Mucrofluidic oil-in-water emulsion and vaccine compositions

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics and represents a vaccine composition for inducing an immune response in animals. The composition contains an antigen and a 40% oil-in-water emulsion diluted to 2.5%, wherein the above 40% oil-in-water emulsion contains 30 vl/vl % of light hydrocarbon non-metabolic oil, 10 vl/vl % of lecithin, 0.6 vl/vl % of sorbitan monooleate, 1.4 vl/vl % of polyoxyethylene sorbitan monooleate; the oil component is dispersed in an aqueous component by emulsification, while the vaccine composition is prepared by a microfluidiser. An average drop size in the composition makes less than 0.3 mcm.

EFFECT: composition possesses improved physical characteristics, enhanced immunising action, as well as high safety.

10 cl, 20 ex, 17 tbl, 11 dwg

 

The scope of the invention

This invention relates in General to the field of vaccines and, in particular, the adjuvant compositions for enhancing the immune response in veterinary medicine. In particular, the invention relates to the use of submicron emulsion of the "oil-in-water adjuvant vaccine to enhance the immunogenicity of antigens. The present invention relates to compositions of submicron emulsion oil-in-water", vaccine compositions containing the antigen included in such emulsions, and to methods for producing emulsions and vaccines.

Prior art

Bacterial, viral, parasitic, mycoplasmal infections are common in veterinary medicine, for example, in cattle, pigs and Pets. Diseases caused by infectious agents, often resistant to antimicrobial pharmaceutical means, leaving no effective means of treatment. Therefore, vaccines are increasingly being used to combat infectious diseases in these animals. Full of infectious pathogen can be made fit for use in the composition of the vaccine after chemical inactivation or appropriate genetic effects. Alternatively, the protein subunit of the pathogen can be expressed in the recombinant expression system and be acidently use in the vaccine composition.

Adjuvant, generally refers to any substance that increases humoral and/or cellular immune response to the antigen. Routine vaccines consist of a crude preparation of killed pathogens and impurities associated with the cultures of pathogenic microorganisms, can act as an adjuvant to enhance the immune response. However, when as antigens for vaccination are applied homogeneous preparations of pathological microorganisms or purified protein subunits, the immune system caused by such antigens, weak, and thus requires the addition of certain exogenous substances as adjuvant. In addition, the production of synthetic and subunit vaccines expensive. So thanks adjuvant may need a lower dose of antigen to stimulate an immune response, thereby saving manufacturing costs of vaccines.

It is known that adjuvants are various ways to enhance the immune response. Many adjuvants modify the cytokine network associated with the immune response. These immunomodulatory adjuvants may exert its effect even without antigen. In General, immunomodulatory adjuvants stimulate the regulation of certain cytokines and simultaneously suppress the regulation of other cytokines, which leads to glue the internal Th1 and/or humoral Th2 response.

Some adjuvants capable of maintaining the conformational integrity of the antigen, and these antigens can be effectively presented to relevant immune effector cells. As a result of this preservation of the antigenic conformation of the adjuvant composition, the vaccine would have a longer shelf life, such as immunostimulating complexes (ISCOM). Ozel, M., et. al.; Quarternary Structure of the Immunestimmulating Complex (Iscom), J. of Ultrastruc. and Molec. Struc. Res. 102, 240-248 (1989).

Some adjuvants have the ability to hold antigen in the form of a depot at the injection site. As a result, the antigen is not so rapidly excreted hepatic clearance. Aluminium salts and emulsion of water in oil" act through effects depo short. For example, you can use the full beta-blockers (FCA), which is an emulsion of water in oil, to obtain long-term depot. FCA usually remains in the injection until his biodegradation, which will ensure the removal of antigen by antigen-presenting cells.

By their physical nature adjuvants can be divided into two broad categories, namely adjuvants in the form of particles and adjuvants, are not composed of particles. Adjuvants in the form of particles exist in the form of microparticles. The immunogen can either be included, or make contact with the microparticles. Aluminium salts, emulsions water in oil" emulsion is AI "oil in water", immunostimulating complexes, liposomes, nano - and microparticles are examples of adjuvants in the form of particles. Adjuvants are not composed of particles are usually immunomodulators, and they are generally used in combination with adjuvants in the form of particles. Muramyldipeptide (induced-active component peptidoglycan extracted from mycobacteria), non-ionic block copolymers, saponins (a complex mixture of triterpenoids extracted from the bark of the treeTypically saponaria), lipid A (a glucosamine disaccharide with two phosphate groups and 5 or 6 chain fatty acids, generally ranging in length from C12 to C16), cytokines, hydrocarbon polymers, derivatives of polysaccharides and bacterial toxins such as cholera toxin and labile toxinE. coli(LT) are examples of adjuvants that are not composed of particles.

Some of the most well-known adjuvants are a combination of immunomodulators, not composed of particles, and particles that can give the effect of a depot adjuvant composition. For example, FCA combines immunomodulatory properties of components ofMycobacterium tuberculosisalong with the short-term effect depo oil emulsions.

The oil emulsion was used as adjuvants in vaccines for a long time. Le Moignic and Pinoy discovered in 1916 that the suspension of killedSalmonella typhimuriumin mineral oil increased the Ala immune response. Then in 1925, Ramon described starch oil as one of the substances that enhance antitoxic response to diphtheria toxoid. However, oil emulsions were not popular until 1937 until the Blockers did not offer adjuvant composition, currently known as the full beta-blockers (FCA). FCA is an emulsion of water in oil consisting of a mineral (paraffin) oil mixed with a killed Mycobacterium andArtacel A. Artacel Amainly represents monooleate manned and is used as an emulsifying agent. Although FCA great induces antitelomerase, it causes severe pain, the formation of abscess, fever, and granulomatoses inflammation. In order to avoid undesirable side reactions, was developed incomplete adjuvant's adjuvant (IFA). IFA similar to the FCA on its composition except for the absence of mycobacterial components. IFA operates through education depot at the injection site and slowly releases the antigen, stimulating antigen prediction of the cell.

Another approach to the improvement of the FCA was founded on the principle that the substitution of mineral oil biologically compatible oil could eliminate reactions associated with the FCA, at the injection site. It was also suggested that the emulsion should be an emulsion of oil-in-water" and not an emulsion water in oil,as the latter creates long-term depot at the injection site. Hilleman et al. described adjuvant oil-based“Adjuvant 65”consisting of 86% of peanut oil, 10%Arlacel Aas the emulsifier, and 4% aluminum monostearate, as a stabilizer. Hilleman, 1966, Prog. Med. Virol. 8: 131-182; Hilleman and Beale, 1983, in New Approaches to Vaccine Development (Eds. Bell, R. and Torrigiani, G.), Schawabe, Basel. For peopleAdjuvant 65was safe and active, but showed less adjuvanticity thanIFA. However, the use ofAdjuvant 65people were discontinued due to reactogenicity of certain quantities of vaccines and reduce adjuvants, using purified or synthetic emulsifier instead ofArlacel A. In U.S. patent No. 5718904 and 5690942 reported that the mineral oil in the emulsion oil-in-water" can be replaced metabolic oil to improve security properties.

In addition adjuvanticity and security, the physical characteristics of the emulsion are also commercially important. Physical signs depend on the stability of the emulsion. The separation of the emulsion, the formation and adhesion are indicators of an unstable emulsion. The separation of the emulsion occurs when oil and water phases of the emulsion have different specific weight. The separation of the emulsion also occurs when the droplets have a large initial size, when they have no Brownian motion. When drops more, then there is megaversity gap, the droplets coalesce into larger particles. Stable stability of the emulsion is determined by several factors such as the nature and amount of the emulsifier, the amount of droplets in the emulsion and the density difference between the phases of oil and water.

Emulsifiers contribute to the stabilization of dispersed droplets, reducing mipomersen free energy and creating a physical and electrostatic barrier for adhesion drops. As emulsifiers used non-ionic and ionic detergents. Nonionic emulsifiers are on the boundary surface and form a relatively-dimensional structures, which leads to a spatial prevention of the formation of dispersed droplets. Anionic or cationic emulsifying agents induce the formation of an electrical double layer, drawing counterions; repulsive force of the double layer causes the repulsion of the droplets from each other at their approach.

In addition to the use of emulsifiers, emulsion stability can also be achieved by reducing the droplet size of the emulsion by mechanical means. Usually for the manufacture of emulsions using propeller mixers, turbine rotors, colloid mills, homogenizers and device for ultrasonic treatment. Microfluidizer represents another way of increasing the uniformity of droplet size in the emulsion. Microfluidizer can create highly chestvennogo, physically stable emulsion with a uniform particle size in the submicron range. In addition to improving the stability of the emulsion, the method of microfluidizer provides final filtration, which is the preferred way of sterilization of the final product. Moreover, sub-micron particles of oil can come from the site of injection into the lymph vessels and then into the lymph nodes drainage network, blood, and spleen. This reduces the probability of formation of the oil depot at the injection site, which can cause local inflammation and significant reaction to the injection.

Microfluidizer currently commercially available. Emulsification occurs in microfluidizer as two fluid flow interact at high speeds inside the camera interaction. Microfluidizer is driven by an air or nitrogen and can operate at values of internal pressure in excess of 20,000 pounds per square inch. In U.S. patent No. 4908154 reported use of microfluidizer to obtain emulsions, essentially free from any emulsifying agents.

The number of submicron adjuvant compositions in the form of an emulsion oil-in-water" have been described in the literature. In U.S. patent No. 5376369 reported submicron adjuvant composition in the form of emulsi the "oil in water", known as adjuvant compositionSyntax (SAF).SAFcontains squalane or squalene as the oil component, forming the emulsion, the amount of the block copolymerPluronic L121(polyoxypropylene-polyoxyethylene) and immunopotency number of muramyldipeptide. Squalene is a linear hydrocarbon precursor of cholesterol, found in many tissues, especially in liver of sharks and other fish. Squalane is produced by hydrogenation of squalene, and he is fully saturated. Squalene and squalane can be metabolised, and was held a sufficient number of their Toxicological studies. Emulsion of squalene or squalane used in anti-cancer vaccines people and had minor side effects and the desired efficiency (see, for example,Anthony C. Allison, 1999, Squalene an Squalane emulsions as adjuvants, Methods 19:87-93).

In U.S. patent No. 6299884 and International patent publication WO 90/14837 it was reported that polyoxy-propylene-polyoxyethylene block copolymers are not essential for the formation of submicron emulsion oil-in-water". Moreover, these references indicate the use of non-toxic metabolic oils, and explicitly excludes the use of mineral oil and toxic oils obtained by distillation of crude oil, in such emulsion compositions.

In U.S. patent No. 5961970 op is Sana another submicron emulsion oil-in-water", which can be used as adjuvant vaccine. In the emulsion described in this patent, a hydrophobic component selected from the group comprising medium chain triglyceride oil, vegetable oil, and mixtures thereof. Surfactant included in the emulsion, can be a natural, biocompatible surfactant such as a phospholipid (e.g., lecithin) or pharmaceutically acceptable non-natural surfactant, such as TWEEN-80. This patent also describes the incorporation of antigen into the emulsion in the formation of emulsions, in contrast to the mixing of the antigen emulsion after independent and indirect formation of an emulsion.

In U.S. patent No. 5084269 reported that adjuvant composition comprising lecithin in combination with mineral oil, reduces irritation in the body of an animal host and simultaneously increases systemic immunity. Adjuvant composition obtained in U.S. patent No. 5084269, industrial uses in vaccines, used in veterinary medicine under the trade nameAMPHIGEN®. The composition ofAMPHIGEN®made from drops of micelles butter, surrounded by lecithin. These micelles provide the coupling of a large number of whole cell antigens compared with conventional adjuvants oil-based. Over the CSOs, the composition of the vaccine on the basis ofAMPHIGEN®contain a low quantity of oil containing from 2.5 to 5% of mineral oil, compared with other vaccine compositions containing oil adjuvants, which usually contain from 10% to 20% oil. The low oil content in the composition of a vaccine based adjuvant causes less tissue irritation at the injection site, resulting in fewer injuries and reduces the amount of trimming in the processing of carcasses of cattle slaughtered. In addition, lecithin coating surrounding the oil droplets, also reduces the reaction at the injection site, which results in obtaining a vaccine that is both safe and effective.

The composition ofAMPHIGEN®used as an adjuvant in many vaccines used in veterinary medicine, but there is a need to maintain the physical characteristics of the vaccine within a short temporary and long-term storage, and when dissolved before injection. In addition, freeze-dried antigen is mixed with the previously received adjuvant composition immediately before injection. This does not always guarantee a uniform distribution of the antigen within the emulsion of the "oil-in-water, and the characteristics of the emulsion can be undesirable. Moreover, after settling into a homogenized emulsion may occur phase separation. Therefore, there is the duty to regulate the need for stable adjuvant compositions in which no phase separation occurs during prolonged storage. One of the ways to prevent phase separation is to reduce droplet size and increase the homogeneity of the particles in the emulsion. Though the way of microfluidizer metabolic compositions in the form of emulsions of oil-based has been described, microfluidized emulsions oil-in-water", such as the composition ofAMPHIGEN®not yet implemented.

In the present invention microfluidizer was used to ensure submicron size surrounded by lecithin drops of mineral oil. Unexpectedly, applicants found that microfluidizer compositions vaccine emulsion oil-in-water", consisting of a mixture of lecithin and oil not only improves the physical characteristics of the compositions, but also enhances the immunizing effect of the compositions. Microfluidizer compositions also feature improved safety characteristics.

Summary of the invention

Applicants unexpectedly found that the adjuvant activity and security properties nematoblastic emulsions oil-in-water oil-based can be improved through microfluidizer. Antigens introduced in microfluidized emulsion that is stable even when the antigen is injected before microfluidizer.

Accordingly, in one of the variations is tov implement the present invention relates to compositions in the form of submicron emulsions oil-in-water", which can be used as adjuvant vaccine. Compositions in the form of submicron emulsions oil-in-water" of the present invention consist of nametables oil, at least one surfactant and a water component, where the oil is dispersed in an aqueous component with an average size of drops of oil in the submicron range. Preferably, nametables oil is light mineral oil. Preferred surfactants include lecithin, Tween 80 and SPAN 80.

The preferred emulsion oil-in-water" of the present invention consists of a compositionAMPHIGEN®.

Emulsion oil-in-water" of the present invention may contain additional components that are appropriate and desirable, including preservatives, osmotic agents, biological molecules adhesives and immunostimulatory molecules. Preferred immunostimulatory molecules include, for example, Quil A, cholesterol, GPI-0100, bromide of dimethyldioctadecylammonium (DDA).

In another embodiment, the present invention relates to a method for producing submicron emulsion oil-in-water". In accordance with the present invention, the various components of the emulsion, including oil, one or more surfactants, a water component and I is th the other component, suitable for use in the emulsion are mixed together. The mixture is subjected to the process of the primary emulsion with an emulsion of the "oil-in-water, which is then passed through microfluidizer obtaining emulsion oil-in-water" with drops of less than 1 micron in diameter, preferably with an average droplet size less than 0.5 micron.

In yet another embodiment, the present invention relates to vaccine compositions that contain the antigen described above submicron emulsion oil-in-water". The antigen is administered either before or after receipt of the emulsion, it is preferable to obtain.

The antigen, which may be included in the vaccine composition of the present invention may be a bacterial, fungal or viral antigen or a combination of both. The antigen may take the form of inactivated whole or partial cell or viral drug or to form an antigenic molecule, obtained from a conventional protein purification, methods of genetic engineering or chemical synthesis.

In another embodiment, the present invention relates to methods of producing compositions of the vaccine containing the antigen or antigens, combined with submicron emulsion oil-in-water".

Upon receipt of the vaccine compositions of the present invention the antigen(s) can be entered or is about (for example, before microfluidizer), or after the formation of the emulsion (for example, after microfluidizer) with the components of the emulsion oil-in-water". Preferably, the antigen is combined with the components of the emulsion oil-in-water".

In yet another embodiment, the present invention relates to vaccine compositions that contain microencapsulating antigen described above submicron emulsion oil-in-water", where microencapsulating antigen is combined with the emulsion after receipt.

Brief description of drawings

In Fig.1 shows the method of obtaining the views of the party amicroprocessor compositions of the vaccine. In this method, the various components of vaccines added to the vessel to add to the left and, ultimately, is pumped into the vessel for mixing, where the components are mixed together by simple mechanical methods.

In Fig. 2 depicts a method of obtaining microfluidized vaccine compositions containing up to obtain emulsion included antigen. The various components of vaccines added to the vessel to add and transferred to the block predimensioning mixing mixing by simple mechanical means. Then the emulsion is passed through microfluidizer and collected in the post-microfluidized the camera.

In Fig. 3 shows the distribution of droplet size vaccines on the basis of necrophiliashaving composition AMPHIGEN®, vaccines based on microfluidizer composition AMPHIGEN® and preparation of the vaccine, mixed in the gas oven.

In Fig. 4 shows a lack of phase separation in drug microfluidized vaccine.

In Fig. 5 shows a comparison of the stability of antigens introduced to obtain emulsion in the preparation of a vaccine based on microfluidizer composition AMPHIGEN® (A) and three controls, vaccine preparations based on microfluidizer composition AMPHIGEN® (A, A and A). All four of the preparation of the vaccines were stored at 4°C for 2 years. At different points in time during storage period (0, 6, 12 or 24 months.) all four compositions were used for the three-month vaccination of calves. Vaccination was performed at 0 and 21 days 2 ml doses of vaccine and serum was taken 2 weeks after the second vaccination. In each of the serum samples was determined the titer of neutralizing antibodies to BVD virus type II (viral diarrhea in cattle). Data are shown as geometric mean for 5 animals.

In Fig. 6 shows the minimum mean square rectal temperature in cattle before and after the introduction of microfluidizers and necrophiliashaving vaccines. T: Group placebo single dose; C: placebo Group - a double dose; T03: Necrophiliajenna composition - single dose; is 04: Necrophiliajenna composition - double dose; T05: Microfluidizer composition - single dose; T: Microfluidizer composition - a double dose.

In Fig. 7 shows the minimum average RMS volume reactions at the injection site observed in cattle after administration microfluidizers and necrophiliashaving compositions of the vaccine. T03: Necrophiliajenna composition - single dose; T04: Necrophiliajenna composition - a double dose; T05: microfluidizer composition - single dose; T: microfluidizer composition - a double dose.

In Fig. 8 shows the geometric mean titers of IgG to recombinant antigen PauA fromStreptococcus aureusafter vaccination with different vaccine compositions containing the recombinant antigen PauA, and the antigen of whole cellsE. coli.

In Fig. 9 shows the geometric mean titers of antigen whole cellsE. colifromStreptococcus aureusafter vaccination with different vaccine compositions containing the recombinant antigen PauA, and the antigen of whole cellsE. coli.

In Fig.10A and 10B shows the distribution of particle size microfluidizer composition AMPHIGEN when the original product (Fig. 10A) and after 22 months. after production (Fig. 10V).

Detailed description of the invention

The authors of the present invention unexpectedly discovered, chemicallyinduced compositions of the vaccine with the addition of emulsion oil-in-water", consisting of a mixture of lecithin and mineral oil not only improves the physical properties of the compositions of the vaccine, but also enhances the immunizing effect of the compositions of the vaccine. Microfluidizer composition of the vaccine are also characterized by improved security properties.

Based on this discovery, the present invention relates to submicron emulsions oil-in-water" that can be used as additives to the compositions of the vaccine. The invention also relates to methods of producing these submicron emulsions oil-in-water" with the help of microfluidizer. In addition, the present invention relates to submicron compositions of vaccines in which the antigen is a combination of sub-micron emulsion oil-in-water". The present invention also relates to methods of producing such compositions vaccine. The present invention also relates to vaccine compositions containing microencapsulation antigens, in combination with sub-micron emulsion oil-in-water", and to methods of producing such vaccines.

For clarity of description, but not limitation, a detailed description of the invention is divided into the following sections that describe or illustrate certain signs, ways to make or use the invention.

Submicron emulsion of the "oil-in-water

is one of the options for implementing the present invention relates to compositions in the form of a submicron emulsion oil-in-water", you can use as an additive to the vaccine. Submicron emulsion oil-in-water" of the present invention enhance the immunogenicity of antigens in vaccine compositions, safe in the introduction of animals and stable during storage.

Submicron emulsion oil-in-water" of the present invention consist of nametables oil, at least one surfactant and a water component, where the oil is dispersed in an aqueous component with an average droplet size in the submicron range.

Under "sub-micron" means that the droplets have a size less than 1 μm (micron) and an average or normal particle size is less than 1 μm. Preferably, the average droplet size of the emulsion is less than 0.8 μm; preferably, less than 0.5 μm and more preferably less than 0.4 μm, or about 0.1-0.3 microns.

Under "average droplet size" means the particle size in average diameter size (VMD). The VMD is calculated by multiplying the diameter of each particle in the volume of all particles of this size and addition. Then this value is divided by the total volume of all particles.

Used in the present description, the term "nametables oil" refers to oils that cannot be metabolised by the body of an animal individual, which is injected emulsion.

Use the established in the present description, the terms "animal" and "animal-individual" refers to all animals, including, for example, cattle, sheep and pigs, are not human.

Nametables oils that can be used in the form of an emulsion of the present invention include alkanes, alkynes and other corresponding acids and alcohols, their ethers and esters, and mixtures thereof. Preferably, a separate connection oils are light hydrocarbon compounds, that is, such components contain from 6 to 30 carbon atoms. The oil may be obtained synthetically or refined petroleum products. Preferred nametables oil that can be used in the emulsions of the present invention, include, for example, mineral oil, paraffin oil and cycloparaffin.

The term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petroleum by distillation. This term is synonymous with the term "liquid paraffin", "vaseline oil, i.e. oil, which is obtained similarly by distillation of petroleum butter, but which has a slightly lower specific weight than white mineral oil. See, for example, Remington''s Pharmaceutical Sciences, 18thEdition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, J. T. Baker (Phillipsburg, PA), USB Corporation (Cleveland, OH). Preferably, the mineral oil which is a light mineral oil, commercially available under the trade name DRAKEOL®.

Usually the oil component of submicron emulsions of the present invention is present in an amount of from 1% to 50% vol., preferably, in the range from 10% to 45%, preferably in an amount of from 20% to 40%.

Emulsion oil-in-water" of the present invention typically contain at least one (i.e. one or more) surfactant. The substance denoted by the terms "surfactant" and "emulsifier" as used in the present description are interchangeable, are substances that stabilize the surface of oil droplets and support of the oil droplets within the desired size.

Surfactants that can be used in the emulsions of the present invention include biologically compatible surfactants and non-natural synthetic surfactants. Biologically compatible surfactants include phospholipid compound or a mixture of phospholipids. Preferred phospholipids are phosphatidylcholine (lecithin), such as soy or egg lecithin. Lecithin can be obtained in the form of a mixture of phosphatides and triglycerides by washing with water, untreated vegetable oils and division and sushi is Oh obtained hydrogenated resins. The purified product can be obtained by fractionation of a mixture insoluble in acetone phospholipids and glycolipids remaining after the removal of triglycerides and vegetable oils while washing with acetone. Alternatively, the lecithin can be obtained from various commercial sources. Other suitable phospholipids include phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, fosfatados acid, cardiolipin, and phosphatidylethanolamine. Phospholipids can be isolated from natural sources or synthesized in the usual way.

Unnatural synthetic surfactants suitable for use in submicron emulsions of the present invention include non-ionic surfactants based on sorbitan, for example surface-active substances with sorbitane, substituted fatty acid (commercially available under the name SPAN® or ARLECEL®), esters of fatty acids polyethoxyethanol sorbitol (TWEEN®), esters of fatty acids of polyethylene glycol from sources such as castor oil (EMULFOR); polyethoxyethanol fatty acid (e.g. stearic acid, available under the name SIMULSOL M-53), the polymer polyethoxyethanol of isooctyl/formaldehyde (TYLOXAPOL), ethers of fatty polyoxyethylene alcohols of (BRIJ®); simple not phenyl esters of polyoxyethylene is a (TRITON®N), simple isooctylphenyl esters of polyoxyethylene (TRITON®X). SPAN® and TWEEN® are the preferred synthetic surface-active substances.

Preferred surfactants for use in the emulsions of the oil-in-water according to the present invention include lecithin, Twen-80 and SPAN-80.

Typically, the amount of surfactant or combination of surfactants using two or more surfactants present in the emulsion is from 0.01% to 10% vol., preferably, from 0.1% to 6.0%, preferably from 0.2% to 5.0%.

The water component is the disperse phase of the emulsion and may consist of water, buffered saline or any other suitable aqueous solution.

Emulsion oil-in-water" of the present invention may contain additional components that are appropriate and desirable, including preservatives, osmotic agents, bioadhesive molecules and immunostimulatory molecule.

I believe that bioadhesive molecules can enhance the delivery and attachment of antigens on mistaway mucosal surface, causing an immune response. Examples of suitable bioadhesive molecules include acidic, non-natural polymers, such as polyacrylic acid and polymethacrylic acid (e.g., CARBOPOL®, CARBOMER; acid synthetically modified natural polymers, such as carboxymethylcellulose; neutral synthetically modified natural polymers, such as (hydroxypropyl)methyl cellulose; main bearing amine polymers, such as chitosan; acid polymers derived from natural sources, such as alginic acid, hyaluronic acid, pectin, resin tragakant and resin karaya; and a neutral non-natural polymers, such as polyvinyl alcohol, or combinations thereof.

Used in the present description, the phrase "immunostimulatory molecules" refers to molecules that enhance the protective immune response induced antigenic component of the vaccine compositions. Suitable immunostimulatory substances include components of the bacterial cell wall, such as derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine acid, such as murabutide, threonyl-MDP and muramyldipeptide; saponine glycosides and their derivatives, for example, Quil A, QS 21 and GPI-0100; cholesterol; and Quaternary ammonium compounds, for example the bromide of dimethyldioctadecylammonium (DDA) and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propandiamine ("avidin").

Saponins are glycosidic compounds that are produced as secondary metabolites by the large number of plant species. Chemicals the structure of saponins gives a wide range of pharmacological and biological activity, including some powerful and effective immunological activity.

Structurally, saponins consist of any aglycone attached to one or more sugar chains. Saponins can be classified in accordance with the composition of their aglycones: triterpene glycosides, steroidal glycosides and steroidal alkaloid glycosides.

Saponins can be distinguished from the bark ofTypically saponaria. For a long time saponins were known as Immunostimulants. Dalsgaard, K., “Evaluation of its adjuvant activity with a special reference to the application in the vaccination of cattle against foot-and-mouth disease”, Acta. Vet. Scand. 69: 1-40 1978. The crude plant extracts containing saponins increased the activity of vaccines against FMD. However, the crude extracts caused adverse side effects when used in vaccines. In the following, Dalsgaard partially purified active component with the properties of the adjuvant, saponin by dialysis, ionoobmennoi and gel-filtration chromatography. Dalsgaard, K., et al., “Saponin called adjuvants III. Isolation of a substance fromTypically saponariaMorina with adjuvant activity in foot-and-mouth disease vaccines”, Arch. Gesamte. Virusforsch. 44: 243-254, 1974. The active component, with properties adjuvant, purified in this way is known as "Quil-A". In terms of weight Quil-A showed increased activity and reduced local reactions compared with saponins. Quil-A is widely used in WACC the fuck used in veterinary medicine.

Subsequent analysis of Quil-A by high performance liquid chromatography (HPLC) revealed a heterogeneous mixture of closely related saponins and led to the discovery of QS 21, which is a potent adjuvant with reduced or minimal toxicity. Kensil, C. R. et al., “Separation and characterization of in saponins with adjuvant activity fromTypically saponariaMolina cortex”,J. Immunol. 146: 431-437, 1991. Unlike other Immunostimulants, QS 21 soluble in water, and it can be used in vaccines containing composition by type emulsions containing no or. It was shown that QS 21 causes a Th1 response in mice by stimulating the production of antibodies IgG2a and IgG2b and induces antigen-specific CD8+CTL (MHC class I) in response to subunit antigens. Clinical studies of people proved its adjuvant properties and an acceptable toxicity profile. Kensil, C. R. et al., “Structural and immunological characterization of the vaccine adjuvant QS-21. In Vaccine Design: the subunit and Adjvuant Approach, ”Eds.Powell, M. F. and Newman, M. J. Plenum Publishing Corporation, New York. 1995, pp. 525-541.

In U.S. patent No. 6080725 describes how to obtain and use conjugate saponin-lipophile. In the conjugate saponin-lipophile, lipophilic part, such as a lipid, fatty acid, polyethylene glycol or a terpene, covalently linked to deacetylating or deacetylating by triterpene saponin through carboxypropyl present at the 3-O-glucuronic acid Proc. of the terpenoid saponin. Attaching lipophilic part to 3-O-glucuronic acid saponin, such as disallusionedTypically,lycosid R or saponin fromGypsophia, saponariaandAcanthophyllumincreases their adjuvant properties on humoral and cellular immunity. In addition, attaching lipophilic part to the remainder of the 3-O-glucuronic acid or deacetylation gives similar saponin, which is easier to clean, is less toxic, chemically more stable and has equal or better adjuvant properties than the original saponin.

GPI-0100 is a conjugate saponin-lipophile described in U.S. patent No. 6080725. GPI-0100 obtained by adding an aliphatic amine to detailspane through the carboxyl group of glucuronic acid.

The Quaternary ammonium compounds.A number of aliphatic nitrogenous bases have been proposed for use as immunological adjuvants, including amines, Quaternary ammonium compounds, guanidine, benzamidine and Toroni. Specific compounds include the bromide of dimethyldioctadecylammonium (DDA) and N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propandiamine ("avidin").

In U.S. patent No. 5951988 described adjuvant composition comprising a Quaternary ammonium salt, for example, DDA, in combination with an oil component. This composition can be used in combination with the all right known immunological substances, for example, viral or bacterial antigens in the vaccine composition, to enhance the angiogenic response. The composition can also be used without the included antigen as nonspecific immunostimulatory composition.

In U.S. patent No. 4310550 described the use of N,N-higher alkyl-N,N'-bis(2-hydroxyethyl)propandiamine and N,N-higher alkyl-xylylenediamine included in the composition with a fat or lipid emulsion as adjuvant vaccine. Method of inducing or enhancing the antigen response antigen in human or animal by parenteral adjuvant compositions described in U.S. patent No. 4310550.

In a preferred embodiment, the present invention relates to submicron emulsion oil-in-water", which consists of composition AMPHIGEN® with drops of size less than 1 μm and a mean droplet size of about 0.25 μm.

Used in the present description, the term "composition AMPHIGEN®" refers to a solution obtained by mixing a solution of lecithin oil DRAKEOL® (Hydronics, Lincoln, NEwith saline solution in the presence of TWEEN® 80 and SPAN® 80. The usual composition AMPHIGEN® contains 40%. light mineral oil (about./vol.), about 25% wt./about. lecithin, about 0.18% of about. TWEEN 80 (about./about.) and about 0,08% about. Span 80 (about./vol.).

Methods of obtaining submicron emulsions oil-in-water"

In another embodiment, the present invention relates to methods of producing the above-described submicron emulsions oil-in-water".

In accordance with the present invention, the various components of the emulsion, including oil, one or more surfactants, a water component and any other component suitable for use in the emulsion are combined and mixed.

The resulting mixture is subjected to the process of emulsification, usually missing one or more times through one or more homogenizers or emulsifiers with emulsion oil-in-water", which has a uniform look and an average droplet size of about 0.5 μm. For this purpose you can use any commercially available homogenizer or emulsifier, for example an emulsifier Ross (Hauppauge, NY), homogenizer (Gaulin(Everett, MA).

Thus obtained emulsion is then subjected to microfluidizer, bringing the droplet size to the submicron range. Microfluidizer can be carried out using industrial microfluidizer, such as model No. 110Y, available from Microfluidics, Newton, Mass; Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.) and Rainnie Minilab Type 8.30 H (Miro Atomizer Food and Diary, Inc., Hudson, Wis.). The action of microfluidizer is forced passing the fluid through a small orifice under high pressure, so that the two streams of liquids and interact at high speeds in the cell interaction with the formation of emulsions with droplets of submicron size.

The droplet size can be defined in various ways known in this field, such as laser diffraction, using a commercially available instrument that defines the size. The size can vary depending on the type of surfactant, the ratio between the surface-active substance and oil pressure, temperature, and other factors. The person skilled in the art can determine the desired combination of these parameters to obtain emulsions with the desired droplet size without undue experimentation. The diameter of the droplets of the emulsions of the present invention is less than 1 μm, preferably with a mean droplet size of less than 0.8 μm, and preferably, with an average droplet size less than 0.5 μm, and more preferably with an average droplet size less than 0.3 microns.

In a preferred embodiment of the present invention a solution of lecithin oil DRAKEOL, commercially available from the company Hydronics (Lincoln, NE) and contains 25% of lecithin in a light mineral oil, are combined and mixed with a saline solution and surface active agents TWEEN® 80 and SPAN® 80 for education solution AMPHIGEN®" or "composition AMPHIGEN®". Then the solution AMPHIGEN® emuleret emulsifier Ross® (Hauppauge, NY 11788) at about 3400 rpm to education the emulsion oil-in-water". Then the emulsion once passed through microfluidizer running at about 4500±500 pounds per square inch. The droplet size microfluidized emulsion oil-in-water" is less than 1 μm with an average droplet size of about 0.25 μm.

The composition of the vaccine containing antigens included in the submicron emulsion of the "oil-in-water

In another embodiment, the present invention relates to vaccine compositions that contain the antigen(s) described above submicron emulsion oil-in-water". These compositions vaccines have increased antigenic response and improved physical characteristics (for example, after a long storage period is not observed phase separation). In addition, the vaccine composition of the present invention are safe for administration to animals.

In accordance with the present invention, the antigen can be combined with the emulsion either before or after it is received, preferably, prior to its receipt. The term "to obtain emulsion" refers to the way in which the antigen is introduced into the emulsion at the stage of microfluidizer. The term "after receiving emulsion" refers to the way in which the antigen is added to the emulsion after the emulsion was microfluidizer. Antigen is added to the emulsion after it is received, can be the free antigen, or he m is et to be encapsulated in microparticles, as further described below in the present description.

Used in the present description, the term "antigen" refers to any molecule, compound or composition, which are antigenic in animals, and which entered into the composition of the vaccine to induce a protective immune response in an animal, which enter the composition of the vaccine.

The term "antigenic" or "immunogenic" as used in connection with the antigen refers to the ability of an antigen to induce an immune response in an animal against the antigen. The immune response may be a cellular immune response mediated primarily cytotoxic T-cells or humoral immune response mediated primarily by T-helper cells, which in turn activates b-cells, leading to the production of antibodies.

A "protective immune response" is defined as any immune response, mediated either by antibodies or cells, or both, arising from the animal, which either prevents or greatly reduced the incidence of, or eliminates, or explicitly reduce the severity of, or obviously slows down the rate of progression of disorders or diseases caused by antigen or pathogen containing the antigen.

Antigens that can be entered into the composition of the vaccine of the present invention include antigens derived from athogenic bacteria, such asMycoplasma hyopneumoniae, Haemophilus somnus, Haemophilus parasuis, Bordetella bronchiseptica, Actinobacillus pleuropneumonie, Pasteurella multocida, Manheimia hemolytica, Mycoplasma bovis, Mycoplasma galanacieum, Mycobacterium bovis, Mycobacterium paratuberculosis,the speciesClostridial,Streptococcus uberis, Streptococcus suis, Staphylococcus aureus, Erysipelothrix rhusopathiae,the speciesCampylobacter,Fusobacterium necrophorum, Escherichia coliserological variantsSalmonella enterica,the speciesLeptospira; pathogenic fungi, such asCandida; protozoa such asCryptosporidium parvum,Neospora canium,Toxoplasma gondiithe speciesEimeria; helminths such asOstertagia,Cooperia,At,Fasciolaor in the form of inactivated whole or partial cell preparation, or in the form of antigenic molecules obtained with conventional protein purification, methods of genetic engineering or chemical synthesis. Additional antigens include pathogenic viruses such as the herpes viruses of cattle-1,3,6, viral diarrhea virus in cattle (BVDV) types 1 and 2, parainfluenza virus bovine respiratory syncytial virus, the virus bovine leukemia virus rinderpest, FMD virus, rabies virus swine fever virus African swine fever, porcine parvovirus, PRRS virus, porcine circovirus, influenza virus, the virus vesicular disease swine fever virus Techen, virus false rabies, or in the form of inactivated whole, or casticin the th cell drug or in the form of antigenic molecules obtained with conventional protein purification, methods of genetic engineering or chemical synthesis.

The amount of antigen should be such that the antigen is combined with the emulsion oil-in-water" would be effective for the induction of protective immune response in the animal. The precise effective amount of an antigen depends on the nature, activity and purity of the antigen, and can be determined by a specialist in this field.

The amount of emulsion oil-in-water" that is present in the compositions of the vaccine, should be sufficient to enhance the immunogenicity of the antigen(s) in the compositions of the vaccine. When it is appropriate and desirable, additional quantities of surface-active substance (s) or additional surface-active agent (substance) can be added to the composition of a vaccine in addition to surface-active substance (s) in the emulsion oil-in-water". Summing up, the oil component is present in the final volume of the composition of the vaccine in the amount of from 1.0% to 20% vol.; preferably, in quantities of from 1.0% to 10%; preferably, in quantities of from 2.0% to 5.0%. Surfactant or combination of surfactants, if there are two or more surfactants present in the final volume of the composition of the vaccine is Alceste from 0.1% to 20% vol., preferably, from 0.15% to 10%, preferably from 0.2% to 6.0%.

In addition to antigen (antigens) and emulsion oil-in-water" vaccine composition may include other components that may be appropriate and desirable, such as preservatives, osmotic agents, biological molecules adhesives and immunostimulatory molecules (e.g., Quil A, cholesterol, GPI-0100, bromide of dimethyldioctadecylammonium (DDA), as described above in connection with emulsion oil-in-water".

The composition of the vaccines of the present invention may also include acceptable in the veterinary carrier. The term "acceptable in the veterinary carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, substances that delay absorption, and the like. Diluents include water, saline, dextrose, ethanol, glycerol and the like. Isotonic substances, along with others, may include sodium chloride, dextrose, lures, sorbitol and lactose. Stabilizers, among others, include albumin.

In a preferred embodiment, the present invention relates to a vaccine composition that includes at least the BVDV antigen of BVDV type I or type II, entered before received the eat emulsion oil-in-water", the droplet size which is less than 1 μm, preferably, the average size of the droplets is less than 0.8 μm; preferably, less than 0.5 μm, and more preferably, the average droplet size is about 0.5 μm. The antigen of BVDV type I and/or II submitted preferably in the form of inactivated viral preparation. Submicron emulsion oil-in-water" preferably includes in its membership composition AMPHINOGEN® (i.e. composition, which contains a light mineral oil, lecithin, TWEEN® 80 and SPAN® 80). The vaccine composition preferably also includes Quil A, cholesterol and thimerosal.

In another preferred embodiment, the present invention provides a vaccine composition, which includes leptospirosis antigen and at least one BVDV antigen of BVDV type I or type II emulsion oil-in-water". Antigens, preferably in the form of inactivated cells or viral drug is administered before receiving emulsion oil-in-water", the size of the droplets is less than 1 μm, preferably, the average size of the droplets is less than 0.8 μm; preferably, less than 0.5 μm, and more preferably, the average droplet size is about 0.5 μm. Submicron emulsion oil-in-water" preferably includes in its membership composition AMPHINOGEN (i.e. composition, Kotor, which contains a light mineral oil, lecithin, TWEEN® 80 and SPAN® 80). The vaccine composition preferably also includes one or more immunostimulatory molecules selected from Quil A, cholesterol, DDA, GPI-100 and aluminium hydroxide.

In another preferred embodiment, the present invention relates to a vaccine composition that includes at least one bacterial antigen, such as recombinant proteinStreptococcus uberis PauAor cellular drugE. Colior a combination of both of them in the emulsion oil-in-water". The antigen(s) is administered before receiving emulsion oil-in-water", the size of the droplets is less than 1 μm, preferably, the average size of the droplets is less than 0.8 μm; preferably, less than 0.5 μm, and more preferably, the average droplet size is about 0.25 μm. Submicron emulsion oil-in-water" preferably includes in its membership composition AMPHINOGEN® (i.e. composition, which contains a light mineral oil, lecithin, TWEEN® 80 and SPAN® 80). The vaccine composition preferably also includes one or more immunostimulatory molecules selected from Quil-A, DDA and GPI-100.

The vaccine composition of the present invention can enter the animal known ways, including oral, intranasal, mucosal, local, transdermal, and parenteral (e.g. intravenous, GNC is rebrushing, intradermal, subcutaneous or intramuscular) route. The introduction can be done in several ways, for example, the first introduction to exercise parenterally, and the subsequent introduction - through the mucous membranes.

Methods of obtaining compositions vaccine

In another embodiment, the present invention relates to methods of producing compositions of the vaccine containing the antigen or antigens and submicron emulsion oil-in-water".

Upon receipt of the vaccine compositions of the present invention the antigen(s) may be entered either before or after receipt of the emulsion oil-in-water". Preferably, the antigen is mixed with the components of the emulsion oil-in-water" before its receipt.

The antigen can be mixed with various components of the emulsions, including oil, one or more surfactants, a water component and any other suitable component to form a mixture. The mixture is subjected to the process of initial mixing, usually by passing one or more times through one or more homogenizers or emulsifiers with the formation of oil-in-water" containing the antigen. For this purpose you can use any commercially available homogenizer or emulsifier, for example an emulsifier Ross (Hauppauge, NY), a Gaulin homogenizer (Everett, MA) or Microfluidics (Newton, MA). Alternatively, different com is ananti emulsion adjuvant, including oil, one or more surfactants and water component can first be combined to form an emulsion of the oil-in-water by using a homogenizer or emulsifier, and then the antigen is added to this emulsion. The average droplet size of the emulsion oil-in-water" after the initial mixing is approximately 1.0 to 1.2 microns.

The emulsion containing the antigen, then subjected to microfluidizer to bring the droplet size to the submicron range. Microfluidizer can be done using industrial microfluidizer, such as model No. 110Y, manufactured Microfluidics, Newton, Mass; Gaulin Model 30CD (Gaulin, Inc., Everett, Mass.) and Rainnie Minilab Type 8.30 H (Miro Atomizer Food and Diary, Inc., Hudson, Wis.).

The droplet size can be defined in various ways known in this field, such as laser diffraction, using a commercially available apparatus for determining the size. The size can vary depending on the type of surfactant, the ratio between the surface-active substance and oil pressure, temperature, and similar factors. You can define a desirable combination of these parameters to obtain emulsions with the desired droplet size. Droplets of the emulsions of the present invention have a diameter less than 1 μm. Preferably the average ramarkable is less than 0.8 μm. Preferably, the average size of the droplets is less than 0.5 μm. More preferably, the average droplet size is approximately 0.3 microns.

In a preferred embodiment of the present invention a solution of lecithin oil DRAKEOL®, which contains 25% of lecithin in a light mineral oil, are combined and mixed with surface-active agents TWEEN® 80 and SPAN® 80 and saline solution with formation of a mixture which contains 40% light mineral oil, lecithin, 0.18% of TWEEN® 80 and 0.08% of SPAN® 80. The mixture is then emuleret emulsifier Ross® (Hauppauge, NY 11788) at about 3400 rpm with the formation of the emulsion product, which is also referred to as "composition AMPHINOGEN®" or "solution AMPHINOGEN®". In subsequent desired(s) antigen(s) are combined with a solution AMPHINOGEN®, and any other appropriate components (e.g., immunostimulating molecules) using an emulsifier such as a homogenizer Ross, with the formation of emulsions of oil-in-water" containing the antigen(s). This emulsion once passed through microfluidizer running at about 10000 ± 500 pounds per square inch. Microfluidized emulsion oil-in-water" has a droplet size less than 1 micron with an average droplet size of about 0.25 μm.

The composition of the vaccine containing microencapsulation antigens in submicron emulsion oil-in-water" pic and the least get

In another embodiment, the present invention relates to vaccine compositions that contain the antigen, encapsulated in microparticles (or "microencapsulating antigen"), and microencapsulating antigen is injected before receiving the above submicron emulsion oil-in-water".

Methods of absorption or capture antigens in the media in the form of particles known in the art (see, for example, Pharmaceutical Particulate Carriers: Therapeutic Applications (Justin Hanes, Masatoshi Chiba and Robert Lander. Polymer microspheres for vaccine delivery. In: Vaccine design. The subunit and adjuvant approach. Eds.Michael F. Powell and Mark J. Newman, 1995 Plenum Press, New York and London). Media in the form of particles represent multiple copies of the selected antigen to the immune system of the animal-the individual and contribute to the capture and retention of antigens in the local lymph nodes. Particles can be fiacitibine by macrophages and can enhance the presentation of the antigen through the release of cytokines. Media in the form of particles have also been described in this area and include, for example, the media obtained from polymers of polymethylmethacrylate and media derived from poly(lactides) and poly(lactide-coglycolide), known as PLG. Polymetylmetacrylate polymers are not biodegradable, whereas the PLG particles can undergo biodegradation random nonenzymatic hydrolysis of clonoey is established ties with lactic and glycolic acids, received the usual metabolic pathways.

Biodegradable microspheres are also used to achieve a controlled release of vaccines. For example, it is possible to achieve continuous release of antigen over an extended period. Depending on the molecular weight of the polymer and the ratio of lactic and glycolic acids in the polymer, the polymer is PLGA can have the speed of hydrolysis of from several days or weeks to several months or years. Slow, controlled release can lead to the formation of high levels of antibodies, similar to antibodies observed after multiple injections. Alternatively, pulsating release of vaccine antigens can be achieved by selecting polymers with different velocities of hydrolysis. The rate of hydrolysis of the polymer is usually dependent on the molecular weight of the polymer and the ratio of lactic and glycolic acids in the polymer. Microparticles derived from two or more different polymers, with varying rates of release of antigen provide a pulsating release of antigens and mimic diagrams vaccination multiple doses.

In accordance with the present invention, the antigen, including any of the antigens described above, can be absorbed on the polymer carrier in the form of particles, the pre is respectfully, the PLG polymer, using any method known in this field (such as the method illustrated in example 17), with the formation of the drug microencapsulating antigen. Then the drug microencapsulating antigen is mixed with the above-described sub-micron emulsion oil-in-water and dispersed therein with the formation of the composition of the vaccine.

In a preferred embodiment, the present invention relates to the composition of the vaccine, which contains the antigen, encapsulated in PLG polymer, and microencapsulating antigen injected into microfluidized emulsion oil-in-water" before its receipt and the emulsion consists of a light mineral oil, lecithin, TWEEN-80, SPAN-80 and saline, and has an average droplet size less than 1.0 microns.

Below are examples of specific embodiments of the present invention. Examples are presented for illustrative purposes only and in no way are intended to limit the scope of the present invention.

EXAMPLE 1

A composition AMPHIGEN®

Composition AMPHIGEN® is produced by way of two stages. In the first stage, 80 l lecithin oil solution Drakeol, 116 l salt solution of tetanus toxoid, 1.2 l SPAN-80 and 2.8 l Tween-80 mixed together and emuleret using emulsifier Ross. Lecithin oil solution Drkeol contains 25% soy lecithin and 75% mineral oil. Emulsified product recycle through the emulsifier Ross to pass at least 5 volumes, or at least 10 minutes Emulsified product stored at 2-7°C for a maximum of 24 h for further processing. The emulsion from the tank emulsifier Ross transferred to the Gaulin homogenizer and homogenized for 20 min at a pressure of 4500 pounds per square inch. Received 40% lecithin oil solution Drakeol (hereinafter referred to as "composition AMPHINOGEN®" or "solution AMPHINOGEN®") then served in sterile carboxypropanoyl containers. Supply perform inside the cap feeder class 100, located in the controlled environment of class 10,000. Containers stored at 2-7°C. In the absence of other indications, composition AMPHINOGEN® is used in the following experiments.

EXAMPLE 2

Primary mixing homogenization instantaneous evaporative mixing vaccine BVD

The device used for this method of homogenization, as shown in Fig.1. Using aseptic technique or steam three-way valves, the bottle containing the antigen BVD type I (the drug is inactivated BVD virus type I), attached to the lower side of an opening in the vessel for mixing. After completion of the transfer of the required amount of antigen BVD type I, the bottle with BVD type I replace the bottle containing the drug is inactivated BVD virus type II (drug and is activated BVD virus type I). After transferring the desired amount of antigen BVD type II completed, a Ross homogenizer attached to the portable tank and start recycling at maximum speed (3300 rpm). Mixing the contents of the vessel support on medium speed.

Using aseptic technique or steam three-way valves, the bottle containing Quil-A at a concentration of 50 mg/ml, added to the intake aperture of the homogenizer in the vessel for mixing. The desired amount of solution Quil-A is passed into the vessel through the vacuum line. After migration solution Quil-A bottle is removed. In the same way, the desired amount of cholesterol in ethanol (18 mg/ml) is transferred into the vessel for mixing. In the following the required amount of the composition AMPHINOGEN®, 10% solution of thimerosal and filling solutions main modified eagle medium (“BME”) is added to the vessel for mixing.

After completion of all additions, mixing continued for another 15 minutes, the resulting composition is divided into aliquot dose of 2 ml, and it is the vaccine BVD based not microfluidizer composition AMPHINOGEN®. Each dose of vaccine contains 500 μg Quil A, cholesterol, 2.5% of the composition AMPHINOGEN® and 0,009% thimerosal. The concentration of antigen for two different strains of BVD determine from the point of view of the ELISA titer (immunofermentnogo analysis) for gp53.

EXAMPLE 3

Secondary mixing microfluidizer

In Fig.2 shows the method used for the secondary mixing by microfluidizer. Microfluidizer subjected to steam sterilization. First auxiliary chamber of the processing module is inserted into the unit and control the camera insert in the second position of the camera. The vessel, containing a fully equipped adjuvants vaccine BVD, obtained as described in example 2, connect with microfluidizer accession highway transfer from the exhaust valve of the supply vessel to the intake opening of microfluidizer. The tank with nitrogen gas connected to the inlet opening of the air filter vessel supply, and installation of pressure in the vessel to regulate the level of 20±5 psi. The exhaust valve of the vessel for collecting attach to the backbone transmission from the exhaust holes of microfluidizer. After making all the required connections for the valves open and microfluidizer begin with working pressure 10000±500 psig. All contained the vaccine is passed through microfluidizer 1 times and collect in the chamber after microfluidizer. The preparation is divided into aliquot dose of 2 ml, and it is the vaccine BVD-based microfluidizer composition AMPHINOGEN®.

EXAMPLE 4

A composition of the vaccine through the your mixing in gas furnace

Composition AMPHINOGEN®, obtained as described in example 1, diluted to 2.5% with the addition of BVD antigens and filler. The resulting solution is mixed in a gas furnace with use of the bar for mixing instead of using a homogenizer. The final product contains the following composition: antigens BVD type 1 and type 2 to 2.5% of the composition AMPHINOGEN® (which contains oil, lecithin, SPAN® and TWEEN® as described in example 1) and saline. TWEEN 80 and SPAN 80 are present in the final preparation of the vaccine, respectively, in the amount of 0.18% and 0.08% vol.

EXAMPLE 5

Comparison of drop size distribution between

the vaccine preparations based not microfluidizer

and microfluidizers composition AMPHINOGEN®

The vaccine is based not microfluidizer composition AMPHINOGEN®, obtained as described in example 2, the vaccine microfluidizer composition based on the composition AMPHINOGEN®, obtained as described in example 3 and the product obtained by mixing in a gas furnace as described in example 4 is used to compare the particle size of the vaccine preparations. 2 ml sample of each drug added to the probe laser diffraction Malvern 2000 and determine the distribution of droplet size. As shown in Fig.3, the results indicate that the drug vaccines microfluidizer songs on the new songs AMPHINOGEN® has a maximum volume of particles of about 0.1 μm, while the preparation of a vaccine not microfluidizer composition based on the composition AMPHINOGEN® has the distribution of the maximum volume of particles of about 1 micron.

EXAMPLE 6

The reduction of phase separation vaccine

Three different preparations of the vaccine: the vaccine is based not microfluidizer composition AMPHINOGEN®, obtained as described in example 2, the vaccine microfluidizer composition based on the composition AMPHINOGEN®, obtained as described in example 3, and the vaccine is obtained by mixing in a gas furnace as described in example 4, compare side by side to determine their properties phase separation during long-term storage. All of these drugs was allowed to stand at 4°C for approximately 1 month., and phase separation of control from the point of view of the external form of a cream layer on top of the vaccine preparations. As shown in Fig.4, when compared with the other two drugs, drug based not microfluidizer composition AMPHINOGEN® no phase separation.

EXAMPLE 7

Getting microfluidizers and not microfluidized vaccine for cattle against viral diarrhea virus in cattle

The antigen of the virus of viral diarrhea in cattle injected to obtain emulsion composition AMPHINOGEN® by microplay is Itachi. The term "include to obtain emulsion" refers to the way in which the antigen is added to the composition AMPHINOGEN® before microfluidizer. Antigen defy the effects of physical forces of the process of microfluidizer along with components of the adjuvant composition. In the control not microfluidizer group drug antigen is dispersed in the composition AMPHINOGEN® by mixing.

The final composition and control, and microfluidizers drugs has the following composition:

BVD type I ELISA titer after inactivation 2535 KE/dose for gp53, BVD type II ELISA titer after inactivation 3290 KE/dose for gp53, Quil-A at a concentration of 1.25 mg/dose, cholesterol at a concentration of 1.25 mg/dose, composition AMPHINOGEN® at a final concentration of 2.5% and thimerosal at a final concentration 0,009%. The vaccine dose is 5 ml

EXAMPLE 8

Long-term stability is enabled after receiving

emulsion BVD viral antigens in the preparation of vaccines

based microfluidizer composition AMPHINOGEN®

The experiment carried out to determine the stability enabled to obtain an emulsion of the antigen during long-term storage. BVD viral antigen type II include to obtain emulsion composition AMPHINOGEN® during microfluidizer to obtain drug microfluidized vaccine (A). Proc. of the other drug, vaccine, containing the same antigen in not microfluidizer composition AMPHINOGEN® (A, A and A), serve as control. Not microfluidizer drugs antigen is mixed with the composition AMPHINOGEN® and mixed by stirring using a homogenizer Ross. All four of the preparation of the vaccine was stored at 4°C for two years. At different points in time during the storage time (0, 6, 12 or 24 months.) all four compositions are used for the three-month vaccination of calves.

At 0 and day 21 three-month calves Vaccinium through subcutaneous route of administration of 2 ml of vaccine composition. Serum from vaccinated animals gather on the 35th day, and serological response to the vaccine is measured from the point of view of antibody titer by ELISA BVDV-E2. As shown in Fig.5, the drug microfluidized vaccine has higher antibody titer in all tested time points (0, 6, 12 and 24 months), suggesting that the stability of the preparation of the antigen is not lost for the inclusion of an antigen to obtain an emulsion during the process of microfluidizer. Moreover, it was found that the drug microfluidized vaccine causes an immune response in all points of time.

EXAMPLE 9

The reduction caused by the vaccine increase

rectal temperature after microfluidizer

Drugs MICROTEL Edizioni and not microfluidized vaccine manufactured as described in example 7, is used for vaccination of cattle in zero day, and rectal temperature control during the period from one day before vaccination to four days after vaccination. The vaccine dose is 2 ml Group Vaccinium either in single or double dose of the vaccine. Values of rectal temperature was measured and recorded daily from the 1st to the 4th day inclusive. Values of rectal temperature at day 0 measured before administration of the test items.

As shown in Fig.6, the results indicate that there is a steep rise of rectal temperature in approximately 24 h after vaccination in animals that were vaccinated either in single or double dose of the composition of the vaccine. However, in animals vaccinated microfluidizer forms of the vaccine, the rise of rectal temperature after vaccination was only minimal and significantly lower than in animals not vaccinated microfluidizer composition (Fig.6).

EXAMPLE 10

The volume of the reaction at the injection site were resolved

faster when vaccination microfluidizer

the vaccine compositions

Drugs microfluidizers and not microfluidized vaccine, manufactured as described in example 7, was used to vaccinate cattle RMSE is in the zero day. Animals included in this study was a hybrid of meat cattle. It was three animals in each treatment group placebo (t and t). In each group with T03 on T was six animals. The vaccine dose was 2 ml, and groups were vaccinated or one or two doses of vaccine at zero day. In zero day test article was administered in the right part of the neck. Animals treated with a double dose (4 ml) of the test items (t, T04 and T), got a double dose as a single injection in the same direction. Monitoring sites of injection, including assessment of the extent of reaction at the injection site, was performed on the right side of the neck with the 0-th to 4-th day, inclusive, and in the 6th, 9th, and 14th days. 0-day sites of injection were observed before the introduction of the tested products. The groups vaccinated with one or two doses of placebo and did not show any significant increase in reaction injection site, and therefore these data are not shown in Fig.7. If not microfluidizer composition of the vaccine was observed proportional increase in reaction injection site between vaccination with one dose and two doses. On the other hand, in the case microfluidizer composition of the vaccine, although a single dose caused a larger amount of reaction injection site, the injection of the second dose did not cause any further increase. B is further, in the case of injections, the animals microfluidizer composition of the vaccine reaction volume of injection site were resolved faster in comparison with those in animals that were injected with not microfluidizer composition of the vaccine. These results are shown in Fig.7.

EXAMPLE 11

Getting the vaccine preparations based on microfluidizer composition AMPHIGEN® enabled to obtain emulsion BVD virus and leptospirosis antigens and immunostimulating molecules, such as Quil-A and DDA

Inactivated by formalin strain CSLLeptospira hardjo-bovisincluded in the composition with an appropriate adjuvant in the amount of approximately 1.4×109organisms/5 ml formalin Inactivated strain TLeptospira Pomonaincluded in the composition in an amount of about 2400 values units/5 ml dose. Values unit was calculated on the basis of the measurement values is pre-processed fermentation liquid. The BVD virus type I included in the composition when the titer Elisa E2 approximately 3000 relative units/5 ml dose. The BVD virus type II included in the composition when the titer Elisa E2 approximately 3500 relative units/5 ml dose. Relative unit was calculated on the basis of the ELISA titer E2 total amount of liquid prior to the preparation of the composition after inactivation. And Quil-A, and cholesterol used at the end of the ation 0.5 mg 1 dose. Thimerosal and composition AMPHINOGEN® was used at final concentrations, respectively 0,009% and 2.5%. Aluminium hydroxide (Rehydragel LV) was used at a final concentration of 2.0%. When DDA was used as an immunomodulator, DDA included into the composition AMPHINOGEN®. Composition AMPHINOGEN® (i.e. 40% of the mother liquor Drakeol-lecithin) contained 1.6 mg/ml DDA, and, when appropriate, was divorced, the final preparation of the vaccine contained 2.5% of the composition AMPHINOGEN® and 0.1 mg/ml DDA.

Upon receipt of the various compositions of the vaccine fractions BVD, Leptos, Quil A, cholesterol, thimerosal, composition AMPHINOGEN® and saline as a filler was added in a Silverson homogenizer and mixed for 15 min at 10000±500 rpm Then the components were microfluidizer through the strainer 200 μm at 10,000 pounds per square inch.

When the composition of the vaccine contains aluminium hydroxide, microfluidizer conducted without aluminium hydroxide. After microfluidizer added aluminum hydroxide and mixed bar mixer overnight at 4°C.

EXAMPLE 12

Getting viral vaccine BVD for research

control of infection

The preparation of the vaccine used in this experiment contain antigens from virus BVD type 1 and BVD virus type 2. Antigen BVD1-5960 used in the ELISA titer after inactivation 2535 KE/dose for gp53. Antigen BVD2-all when ELISA titer after inactivation 3290 KE/dose for gp53. Quil A and cholesterol were used at a concentration of 0.5 mg/ml of Thimersol and composition AMPHINOGEN® was used at a final concentration respectively 0,009% and 2.5%. When DDA was used as an immunomodulator, DDA included into the composition AMPHINOGEN®. The composition of the mother liquor AMPHINOGEN® (40% solution Drakeol-lecithin) contain varying amounts of DDA, and, when appropriate, was divorced, the final preparation of the vaccine contained 2.5% of the composition AMPHINOGEN® and DDA in concentrations ranging from 0.5 mg/dose up to 2.0 mg/dose. Aluminum gel (Rehydragel-LV) was used at a final concentration of 2%. GPI-0100 used in the range of 2, 3 and 5 mg/dose.

All components were added in a Silverson homogenizer and mixed for 15 min at 10500 rpm, and then microfluidizer passing through the chamber 200 μm at 10,000 pounds per square inch. When the drug vaccines contain aluminium hydroxide, microfluidizer conducted without aluminium hydroxide. After microfluidizer added aluminum hydroxide and mixed bar mixer overnight at 4°C.

EXAMPLE 13

Protection against infection control leptospirae after vaccination microfluidized vaccine composition

Amphinogen with leptospirosis antigens

Table 1
Group treatment
The treatment groupThe adjuvant composition
TSaline
TQuil A, cholesterol and composition AMPHINOGEN® (QAC)
T03Quil A, cholesterol, composition AMPHINOGEN® and AlOH (QAC-AlOH)
T04DDA, cholesterol and composition AMPHINOGEN® (DDA)
T05DDA, cholesterol, composition AMPHINOGEN® and AlOH (DDA-AlOH)

Table 1 shows the composition of the adjuvant compositions in vaccine preparations tested in this study. The vaccine preparations were obtained as described in example 11. Each group contained six animals. This study used a hybrid heifers at the age of about 6-7 months. Vaccination was performed at 0 and 21 days by subcutaneous volume of 5 ml. vaccine Control infection with a strain ofL. hardjo-bovisfrom NADC (National center for diseases of agricultural animals). Control contamination produced during 57-59 th days inoculate 1 ml Control the infection was carried out by conjunctival introduction to the eyes and vaginally. Material control infection contained 5,0×106leptospires/ml M the Chu took weekly to obtain a culture of leptospires, FA (fluorescence analysis) and PCR (polymerase synthesis reaction chain). The kidney tissue was taken for 112 and 113 days.

Table 2
The results of the study of the control of infection leptospirae
The treatment% of calves that have ever been discovered Leptospira in the urine and renal tissue according to crops% of calves that have ever been discovered Leptospira in the urine and renal tissue according to FA% of calves that have ever been discovered Leptospira in the urine and renal tissue according to PCR% of calves that have ever been discovered Leptospira in the urine and renal tissue according to all analyses
Saline10083,383,3100
QAC0000
QAC/AlOH050,00 50,0
DDA0000
DDA/AlOH033,316,750,0

Table 2 shows the research data control infection leptospirae. When determining the percentage of infection leptospirae have been subjected to control and infected animals used the following criteria. If kidney culture was positive in only one sample, the animal was considered positive for Leptospira. If the animal is positive only in one sample, according to the FA or PCR, the animal is considered negative. If the sample is positive and according to FA, and PCR, only one sample, it was considered positive for Leptospira.

The results, shown in table 2, indicate that there were significantly shorter duration of allocation of leptospires in the urine in all groups of vaccines based on all three tests. With regard to the colonization of the urine and kidneys, the effectiveness of compositions containing QAC and DDA without AlOH, were comparable. AlOH did not improve and even reduced the effectiveness of vaccines containing QAC and DDA in the study of the control of infection.

Table 3
The range of titer microscopic agglutination per day maximum geometric mean titer before control infection (35-day)
The treatmentL. HardjoL. pomona
Saline<20<20
QAC160-6401280-10240
QAC/AlOH160-25608-10240
DDA40-1280320-2560
DDA/AlOH320-6401280-5120

Serological reactions against both leptospirose antigens in the composition of the vaccine were detected in vaccinated animals, and the maximum response was observed at the 35th day. No correlation between serological response and protection against infection control. The presence of aluminum gel in the composition of the vaccine reduced the level of protection, although serological reaction was intensified by the presence of aluminum gel in the vaccine.

b> EXAMPLE 14

The immune reaction caused by BVD viral antigen,

and protection against the control of infection with BVD virus

type 2 after immunization drug microfluidized vaccine containing composition AMPHIGEN® and DDA

In this experiment used a 4-7-month-old seronegative calves. There were six different groups, and each group had ten animals (table 4). In the 0-th and 21-St day, each animal received one dose of vaccine or placebo 2 ml subcutaneously in the lateral surface of the neck about halfway between the shoulder and the nape of the neck.

Table 4
Group treatment
The treatment groupThe adjuvant composition
TSaline
TQuil-A, composition AMPHINOGEN® and cholesterol
T03composition AMPHINOGEN®, cholesterol, DDA (0.5 mg/dose) and AlOH
T04composition AMPHINOGEN®, cholesterol and DDA (0.5 mg/dose)
T05composition AMPHINOGEN®, cholesterol and DDA (1,0 mgdose)
Tcomposition AMPHINOGEN®, cholesterol and DDA (2.0 mg/dose)

Dose 5 ml of virus for the control of infection (approximately 2.5 ml per nostril) were administered intranasally at the 44th day of the study. No cytopathic BVD virus type 2 isolate #24515 (strain Ellis), lot #46325-70 used in this study as control strain infection. Retained samples of the material control of infection was titrated (2 repetitions for titration) at the time when he started controlling infection, and immediately after its completion. The average titer of live virus at a dose of 5 ml was 5.3 log10FAID50/5 ml prior to infection control and 5,43 log10FAID50/5 ml after control of infection (FAID represents the equivalent TCID50average cytopathogenic dose).

Animals were monitored daily from the 3rd to the 58th day. Scoring of clinical disease, 0, 1, 2 or 3 on the basis of clinical signs that could be attributed to infection with BVD 2, was determined for each animal with 42nd on the 58th day. Scoring in the 44th day were recorded before the test contamination. Blood samples (2 tubes with a capacity of 13 ml to separate the serum, SST) were taken from each animal at 0, 21st, 35th, 44th and 58th day to determine serum antibody titers, neutralizes the virus BVD type 1 and BVD type 2.

Blood samples were taken from each animal with the 42nd on day 58, inclusive, and determined the presence of BVD virus in cells leukocyte film. On the 44th day the samples were obtained prior to infection control.

To determine the leukocyte blood samples (one test tube and 4 ml EDTA) were taken from each disease with 42nd on day 58, inclusive. On the 44th day the samples were obtained prior to infection control.

Leukopenia was defined as a reduction by 40% or more in the number of cells, compared to the baseline level (average number of cells before infection control for the two days prior to infection control and the day of infection control).

Scoring of clinical disease used to determine a painful condition as follows: if the score is ≤1, then disease=no; if the score is >2, then disease=Yes.

As shown in tables 5 and 6, the groups vaccinated with vaccines containing viral antigens BVD, along with microfluidizer composition AMPHINOGEN®, Quil-A, or DDA, was seroprevalence with significant neutralization titres in serum for virus and BVD type 1, BVD type 2. In these groups was also significant decrease in the percentage of animals exhibiting viraemia after control of infection, while in the control group 100% alive is the shaft had viremia (table 7). In addition, these vaccinated groups, the incidence was also significantly reduced (table 8). Similarly, the percentage of animals exhibiting leukopenia, was also reduced in the vaccine groups, and decreased leukopenia was more significant in the group containing the DDA than the group containing Quil-A (table 9). In the control group there was a significant drop weight gain, when compared with vaccinated groups (table 10).

Serology

Before vaccination in the 0-th day of the animals in the study were seronegative (SVN<1:2) for antibodies to BVD virus types 1 and 2 (data not shown). Through day 14 after the second vaccination (35th day) all the animals who were given a placebo (t) remained seronegative in respect of antibodies to BVD virus types 1 and 2; and all animals vaccinated ITA (research test antigen) (t, T03, T04, T05 and T) were seropositive (SVN≥1:8) in respect of antibodies to BVD virus types 1 and 2. In one animal, which had introduced the vaccine compositions with additives AMPHINOGEN® 2 mg/dose DDA, on the 35th day titer SVN antibodies to BVD virus types 1 and 2 was 3 (tables 11 and 12).

Before controlling contamination at the 44th day, all the controls (t), with the exception of one, were seronegative (SVN<1:2) in respect of antibodies to BVD virus types 1 and 2 (data not shown). One control (#2497) was seropositive (SVN=10), who compared the antibodies to BVD virus types 1 and seronegative in respect of antibodies to BVD virus type 2. 14 days after control of infection all animals in the study were seropositive against antibodies to BVD virus types 1 and 2.

Table 5
The geometric mean of the titers of neutralizing
the BVD virus type 1 in serum
The treatmentThe geometric mean of the titers SVN BVD virus type 1
021354458
TSaline<2<2<2<223,9
TAntigen Quil-A<239,119824,514018,227554,5
T03Amphigen 0.5 mg, DDA, Al<251,832204,8 22381,123170,4
T04Amphigen 0.5 mg DDA<227,014512,48932,021996,2
T05Antigen 1.0 mg DDA<226,711585,28194,620882,0
TAmphigen 2.0 mg DDA<223,58778,76769,316961,1

Table 6
The geometric mean of the titers of neutralizing
the BVD virus type 2 in serum
The treatmentThe geometric mean of the titers SVN BVD virus type 2
021354458
T Saline<2<2<2<2522,0
TAntigen Quil-A<28,92272,42048,224833,6

T03Amphigen 0.5 mg DDA, Al<29,53565,72702,220881,8
T04Amphigen 0.5 mg DDA<24,11260,7989,118496,2
T05Antigen 1.0 mg DDA<26,41398,81453,930047,8
TAmphigen 2.0 mg DDA<27,71673,2 1428,916384,0

Table 7
Virus isolation after controlling BVD infection
The treatmentThe selection of BVD virus
In the days of research
of
Frequency
occurrence (%) of animals with viremia
The average number of days with the presence of viremia
TSaline47 5810/10 (100,0)10,4
TAntigen Quil-A50 531/10 (10,0)0,4
T03Amphigen 0.5 mg DDA, Al--0/10 (0,0) 0,0
T04Amphigen 0.5 mg DDA48, 50, 52, 573/10 (30,0)0,5
T05Antigen 1.0 mg DDA49 through 512/10 (20,0)0,4
TAmphigen 2.0 mg DDA48 522/10 (20,0)0,5
Table 8
Clinical signs of disease caused by BVD,
after controlling infection
The treatmentThe frequency of vstreche-
on (%) of animals with diseases
of
Frequency (%) of cases observed in animals with clinical signs of disease caused BVDThe total number of nab-
ludeni
0 123
TSole-
howl
Rast-
thief
9/10(90,0)75(46)63(37,5)29(17,3)1(0,6)168
TAmphi
gene Quil-A
1/10(10,0)105(61,8)63(37,1)2(1,2)0(0)170
T03Amphi
gene 0.5 mg DDA, Al
2/10(20,0)99(58,2)67(39,4)4(2,4)0(0)170

T04Amphi
gene 0.5 mg DDA
0/10(0,0)118(69,4)52(30,6)0(0)0(0)170
T05Amphi
gene 1.0 mg DDA
0/10(0,0)101(59,4)69(40,6)0(0)0(0)170
TAmphi
gene 2.0 mg DDA
0/10(0,0)104(61,2)66(38,8)0(0)0(0)170

Table 9
Leukopenia after controlling infection
The treatmentLeukopenia
Frequency (%) detect animals with leukemiaThe minimum root mean square value of the days of the presence of leukemia
TSaline10/10 (100,0)7,8
TAntigen Quil-A6/10 (60,0)1,2
T03mygen 0.5 mg DDA, Al2/10 (20,0)0,2
T04Amphigen 0.5 mg DDA4/10 (40,0)0,8
T05Antigen 1.0 mg DDA3/10 (30,0)0,9
TAmphigen 2.0 mg DDA2/10 (30,0)0,5

Table 10
Body weight and weight gain during the study
The treatmentThe average body weight (pounds) per day researchThe increase of body weight (pounds)
-1435058
TSaline378,0484,9491,0of 476.9the 98.9
TAmpage the Quil-A 428,0526,5546,7579,0151,0
T03Amphigen 0.5 mg DDA Al410,5514,4534,2579,0168,5
T04Amphigen 0.5 mg DDA373,7472,3492,6538,1164,4
T05Antigen 1.0 mg DDA358,9451,4478,9507,1148,2

TAmphigen 2.0 mg DDA408,0was 513.3533,9560,3USD 151.6

Virus isolation

According to the data presented in table 13, during the period of control of infection (from 44th on the 58th day) in all animals in the control (C) was viremia (virus BVD was granted the Yong in 1 or more days). The groups, which were introduced ITA, the frequency of detection of viremia in animals was 1, 0, 3, 2 and 2 in each group of 10 animals (respectively t, T03, T04, T05 and T). The difference between the control and groups, which were introduced ITA, was statistically significant (P≤0,05). The minimum root mean square value of the days of viremia was also significantly higher (10.4 days) in the control group compared with the groups, which were introduced ITA (from 0.0 to 0.5 days).

Clinical disease

Animals with point estimates of clinical signs 2 or 3 was considered to be showing signs of disease caused by BVD. As shown in table 14, the incidence of animals with clinical signs of disease caused by BVD was 9 out of 10 in the control (C) and 1, 2, 0, 0 and 0 out of 10 in each group, which was introduced ITA (respectively t, T03, T04, T05 and T). The difference between the control and groups, which were introduced ITA, was statistically significant (P≤0,05).

Leukopenia

As shown in table 15, during the period of control of infection (from 44th on the 58th day) in all 10 animals in the control (C) was leukopenia (reduction of 40% in the number of cells compared with baseline levels prior to infection control, 42nd-44th days). The number of animals with leukopenia was 6, 2, 4, 3 and 2 out of 10 animals in each group, which was introduced ITA (respectively t, T03, T04, T05 and T). The difference between the control and the group, which have introduced the vaccine, which had an additive composition AMPHINOGEN® in the amount of 0.5 mg/dose and aluminium hydroxide (T03), was statistically significant (P≤0,05). The minimum root mean square value days leukopenia was significantly greater (7.8 days) in the control group compared with the groups, which were introduced ITA (from 0.2 to 1.2 days).

EXAMPLE 15

The immune reaction caused by BVD viral antigen,

and protection against the control of infection with BVD virus

type 2 after immunization microfluidizer

the composition of the vaccine containing GPI-0100

Observed set of experimental conditions as described in example 14, and carried out a direct comparison between Quil-A and GPI-01000. As shown in tables 11 and 12, in animals vaccinated for BVD antigens in the preparation on the basis microfluidizer composition AMPHINOGEN®, containing or Quil-A, or GPI-01000, there was a significant antibody titer to the virus BVD type 1 and BVD virus type 2. The titer of antibodies to BVD virus type 1 was much higher than for BVD virus type 2. However, subsequent control infection with BVD virus type 2 showed strong protection, and the frequency of the disease was significantly reduced in calves vaccinated microfluidizer drug vaccines based on the composition AMPHINOGEN® containing GPI-01000.

table 11
Geometric average neutralization titers in the serum of BVD virus type 1
The treatmentThe geometric mean of the titers of neutralizing serum virus
021354357
TSaline<2<2<2<235,5
TAmphigen, Quil-A<298,720171,012203,444762,4
T03Antigen 2 mg, GPI-0100, AlOH<284,610998,57383,225709,2
T04Antigen 2 mg GPI-0100<2to 106.018179,2 8933,228526,2
T05Antigen 3 mg GPI-0100<262,915024,38780,119824,4
TAntigen 5 mg GPI-0100<271,112203,37512,016670,2

td align="center"> 14,7
Table 12
Geometric average neutralization titers in the serum of BVD virus type 2
The treatmentGeometric average neutralization titers in the serum of BVD virus type 2 in day research
021354458
TSaline<2<2<2<2
TAmphigen, Quil-A<212,92312,01692,51663,4
T03Antigen 2 mg, GPI-0100, AlOH<213,21663,51116,81562,3
T04Antigen 2 mg GPI-0100<220,52610,21978,22478,7
T05Antigen 3 mg GPI-0100<211,41752,81305,22435,4
TAntigen 5 mg GPI-0100<2to 12.03158,42120,21845,6

Table 13
The separation is of the BVD virus after infection control
The treatmentThe selection of BVD virus
Number (%) of animals with viremiaThe minimum root mean square value of the days of the presence of viremia
TSaline10/10(100,0)8,4
TAntigen Quil-A3/10(30,0)0,3
T03Antigen 2 mg, GPI-0100, AlOH0/10(0,0)0,0
T04Antigen 2 mg GPI-01001/10(10,0)0,1
T05Antigen 3 mg GPI-01003/10(30,0)0,3
TAntigen 5 mg GPI-01002/10(20,0)0,2

Table 14
Clinical signs of disease, you are the bathing virus BVD,
after controlling infection
The treatmentThe number-
in (%) of animals with diseases
of
Number (%) of observations with a point estimate of clinical diseaseTotal coliform
chest
in the nab-
ludeni
012
TSaline5/10(50,0)103(60,6)55(32,4)12(7,1)170
TAmphigen, Quil-A5/10(50,0)115(67,6)48(28,2)7(4,1)170
T03Antigen 2 mg, GPI-0100, AlOH0/10(0,0)128(75,3)42(24,7)0(0)170
T04Antigen 2 mg GPI-01000/10(0,0)124(72,9) 46(27,1)0(0)170
T05Antigen 3 mg GPI-01000/10(0,0)104(61,2)66(38,8)0(0)170
TAntigen 5 mg GPI-01000/10(0,0)128(75,3)42(24,7)0(0)170

2 mg, GPI-0100, AlOH
Table 15
Leukopenia after controlling infection
The treatmentLeukopenia
Number (%) of animals with leukopeniaThe minimum root mean square value of the days of the presence of leukopenia
TSaline9/10(90,0)8,7
TQuil-A6/10(60,0)1,6
T037/10(70,0)2,6
T042 mg GPI-01004/10(40,0)1,5
T053 mg GPI-01007/10(70,0)2,6
T5 mg GPI-01008/10(80,0)2,9

In conclusion, the safety of each vaccine was demonstrated by the absence of adverse reactions in vaccinated animals. The activity of each vaccine was demonstrated by seroprevalence (SVN antibody titers to BVD 1 and BVD-2>1:8) in 100% of vaccinated animals. Satisfactory resistance to infection control was demonstrated vaccine with the addition of only 2 mg GPI-0100.

EXAMPLE 16

The preparation of vaccines containing microencapsulating

antigen, microfluidized emulsion oil-in-water"

3 g of Trehalose (Fluka) are added to water to obtain a stock solution containing 333 mg/ml trehalose. Recombinant antigen PauA, the solubilized 0.8% solution of SDS (sodium dodecyl sulfate) (SDS/rPauA) are added to the trehalose to obtain a final concentration 494 ág rPauA/ml In the following with the adiya's 10 g polylacticacid acid (PLG-Resomer RE 503H, Boeringher Ingelheim) is dissolved in 200 ml of methylene chloride (MeCl2). The resulting solution PLG/MeCl2combined with a solution of SDS-rPauA/trehalose obtained in the first stage. The combined solution is subjected to microfluidizer using microfluidizer (Microfluidics model MAN), and microfluidizers the product is dried by spraying, using a device Temco Spray Dryer, model SD-05. Dried by spraying the material collected using a strainer 500 microns.

The concentration rPauA this spray dried material is quantitatively determined by analysis of the Western blot. 1,04 mg spray dried material was dissolved in 50 μl of acetone and centrifuged at 13200 rpm at room temperature for 10 minutes the Supernatant removed. The supernatant and obtained after centrifugation of sediment fractions are dried in the hood biological safety for 2.5 hours the Precipitate after centrifugation resuspended in 47,43 ál of the sample solution (25 μl of sample buffer + 10 μl of the reducing substances + 65 μl of water). The dried supernatant fraction resuspended 20 ál of the sample solution. In the analysis of the Western blot of purified PauA used as a standard for the quantitative determination of the content of rPauA in the spray dried material.

The mother solution of 20% mannitol is produced by dissolving 100 g of mannitol (Sigma 500 ml of water for injection (WFI). The solution is heated to 40°C hot plate/stirrer and cooled to 30°C. the Solution is subjected to sterile filtration through a sterile 0.22 μm filter (Millipore). 2.5% solution of carboxymethyl cellulose is prepared by dissolving 12.5 g of carboxymethyl cellulose (Sigma) in 500 ml of WFI and mixed overnight at 4°C. the Solution autoclave at 121°C.

The powder obtained by spray drying, dissolved in a solution containing 5% mannitol, 0.3% of carboxymethylcellulose and 1:5000 thimerosal. The final solution is divided into aliquot quantities in bottles for 3 ml and lyophilizers using lyophilizator (USIFROID). Dried powder is microencapsulating rPauA. Microencapsulating subunit protein antigen resuspended in 2 ml microfluidized emulsion oil-in-water" containing composition AMPHINOGEN® (such as microfluidizer emulsion described in example 20), and used as vaccines.

EXAMPLE 17

Getting microfluidizer composition vaccines containing

and the antigen of whole bacterial cells, and recombinant protein antigen in the emulsion of the "oil-in-water

Made two drug vaccine, which contained and recombinant protein PauAStreptococcus uberisand bacterial cellsEscherishia coliadded to obtain an emulsion of oil-in-water", as is written in examples 2 and 3. Recombinant antigen PauA was present at a concentration of 100 μg per 1 dose, and cellsE. coliwas in target number of 4×1091 dose. Emulsion adjuvant compositions of the two vaccine compositions shown in table 16.

Table 16
The composition of the vaccine containing the recombinant protein, and whole cellsE. coli
The treatmentAntigenAdjuvant
TPlaceboSaline
TPau A/E. coliSEAM-14
T03Pau A/E. coli2.5% amphigen, 0.5 mg GPI-0100, 0.5 mg of cholesterol
T04Pau A/E. coli2.5% amphigen, 0.5 mg of bromide of dimethyldioctadecylammonium (DDA), 0.5 mg of cholesterol

EXAMPLE 18

The immune response to microfluidized vaccine

containing rPauA and bacterial antigens of whole cells

in the emulsion of the "oil-in-water

This expertise is ment used Mature dairy cows. At the time of inclusion in the study animals was the end of their first or second lactation. 2 ml of each composition of the vaccine was administered subcutaneously 3 times, 1 time during the launching cows (D-0), after 28 days (D=28) and again 4 to 10 days after calving (With+4+10). The first and the third dose was administered in the left side of the neck, and the second dose was injected into the right side of the neck. Blood was taken before each vaccination and after about 14 days and 32 days after the third vaccination. The titer of antibodies to theE. coliand to the antigen rPauAwere determined using ELISA. As shown in Fig.8, the results indicate that the titer of antibodies to rPauA was higher in the group vaccinated with a vaccine composition containing GPI-0100 as immunostimulant, and he reached the peak on the 70th day after the initial vaccination. The titer of antibodies toE. coliit is shown in Fig.9. The titer of antibodies toE. coliwas comparable in both compositions, vaccines, although the presence of the GPI-0100 as immunostimulant caused relatively higher antibody titer compared to the composition with DDA as immunostimulant.

EXAMPLE 19

Analysis viricidal activity microfluidizer

the vaccine preparations based on the composition AMPHINOGEN®

To determine inactivates whether the virus microfluidizer, defined viricidal activity three microfluidizer drugs vaccines cos the ve compositions AMPHINOGEN®. These three drugs contained three different infectious virus of cattle, namely herpes virus of cattle (BHV), the virus parainfluenza 3 (PI3) and respiratory syncytial virus in cattle (BRSV).

Identifying viricidal activity three vaccine preparations were carried out in accordance with the requirements of the provisions of the USDA 9CFR.113.35.

The results, shown in table 17, indicate that microfluidizer drugs vaccines based on the composition AMPHINOGEN® does not cause any significant inactivation of the drug vaccine.

Table 17
Analysis viricidal activity microfluidizer vaccines
SeriesBRSVBHVPI3
A00,20
AV200-0,20-0,2
AM750-0,3-0,3
AM75@37Ca 0.1 -0,3-0,2
B0-0,1-0,2
BM20000-0,2
BM75-0,2-0,50
BM75@37C0,5-0,50
C0,1-0,1-0,2
CM200-0,2-0,1-0,2
CM750,10,5-0,2
CM75@37C0,50,5-0,2

A = Cholesterol, added at a concentration of 650 ml/min

In = Cholesterol, added at a concentration of 28 ml/min

C = Cholesterol, added at a concentration of 5 ml/min

M200 = Microfluidizer mesh filter 200 microns

M75 = Microfluidics is by mesh filter 75 micron

M75@37C = Liquidheated to 37°C before microfluidizer

Value above 0.7 is an indication viricidal effect.

EXAMPLE 20

Getting microfluidizer composition AMPHINOGEN®

Composition AMPHINOGEN® was obtained by combining a solution of lecithin oil DRAKEOL (light mineral oil with 25% of lecithin and TWEEN-80 (final concentration of 0.18%) and Span 80 (final concentration of 0.08%) when mixed within 8-22 h at 36±1°C. Then the mixture was added to the salt solution with an emulsifier Ross® (Hauppauge, NY 11788) at approximately 3400 rpm In subsequent mixture was passed once through microfluidizer camera interaction 200 μm at 4500±500 pounds per square inch. In Fig.10A and 10B show the stability of microfluidizer composition AMPHINOGEN®. The distribution of particle size as measured by laser diffraction, in the beginning, at the starting time point (Fig.10A) was almost identical to the size distribution of particles through 22 months of storage at 4°C (Fig.10V).

1. The composition of vaccines for the induction of immune responses in animals, where this composition comprises an antigen and a 40% emulsion of the oil-in-water, diluted to 2.5%, where this 40% emulsion of oil-in-water" contains:
a) 30% vol./about. light petroleum nametables oil;
b) 10% vol./about. lecithin;
c) 0,6% vol./about. sorbitane of monooleate (SPAN),
d) of 1.4%.about. p is liuxiaolingtong of monooleate,
where is the oil component is dispersed in the aqueous component by emulsification,
where the vaccine composition obtained using microfluidizer and has an average droplet size less than 0.3 microns.

2. The composition of the vaccine for p. 1, additionally containing a salt solution of tetanus toxoid and where the maximum size of the oil particles is 0.1 μm.

3. The composition of the vaccine under item 1, where the specified antigen is a viral antigen or a bacterial antigen.

4. The composition of the vaccine under item 3, where the specified viral antigen comprises a killed virus diarrhea, bovine type 1 or type 2, and where specified bacterial antigen comprises at least one component selected from the inactivated bacteria Leptospira, the recombinant protein of Streptococcus uberis PauA or preparation of E. coli cells.

5. The method of obtaining the composition of the vaccine under item 1, including:
(a) obtaining a mixture by combining a light hydrocarbon nametables oil, a surfactant and a water component;
(b) adding antigen to the mixture obtained in stage (a);
(c) the primary emulsification of a mixture containing a specified antigen, which is obtained in stage (b) to obtain the emulsion oil-in-water", with an average droplet size of the oil from 1.0 μm to 1.1 μm; and
(d) microfluidized emulsion obtained in stage (C) with what rucenim the specified composition of the vaccine, where the average size of the droplets of the composition is less than 0.3 microns.

6. The method according to p. 5, where the composition further comprises a salt solution of tetanus toxoid and where the maximum size of the oil particles is 0.1 μm.

7. The method according to p. 5, where the specified antigen is a viral antigen or a bacterial antigen.

8. The method according to p. 7, where the specified viral antigen comprises a killed virus diarrhea, bovine type 1 or type 2, and where specified bacterial antigen comprises at least one component selected from the group inactivated bacteria Leptospira, the recombinant protein of Streptococcus uberis PauA or preparation of E. coli cells.

9. The composition of vaccines for the induction of immune responses in animals, including microencapsulating antigen and 40% emulsion of the oil-in-water, diluted to 2.5%, where this 40% emulsion of oil-in-water" contains:
a) 30% vol./about. light petroleum nametables oil;
b) 10% vol./about. lecithin;
c) 0,6% vol./about. sorbitane of monooleate (SPAN),
d) of 1.4%.about. polyoxyethylenesorbitan of monooleate,
where is the oil component is dispersed in the aqueous component by emulsification,
where the vaccine composition obtained using microfluidizer and has an average droplet size less than 0.3 microns,
where specified microencapsulating antigen dispersed in the specified Amul the FIC.

10. The composition of the vaccine for p. 9, where the composition further comprises a salt solution of tetanus toxoid and where the maximum size of the oil particles is 0.1 μm.



 

Same patents:

FIELD: medicine.

SUBSTANCE: as an active substance, the composition contains butoconazol, a base that is a combination of a hydrophobic ingredient, a hydrophilic ingredient and an emulsifier, and also a gel-forming polymer. Hydroxypropylstarch phosphate is preferentially used as the gel-forming polymer. A method for preparing the declared composition consists in the fact that a mixture of butoconazol with a portion of the hydrophilic ingredient, the hydrophobic ingredient and emulsifier is added with a dispersion of the gel-forming polymer in the rest of the hydrophilic ingredient; the produced mixture is agitated homogenously with a preserving agent added where it might be necessary.

EFFECT: new pharmaceutical composition is characterised by a high level of antifungal activity, stability both at a storage temperature, and at a use temperature, and good pack extrusion.

14 cl, 2 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: claimed is application of fat emulsion for parenteral feeding as solvent for compounds which are poorly soluble in water. Fat emulsion contains in 1 l of solution: 30 g of refined soybean oil, 30 g of triglycerides with the average chain length, 25 g of olive refined oil, 15 g of purified fish oil.

EFFECT: obtaining solvent for compounds, poorly soluble in water, which makes it possible to determine parameters and spectrum of biological activity of novel compounds of chemical nature at the stages of pre-clinical and clinical tests, which does not change basic biological constants and possesses biological inertness.

2 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a producing method of cream containing fusidic acid, which involves a stage of application of sodium fusidate as an initial active ingredient and conversion of the above sodium fusidate in situ to fusidic acid in an oxygen-free medium by immediate addition of the acid to a cream base containing a preservative, an acid, a cosolvent, an emulsifier, a wax-like product and water.

EFFECT: obtaining cream having high stability at storage and smaller particles of an active ingredient.

9 cl, 11 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents dermatological cream, intended for local treatment of bacterial skin infections and for healing wounds associated with them, which contains framycetin sulfate and biopolymer, included into cream base, which contains at least one substance from each of the following groups: preservative, primary and secondary emulsifier, selected from the group which contains ketostearyl alcohol, ketomacrogol 1000, polysorbate-80 and Span-80; paraffin as wax-like product, cosolvent, selected from the group, including propylene glycol, hexylene glycol and polyethylene glycol-400; nitric acid or lactic acid and water, with said biopolymer preferably being chitosan.

EFFECT: invention provides higher therapeutic effect.

8 cl, 10 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmaceutics and represents a pharmaceutical composition for parenteral administration containing sub-micron particles of dosocahexaenoic acid ester dispersed in a water phase with the use of mixture of at least two surfactants specified in a) at least one fatty acid polyoxyethylene ester and b) at least one phospholipide derivative, as well as a method for preparing the above pharmaceutical composition.

EFFECT: invention provides higher pharmacological activity.

14 cl, 3 dwg, 3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the pharmaceutical and cosmetic industry, in particular to nanoemulsions of a water-in-oil type for transdermal application with biologically active compounds.A nanoemulsion of the water-in-oil type contains 35-80% of a hydrophobic phase, 1-15% of a hydrophilic phase, and a surface-active substance.

EFFECT: nanoemulsion of the water-in-oil type for transdermal application with biologically active compounds possesses good storage stability.

8 cl, 1 dwg, 1 tbl

FIELD: biotechnology.

SUBSTANCE: aqueous composition for anaesthesia is proposed, which comprises propofol as an active agent, the PEG-660-12-hydroxystearate as a solubiliser, benzyl alcohol, or chloroethanol or parabens as preservative, the tocopherol and arginine or glycine at a specific content of components wt %. The GABA agonists can be additionally added to the composition, e.g. aminophenyl-butyric acid, local anesthetics such as lidocaine, alpha-2-adrenoceptor agonists such as xylazine. The method is proposed for implementing anaesthesia comprising administering to a patient of an effective amount of the claimed composition.

EFFECT: invention provides low toxicity of dosage form and high efficiency.

5 cl, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: composition containing curcumine, an acid specified in a group consisting of citric acid, malic acid, acetic acid, tartaric acid, lactic acid, alginic acid or a mixture thereof, and an edible emulsifying agent with specific characteristics and taken in a certain amount wherein the above composition is applicable as a therapeutic agent. The composition containing curcumine, the edible emulsifying agent or a mixture thereof, the acid specified in a group consisting of citric acid, malic acid, acetic acid, tartaric acid, lactic acid, alginic acid or a mixture thereof taken in certain proportions, and additionally encapsulated into a gelatine capsule, wherein the above composition is applicable for treating or preventing an inflammation and/or diseases caused by the inflammation.

EFFECT: compositions improve the curcumine bioavailability effectively.

13 cl, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: hydrophilic therapeutic agent is specified in a group consisting of albuterol, bendamustine, captopril, carboplatin, ciprofloxacin, gemcitabine, ibandronate, lamivudine, metformin, niacin, oxycodone, ranitidine and sumatriptan. The composition also contains a solvent, a surfactant and a hydrophilic carrier. The above hydrophilic carrier is compatible to the therapeutic agent solution and the surfactant.

EFFECT: good stability and bioavailability compatible to that of injection formulations when administered orally.

21 cl, 2 dwg, 13 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: pharmaceutical oil-in-water emulsion contains mometasone or mometasone furoate, propylene glycol and water. The propylene glycol concentration makes from 20 to approximately 45 wt %. A mass ratio of propylene glycol and water in the oil-in-water emulsion makes from 1:1 to approximately 1:3. A portion of mometasone or mometasone furoate is found insoluble in the emulsion.

EFFECT: composition is characterised by stability and therapeutic effect.

27 cl, 6 dwg, 5 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to a combination of a type II anti-CD20 antibody and an anti-Bcl-2 active agent for treating cancer, first, CD20 expressing cancer, wherein the above type II anti-CD20 antibody is a humanised B-Lyl antibody and the above active agent is a Bcl-2 protein binding inhibitor and thereby destroys the Bad/Bcl-2 complex with the IC50 anti-Bcl-2 inhibitory activity of 1 mcM or less, as well as to using the type II anti-CD20 antibody for producing a medical drug applicable for treating cancer, first, CD20 expressing cancer, in a combination with the anti-Bcl-2 active agent.

EFFECT: group of inventions in effective in treating cancer, first, CD20 expressing cancer, in a combination with the anti-Bcl-2 active agent.

10 cl, 3 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. What is presented is a prophylactic or therapeutic agent for IL-31 related itching, containing an effective amount of anti-NR10 antibody, possessing NR10-neutralising (alternative name IL-31RA) activity as an active ingredient, and pharmaceutically acceptable additives, wherein the anti-NR10 antibody represents an antibody, which contains the heavy-chain amino acid sequence SEQ ID NO: 17 and the light-chain amino acid sequence SEQ ID NO: 18, or its humanised antibody. What is presented is using the anti-NR10 antibody containing the heavy- and light-chain amino acid sequences SEQ ID NO: 17 and SEQ ID NO: 18 respectively, or its humanised antibodies for preparing the prophylactic or therapeutic agent for IL-31 related itching. What is described is a method for preventing or treating IL-31 related itching, which involves the stage of administering the anti-NR10 antibody containing the heavy-chain amino acid sequence SEQ ID NO: 17 and the light-chain amino acid sequence SEQ ID NO: 18, or its humanised antibodies.

EFFECT: invention enables suppressing IL-31 overexpression itching.

3 cl, 5 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology. What is presented is a composition of antibodies possessing an IGF-IR binding capacity and prepared by culturing a CHO DSM ACC 2795 cell line with an amount of fucose residues in a sugar chain of the antibodies making at least 99%. What is also disclosed is a pharmaceutical composition for tumour growth inhibition and a method for preparing the above pharmaceutical composition by mixing a composition of the antibodies according to the invention with a pharmaceutically acceptable carrier.

EFFECT: composition of the antibodies possesses a low antibody-dependent cell-mediated cytotoxicity (ADCC) and can be used in the therapy of IGF-IR associated diseases.

5 cl, 1 dwg, 4 tbl, 15 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to experimental cardiology and pharmacology, and concerns the endothelial dysfunction correction. That is ensured by a 28-day intragastric administration of a mixture of homeopathic dilutions of vascular endothelial growth factor (VEGF) monoclonal antibodies 2 times a day in a dose of 4.5 mg/kg with underlying intraperitoneal administration of L-NAME 12.5 mg/kg a day into white Wistar rats.

EFFECT: administering the homeopathic dilutions of the VEGF monoclonal antibodies prevents developing the L-NAME induced endothelial dysfunction in the specific experimental environment.

5 cl, 3 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: invention concerns using humanised anti-interleukin-6 receptor (IL-6R) antibodies, which block the IL-6 signal transport and inhibit the biological activity of Il-6 for producing a therapeutic agent for systemic-onset juvenile rheumatoid arthritis.

EFFECT: more effective treatment of systemic-onset juvenile rheumatoid arthritis.

8 cl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology and immunology, and can be used in treating an individual with a stable pathogenic infection or a tumour. That is ensured by administering a therapeutically effective amount of a programmed death (PD-1) peptide antagonist and a therapeutically effective amount of a pathogen or tumour antigen molecule (vaccine).

EFFECT: invention provides the synergetic action of the antigen and PD-1 antagonist (anti-PD-L1 antibody) combination by intensifying the T-cell immune response to the pathogen or tumour.

14 cl, 29 dwg, 5 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biochemistry, in particular to single variable domain, aimed against IL-6R, to polypeptide and construction, directed against IL-6R, containing said single variable domain, as well as to methods of obtaining them. Disclosed are nucleic acids, coding said single variable domain, polypeptide and construction, as well as genetic constructions, containing said nucleic acids. Described are host cells and host organisms, containing said nucleic acids. Invention also deals with composition for blocking interaction of IL-6/IL-6R, containing effective quantity of described single variable domain, polypeptide, construction, nucleic acid or genetic construction. Also disclosed is method of prevention and/or treatment of at least one of diseases or disorders, associated with IL-6, IL-6R, complex IL-6/IL-6R and/or signal pathways, in which IL-6, IL-6R or complex IL-6/IL-6R is involved and/or biological functions and reactions, win which IL-6, IL-6R or complex IL-6/IL-6R takes part with application of described single variable domain, polypeptide, construction or composition.

EFFECT: invention makes it possible to block interaction of IL-6/IL-6R effectively with increased affinity and biological activity.

25 cl, 70 dwg, 56 tbl, 61 ex

Siglec-15 antibody // 2539790

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is described is a pharmaceutical composition used for treating and/or preventing pathological bone metabolism and containing this antibody. The invention can be used in medicine.

EFFECT: antibody and its functional fragment specifically recognising human Siglec-15 and possessing the osteoclast inhibitory activity are described.

73 cl, 57 dwg, 4 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is described is a recovered human antibody or its antigen-binding fragment. The antibody binds to human interleukin-4 alpha-receptor (hlL-4R). There are also described a nucleic acid molecule coding this antibody, an expression vector, a host cell, a method for producing such antibody and a therapeutic composition containing this antibody.

EFFECT: presented group of inventions can be used in medicine for treating asthma and atopic dermatitis.

15 cl, 3 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry.

EFFECT: method of identifying a candidate substance that inhibits hepsin activation of pro-macrophage-stimulating protein (pro-MSP) is provided.

10 cl, 10 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented inventions refer to a lyophilised composition for inducing an immune response to flavivirus, compositions for preparing the above lyophilised composition, and a method for preparing the lyophilised composition. The characterised lyophilised composition contains an effective amount of live attenuated flavivirus, one or more stabilising agents, one or more buffer components, lactose and amorphous mannitol, which is prepared by lyophilising mixture containing an effective amount of live attenuated flavivirus, one or more stabilising agents, one or more buffer components, lactose and mannitol; flavivirus can be chimeric flavivirus. Preparing the above lyophilised composition involves freezing the components and drying them thereafter.

EFFECT: inventions enable preparing the transportation and storage stable compositions containing flavivirus.

31 cl, 13 dwg, 10 tbl, 2 ex

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