toThe solution (0.05% v/v) was added to the microfluidized composition and the pH adjusted to about 6.9 to about 7.5 with NaOH or HCl. Bay is prepared as in example 7Stock solution, after DDA addition, BayThe components are added to the aqueous phase.

Example 13c. antigen, Quil A, Cholesterol, DDA, DEAE dextran

To prepare an immunogenic composition or vaccine composition comprising antigen, Quil a, cholesterol, DDA and DEAE dextran, the desired antigen is added to a suitable buffer. Stock solutions of Quil a were prepared as in example 1 and slowly added to the antigen solution. The composition is homogenized. A stock solution of cholesterol was prepared as in example 1 and slowly added to the antigen/Quil a solution during homogenization. A DDA stock solution was prepared as in example 2 and added slowly to the antigen/QuilA/cholesterol solution during homogenization. A DEAE dextran solution was prepared as in example 9.DEAE dextran solution was added during homogenization and the resulting composition was brought to final volume.

Example 13d. antigen, Quil A, Cholesterol, DDA, DEAE dextran, oil

To prepare an immunogenic or vaccine composition comprising antigen, Quil a, cholesterol, DDA, DEAE dextran and oil, the desired antigen is added to a suitable buffer. Stock solutions of Quil a were prepared as in example 1 and slowly added to the antigen solution. The composition is homogenized. A stock solution of cholesterol was prepared as in example 1 and slowly added to the antigen/Quil a solution during homogenization. A DDA stock solution was prepared as in example 2 and slowly added to the antigen/Quil a/cholesterol solution during homogenization. A DEAE dextran solution was prepared as in example 9. During homogenization, a DEAE dextran solution is added. An oil composition was prepared as in example 11. During homogenization, the oil composition is added by adding the aqueous phase to the oil phase while homogenizing and bringing the resulting composition to final volume.

Example 14 feline leukemia Virus (FeLV) vaccine

Animals were randomly assigned to treatment groups using a Randomized complete block design (Randomized complete block design). Table 1 shows the study design. The blocks are based on birthdays and birth times. Animals were classified according to birth date, then according to parity. 4 blocks were used. Animals were randomly assigned to receive treatment within the same block. In the vaccination phase of the study, two consecutive blocks were combined to form a group of 8 animals. Groups of animals were randomly assigned to two rooms, each containing 5 groups (10 blocks) of animals. In one group of animals, animals were randomly assigned to four cages in close proximity to each other, such that each cage contained two animals receiving the same treatment. In the challenge phase of the study, animals from one vaccination room were randomly assigned to one or two challenge rooms. The vaccination room selected as divided into two challenge rooms had five blocks randomly assigned to each challenge room (2.5 groups; 20 animals). Another challenge room contained ten blocks (5 groups; 40 animals). Animals from the same group were randomly assigned to four cages in close proximity to each other in one challenge room.

The vaccine used in this study was prepared as in example 13, but using 1.5% ofStock solutions. Specifically, FeLV subpopulations A, B and C were propagated in FeLV-transformed lymphoid cells to make2(Pfizer, Inc.). Viral antigens are chemically inactivated, combined with sterile adjuvants to enhance immune response, and packaged in liquid form. The total amount of the preparation is 100 ml, and the preparation contains feline leukemia virus and 25 micrograms of Quil A/aluminum hydroxideFor Veterinary products (IVP.) A total of 94.5 ml of 1.106 × 10⁵The nanolg/ml FeLV stock solution was mixed slowly for 15 minutes. If necessary, the pH is adjusted to 5.9 to 6.1 with 4N HCl or 18% NaOH. 0.5 ml of 5.0 mg/ml Quil A solution was added to the antigen solution with stirring. Then 5.0 ml of 100% (v/v) was slowly addedThe composition was stirred at 4 ℃ for at least 2 hours. The pH is adjusted to 7.0 to 7.3 with 18% NaOH or 1N HCl, as required.

A preparation containing feline leukemia Virus and 37.5 micrograms Quil A/aluminum hydroxide was prepared in the same manner as for 25 micrograms Quil A IVPBut to this antigen solution 7.5 ml of Quil a stock solution was added.

Prepared in a total volume of 350 ml and containing feline leukemia virus, Quil A, cholesterol, DDA andwhile stirring 349.3 ml of 1.106 × 10⁵Ng/ml FeLV stock solution, 0.14 ml of 50.0 mg/ml Quil A solution was slowly added to the antigen solution. Then, 0.39 ml of 18 mg/ml cholesterol/ethanol solution was slowly added. The composition was homogenized at 10,000rpm for 3 minutes. A total of 0.19 ml of an 18.0 mg/ml DDA/ethanol solution was added to the composition with stirring. The total amount is 1.5 percent of 5.0 mlThe solution was slowly added to 145.0 ml of the feline leukemia virus, QuilA, cholesterol, and DDA composition. The pH is adjusted to 7.0 to 7.3 with 18% NaOH or 1N HCl, as required.

Table 1: design of experiments

^aVeterinary product for research

^bMixed to contain relative potency comparable to that of a Reference vaccine (FeLV Reference Lot No.12)

^cSubcutaneous (SC ═ subcutaneous)

^dDepo-On day 42 (about 5.0 mg/kg) by intramuscular route

^eMouth and nose

Quil A-Cholesterol ═ Sail A, adjuvant, incorporated into lipid particles of Cholesterol

Carbomer (Carbomer)

DDA ═ dimethyl dioctadecyl ammonium bromide

All animals were observed daily and observations were recorded. All animals were body temperature recorded by measuring ear temperature on day-1 prior to administration of the first vaccine dose and day 20 prior to administration of the second vaccine dose. Blood samples (1.0-2.0 ml) were collected from each animal on day-2 by venipuncture of the jugular vein. The sedative dose (about 5.0 mg/kg) is administered intramuscularly depending on body weight(Fort dog Animal Health) to minimize Animal pressure and avoid injury to Animal handlers during blood collection. Blood was collected in a Serum Separation Tube (SST) and subjected to a serum separation process. The serum was stored at-20 ℃ or lower until testing.

The cats were administered a 1.0 ml dose of placebo or FeLV vaccine by subcutaneous route. The first vaccination was performed on day 0 and the second vaccine administration was performed on day 21. All animals were observed for an immediate local pain response (sting response) of about 1 hour after the first and second vaccination. The observations are recorded on a file. All animals were body temperature measured by measuring ear temperature on days 1 and 2 after administration of the first vaccine dose and on days 22 and 23 after administration of the second vaccine dose. Injection site reactions (swelling) were also determined on day 1 after the 1 st vaccination and on days 22 and 23 after the 2 nd vaccination. On day 35, blood samples (1.0-2.0 ml) were collected from each animal by venipuncture of the jugular vein, processed for serum separation and stored at-20 ℃ or lower until testing.

Animals were placed in individual cages on day 35. The challenge virus was virulent feline leukemia virus (FeLV), the Rickard strain, with a titer of about 10^6.1TCID₅₀Per milliliter. FeLV challenge material was thawed and kept on ice prior to administration. Animals were challenged by nasal administration of 1.0 ml undiluted challenge substance on days 37, 40, 42 and 44. A 1 ml needle-free tuberculin syringe was filled with the challenge material. Approximately 0.5 ml was administered to each nostril of each kitten. On day 42, DEPO-Challenge administration was performed after about 5 hours. After each day of challenge, a sample of the challenge material was retained for confirmation of titer.

Blood samples (1.0-2.0 ml) were collected from each animal by venipuncture of the jugular vein on days 64, 85, 106, 127, 134, 141, 148 and 155 after challenge. Sedative doses as described above(Fort Dodge). Blood was collected in a Serum Separation Tube (SST), subjected to a serum separation treatment and stored at a temperature of-20 ℃ or lower until the test was performed. Serum samples were tested for the presence of the FeLVp27 antigen (marker of FeLV infection) by ELISA (IDEXX; Westbrook, Maine). The final results were evaluated by the color intensity produced and the optical density measured by a spectrophotometer at 405/490 nm. In terms of a valid test, the optical density of the positive control group must be between 0.131 and 2.999, while the optical density of the negative control group must be less than or equal to 0.0039.

Virus isolation was performed using serum samples collected on days-2 and 35. Serum samples from days 127 to 155 are considered for evaluation of FeLV vaccine efficacy. Serum samples from day 127 (week 12), day 134 (week 13), day 141 (week 14), day 148 (week 15) and day 155 (week 16) were tested for the presence of FeLV p27 antigen. An animal is considered to be persistently infected if it has three or more positive FeLV p27 antigen test results during days 127 (week 12) to 155 (week 16).

Temperature analysis using a generally linear-repeat measured mixture model, pairwise treatment comparisons were made between treatment groups T01 and T02, T03 and T04 at each time point if the overall treatment and/or time point treatment effect was significant. The least squares means, 95% confidence intervals, minimum and maximum values for each set of treatments at each time point were calculated.

The frequency distribution of the occurrence of sting response for each treatment was calculated and time point data was collected. The frequency distribution of injection site swelling for each treatment was calculated and time point data was collected. The frequency distribution of the systemic response after vaccination was calculated for each treatment.

No immediate response was observed in either treatment group during the first and second vaccinations. No adverse reactions were observed in any of the treatment groups at about 1 hour after the first and second vaccinations. Neither fever (body temperature ≥ 39.5 ℃) nor low temperature (body temperature <37 ℃) were observed in either treatment group after the first and second vaccination. At any time point, there was no significant difference in mean body temperature between treatment groups (p > 0.08). No injection site swelling was observed in either treatment group after the first and second vaccination.

The final results from weeks 12 to 16 after challenge indicated that 16 of the 19 animals (84%) receiving the placebo vaccine (group T01) continued to have viremic properties against FeLV. 13 of the 19 animals of the T02 group (68%) were protected from challenge by FeLV toxicity. The level of protection was statistically significant (p ═ 0.0004) compared to kittens receiving placebo vaccination. 12 of 19 animals in the T03 group (63%) were protected from challenge by FeLV toxicity. The level of protection was statistically significant compared to kittens receiving placebo vaccination (p ═ 0.0013). 19 of 20 animals (95%) in the T04 group were protected from challenge by FeLV toxicity. The level of protection was statistically significant compared to kittens receiving placebo vaccination (p ═ 0.0001).

Thus, vaccines administered to groups T02, T03, and T04 all showed safety in kittens of the smallest age when administered in a two-dose regimen separated by 3 weeks. In addition, vaccines administered to these groups also significantly reduced the level of FeLV persistent viremia in kittens of the smallest age when administered in a two-dose regimen divided by 3 weeks. There was a statistically significant reduction in FeLV persistent viremia established in kittens of the T02, T03 and T04 groups. In addition, there were statistically significant differences between T04 and the other vaccine groups (T02, T03). Surprisingly and unexpectedly, vaccines containing the novel adjuvant formulations proved to be more effective than vaccines containing adjuvant components commonly used in cats.

Example 15 feline leukemia Virus vaccine

Kittens were acclimatized for 16 days after arrival. Animals were then randomly assigned to one room and again to treatment groups (one animal per treatment group in each room). On day-1 of the study, blood samples (1.0-2.0 ml) were collected from each animal by venipuncture of the jugular vein. The sedative dose (about 5.0 mg/kg) is administered intramuscularly depending on body weight(Fort dog Animal Health) to minimize Animal stress and avoid injury to Animal handlers during blood collection. Blood was collected in a serum separation tube and subjected to a serum separation treatment. All animals were also observed daily and observations were recorded.

The vaccine was prepared as in example 13, but 1.5% was usedStock solutions. Propagation of FeLV subpopulations A, B and C in FeLV-transformed lymphoid cells to produce2. Viral antigens are chemically inactivated, combined with sterile adjuvants to enhance immune response, and packaged in liquid form. IVP containing feline leukemia Virus, Quil A, cholesterol and DDA at a Relative Potency (RP) of 2 was prepared in a total volume of 500.0 ml in the following manner. A total of 20.7 ml of FeLV stock solution (50.0 RP/ml, where 1RP ═ 3,624 ng/ml of antigen) was added to 478.2 ml of 0.063% PBS buffer. 0.21 ml of 50.0 mg/ml Quil A solution was slowly added to the antigen solution with stirring. Then, 0.58 ml of 18 mg/ml cholesterol/ethanol solution was slowly added. A total of 0.29 ml of 18.0 mg/ml DDA/ethanol solution was slowly added to the composition with stirring. The composition was homogenized at 10,000rpm for 3 minutes. The composition was then microfluidized in one pass through a 200 micron restricted-size chamber at 10,000(+500) psi. 10.0 ml of 1.5% was added under stirringThe solution was slowly added to 290.0 ml of the feline leukemia virus, Quil A, cholesterol and DDA composition. The pH is adjusted to 7.0 to 7.3 with 18% NaOH or 1N HCl, as required.

IVP containing feline leukemia virus with RP 5 was prepared in the same manner as for IVP with RP 2 using 51.7 ml of FeLV stock solution and 447.2 ml of 0.063% PBS buffer, with the remaining components maintained in the same amounts.

In the same manner as for IVP with RP 2, 93.1 ml of FeLV stock solution, 355.9 ml of 0.063% PBS buffer, 0.19 ml of Quil A solution, 0.52 ml of cholesterol solution and0.26 ml DDA solution (total volume 450 ml) IVP containing feline leukemia virus with RP 10 was prepared. Then, 1.5% of 8.3 ml of the solution was addedThe solution was slowly added to 241.7 ml of the feline leukemia virus, Quil A, cholesterol and DDA composition.

IVP containing feline leukemia virus with RP 15 was prepared in the same manner as for IVP with RP 10 using 139.7 ml of FeLV stock solution and 309.4 ml of 0.063% PBS buffer, with the remaining components remaining in the same amounts.

IVP containing feline leukemia virus with RP 20 was prepared in the same manner as for IVP with RP 2 using 206.9 ml of FeLV stock solution and 292.0 ml of 0.063% PBS buffer, with the remaining components maintained in the same amounts.

To administer a 0.5 ml dose, 300.0 ml of feline leukemia virus having an RP of 5, Quil A, cholesterol, DDA andthe IVP of (1). A total of 21.7 ml of FeLV stock solution (35.8 RP/ml, where 1RP ═ 1,864 μ g/ml of antigen) was added to 277.7 ml of 0.063% PBS buffer. 0.12 ml of 50.0 mg/ml Quil A solution was slowly added to the antigen solution with stirring. Then, 0.35 ml of 18 mg/ml cholesterol/ethanol solution was slowly added. A total of 0.17 ml of 18.0 mg/ml DDA/ethanol solution was slowly added to the composition with stirring. The composition was homogenized at 10,000rpm for 3 minutes. The composition was then microfluidized in one pass through a 200 micron restricted-size chamber at 10,000(+500) psi. 3.3 ml of 1.5% of the total weight was added under stirringThe solution was slowly added to 96.7 ml of the feline leukemia virus, Quil A, cholesterol and DDA composition. If necessary, the pH is adjusted to 18% NaOH or 1N HClBetween 7.0 and 7.3.

IVP was prepared in the same manner and with appropriate adjustments for the 0.5 ml dose for administration of a 1.0 ml dose comprising feline leukemia virus with PR 5, QuilA, cholesterol, DDA and

preparation of a Total amount of 300.0 ml of feline leukemia Virus containing RP 10 andthe IVP of (1). A total of 62.1 ml of FeLV stock solution (50.0 RP/ml, where 1RP ═ 3,624 μ g/ml of antigen) was added to 237.9 ml of 0.063% PBS buffer. The composition was homogenized at 10,000rpm for 3 minutes. The composition was then microfluidized in one pass through a 200 micron restricted-size chamber at 10,000(+500) psi. 3.3 ml of 1.5% of the total weight was added under stirringThe solution was slowly added to 96.7 ml of the feline leukemia virus composition. The pH is adjusted to between 7.0 and 7.3 with 18% NaOH or 1N HCl, as required.

On study day 0 and study day 20, the kittens were administered placebo and FeLV vaccine subcutaneously using a 22 x 3/4 "needle and a 3cc syringe (table 2). Treatment group T01 was administered a 1.0 ml dose of placebo. The treatment groups T02, T04, T05, T06, T07, T08 and T09 were administered with a 1.0 ml dose of FeLV vaccine. The treatment group T03 was administered a dose of 0.5 ml of FeLV vaccine. Treatment group T10FeLV was administered canarypox vaccine (Merial) by intradermal route administration using an intradermal gun syringe.

Table 2: design of experiments

All animals were observed after the first vaccination (study day 0) and the second vaccination (study day 20) for signs of pain including sounding, scratching/biting and aggressiveness or attempted escape at the time of administration of the test vaccine. The attitude (normal or abnormal) after vaccination was also recorded on the file. All animals were observed for the occurrence of adverse systemic reactions for approximately 1 hour after vaccination on study day 0 and study day 20. The observations are recorded on a file. The site of vaccination was contacted and the pain at the injection site, redness at the injection site, swelling at the injection site and the magnitude of swelling were recorded. Observations were made on days 2,5 and 9 of the study after the 1 st vaccination and on days 25, 28 and 32 of the study after the 2 nd vaccination. The observations are recorded on a file.

Blood samples (1.0-2.0 ml) were collected from each animal by venipuncture of the jugular vein on study day 32 (before challenge). Animals were challenged by nasal administration of 1.0 ml undiluted challenge material on study days 34, 36, 39 and 41. A 1 ml needle-free tuberculin syringe was filled with the challenge material. Approximately 0.5 ml was administered to each nostril of each kitten. Mean titer of FeLV challenge material was 10^6.1TCID₅₀Per milliliter. Blood samples (1.0-2.0 ml) were collected from each animal on study days 61, 83, 106, 126, 133, 138, 146 and 152 by venipuncture of the jugular vein.

Result-safety

Three animals in the treatment group T09 showed an immediate sting-type response during the first vaccination (study day 0). During the second vaccination (study day 20), one animal in treatment group T05, four animals in treatment group T08 and two animals in treatment group T09 showed an immediate sting-type response.

Three animals in treatment group T09 produced minimal sounds during the first vaccination. Animals that were painful at the first vaccination also produced a slight sound at this time. During the second vaccination, one animal in treatment group T05, four animals in treatment group T08, and two animals in treatment group T09 produced minimal sounds. Animals that were painful at the second vaccination also produced a slight sound at this time.

Three animals in treatment group T09 showed a challenge/escape attempt during the first vaccination. During the second vaccination, one animal in treatment group T05, four animals in treatment group T08 and two animals in treatment group T09 showed an aggressive behavior/escape attempt.

None of the treatment groups presented a bite/scratch at the injection site during the first or second vaccination. No injection site reactions were observed in either treatment group after the first or second vaccination. No adverse reactions were observed in any of the treatment groups.

Results-efficacy

The results of the FeLVp27 antigen test were negative for all animals in serum samples collected on day-1 prior to vaccination. All animals in the serum samples collected on day 32 before challenge had negative results for the FeLVp27 antigen test.

The final results from weeks 12 to 16 after challenge (table 3) indicated that 9 of 10 animals of treatment group T01 (placebo) (90%) had persistent viremia properties on FeLV. Results during the same period indicated that 6 of 10 animals (60%) in treatment group T02 were protected from virulent challenge by FeLV; this level of protection was not statistically significant (p ═ 0.0573) compared to kittens receiving placebo vaccination. 9 of 10 animals of treatment group T03 (90%) were protected from virulent challenge by FeLV; this level of protection was statistically significant (p ═ 0.0011) compared to kittens receiving placebo vaccination. 10 of the 10 animals of treatment group T04 (100%) were protected from virulent challenge by FeLV; this level of protection was statistically significant (p ═ 0.0001) compared to kittens receiving placebo vaccination. 10 of the 10 animals of treatment group T05 (100%) were protected from virulent challenge by FeLV; this level of protection was statistically significant (p ═ 0.0001) compared to kittens receiving placebo vaccination. 7 of 10 animals of treatment group T06 (70%) were protected from virulent challenge by FeLV; this level of protection was statistically significant (p-0.0198) compared to kittens receiving placebo vaccination. 10 of the 10 animals of treatment group T07 (100%) were protected from virulent challenge by FeLV; this level of protection was statistically significant (p ═ 0.0001) compared to kittens receiving placebo vaccination. Of the 10 animals of treatment group T08 8 animals (80%) were protected from virulent challenge by FeLV; this level of protection was statistically significant compared to kittens receiving placebo vaccination (p ═ 0.0055). Of the 10 animals of treatment group T09, 5 animals (50%) were protected from virulent challenge by FeLV; this level of protection was not statistically significant (p-0.1409) compared to kittens receiving placebo vaccination. Finally, 6 of 10 animals of treatment group T10 (60%) were protected from virulent challenge with FeLV; this level of protection was not statistically significant (p ═ 0.0573) compared to kittens receiving placebo vaccination.

TABLE 3 Abstract of protection level

Treatment group	Relative potency of vaccines	Level of protection	Prevention score
				T01	NA	10％
T02	4.58	60％	55.6％
				T03	4.58	90％	88.9％
T04	26.32	100％	100％
				T05	18.58	100％	100％
T06	11.16	70％	66.7％
				T07	4.77	100％	100％
T08	1.64	80％	77.8％
				T09	11.12	50％	44.4％

Discussion of the related Art

The vaccines used in treatment groups T02, T03, T04, T06 and T07 showed a satisfactory safety profile during the first vaccination, since no reaction was observed at that time. 1 animal in treatment group T05 showed an immediate response (pain on administration, minor sounds, aggressiveness/attempt to escape) during the second vaccination. This may be associated with an adverse response of a particular animal to a vaccination, rather than problems with vaccine formulations. All vaccines showed a satisfactory post-vaccination safety profile, as no local and adverse reactions associated with vaccination were observed.

Since > 80% protection (> 75% prevention score) was achieved after challenge with virulent FeLV, FeLV vaccines administered to treatment groups T03, T04, T05, T07 and T08 showed satisfactory efficacy. The vaccine administered to group T07 provided a 100% protective effect, which was quite surprising and unexpected, since animals in this group received 25% and 33% of the antigen dose in animals in groups T04 and T05, respectively. A clear advantage of the adjuvants disclosed and tested herein is that they allow for the use of smaller doses of antigen, but can still induce a complete protective immune response. The vaccines administered to treatment groups T02, T06 and T09 showed a more or less reduced efficacy (< 80% protection; prevention score < 75%) after challenge with virulent FeLV. The reduced efficacy of the vaccine administered to treatment group T02 may be due to the presence of low-response animals in this group.

Example 16 in ovo vaccination against Eimeria (Eimeria) in chickens

Avian coccidiosis is an intestinal disease that is generally caused by protozoa of the genus eimeria and represents a serious problem throughout the world in the poultry industry. Parasites fed during feeding colonize the intestinal tract, where they cause severe damage to the intestine and underlying tissues. The economic losses to the poultry industry are very significant, as both feed conversion rates and weight gain of broiler and laying birds decrease. A general summary of the state of the art (including vaccination against Eimeria using, for example, recombinant Eimeria protein as an antigen and various adjuvant systems) is described in the following documents, which are all incorporated herein by reference: (1) h.s.lillehoj et al, j.paritol, 91(3),2005, pp.666-673; (2) H.S. Lillehoj et al, Avian Diseases,492005, 112-; and (3) R.A. Dalloul et al, Expert Rev.vaccines,5(1),2006, pp.143-163. This example designs the use of a novel vaccine composition for coccidiosis using an adjuvant component that provides superior performance.

The highly effective adjuvants of the present invention may be used with antigenic material from all Eimeria species, including purified or partially purified protein extracts of the antigenic material, or one or more recombinantly expressed proteins thereof, or fragments of any or all such proteins, and thus include antigens from Eimeria acervulina, Eimeria acervulina (Eimeria ahsita), Eimeria bovis (Eimeria bovis), Eimeria brunetti (eimeriabanetti), Eimeria freudenrea (Eimeria fragularia), Eimeria maxima (Eimeria maxima), Eimeria turkey (Eimeria melegardis), and Eimeria mitis (Eimeria mitis), Eimeria necatrix (Eimeria nerix), Eimeria praecox (Eimeria praesox), Eimeria tenella (Eimeria tenella), and Eimeria brunetti, and the like.

Adjuvanted vaccines provided by the present invention may be provided against any protein or macromolecule produced at one or more time points in the life cycle of the protozoan, including but not limited to: oocysts (whether sporulated or not), sporangia, sporozoites (sporozoites), schizonts, merozoites, male or female gametocytes. In a preferred embodiment, proteins excreted in large amounts into the faeces in the oocyst stage or samples of such proteins partially or completely purified by conventional means are preferred substances for use as sources of recombinant protein antigens.

Other examples of Eimeria proteins that may be used as sources of antigen for the present vaccine formulations include those described in Karkhanis et al, Infection and Immunity,1991, pp.983-989, including protective antigens of about 20 to about 30kDA in mass as described therein. Other examples include Eimeria 23kDA3-1E protein and Etp100 protein (as recovered from Eimeria tenella).

The high-potency adjuvant of the present invention can be used with antigenic substances from Neurospora caninum.

Furthermore, the high potency adjuvants of the present invention may be used with any of the following protozoan pathogens: cryptosporidium parvum (cryptosporidiosis), chlamydosporium cyclo (cyclosporozoosis), Isospora bellidii (Isospora belli) (isosporozoosis), Toxoplasma gondii (Toxoplasma gondii) (toxoplasmosis), Plasmodium (Plasmodium) (malaria) and Babesia spp (Babesia spp) (babesiosis), and protozoa related animals causing these or related diseases, usually apicomplexan (Apicomplexen).

The effectiveness of in ovo delivery of vaccines containing specific adjuvant systems was evaluated as follows.

Materials and methods:

1. materials:

recombinant E.maxima protein (protein 3-1E) was expressed in E.coli and purified by affinity column. Crude preparations of whole cell E.maxima macromolecules, which are eluted from the disrupted cells with detergent, are also used as antigen, and this crude antigen is referred to as "EM". In a preferred embodiment, the adjuvant is as described above in example 8 and prepared according to the procedures provided in the scheme of this example (see page 41). Thus, in a typical embodiment, each embryo will receive an injection of about 50 to about 100 microliters of a vaccine solution comprising per milliliter: about 50 or 100 micrograms of recombinant 3-1E protein or other protein species, or, about 50 or 100 micrograms of crude cell "EM" extract; about 20 micrograms of Quil a; about 20 micrograms of cholesterol; (iv) about 0.075% (v/v) of CARBOPOL; about 10 micrograms of DDA; and about 250 micrograms of R1005, all provided, for example, in 20mM PBS.

In selecting saponins for use herein, the following additional information is used for guidance. The term saponin as defined refers to a glycoside derived from a Plant, and The biological properties of a number of saponins have been extensively studied (The Plant Glycosides, mclroy, r.j., Edward Arnold and co., London, 1951). The saponins most mainly used in the art for the manufacture of vaccines are saponins derived from the plants quillaja saponaria, Aesculus hippocastanum or Gyophilla struthium. Extracts of the bark of the Chilean Gleditsia sinensis known to have adjuvant activity are known, such as Quil A. Purified fractions of Quil a have also been described which retain adjuvant activity but are less toxic than Quil a, such as QS 21. QS21 is also described in Kensil et. (1991.J. immunology vol146, 431-437). Such saponin-containing materials are highly effective materials when mixed with other adjuvant components of the invention (as described hereinbefore and hereinafter). Other useful formulations include those using aescin, which is described in the Merck index (Merck index; 12th edition: item 3737) as a mixture of saponins found in horse chestnut tree (horse chestnut tree) seeds (Escin). In a preferred embodiment of the present invention, the saponin is "Quil a" sold by the company E.M Sergeant, usa.

It must further be understood that the saponin extract may be used as a mixture or as a purified individual component from such fractions/preparations, including QS-7, QS-17, QS-18 and QS-21 from Massachusetts, Antigenetics, USA, or similar crude, fractionated or refined saponin products provided by Isconova, Sweden and mixtures thereof. In one embodiment, Quil a is at least 85% pure. In other embodiments, Quil a is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97 percent. 98% or 99% pure.

2. Embryo vaccination:

eggs were purchased from Moyers Hatchery, Inc. of Quakertown, PA. In the context of in ovo immunization, broiler eggs are incubated for 18 days, fertilized eggs are then candled (at 18 days of embryonic development), and then injected with 20mM PBS and adjuvant alone or formulated with recombinant 3-1E protein or "EM" preparations. Injections were performed according to the manufacturer's instructions using an "Intelliject" in ovo syringe (Avitech, Hebron, MD). 100 microliters of sample was injected into the amniotic cavity of each egg using a 17.5 cm long 18 gauge needle supplied by Avitech (Hebron, MD). A 50 microliter dose may also be used in carrying out the present invention.

3. Chicken:

once the broilers are hatched (about day 21-22), the chickens are shipped to the laboratory using disposable cartons that transport the chickens (frederick packaging, inc., Milwaukee, WI), then raised in the Petersime unit and provided with feed and water that is freely available.

The chickens were kept in an Eimeria-free facility in a brooder pen and, when infected with live oocysts of Eimeria maxima, were transferred into large cages at separate sites until the end of the experimental period.

4. Parasite:

the USDA BARC strain of E.maxima #41 (which was maintained in Animal Parasitic diseases laboratory-BARC and propagated according to the protocol established in Drehoz laboratory) was used. Freshly produced oocysts from eimeria maxima (Beltsville #41) were cleaned by floating on 5% sodium hypochlorite and washed three times with PBS and viability was calculated by trypan blue staining using a hemocytometer.

5. Eimeria challenge infection:

the wings of 7-day-old chicks were labeled, and all the chickens of the experimental groups were inoculated with eimeria maxima through the esophagus using an inoculating needle except for uninfected control groups, and then the chicks were placed in oocyst collection cages.

6. Determination of weight gain:

the body weight of the individual chicks was determined on day 0 (uninfected), on days 6 and 10 after infection with E.maxima.

7. Assessment of fecal oocyst production

Care animals were instructed not to clean the cages and allowed to collect the fallen chicken manure. Starting on day 6 post-infection, the collection tray was placed under each cage for 5 days and fecal material was collected in large plastic pots (2 liters). The chicken manure soaked in tap water in each tank was ground in a blender together with more water (total volume 3 liters) and two 40 ml random samples were taken from each sample and stored in a refrigerator until counted. To count coccidial oocysts, multiple dilutions were first made to determine the optimal dilution for calculating oocysts for each sample. Oocysts were counted under a microscope using a sucrose floatation method (established by the bollehoj laboratory) using a McMaster counting chamber (counting chamber). The total number of oocysts excreted by each chicken was calculated using the following formula: total oocysts/chicken (oocyst count x dilution x stool sample volume/counting chamber volume)/number of chickens in each cage.

8. Collecting samples:

blood was collected on day 6 post infection and serum antibody responses were measured. Blood samples were taken from individual chicks (N-4-5/group), allowed to clot at 4 ℃ for 4 hours and sera collected. Serum samples were tested for anti-eimeria antibodies using ELISA. Briefly, microtiter plates were coated with 10. mu.g/well of recombinant coccidian antigen Ea3-1E, EtMIF or EtMIC2 overnight, washed with PBS-0.05% Tween and blocked with PBS-1% BSA. Serum dilutions (1: 20, 1: 40, 1: 80, 1: 160; 100. mu.l/well) were added, incubated with gentle shaking, washed and bound antibodies detected with peroxidase-coupled rabbit anti-chicken IgG (Sigma) and peroxidase-specific substrate. The Optical Density (OD) was determined at 450nm in a microtiter plate reader (Bio-Rad, Richmond, Calif.).

Intestinal tissue was collected at and 6 and 10 days after hatch and cytokine (IFN-. gamma., IL-2) production was tested using real-time RT-PCR as a measure of Th1 stimulation.

cDNA Synthesis

Total RNA was extracted from intestinal IEL using TRIzol (Invitrogen, Carlsbad, Calif.). 5 micrograms of RNA were treated with 1.0U DNase I and 1.0 microliter of 10 × reaction buffer (Sigma), incubated at room temperature for 15 minutes, 1.0 microliter of stop solution was added to inactivate the DNase I, and the mixture was heated at 70 ℃ for 10 minutes. The RNA was reverse transcribed using the StrataScript first Strand Synthesis System (Stratagene, La Jolla, Calif.) according to the manufacturer's recommendations.

10. Quantitative RT-PCR

The quantitative RT-PCR oligonucleotide primers used for chicken interferon-gamma (IFN-. gamma.) and GAPDH controls are listed in Table 4. Amplification and detection were performed using an equal amount of total RNA from intestinal IEL using the Mx3000P system and the Brilliant SYBR Green QPCR master mix (Stratagene). Using warp logarithm (log)₁₀) Diluted standard RNA to generate a standard curve and the amount of individual transcripts was normalized to the amount of GAPDH analyzed by the Q-gene program. Each assay was performed in triplicate. To normalize RNA levels between samples in the same experiment, values from all samples from the experiment were pooled to calculate the mean threshold cycle (C) of the amplification product_t) The value is obtained.

TABLE 4 oligonucleotide primers for quantitative RT-PCR of chicken IFN-. gamma.and GAPDH.

Before and 10 E.maxima inoculation^thSpleen was collected for analysis of splenocyte proliferation by DPI (day after infection). Place spleen in a dish containing 10 ml of Hank's Balanced Salt Solution (HBSS) supplemented with 100 units/ml penicillin and 100. mu.g/ml streptavidin (Sigma, St.Louis, Mo.) Single cell suspension of splenic lymphocytes was prepared and lymphocyte proliferation was performed.briefly, splenic cells were conditioned to 5 × 10 in IMDM medium (Sigma) supplemented with 10% Fetal Bovine Serum (FBS) (Hyclone, Logan, UT), 100 units/ml penicillin, and 100. mu.g/ml streptavidin (Sigma) (referred to as 10% intact IMDM medium)⁶Or 1 × 10⁷Cells/ml. Splenocytes (100. mu.l/well) were plated in 96-well flat bottom plates at 41 ℃ with 5% CO₂And incubated in a humidified incubator (Forma, Marietta, OH) with 95% air for 48 hours. Using 2- (2-methoxy tomb-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt (WST-8, Cell-Counting)Dojindo Molecular Technologies, Gaithersburg, Md.) measures the proliferation of cells. The Optical Density (OD) was determined at 450nm using a microtiter plate spectrophotometer (BioRad, Richmond, Calif.).

Results

The results show that broiler chickens vaccinated with 100 microliters of adjuvant formulation (i.e., 100 microliters comprising recombinant 3-1E protein at a dose as previously defined) increased weight by an additional 45-85 grams compared to unvaccinated, but eimeria maxima-infected chickens.

The vaccine of the invention also showed a clear effect on cell-mediated immunity as measured by the mitogenic lymphocyte proliferation assay, 1 × 10 incubated with Con A for 48 hours⁷Proliferation results of cells/ml splenic lymphocytes show that splenocytes from E.maxima infected chickens immunized with Pfizer adjuvant with or without antigen show substantially higher lymphocyte proliferation levels, especially when 50 microgram doses are used. In administering the adjuvanted vaccine combination of the inventionAfter this, a significant increase in IL-1B production, IFN- γ production and IL-15 production was observed, most particularly in the spleen. Taken together, these results clearly indicate the effect of the present adjuvant on cytokine responses and support the effect of cytokines on increasing cell-mediated, rather than humoral, immune responses.

The vaccines of the present invention also show a clear effect on fecal oocyst output. The uninfected control chickens did not shed any oocysts. Fecal oocyst output was significantly reduced in groups treated with Pfizer adjuvant alone after infection with eimeria maxima. Chickens in ovo vaccinated with crude eimeria maxima and adjuvant showed much less fecal oocyst excretion than those groups vaccinated with the crude eimeria maxima formulation alone (EM group).

It is noted that while purified recombinant Eimeria maxima protein 3-1E has been used to perform the foregoing experiments, the use of recombinant Ea3-1E, EaMIF and EtMIC2 antigens (either alone or in combination with 3-1E, or with each other, or as any combination of any of them) is also a preferred embodiment of the invention, and in general, all Eimeria protein antigens can be used to perform the invention, as long as they are mixed with the adjuvants of the invention.

Example 17 evaluation of E.coli strain J5 vaccine in cattle

One of the objectives of this study was to evaluate in cattle the immune response of cattle to an E.coli (strain J-5) antigen when administered in different novel formulations. The commercial vaccine J5 is sold as a prophylactic vaccine for enterobacter calves mastitis (coliformestimastitis), which is moderately effective in current formulations. The animals were determined to have low titers of E.coli J5 antibody prior to vaccination (analysis was based on serum blood samples taken prior to vaccination, respectively).

Beef cattle

Experimental vaccine prepared by using inactivated Escherichia coli J5 vaccine as antigenAnd was made according to example 13 above. Each treatment group initially contained 7 animals (table 5). One treatment group received saline (T01) and the other received the commercial J5 vaccine (T02-Enviracor)^TMPfizer J-5 E.coli bacterins). Other treatment groups received different formulations containing the adjuvants specifically identified in table 5. All vaccinations were administered by subcutaneous injection on study days 0 and 21. The volume administered was 5 ml.

TABLE 5 vaccine groups-beef cattle

In Table 5, QC is an abbreviation for Quil A/cholesterol, D is an abbreviation for DDA, C is an abbreviation for Carbopol, R is an abbreviation for R1005, X is an abbreviation for DEAE-dextran, O is an abbreviation for oil.

The following stock solutions were prepared according to the above examples 1 to 13, E.coli being about 4-5 × 10 per dose⁹Individual organisms (this was determined by direct counting by light microscopy). Quil A was dissolved in 50 mg/ml water, cholesterol in 17 mg/ml ethanol, DDA in 17 mg/ml ethanol, R1005 in 5 mg/ml phosphate buffered saline, DEAE dextran in 200 mg/ml water, TLR agonist in 20 mg/ml TE buffer and Iscomatrix in 5.4 mg/ml water. The individual components (v/v) are added in alphabetical order from left to right. For example: QCDC, add appropriate volume of Quil a, then cholesterol, DDA and finally Carbopol. When oil is included in the formulation, the separate components are mixed and added, followed by5LT mineral oil is emulsified in a mixture of Span 80 and Tween 80(QCDO) or Span85 and Tween 85 (QCDXO).Is a commercial light mineral oil.

Blood samples were collected on study days 0, 21 and 49 for serological testing. Antibody titers against E.coli J5 in serum samples were determined by J5 specific, indirect ELISA assay. IgG antibody isotypes were determined with sheep anti-bovine antibody conjugates (Bethy Labs). Titers were determined and expressed as geometric means thereof.

Results

The serological results of this study are shown in tables 6-8. Higher antibody titers are generally associated with better vaccine protection. The total J5-specific IgG titers are shown in table 6. Several formulations of the present invention produced significantly higher titers than the commercial product, although similar amounts of J5 antigen were added to these formulations. QCDO, QCDX and QCDXO formulations were particularly effective in inducing a good immune response in these cattle.

TABLE 6 IgG antibody titers

The J5-specific IgG1 antibody isotype was determined. These results are shown in table 7. Likewise, QCDO, QCDX and QCDXO formulations were particularly effective in inducing a good immune response in these cattle. Even with only one vaccine, these formulations can still produce much higher titers than when two commercial vaccines are injected.

TABLE 7 IgG1 antibody titers

IgG2 antibody titers are shown in table 8. This antibody isotype is generally associated with better milk neutrophil phagocytosis and protection of animals. QCDO, QCDX and QCDXO formulations were particularly effective in inducing a good immune response in these cattle.

TABLE 8 IgG2 antibody titers

Dairy cattle

An experimental vaccine was formulated using inactivated E.coli J5 vaccine as the antigen and produced according to example 13 above. Each treatment group initially contained 7 animals (table 9). One treatment group received saline (T01) and the other received the commercial J5 vaccine (T02-Enviracor)^TMPfizer J-5 E.coli bacterins). Other treatment groups received different formulations containing the adjuvants specifically identified in table 9. All vaccinations were administered by subcutaneous injection on study days 0 and 21. The volume administered was 5 ml.

TABLE 9 vaccine group-cows

In Table 9, QC is abbreviation for Quil A/cholesterol, D is abbreviation for DDA, C is abbreviation for Carbopol, R is abbreviation for R1005, X is abbreviation for DEAE-dextran, T is abbreviation for TLR agonist (CpG-ODN) and O is abbreviation for oil the following stock solutions were prepared, E.coli is about 4-5 × 10 per dose⁹Individual organisms (determined by direct counting by light microscopy). QuilA was dissolved in 50 mg/ml water, cholesterol in 17 mg/ml ethanol, DDA in 17 mg/ml ethanol, R1005 in 5 mg/ml phosphate buffered saline 20mM, DEAE dextran in 200 mg/ml water, and TLR agonist in 20 mg/ml TE buffer. The individual components (v/v) are added in alphabetical order from left to right. For example: QCDCR, add appropriate volume of Quil a, add cholesterol, DDA, and finally Carbopol. When in the formulationWhen oil is contained, the separated components are mixed and added, and then5LT mineral oil is emulsified in a mixture of span 80 and Tween 80(TXO, QCDO) or span85 and Tween 85.

Collecting blood

Blood samples were collected on study days 0, 21 and 49 for serological testing. Antibody titers against E.coli J5 in serum samples were determined by J5 specific, indirect ELISA assay. IgG antibody isotypes were determined with sheep anti-bovine antibody conjugates (Bethy Labs). Titers were determined and expressed as geometric means thereof.

Results

The serological results of the study are shown in table 10. Higher antibody titers are generally associated with better vaccine protection. Total J5 specific IgG titers are shown in table 10. Several formulations of the present invention produced significantly higher titers than the commercial product, although similar amounts of J5 antigen were added to these formulations. QCDO, TXO and QCDXO formulations were particularly effective in inducing a good immune response in these cattle.

TABLE 10 IgG antibody titers

The J5-specific IgG1 antibody isotype was determined. These results are shown in table 10. Likewise, QCDO, TXO and QCDXO formulations were particularly effective in inducing a good immune response in these cattle. Even with only one vaccine, these formulations can still produce much higher titers than when two commercial vaccines are injected.

This antibody isotype is generally associated with better milk neutrophil phagocytosis and protection of animals. The QCDXO formulations were particularly effective in inducing a good immune response in these cattle.

Example 18 bovine viral diarrhea Virus vaccine

Purpose of study

This study compared the safety, efficacy and cross-protection against challenge with BVDV-1 in naive (naive) calves against BVDV-1 in two killed type 1 and type 2 bovine viral diarrhea virus (BVDV-1 and BVDV-2 or BVDV-1/2) vaccines and a BVDV-1 and BVDV-2 extract vaccine formulated with the adjuvant of the present invention with a negative (saline) and two positive controls (modified live BVDV-2 vaccine and currently available killed BVDV-1/2 vaccine). The study design is shown in table 11.

The present study also shows that the adjuvants of the present invention can be used to distinguish between animals vaccinated with the vaccine composition of the present invention and animals naturally exposed to BVDV.

Animal(s) production

Healthy weaned beef cattle of either sex, 7 to 15 months old, seronegative for BVDV-1 and BVDV-2 were used.

TABLE 11 study design

QC for Quil A/cholesterol, D for DDA, C forAbbreviation of (1), R is BayAbbreviations of (a).

Vaccination

On study days 0 and 21, animals (N10/group) were vaccinated as described in table 11. The administered antigen (BVDV) was 5,500 relative potency units (RU) per dose (determined by ELISA assay). Calves in the T01 group served as control groups. The control group received a sterile solution of 0.9% sodium chloride. Groups T02 through T06 received experimental BVDV1/2 vaccine with the adjuvants shown in table 11. The T02 group received only one vaccination (study day 0). It received a Modified Live Virus (MLV) BVDV-2 vaccine without adjuvant. Group T03 received a solution containing 2.5% oil-in-water emulsionAnd Quil a/cholesterol adjuvantThe killed viral BVDV-1/2 vaccine. Group T04 received killed viral BVDV-1/2 vaccine containing Quil A/cholesterol, DDA and Carbopol. Group T05 received killed viral BVDV-1/2 vaccine containing Quil A/cholesterol, DDA, Carbopol and R1005. Group T06 received a killed virus BVDV-1/2, a high titer extract vaccine containing Quil A/cholesterol, DDA and Carbopol on day 0 and a similar low titer extract vaccine on day 21. All treatments, except group 2, were given a 2 ml dose by the intradermal route on days 0 and 21.

QCDC +/-R contains 100 micrograms Quil A, 100 micrograms cholesterol, 50 micrograms DDA, and 0.075% Carbopol, with 1000 micrograms of R1005 per 2 milliliter dose, as previously described.

Attack of

5.4log per 5ml dose by intranasal route at a concentration of about 4 ml (about 2 ml per nostril) on day 42¹⁰TCID₅₀The non-cell-diseased BVDV-1 strain (NY-1 strain; CVB, USDA, Ames, IA) of (A) challenged all animals.

Observation of

Injection site observations were recorded at the first injection site (left neck) on study days 0 (pre-vaccination), 1, 2, 3, 7 and 21. Observations of the second injection site (left neck) were recorded on study days 21 (pre-vaccination), 22, 23, 24, 28 and 35. All palpable injection site responses (LxWxH, cm) were measured. Rectal temperatures at the time of initial vaccination were recorded at study days-1, 0 (pre-vaccination), 1, 2 and 3. The temperature of the booster vaccination was recorded at study days 20, 21 (before vaccination), 22, 23 and 24.

Taking a blood sample

Blood samples from each available animal were collected on study days-1, 20, 34 and 49 using Serum Separation Tubes (SSTs). Blood samples were collected using EDTA tubes on study days 33 to 35 (pre-challenge) and 36 to 49. Blood samples were collected using Cell Preparation Tubes (CPT) on study days 34 (pre-challenge) and 36 to 49.

Results

Table 12 shows Geometric Least Squares Mean (GLSM) serum neutralizing antibody titers against BVD virus as determined by coagulation analysis on the day of the study. The results show that the adjuvants of the invention increase the potency against BVDV-1 and BVDV-2 as the study progresses. An acceptable titer for UDSA is a titer above 8. These data show titers above 5,000, indicating that large amounts of antibodies capable of stopping live virus can be produced when the virus enters potentially infected and diseased animals.

TABLE 12 serum antibody neutralization titers

Table 13 presents the leukopenia data at study days 43-56. The results of the daily leukopenia studied demonstrated that the MLV vaccine (T02) could prevent infection by specific viral challenge. The measurement of leukopenia is the standard for USDA nuclear MLV product licensing. However, for inactivated viruses, leukopenia is not the standard for USDA, and this data suggests that the adjuvants of the invention cause leukopenia only in up to 20% of animals, whereas most inactivated virus vaccines can cause 100% leukopenia. This indicates that the adjuvants of the invention can drive a strong Th1 response with inactivated antigen. This is difficult and rare in inactivated preparations.

TABLE 13 leukopenia on the following study date

Table 14 presents the serum neutralization titers at day 41 (20 days after the second vaccination, before challenge). Modified live viruses can only develop an antibody response to the exact virus in the vaccine. This is evident from the fact that group T02 only shows protection against BVDV-2. However, adjuvanted inactivated vaccines of T03(PreZent-a), T04(QCDC) and T05(QCDCR) can generate strong antibody responses early in the initiation of immunization and throughout the life of animal studies against a serologically divergent panel of BVDV. This shows the ability of these adjuvants to provide safety and efficacy in a challenge model, thereby protecting cattle in both homologous and heterologous challenges.

TABLE 14 serum neutralization titers at day 41

The activity of the marker. The data presented herein show that the adjuvants of the invention can be used to distinguish between animals vaccinated with the vaccine compositions of the invention and animals naturally exposed to BVDV. This can be seen by determining the difference in antibody profiles between the constitutive and non-constitutive gene products of the virus. This marker activity was demonstrated by gel electrophoresis using radioimmunoprecipitation analysis (FIG. 1). The antibody responses to NS2/3 and E2 proteins of BVDV were very significant in animals vaccinated with MLV vaccine or in animals naturally exposed to adjuvanted inactivated vaccine of BVDV or PreZent-A. However, the adjuvants of the present invention showed antibody responses only to the E2 protein, and not to the NS2/3 protein. Thus, animals vaccinated with an inactivated BVDV vaccine comprising the adjuvant of the present invention can be distinguished from animals naturally infected, or vaccinated with MLV, or vaccinated with the PreZent-a vaccine. This can be considered as a valuable marker vaccine for eradicating these disease types in a population of animals.

Example 19 Mycoplasma hyopneumoniae in Swine

Background

Mycoplasma hyopneumoniae (MPS) or epidemic pneumonia is a widespread chronic disease characterized by cough, growth retardation, and reduced feeding efficiency. The pathogenic agent is mycoplasma hyopneumoniae; however, naturally occurring diseases are often caused by infection of bacteria in combination with mycoplasma.

MPS causes considerable economic losses in all pig farming areas. Investigations conducted in various parts of the world have indicated that typical lesions seen in MPS can occur in 30% -80% of slaughter-weight (slauguer-weight) pigs. The actual incidence may be higher since the damage caused by mycoplasma may be eliminated before the pig reaches slaughter weight only. The prevalence of mycoplasma pneumoniae infection in chronic swine pneumonia is reported to be 25% to 93%. Pigs of all ages were susceptible to MPS only, but the disease was most common in growing and finishing pigs. Current evidence indicates that the mycoplasma pneumoniae line is transmitted by aerosol or direct contact with respiratory secretions from infected pigs. It is possible to transmit from the sow to the piglet during lactation. Once infection is established, MPS develops year-to-year in infected herds, varying in severity with environmental factors such as season, ventilation, and pig density.

Purpose of study

The aim of this study was to compare the efficacy of a mycoplasma hyopneumoniae vaccine formulated with the novel adjuvant of the present invention with the efficacy of a commercial experimental series of mycoplasma hyopneumoniae bacterins after an intratracheal challenge with a toxic mycoplasma hyopneumoniae lung homogenate.

Animal(s) production

Sixty-six (66) days old, no history of disease caused by mycoplasma hyopneumoniae and PRRSV, or clinically healthy crossbred pigs vaccinated against the same organism, were used in the study. Before delivery to the study site and 2 days after arrival, toPigs were treated intramuscularly in the hind leg (as indicated by the label) to prevent stress-related diseases (such as streptococcus suis). Animals were assigned to treatment groups and pens according to a randomized schedule. The study design is shown in table 15.

TABLE 15 Experimental design

QAC is an abbreviation for Quil A/cholesterol.

Veterinary preparation for research (IVP)

Antigens and veterinary preparations for study (IVP) are shown in table 16. Vaccines for treatment groups T02, T03 and T04 (all but T05) were prepared according to example 13 using the component concentrations shown in table 16 below. The components were added in the order listed in the table.

The brine synergist was added to the vessel and homogenization was started and continued through the whole preparation step. Inactivated mycoplasma hyopneumoniae was prepared from a mixed volume of 75 liters of fermentate per 800 liters of final formulated product and added to a concentration of 0.09375ml per dose. Quil A was added to the concentrations listed in Table 16. Then, a cholesterol/ethanol solution was added. DDA/ethanol solution was added, followed by BayR1005 glycolipid solution. Carbopol was then added and the solution brought to final volume with saline extender.

The Vaccine used to treat group T05 (Amphigen Based Vaccine formulation) was a commercially available product(Pfizer,Inc)。

TABLE 16 veterinary products for research (IVP)

Vaccination

Animals in the NTX-treated group were not vaccinated or challenged. Animals in T01, T02, T03, T04, and T05 were vaccinated intramuscularly at 2 ml per dose by qualified individuals blinded to the treatment group at approximately 3 weeks of age (day 0-right neck) and 5 weeks of age (day 14-left neck).

Attacking material

Animals from T01 to T05 were challenged by the intratracheal route 3 weeks after the second vaccination (approximately 8 weeks old-study day 35). Animals were challenged with a 5ml dose of a frozen lung homogenate (1: 50 dilution in Friis medium) of 10% mycoplasma hyopneumoniae strain 11(LI 36).

Blood sampling sample

Blood samples (approximately 5 to 10 ml in serum separator tubes) were collected from all pigs and tested for mycoplasma hyopneumoniae serology (ELISA-IDEXX) on day-1 or 0 (prior to first vaccination), day 13 or 14 (prior to second vaccination), day 34 or 35 (prior to challenge) and day 63 (at necropsy).

Weight (D)

All animals were weighed at the time of arrival for grouping purposes, on day 34 or 35 (pre-challenge), and on day 62 or 63 (pre-necropsy).

Necropsy examination

On day 63, all surviving animals were euthanized according to site-specific procedures. The lungs were visually assessed for characteristic lesions caused by mycoplasma hyopneumoniae infection and scored for lesions caused by mycoplasma hyopneumoniae challenge. The lung injury score was recorded as the percentage of lung injury of each lung lobe. The percent solidity of each lobe (left anterior, left middle, left posterior, right anterior, right middle, right posterior, and appendages) was scored as an actual number between 0-100%. The percentage of each lobe can be used in a weighted formula to calculate the total percentage of lungs with lesions. Six (6) NTX animals were necropsied and their lung scores were recorded prior to challenge on day 34 or 35.

Lung injury scoring

The percentage of total lung with lesions was calculated using the formula: the percentage of total lung with lesions { (0.10 × left anterior leaf) + (0.10 × left middle leaf) + (0.25 × left posterior leaf) + (0.10 × right anterior leaf) + (0.10 × right middle leaf) + (0.25 × right posterior leaf) + (0.10 × accessory site) }. Arcsine square root transformation (arcsine square root transformation) was applied to the percentage of total lungs with lesions prior to analysis. The transformed lung lesions were analyzed in a general linear mixture model. Linear combinations of parameter estimates are used in prior comparisons (prior comparisons) after testing the effect of the treatment. The back transformed least squares means (backshifted least squares means), its standard deviation and its 90% confidence interval, as well as the minimum and maximum values, were calculated as a significant (P ≦ 0.10) percentage of total lungs with lesions.

Results

As shown in the results of Table 17 below, the adjuvants of the present invention are mixed with adjuvantsThe oil-adjuvanted T05 group showed equal performance. Typically, a lung injury score of less than 3 is considered to have had the efficacy provided by the vaccine treatment. The adjuvant combinations of the invention all meet this criteria and QCDCR performs best in terms of score and range between individual animals.

Each group N-12 except T05 (where N-11)

Example 20 Feline Avian Influenza Virus (FAIV)

The study evaluated the efficacy of influenza vaccines using the adjuvants of the invention in cats by challenge with virulent strains of avian influenza virus.

Method and results

Before vaccination, animals were determined to be both influenza virus negative and anti-influenza virus antibody negative based on oropharyngeal swabs taken before vaccination and serum blood samples analysis, respectively.

An experimental vaccine is prepared by using inactivated pathogenic avian influenza virus and purified Hemagglutinin (HA). Each treatment group initially contained 6 animals (table 18). Two treatment groups received experimental FAIV vaccine (T01 vaccine antigen is purified H5HA protein; T02 vaccine antigen is inactivated H5N2 strain), one treatment group received inactivated modified H5N1 strain vaccine (T03), one placebo control group received adjuvant-only vaccine (T04) and one negative control group received saline-only (T05). All vaccines were administered by subcutaneous injection on study days 0 and 21. The administration volume was 1 ml. After vaccination, the animals were observed continuously until they recovered and allowed to sit upright to determine no adverse reactions. The observations were recorded about 1 hour after vaccination and any other complications observed after vaccination.

The adjuvant composition was previously described by example QCDC above using QuilA (20 micrograms), cholesterol (20 micrograms), DDA (10 micrograms) and Carbopol (0.05%) per dose. The antigen is inactivated whole virus or purified H5HA protein.

The animals were evaluated for response at the injection site and serological response to the vaccine. Three animals (two in the T02-inactivated H5N2 group and one in the T05-saline group) were euthanized prior to challenge due to congenital hyperoxaluria. On study day 49, all surviving cats were challenged with the virus strain H5N 1A/Vietnam/1194/04 via the intratracheal route to evaluate the efficacy of the vaccine candidates. 10 in 5.0 ml⁵TCID₅₀The animal is attacked and the material is released just above the bifurcation using a bronchoscope, a small tube introduced into the trachea. After challenge, animals were observed and sampled for 5 days. At the end of the animal phase (study day 54), all animals were euthanized and necropsied for each.

TABLE 18 vaccine groups

Blood samples were collected for serological testing prior to vaccination on study day-14, on days 0, 21 and 49. Blood samples were collected for virology testing at study days 49 and 54. Unplanned (unscheduled) blood samples were taken from all surviving animals on study day 42 to test kidney function against pre-challenge sera.

Oropharyngeal swabs were collected from all animals on study day-14, study day 49 prior to challenge, and on days 50 to 54. Rectal swabs were collected from all animals on day 49 and from days 50 to 54 of the pre-challenge study. Swab collection was completed on study day 49 just prior to challenge.

During necropsy, all lung lobes were removed aseptically, weighed, and visually assessed for characteristic lesions due to FAIV infection. The percentage was used to identify the degree of lung consolidation. The left lung was fixed in 10% neutral-buffered formalin for histopathological examination. The right lung was collected and sampled for virological testing. In addition to the lung, kidney samples and any macroscopic tissues were also taken and stored in 10% neutral-buffered formalin for histopathological examination.

Viral titers were determined in blood samples, oropharyngeal and rectal swabs, lung tissue samples by H5N 1-specific TaqMan PCR. Briefly, RNA was isolated using the MagnaPure LC System with the MagnaPure LC Total nucleic acid isolation kit (Roche diagnostics; Albere, Netherlands) and influenza A virus was detected using real-time RT-PCR analysis. Data are expressed in Control Dilution Units (CDU). CDU was determined from a standard curve generated from serially diluted virus stocks, each dilution having been subjected to nucleic acid extraction and TaqMan PCR amplification in the same manner as the test sample.

RT-PCR positive oropharyngeal swabs and lung tissue samples were also analyzed by virus isolation and titration on Madine-Darby canine kidney (MDCK) cells. Results are expressed as log per milliliter or gram of sample₁₀50% tissue culture infectious dose (log)₁₀TCID₅₀Per ml or log₁₀TCID₅₀/g)。

Plasma samples were analyzed by virus neutralization and hemagglutination inhibition. In the Hemagglutination Inhibition (HI) assay, viral suspensions of the influenza strains vietnam 1194/04(H5N1, clade 1) or indonesia 05/2005(H5N1, clade 2) were incubated with serial (2-fold) dilutions of serum samples pre-treated with cholera filtrate (taken from vibrio cholerae cultures). Next, erythrocytes were added to the dilution and after incubation, the maximum dilution of the agent showing complete inhibition of hemagglutination was defined as the HI titer.

Virus Neutralization (VN) analysis was based on endpoint titration of sera. Briefly, a fixed amount of virus was mixed with a series (2-fold) dilution of the serum sample. Virus neutralization was read using MDCK cells as indicator cells and visualized by erythrocyte agglutination. VN titers were scored using the highest dilution of serum in which 50% of the inoculated cell cultures showed hemagglutination.

The left lung was collected at necropsy and fixed in 10% neutral-buffered formalin for histopathological examination. After fixation, the tissues were embedded in paraffin, and tissue sections were prepared and stained with hematoxylin and eosin for histological examination. The description and extent of the pathological changes observed were recorded.

Results

None of the animals in the 5 treatment groups showed any pain or swelling at the injection site after the first and second vaccination. Furthermore, no skin abnormalities were recorded at the injection site. Body temperature did not differ significantly at the 0.1 significant level between treatment groups by linear mixed model analysis after vaccination and before challenge. One T01 animal developed a fever (. gtoreq.40 ℃) before and several days after the first vaccination on day 0. After vaccination (days 0 and 21), a burst temperature increase of up to 40 ℃ or more was recorded in individual animals. No abnormal health associated with vaccination was observed during the study. Three animals (two in the T02-H5N2 group and one in the T05-saline group) were euthanized by congenital hyperoxaluria prior to challenge. Several animals from all treatment groups developed wound complications after implantation of the thermograph. No concomitant treatment was applied from day 0 to study completion.

Vaccinated T01, T02 and T03 animals showed fewer clinical signs and no mortality after challenge compared to control T04 and T05 animals. In T01, one animal showed depression and increased respiratory effort 2 days after challenge. None of the remaining five T01 animals showed any abnormal health after challenge. In T02 (n-4) and T03 (n-6), all animals remained healthy after challenge. In T04(n ═ 6), the first clinical signs of abnormalities (depression and increased respiratory effort) were seen in both animals 2 days after challenge. All 6 animals in T04 seen depression and increased respiratory effort 3 days after challenge. Therefore, for reasons of euthanasia, 2 animals had to be euthanased. 4 days after challenge (day 53), one animal was found to have died, the remaining 3T 04 animals showed depression, increased respiratory effort, third eyelid prolapse (third eyelid prolapse), and runny nose, and had to be euthanized for reasons of peace. In T05(n ═ 5), the first clinical signs of abnormalities (depression and increased respiratory effort) were seen in 1 animal 1 day after challenge. 2 days after challenge, another 2 animals began to show these signs. One animal was found to have died 3 days after challenge, and the remaining 4 animals showed depression, increased respiratory effort and third eyelid prolapse. One animal was then euthanized for euthanasia reasons. 4 days after challenge, 1 out of the remaining 3 animals had deteriorated respiratory effort, and another animal additionally showed rhinorrhea. 4 days after challenge (day 53), all remaining 3 animals received euthanasia for euthanasia reasons.

After challenge, the mean body temperature of vaccinated animals (T01, T02 and T03) was kept below 40 ℃. The mean temperature of the control animals (T04 and T05) rose to ≥ 40.0 ℃ the day following challenge. The difference in mean body temperature between treatment groups was significant by linear mixed model analysis (p ═ 0.0001). Individual animal data showed that the body temperature of a small number of T01, T02, and T03 animals rose to 40.0 ℃ and higher at sporadic time points on day 53. 2 animals in T01 developed fever at one time point (ranging from 40.0 to 40.1 ℃). In T02 2 animals developed fever (ranging from 40.0 to 40.3 ℃) at one and three time points, respectively. In T03, 1 animal had a fever at three time points (ranging from 40.0 to 40.3 ℃). All animals in T04 and T05 had a fever of at least 12 hours between days 50 and 51.

HI antibody titers were determined against influenza strains vietnam 1194/04(H5N1, clade 1) and indonesia 05/2005(H5N1, clade 2) prior to the first and second vaccinations and prior to challenge. The lower limit of detection is 5. Before vaccination, titers in all 5 treatment groups were below the lower limit of 5. All vaccinated (T01, T02 and T03) animals developed HI antibody titers above 5 after vaccination and showed at least a 6-fold increase in titer compared to the values before vaccination. In T01 and T03, titers against vietnam 1194/04 ranged from 20 to 160 after the first vaccination and from 140 to 960 after the second vaccination. In T02, titers against vietnam 1194/04 were lower than those measured in T01 and T03, ranging from 5 to 30 after the first vaccination and from 5 to 70 after the second vaccination. The HI antibody titers against indonesia 05/2005 were similar to those against vietnam 1194/04.

Plasma samples taken before and after challenge were tested for viral load by H5N1 specific real-time RT-PCR. All animals had virus negative samples prior to challenge. After challenge, no virus was detected in the plasma of T01 and T03 animals. In contrast, 25% (one-fourth) of the T02 animals, 67% (four-sixths) of the T04 animals, and 60% (three-fifths) of the T05 animals were virus-positive in plasma after challenge. The differences between treatment groups were significant by linear mixture model analysis (p ═ 0.0247).

Viral shedding was assessed in challenged throat swab samples by real-time RT-PCR and virus titration, and rectal swab samples were tested for viral shedding (shedding) by real-time RT-PCR. After challenge, no viral shedding was detected in the throat of T01 animals. In T02, all 4 animals (100%) shed virus at one point after challenge. In T03, 2 of 6 animals (33%) shed virus after challenge. In T04, 3 out of 6 animals (50%) shed virus after challenge. In T05, 4 of 5 animals (80%) shed virus after challenge. No samples were taken from T04 and T05 animals 5 days after challenge, as all animals began to die since then. However, for statistical analysis, the final test results for animals that had died or were euthanized prior to the last day of the study were performed prior to the last day of the study.

Throat samples with RT-PCR positive results (. gtoreq.1.8 CDU) were also used in the virus titration analysis. Virus titration confirmed that all RT-PCR positive samples contained infectious influenza virus (data not shown). In vaccinated animals (T02 and T03), the infectious virus titer was lower than in control animals (T04 and T05). These differences were significant when T02 or T03 was compared to T04 3 days post challenge, and when T02 or T03 was compared to T05 3, 4 and 5 days post challenge. Titers in T02 and T03 animals were 0.5log₁₀TCID₅₀. Titers observed in T04 ranged from 2.3 to 4.3log₁₀TCID₅₀. Titers in T05 ranged from 1.5 to 3.8log₁₀TCID₅₀。

Viral shedding assessed by rectal swabs in feces was detected in all treatment groups (except T02) 3 or 4 days post challenge. The amount of virus detected by RT-PCR was 2.2 to 2.3log in T01₁₀3.2log in CDU and T03₁₀2.0 to 2.7log in CDU, T04₁₀CDU and 2.2log in T05₁₀The CDU. At a significant level of 0.1, there was no significant difference between treatment groups on any day post challenge.

In vaccinated animals (T01, T02 and T03), lung disease was less severe than in control animals (T04 and T05). All vaccinated animals developed mild, multifocal, subacute bronchiogenic interstitial pneumonia. The control animals showed subacute bronchogenic interstitial pneumonia either moderate (two T04 animals and one T05 animal) or severe (four T04 animals and four T05 animals), all animals showed multifocal distribution, except two control animals (one T04 animal and one T05 animal) showed diffuse distribution. The extent of consolidation of the entire lung was assessed, expressed as a percentage of consolidation of total lung tissue. Consistent with the discovery of lung lesions, the percent of solid lesions in vaccinated animals (T01, T02, and T03) was significantly lower than in control animals (T04 and T05).

Viral load in lung tissue collected at the time of death or euthanasia of the animals was assessed by viral titration and H5N1 RT-PCR. Lung tissues from vaccinated animals (T01, T02 and T03) had significantly lower mean viral titers than from control animals (T04 and T05). There was no significant difference between the mean titers of lung tissue from vaccinated animals (T01, T02, and T03). The same results were generated by RT-PCR analysis.

Clinical signs observed in control animals receiving adjuvant (T04) or saline (T05) after challenge with a highly pathogenic H5N1 avian influenza strain include fever, death, viremia, virus shedding from the throat and stool, viral infection of the lungs, and lung lesions including consolidation.

Vaccination with 6 kittens of purified H5HA protein (T01) prevented viremia, throat shedding of virus and death following challenge with the highly pathogenic H5N1 avian influenza virus strain. Furthermore, vaccination with purified H5HA protein (T01) reduced clinical signs including fever, viral load in the lungs, and lung lesions including consolidation.

Vaccination with 4 kittens of inactivated H5N2 strain (T02) prevented clinical signs, virus excretion from feces, and death following challenge with the highly pathogenic H5N1 avian influenza virus strain. Furthermore, vaccination with the inactivated H5N2 strain (T02) reduced viremia, fever, virus shedding from the throat, viral load in the lung, and changes in lung disease (including consolidation).

Vaccination with the inactivated H5N1 strain (T03) as 6 kittens could prevent clinical signs, viremia and death following challenge with the highly pathogenic H5N1 avian influenza virus strain. Furthermore, vaccination with the inactivated strain H5N1 (T03) reduced fever, virus shedding from the throat, viral load in the lungs, and changes in lung disease (including consolidation).

Abstract

When using vaccines formulated with inactivated or purified HA antigen with QC/DC adjuvant, no injection site reactions were observed. The vaccine can provide complete protection against clinical disease and death in vaccinated cats, significantly reducing viral load in blood and tissues and significantly reducing viral shedding.

Example 21 cancer

Background

The study was performed in immunodeficient and immunocompetent (immunocompetent) rats using human and rat hepatocellular carcinoma cells to generate ectopic and orthotopic models.

Animal(s) production

Nude (Crl: NIH-rnu) male mice 6-8 weeks old were purchased from Charles River (Wilmington MA). Rats were placed in pairs in small isolation cages of polycarbonate and provided with randomly accessible reverse osmosis water (reverse osmosis water) and irradiated standard rat food; all water and beds were autoclaved. Record body weight twice a week; animals were maintained for approximately 7 weeks by CO inhalation at the end of the experiment₂Euthanasia was performed.

The experimental design incorporates two stages. In phase I, rats are randomly divided into two groups according to their body weight. Rats in group 1 received no tumor cell injection, while rats in group 2 received subcutaneous tumor cell injection. Group 2 rats were randomized (by tumor size and rate of arrival (see table 19)) into two groups for use in stage ii 3 weeks after tumor injection, one of which included two subgroups: 1) non-tumor bearing control group (control group) receiving saline; 2) tumor-bearing control group treated with adjuvant only (tumor group); and 3) tumor bearing subjects (tumor treatment groups) given vaccine doses (two subcutaneous injections given two weeks apart). All animals were necropsied 14 days after the second vaccine administration.

Vaccine

0.2 ml of vaccine per dose was administered by subcutaneous injection.

TABLE 19 vaccine groups

QC for Quil A/cholesterol, D for DDA, C forAbbreviation of (1), R is BayAbbreviation of (1), abbreviation of PBS for phosphate buffered saline

Vaccine preparation

Quil-A (20. mu.g/dose), cholesterol (20. mu.g/dose), DDA (10. mu.g/dose), Carbopol (0.05%), with or without the glycolipid Bay(1000 microgram/dose) and antigen. The compositions were blended using a homogenizer and added in the order described above.

Antigen preparation

HepG2 cells (clone HB-8065) were obtained from the American Type Culture Collection (ATCC, Manassas, Va.) HepG2 is an immortalized cell line derived from the liver tissue of well-differentiated hepatocellular carcinoma of the 15-year-old Caucasian male (Caucasian male). cells were expanded under standard cell Culture conditions and made 1 × 10 in Matrigel (Matrigel)⁷Cell/ml concentration for injection. Each rat was injected with 0.5 ml of cell suspension at the second treatment site by subcutaneous route.

Measuring

Tumor size was measured twice weekly throughout the study by caliper, where volume (in cubic centimeters) is { [ W (millimeters) × W (millimeters)]/2 × L (millimeters) }/1000 blood was collected by post-ocular sampling for serum chemical and biomarker measurements with CO during the sampling procedure₂/O₂Animals were lightly anesthetized. The chemical end point was analyzed using a Hitachi917 automatic analyzer (Roche, Indianapolis, IN). The final blood sample line is CO₂Under anesthesia it was taken by cardiac puncture. Analysis using commercially available ELISA: Alpha-Feto Protein (R)&D Systems, Minneapolis MN) and Human Albumin (Bethy Laboratories, Montgomery TX) to assess serum endpoints. By inhaling CO₂Animals were euthanized. Tumors were excised, weighed and placed in formalin for histological examination.

Student unpaired T-test (Student's unpaired T-test) was used to compare multiple parameters between treated and control rats. All values are expressed as mean ± SD and p <0.05 are considered statistically significant.

Results

The measurements of body weight were corrected by subtracting the tumor weight (1 gram from the volume data and assuming 1 cc). Data were analyzed in two ways: by treatment group and by tumor bearing animals or non-tumor bearing animals. When comparing the body weights of tumor-bearing animals and non-tumor-bearing animals, there was a significant difference between the groups at the final time point; there was no difference at baseline. Even though there was no significant difference in body weight when comparing treatment groups, possibly due to the short study period, there was a perceptible tendency for weight loss in both tumor-bearing groups relative to the control group, and the animals receiving the vaccine had a positive tendency towards recovery when compared to tumor-bearing animals receiving vehicle without antigen (table 20). Furthermore, the percentage change in body weight during the experiment and the tumor volume at the end (r)²0.72) or tumor weight (excised) (r)²0.73) has a reasonable relationship.

Table 20. body weight change between experimental groups after a period of time. The shaded area indicates the date of vaccine administration.

Tumor size

Tumor size of control rats and tumor size of vaccine-treated rats were compared over a four week period. Although no statistically significant difference was found between the groups compared (due to the large change in tumor size), the total tumor volume tended to decrease strongly (table 21) and the mean excised tumor weight of the rats receiving the vaccine (table 22) also decreased.

Table 21. tumor size change between vehicle-treated (tumor) and vaccine-treated (treated tumor). The shaded area indicates the date of vaccine administration.

Date	Tumor(s)	Treated tumors
			6	4.6±2.1	4.7±2.0
9	4.9±2.0	4.8±1.8
			13	4.6±1.8	5.0±2.2
16	12.2±2.7	12.3±2.5
			20	22.6±3.8	13.4±2.6
23	33.3±4.8	14.1±2.8
			27	34.5±4.6	13.8±2.8
30	35.6±4.6	13.5±3.4
			34	37.7±8.0	14.1±2.9

37	38.6±10.2	13.7±2.3
			41	44.5±12.1	12.5±2.1
44	52.9±13.9	13.0±2.1
			48	61.7±15.1	16.9±2.9

Table 22 tumor weight differences at necropsy.

Serum analysis

Human alpha-fetoprotein (AFP) was measured by ELISA at different time points during the study. These data were used together with body weight and tumor size data to arbitrarily assign animals to treatment groups. Data from this study and historical data indicate that AFP can only be detected in animals with tumors. Comparison of the longitudinal AFP data for the tumor control and treatment groups indicated that the AFP of the animals of the treatment group was reduced after the first vaccine injection and at the end of the study, the AFP of the animals of the treatment group was much lower relative to the AFP of the animals of the tumor-bearing control group; 4.78. + -. 3.2 ng/ml for vehicle treated rats relative to 0.97. + -. 2.5 ng/ml for vaccine treated rats, respectively. In addition, AFP appears to be related to both tumor volume and excised tumor weight.

Human albumin (hALB) was measured by ELISA at various time points during the study. Data from this study and historical data indicate that hALB can only be detected in tumor bearing animals. Comparison of the hALB data for the tumor control and treated groups indicated that at the end of the study, the hALB of the rats in the treated group was much lower relative to the rats in the tumor-bearing control group (data not shown). Furthermore, like AFP, hALB showed correlation with both tumor volume and excised tumor weight (data not shown).

The core serochemistry groups were analyzed at different time points throughout the study. This group includes AST, ALT, cholesterol, alkaline phosphatase, GGT, BUN, glucose, creatine, total bilirubin, total protein, albumin, globulin, and the minerals Ca, P, Na, K, and Cl. Similar to body weight, data were analyzed in two ways: by treatment group and by tumor bearing animals or non-tumor bearing animals. The only endpoints where a difference was observed were: AST, ALT and cholesterol. There were no significant differences between groups at the baseline time point by both comparisons (data not shown). When comparing the chemical indices of tumor-bearing animals to non-tumor-bearing animals, there was a significant difference between the groups at the final time point, and AST, ALT, cholesterol was increased in tumor-bearing animals (data not shown).

Conclusion

Our data were pooled to demonstrate that the tumor burden was reduced in animals treated with the vaccine prepared against HepG2 tumor cell line relative to the tumor-bearing control group that received the vehicle.

Example 22 CpG

Background

The adjuvant described herein is a strong vaccine adjuvant platform that can be enhanced by using the adjuvant as a delivery system for CpG, by boosting the immune response using ORN/odn (CpG).

Materials and methods

Female C57BI/6 mice weighing approximately 18-20 grams (n-10/group) were used in the study. On days 0, 14 and 21 of the study, the mice were immunized by injecting a total volume of 50 microliters by intramuscular route (IM) into the left tibialis anterior muscle.

Dosage of reagents

One dose of the composition comprises one or more of the following in various combinations:

buffer solution: NaH₂PO₄·2H₂O (229.32 mg/L), NaCl (1168.00 mg/L) and Na₂HPO₄(1144.00 mg/l), dissolved in WFI and sterile filtered with a 0.1 micron filter

Ovalbumin (OVA-antigen): 10 microgram of

CpG ODN: 10 microgram of

Cholesterol: 1 microgram of

Quil A: 1 microgram of

DDA: 0.5 microgram

Carbopol：0.0375％

R1005: 50 microgram

Vaccine preparation

The buffer was placed in a 50 ml flask with a stir bar and stirred at a fixed speed throughout the following steps. The components were added in the following order: antigen (OVA); CpG ODN; quil A; cholesterol (dropwise); DDA (dropwise);and BayThe composition was stirred at room temperature (about 25 ℃) for a minimum of 30 minutes while being shielded from light by covering with aluminum foil paper. The solution was forced through a 25G needle into a syringe to break up any large floating particles, and a homogeneous (cloudy) suspension was taken, which was then transferred to a sterile glass vial for storage.

Sample collection

The following samples were collected:

plasma: 4 weeks after the initial vaccination (1 week after the second booster vaccination)

Cytotoxic T Lymphocytes (CTL) (6 weeks after primary vaccination (3 weeks after secondary booster vaccination))

Cytokine secretion in supernatant (4 weeks after initial vaccination)

24-hour supernatant (IL-2, IL-4, IL-10; TNF)

72-hour supernatant (IFN-g)

Tetramer (4 weeks after initial vaccination)

Cytokine-producing T cells (6 weeks after initial vaccination)

The results are provided as relative scores for each adjuvant, showing the effect of the adjuvant. The endpoints are relative scales based on the sum of individual cytotoxic T lymphocyte responses.

Results and discussion

As shown in table 23, QCDCR plus OVA can produce stronger CTL responses than their subcomponents, however, the overall response was lower (< 20%). Combining QCDCR or its subcomponents with CpG can significantly improve OVA-specific CTL responses. Overall QCDCR/CpG plus OVA produced the strongest CTL responses, however, there was no significant difference in response between this group and cholesterol/CpG plus OVA (ratio 25: 1). Culture supernatants from spleen cells stimulated with OVA (1 mg/ml) were analyzed for cytokines by ELISA. QCDCR alone or its subcomponents produced only very weak cytokine responses. Combining QCDCR or its subcomponents with CPG increased the secretion of antigen-specific IL-2 and IFN-g (Th1 shifted cytokine). QCDCR is equally potent as CpG in amplifying cellular immune responses. The combination of the two shows a synergistic effect. When the subcomponents of QCDCR were analyzed with CpG, the combination with Quil a gave the best response, followed by the inclusion of cholesterol and CpG.

TABLE 23 relative CTL responses

Group of	CTL	IFN-g	Tetramer	IL-2	Total of
						QCDCR-CpG+OVA	18	16	18	18	70
QCDC-CpG+OVA	15	18	17	16	66
						Ch+CpG+CR+OVA	12	14	15	17	58
Ch+CpG+DC+OVA	8	17	13	15	53
						Ch+CpG+DCR+OVA	16	9	14	13	52
C+CpG+OVA	9	13	16	11	48
						DCR+CpG+OVA	13	13	12	9	47
CR+CpG+OVA	10	10	11	8	39
						DC+CpG+OVA	11	11	8	6	36
CpG+OVA	14	8	9	3	34
						QCDCR+OVA	7	5	7	14	33
CR+OVA	5	7	4	7	23
						QCDC+OVA	3	6	2	10	21
CR+OVA	4	3	5	4	16
						DCR+OVA	6	2	6	2	16
DC+OVA	2	4	3	5	14
						OVA	1	1	1	1	4

QC for Quil A/Cholesterol, Ch for Cholesterol, D for DDA, C forAbbreviation of (1), R is BayAbbreviations of

Example 23 Canine Coronavirus (CCV)

Range of

The adjuvant potency with the indicated antigenic component was assessed using a murine model using Canine Coronavirus (CCV) and a novel combination adjuvant.

Animal(s) production

There were 10 CF-1 mice in each treatment group, and 0.2 ml was administered to each animal in each treatment group by subcutaneous route.

Treatment group

The test formulations shown in table 24 were prepared in 1.0 milliliter surfactant (fielddose) volumes with the following indicated concentrations. Only 0.2 ml of vaccine was administered per mouse.

TABLE 24 test formulations

Vaccine preparation

Vaccine preparation of the adjuvants of the invention is described in examples 1-13 above. The concentrations of the adjuvant components are provided in table 24. Adjuvants were added in the order in the table.

Saline synergist was added to the tube and homogenization was started. The concentrations of inactivated CCV shown in table 24 were added. Quil A was added at the concentrations listed in Table 24. Then, cholesterol in ethanol solution was added and homogenization continued. Then, DDA/ethanol solution was added during homogenization. The mixture was microfluidized at 10,000 psi. Carbopol was then added with mixing to adjust the pH to 6.8 to 7.2. Then, Bay was added while mixingGlycolipids. Finally, the composition is brought to final volume with saline synergist.

Vaccines for treatment groups receiving commercially available AbISCO products (Isconova, sweden) were manufactured according to the label instructions. The AbISCO product is based on quillaja saponins and ISCOM technology using highly purified saponins.

The analysis method comprises the following steps: beta CCV seroneutralization

The serum was heat inactivated at 56 ℃ for 30 to 40 minutes. Serial dilutions of various sera (undiluted, 2,4, 8 …) were prepared by adding 120 microliters to 120 microliters of diluent in a clean sterile tray. At least two replicates of wells/dilutions were used. If desired, a 1: dilution factor of 16. Working challenge stock was prepared by diluting live CCV to a level containing about 240 viral particles in 120 microliters. Then, 120 microliters of each serum dilution was combined with 120 microliters of the virus solution to a total volume of 240 microliters. The solution was mixed and kept at room temperature (about 25 ℃) for 30 to 60 minutes to neutralize it. Then, 120 microliters of each dilution series were transferred to a naked resting monolayer (bathing naked monolayers) of NLFK cells implanted 7 to 12 days ago. CPE was evaluated after 4 to 6 days. Back titration confirmed that 50 to 316 virus particles hit each monolayer.

Results

TABLE 25 serum neutralization

Treatment group	Serum neutralizing titer
		Salt water	2
Antigen only	64
		AbISCO-100	256
AbISCO-200	23
		AbISCO-300	11
Quil-A/cholesterol	315
		R	512
RC	11
		DRC	630
QCR	1024
		QCDC	630
QCRC	724
		QCDRC	1448

SUMMARY

The combined effect of adjuvants formulated with CCV may provide superior properties to vaccine adjuvants, taking into account the chemical nature of the components.

The serological results of this study are shown in table 25. Higher serum neutralizing antibody titers are often associated with better protection provided by the vaccine. Several adjuvant formulations of the invention can produce much higher potency than commercially available adjuvanted products, even though these formulations have similar amounts of CCV antigen added. QCDC, QCR, DRC, QCRC and QCDRC formulations are particularly effective in inducing a good immune response in mice.

Example 24 bovine rotavirus antigens

Range of

A murine model was used to evaluate the adjuvant performance of a given antigen component using bovine rotavirus and the combination adjuvant of the invention.

Animal(s) production

There were 10 CF-1 mice in each treatment group, and 0.2 ml was administered to each animal in each treatment group via the subcutaneous route.

Treatment group

The test formulations shown in table 26 were prepared to 2.0 ml surface dose volumes with the following indicated concentrations. Only 0.2 ml of vaccine was administered per mouse.

TABLE 26 test formulations

Vaccine preparation

Vaccine preparation of the adjuvants of the invention is described in examples 1-13 above. The concentrations of the adjuvant components are provided in table 26. Adjuvants were added in the order in the table.

Saline synergist was added to the tube and homogenization was started. Inactivated bovine rotavirus was added at the concentrations shown in table 26. Quil A was added at the concentrations listed in Table 26. Then, the cholesterol/ethanol solution was added and homogenization continued. Then, DDA/ethanol solution was added during homogenization. The mixture was microfluidized at 10,000 psi. Then adding while mixingThe pH was adjusted to 6.8 to 7.2. Then, Bay was added while mixingGlycolipids. Finally, the composition is brought to final volume with saline synergist.

Vaccines for treatment groups receiving commercially available AbISCO products (Isconova, sweden) were manufactured according to the label instructions. The AbISCO product is based on quillaja saponins and ISCOM technology using highly purified saponins.

Results

TABLE 27 serum neutralization titers

Test formulations	Neutralizing titer in Serum (SN)
		Salt water	≤3
Antigen only	23
		AbISCO-100	16
AbISCO-200	16
		AbISCO-300	14
Quil-A/cholesterol	14
		QCDC	16

R	10
		QCR	16
QCDCR	16
		QCDC	3
QCCR	5
		QCDCR	39
DRC	20
		RC	3

QC for the abbreviation of Quil A/cholesterol, D for DDA,

C isAbbreviation of (1), R is BayAbbreviations of

The combined effect of adjuvants formulated with bovine rotavirus may provide superior properties to vaccine adjuvants, taking into account the chemical nature of the components (see table 27).

While several of the adjuvant formulations can provide similar levels of serum neutralizing antibody titers, QCDCR adjuvant provides the highest levels.

Example 25 Canine influenza Virus

Range/study design

A canine model was used to evaluate adjuvant performance with a specified antigen component using Canine Influenza Virus (CIV) and a novel combination adjuvant.

The study had a randomized complete block design (see table 28). Animals were classified according to birthday, forming blocks of size 5. Animals within the same block were randomly assigned to treatment groups. Animals in the same block were randomly assigned to pens (cages) close to each other. Animals were well-kept and had no history of hypersensitivity to the commercial vaccine. Animals did not receive a vaccine against CIV.

TABLE 28 study design

QC for Quil A/cholesterol, D for DDA, C forAbbreviations of

TABLE 29 vaccine compositions

Vaccine preparation

Vaccine preparation of the adjuvants of the invention is described in examples 1-13 above. The concentrations of the adjuvant components are provided in table 29. Adjuvants were added in the order in the table.

Saline synergist was added to the tube and homogenization was started. Inactivated canine influenza virus was added at the concentrations shown in table 29. Quil A was added at the concentrations listed in Table 29. Then, the cholesterol/ethanol solution was added and homogenization continued. Then, DDA/ethanol solution was added during homogenization. The mixture was microfluidized at 10,000 psi. Then adding while mixingThe pH was adjusted to 6.8 to 7.2. Finally, the composition was brought to final volume with saline extender.

Testing

Serology was assessed by standard analytical methods according to USDA using the hemagglutination inhibition (HAI) assay.

Results/summaries

THE hae serological results for THE average titer of HAI geo at day 42 and 180 are presented in table 30.

TABLE 30 HAI Titers

The combined effect of adjuvants formulated with influenza viruses may provide superior properties to vaccine adjuvants, taking into account the chemical nature of the components.

Higher antibody titers are often associated with better protection provided by the vaccine. In general, the aluminum adjuvant (T02) and the adjuvants of the invention (T03, T04 and T05) resulted in an increase in HAI titers, but the response elicited by the adjuvants of the invention was superior and had higher titers at day 180 in the high dose group (T05). The potency of the low and medium dose adjuvants of the invention is equivalent to the potency of conventional aluminium-containing adjuvants for influenza. In addition, since the adjuvants of the present invention provide T helper 1 immune responses, whereas aluminum does not, longer immune phases are expected and faster memory mechanisms are provided.

Claims (3)

1. A vaccine composition comprising an antigen component and an adjuvant component, wherein the antigen component comprises a J-5 escherichia coli bacterin, and the adjuvant component comprises:

a) at least one of DEAE dextran and oil, and Quil a, cholesterol, and DDA; or

b) DEAE dextran, CpG-ODN, and oil.

2. The vaccine composition of claim 1, wherein the adjuvant component comprises DEAE dextran, CpG-ODN and oil, and the oil is light mineral oil.

3. Use of a vaccine composition according to any one of claims 1-2 for the manufacture of a medicament for the treatment or prevention of mastitis in cattle.