Adjuvant based on polyinosinic acid-polycytidylic acid

FIELD: chemistry.

SUBSTANCE: present invention relates to a kit for an immunogenic composition, and an immunogenic composition which contains a polynucleotide adjuvant composition together with an antigen (for example as a vaccine). The disclosed adjuvant compositions (versions) have special physical properties (for example molecular mass, concentration and pH), which meet requirements for a safe adjuvant for inducing intense immune response. The invention also relates to a method of preparing an immunogenic composition and using immunogenic adjuvant compositions, particularly for inducing immune response to an antigenic compound.

EFFECT: disclosed adjuvant compositions are safe and efficiently provide the desired immunogenic effect in humans.

25 cl, 2 dwg, 15 tbl, 16 ex

 

The scope to which the invention relates

The invention generally relates to adjuvant compositions and methods for their use in enhancing the immune response, specifically to compounds, vaccines and methods for enhancing immunogenicity of an antigen, and more specifically to a polynucleotide adjuvant compositions, vaccines, comprising the polynucleotide adjuvant composition, and to methods of use of such polynucleotide adjuvant compositions and vaccines to enhance the immune response in the host.

Background of the invention

1. Description of the prior art

The immune system can be both specific and nonspecific immunity. In General, b - and T-lymphocytes, which Express specific receptors for the antigen on their cell surface, provide specific immunity. The immune system can respond to different antigens in two ways: 1) humoral-mediated immunity, which involves the stimulation of b-cells and production of antibodies or immunoglobulins, representing the antigen cells (APCs) and T-cell helper cells (Th1 and Th2), and 2) cell-mediated immunity (CMI), which generally involves T cells, including cytotoxic T lymphocytes (CTLs), although the generation of a CTL response is also involved other cells (e.g. cells of the Th1 and/or Th2 and APCs).

Nespe the specific immunity covers various cells and mechanisms along with others, such as phagocytosis (uptake of foreign particles or antigens) by macrophages or granulocytes and cell activity of natural killer cells (NK). Nonspecific immunity is based on mechanisms that are less evolutionarily advanced, and does not show the acquired nature of specificity and memory, which are illustrative milestones specific immune response. The key differences between specific and nonspecific immunity is based on the specificity of b - and T-cells. These cells mainly acquire their reactivity after activation by specific antigen and have mechanisms for manifestations of memory in case future contact with that specific antigen. In the vaccination (involving specificity and memory) is an effective Protocol to protect against harmful pathogens.

Adjuvants are generally compounds which when introduced to the antigen (or mixed with the antigen or introduced before the introduction of the antigen) amplify or modify the immune response to the specific antigen.

Examples of adjuvants that have been used to enhance an immune response include aluminum compounds (all generally referred to as “Alum”), emulsion oil in water (complete adjuvant's adjuvant (CFA) is an oil-water emulsion containing dried, is broken by heating organisms Mycobacterium tuberculosis), saponin (isolated from the bark Typically Saponoria, induced active component known as Quile A), one CpG (synthetic oligodeoxynucleotide containing neetilirovannye the CpG dinucleotides), MPL (derived from the polysaccharide of Salmonella Minnesota Re595), liposomes (usually made of biodegradable materials such as phospholipids) and microparticles of biodegradable polymers (made of various polymers, such as PLGA, polyphosphazene and polyanhydride). Estimated adjuvant properties of these compounds, each adjuvant showed advantages and disadvantages.

The biggest problem of the use of adjuvants for human vaccines, in particular for routine childhood vaccines, is the toxicity and side effects of most adjuvant preparative forms. The application of new technologies in vaccine development leads to the production of purified subunit and synthetic antigens, which tend to have low immunogenicity. The development of new adjuvants to enhance the immunogenicity/efficacy and reducing side effects is one of the main problems of the research and development of vaccines.

Polynucleotide complexes investigated in terms of their various uses, including acting as adjuvants. Double-strand RNA ((dsRNAs) are very active biologists who definition modifiers, which in nanomolar concentrations can have a profound effect on the cells. Modulating effects of dsRNA include a wide spectrum of action at the molecular and cellular level.

At the molecular level dsRNAs can induce biological effects, such as interferon synthesis, induction of protein kinase, increased antigen tissue compatibility and inhibition of metabolism. And at the cellular level dsRNAs can induce biological effects, such as progenote, mitogenome, activation of macrophages, activation of cell-mediated immunity and induction of an antiviral state. One promising potential dsRNAs is their immunomodulating effect in the ways of antimicrobial treatment. In U.S. patent No. 4124702 revealed that double-strand polynucleotide caused the induction of interferon in cells of living animals. In U.S. patent No. 3906092 disclosed that the reaction of antibodies to the vaccine adjuvant type amplified by the inclusion in the vaccine polynucleotide or complex polynucleotides. Houston et al. established PICLC (complex polyinosinic acid, polycytidylic acid, poly-L-linkermakeexternalimage) as a potent adjuvant to increase the primary antibody response without the assistance of additional adjuvant (Houston et al., Infection and Immunity, 14:318-9, 1976C). It was found that mycoviruses dsRNA significantly strengthening the em hemagglutinins reaction of antibodies to sheep erythrocytes (sRBC) (Wright and Adler-Moore, Biochemical and Biophysical Research Communications, 131:949-45, 1985).

However, PIC (polyinosine acid, policitally acid) manifests severe toxicity when applied in animals. For example, Phillips et al. reported that severe toxic manifestations were caused by dogs after sub-chronic injection PIC at a dose of 2.0 mg/kg Toxicity was characterized by reduced spontaneous activity, poor coordination, vomiting, anorexia, loss of body weight, hematological changes, reflecting reduced hematopoiesis, decreased activity of alkaline phosphatase and transaminases, degeneration of the thymus, the destruction of bone marrow, increased hepatic sinusoidal capillaries in the Central equity areas, liver cell necrosis, collapse of the structures of the liver and generalized arthritis (see Phillips et al., Toxicology and Applied Pharmacology, 18:220-30, 1971).

PIC, one of the most studied polynucleotide complexes, was ineffective when used in monkeys and human beings due to its instability in the body after injection. Thus, the PIC has been modified multiple ways to overcome one or another disadvantage. For example, the complex polyribosomes-polyribosylribitol acid with poly-L-lingeroption about 5-15 times more resistant to hydrolysis by pancreatic ribonuclease than maternal PIC. Another example is ICLC dsRNA, and the and abbreviated PICLC, which, as has been found highly effective as antiviral or antitumor agents. PICLC is a synthetic dsRNA composed of threads polyribosomes and polyribosylribitol acid (PIC). Although PICLC is a promising immunostimulant which has great potential in ways antimicrobial and anticancer treatment, it was shown that it causes serious side effects in humans, especially with the introduction of the drug in multiple, high doses. Some of the reported side effects include fever, hypotension, leukopenia, myalgia, thrombocytopenia and polyarthralgia. The inherent problem of toxicity need to be overcome to make PICLC security people. In addition, therapeutic efficacy of poly ICLC is limited by the resistance in vivo.

Antiviral agent consisting of polyinosinic acid-polycytidylic acid (poly I:C), kanamycin and calcium (Av PICKCa), used to treat viral infections. It was shown that Av PICKCa able to induce production of interferon and interleukin-2. Av-PICKCa entered separately as an antiviral drug that stimulates the nonspecific immune response, i.e. it stimulates a form of interferon that is not specific for any given antigen. This action is virusna reaction is profoundly different from the specific to the antigen of the immune response, generated with the introduction of adjuvant in combination with the antigen.

Importantly, the author of the present invention have found that Av PICKCa have adjuvant properties, i.e. the ability to induce a specific immune response when introduced to the antigen. Further, the inventor also found that the Av-PICKCa was an effective adjuvant when applying together with antigens, rabies and dengue haemorrhagic fever.

Lin et al. described what Av PICKCa can be used as adjuvant (Lin, et al., A new immunostimulatory complex (PICKCa) in experimental rabies: antiviral and adjuvant effects, Arch Virol, 131:307-19, 1993; and China patent No. 93105862.7). In China patent No. 93105862.7 disclosed the application of the General composition of poly I:C, kanamycin and calcium (PICKCa) as adjuvant in the vaccine for use in humans and mammals.

Samples Av-PICKCa are heterogeneous in terms of size and mass of the molecules. Av-PICKCa described in the literature from the point of view of the average or range of values of the coefficient of deposition, as measured Svedberg S. Antiviral drug Av PICKCa there in one embodiment, from 5S to 8S (source A), see Zhung J.C., Research recollection of polyinosinic-polycytidylic acid (PIC). Paper presented at the 5th Chinese conference “fifth Chinese interferon conference in clinical application and theory”, Siam 1985, pp.23-28. In other embodiments, implementation of the Av-PICKCa there with a coefficient of deposition from 4S to 12S with an average ratio 6S (source or from 5S to 12S with an average coefficient of 7S (source) or from 8S to 10S (source D), see Hu Q.G., Tianjin Av PICKCa''s laboratory research and clinical application, Fujian Medical Journal, 1983.12; (6) 28-30 and Hu Q.G. Chinese Medical and Pharmaceutical Industry Journal, 1983 (9) 3134.

The ratio of deposition of this heterogeneous collection of molecules in Av PICKCa can be translated into an equivalent molecular mass (mm daltons) using conversion factors mm = 1,100×S2,2Su B.X. et al.; Introduction of Biochemical Technology, 1stEdition, Zhongshan University, 1978, 356-357). The results of the translation in the daltons are presented in the table below.

Table And
Specifications Av-PICKCa
Source Av PICKCaThe deposition coefficient SThe molecular mass in daltons
MinimalMaximumAverageMinimalMaximumAverage
And5838,000107,000
In412 623,000260,00057,000
C512738,000260,00079,000
D810107,000174,000

Original research Av-PICKCa adjuvant Lin et al. was conducted with a sample having molecules that have properties similar to source A, i.e. the ratio of deposition from 5S to 8S, that is equivalent to the molecular weight of the molecules in the range from 38000 daltons to 107000 daltons (see Lin et al. above).

It was believed that all forms PICKCa equally safe and effective, provided that the Av PICKCa represents essentially a form PICKCa, and, in addition, provided traditional use Av PICKCa as an antiviral drug. However, it turned out that this is not the case. The study, conducted by the author of the invention, demonstrates that the efficacy and toxicity of PICKCa when used as an adjuvant in combination with an antigen, in fact, varies depending on various molekulyarnoi mass. The inventor has found that the Av-PICKCa provides optimal efficacy/safety for use as adjuvant and that PICKCa in certain conditions causes unacceptable adverse side effects. Thus, there remains a need in Freund, which is more suitable for use in humans and which is safe and effective in providing the desired immunogenic effect. The present invention addresses this need and provides other advantages which will be apparent by reference to the detailed description.

Literature

Interest can imagine the following links:

The Japan patent A;

U.S. patent No. 4124702;

U.S. patent No. 3692899;

U.S. patent No. 3906092;

U.S. patent No. 4389395;

U.S. patent No. 4349538;

U.S. patent No. 4024241;

U.S. patent No. 3952097;

Houston et al., Infection and Immunity, 14: 318-9, 1976C;

Wright and Adler-Moore, Biochemical and Biophysical Research Communications, 131: 949-45, 1985;

Phillips et al., Toxicology and Applied Pharmacology, 18: 220-30, 1971;

Lin, et al., A new immunostimulatory complex (PICKCa) in experimental rabies: antiviral and adjuvant effects, Arch Virol, 131: 307-19, 1993;

China Patent 93105862.7;

Zhung J.C., Research recollection of polyinosinic-polycytidylic acid (PIC). The paper of fifth Chinese interferon conference in clinical application and theory, Siam, 1985, pp.23-28;

Hu Q.G., Tianjin Av PICKCa''s laboratory research and clinical application, Fujian Medical Journal, 1983,12; (6): 28-30;

Hu Q.G. Chinese Medical and Pharmaceutical Industry Journal, 1983 (9) 3134;

Su B.X. et al; Introduction of Biochemical Technology, 1st Edition, Zhongshan University, 1978, 356-357;

Gupta R.K. et al., Adjuvants - a balance between toxicity and adjuvanticity, Vaccine, 11:293-306, 1993;

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Sela, M., Science 166:1365-1374 (1969);

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Klein, J., et al., Immunology (2nd), Blackwell Science Inc., Boston (1997);

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Richard T., Kenney et al., Meeting Report - 2ndmeeting on novel adjuvants currently in/close to human clinical testing, Vaccine 20 2155-2163, 2002;

Laboratory Techniques in Rabies Edited by F.X.Meslin, M.M.Kaplan, H.Koprowski 4thEdition ISBN 92 4 1544 1.

A brief description of the invention

The present invention relates to polynucleotide adjuvant composition and methods of its use in the induction of immune responses. The present invention also relates to immunogenic compositions comprising a composition of polynucleotide adjuvant with the antigen (e.g., as a vaccine). Adjuvant compositions according to the invention have certain physical properties (in the example, the molecular weight, size, concentration and pH), which aims to meet the needs in an efficient and safe Freund for the induction of enhanced immune response. The present invention also relates to methods of applying such adjuvant compositions, in particular, when the induction of an immune response to antigenic connection.

In one embodiment, the invention relates to a polynucleotide adjuvant composition comprising polyribosome-polyribosylribitol acid (PIC), an antibiotic and a positive ion, where the antibiotic can be a kanamycin, and the positive ion may be a divalent ion such as calcium. The present invention also relates to immunogenic compositions comprising polynucleotide adjuvant composition together with the antigen or vaccine.

The present invention is to deepen knowledge by defining a new composition, which can safely and effectively be used as an adjuvant to enhance and/or modify the immune response in the host animal or human. Although in the foregoing description illustrates the use of the antiviral drug Av PICKCa for use as adjuvant, this form PICKCa, according to the observations, called a limited specific immune response is ri introduction to the antigen. In addition, it was found that PICKCa in certain conditions causes unacceptable adverse side effects.

The present invention is directed to solving these problems by providing adjuvant compositions, generally referred to here as the “PIKA”, which can most efficiently and safely enter as adjuvant in animals, including humans.

PIKA is a composition comprising polynucleotide, an antibiotic and a positive ion, which was specially developed as an adjuvant. In the invention include compositions having unusual properties of the product that make it most suitable for use as adjuvant in an immunogenic composition, subject to the introduction of animals and/or people.

More specifically, the present invention relates to a polynucleotide adjuvant composition comprising polynucleotide, an antibiotic and a positive ion, where polynucleotide can be polyribosome-polyribosylribitol acid (PIC), the antibiotic is a kanamycin, anthracyclin, bucerotidal, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, neomycin, puromycin, streptomycin or streptozocin and ion is a calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, or zinc.

More specifically, the present invention relates to the specification, including the molecular weight, concentration and pH, composition, comprising polynucleotide, an antibiotic and a positive ion, which is aimed at satisfying the needs of safe Freund, which causes the maximum desired immune response.

The present invention also relates to immunogenic compositions comprising polynucleotide adjuvant composition and an antigen or vaccine.

In certain embodiments implementing the present invention are presented in the form of a set that includes a polynucleotide adjuvant and immunogenic connection.

In addition, the present invention relates to a method of enhancing immune response to antigenic connection with the introduction of the master of the immunogenic composition. The host may be a human or an animal. The introduction can be accomplished by injection, such as intramuscular, intraperitoneal, intravenous or subcutaneous injection, or by inhalation. In other embodiments, implementation of the immunogenic composition can be delivered rectally, vaginally, intranasally, orally, in the eye, topical, transdermal or intradermal.

Accordingly, the present invention relates to adjuvant and immunogenic compositions that can be safely used in humans and animals.

Accordingly, in one aspect the invention is characterized palynol amidou adjuvant composition, including polyribosome-polyribosylribitol acid (PIC), an antibiotic and a positive ion, where the composition comprises molecules adjuvant, are heterogeneous in molecular weight, and molecular weight is from about 66000 to 1200000 Dalton.

In related embodiments, the implementation of the molecules of the polynucleotide adjuvant composition of heterogeneous molecular weight, and molecular weight is from about 300,000 to 1200000 daltons, or from about 66000 to 660000 daltons, or from about 300,000 to 660000 daltons, or from about 300000, up to 2,000,000 daltons, or from about 300,000 to 4000000 daltons, or from about 500000 to 1000000 daltons, or from about 1000000 to 1500000, or from about 1500000 up to 2,000,000 daltons, or from about 2500000 2000000 to daltons, or from about to 3000000 2500000 daltons, or from about 3000000 to 3500000 daltons, or from about 4000000 3500000 to daltons, or from about 4,000,000 to 4500000 daltons, or from about 4500000 to 5000000 Dalton.

In related embodiments, the implementation of the molecules of the polynucleotide adjuvant composition in the composition have an average molecular weight equal to or greater than 150,000 daltons, or equal to or greater than 250,000 daltons, or equal to or greater than 350,000 daltons, or equal to or greater than 500000 daltons, or equal to or greater than about 650,000 daltons, or equal to or greater than the 750,000 daltons, or equal to or greater than 1,000,000 daltons,or equal to or more than 1200000 Dalton, or equal to or more than 1500000 daltons, or equal to or greater than 2000000 Dalton.

Accordingly, in one aspect, the invention characterizes the polynucleotide adjuvant composition comprising polyribosome-polyribosylribitol acid (PIC), an antibiotic and a positive ion, where the composition comprises molecules adjuvant, heterogeneous in size of the molecules, with a coefficient of deposition in Svedbergs (sedbergh) (S) approximately 6,43S to 24,03S.

In related embodiments, the implementation of the molecules of the polynucleotide adjuvant composition are heterogeneous in molecular size and molecular size of approximately 12,8S to 24,03S, or from about 6,43S to 18,31S, or from about 12,8S to 18,31S, or from about 12,8S to 30,31S, or from about 12,8S to 41,54S, or from about 13,5S to 18,31S, or from about 13,5S to 24,03S, or from about 16,to 14S 22,12S, or from about 22,12S to 26,6S, or from about 26,6S 30,31S, or from about 30,31S to 35,55S, or from about 33,55S to 36,45S, or from about 36,45S to 39,1S, or approximately 39,1S to 41,54S, or from about 41,54S to 43,83S, or from about 43,83S to 45,95S.

In other related embodiments, the implementation of the polynucleotide adjuvant composition has an average coefficient of deposition (sedbergh) more than 9 or more than 12 or more than 13,5 or more than 15, or greater than 16, or more than 17 or greater than 18, or greater than 19, or the pain of the e than 20, or more than 21 or more than 22 or more than 25, or greater than 30.

In a related embodiment, the antibiotic in the composition is a kanamycin, neomycin, anthracyclin, butirosin sulfate, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, puromycin, streptomycin or streptozocin.

In yet another related embodiment, the adjuvant composition also includes a source of calcium ions.

In another related embodiment, the positive ion in the composition is a calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, or zinc. Positive ion can be represented in the form of inorganic salts or organic complexes.

The source of calcium ions may be provided, for example, calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.

In one aspect of particular interest, the invention relates to a polynucleotide adjuvant composition comprising polyribosome-polyribosylribitol acid (PIC), kanamycin and calcium, and the composition includes an adjuvant molecules, heterogeneous molecular weight of approximately from 66000 to 1200000 Dalton.

In related embodiments, the implementation of molecules polyrib Yasinovka-polyribosylribitol acid (PIC), kanamycin and calcium have a molecular weight from about 300,000 to 1200000 daltons, or from about 66000 to 660000 daltons, or from about 300,000 to 660000 daltons, or from about 300000, up to 2,000,000 daltons, or from about 300,000 to 4000000 daltons, or from about 500000 to 1000000, or from about 1000000 to 1500000 daltons, or from about 1500000 up to 2,000,000 daltons, or from about 2500000 2000000 to daltons, or from about to 3000000 2500000 daltons, or from about 3500000 3000000 to daltons, or from about 4000000 3500000 to Dalton, or about to 4000000 4500000 daltons, or from about 4500000 to 5000000 Dalton.

In other related embodiments, the implementation of molecules polyribosomes-polyribosylribitol acid (PIC), kanamycin and calcium adjuvant, are heterogeneous in molecular weight, have a molecular mass equal to or greater than 150,000 daltons, equal to or greater than 250,000 daltons, or equal to or greater than 350,000 daltons, or equal to or greater than 500000 daltons, or equal to or greater than about 650,000 daltons, or equal to or greater than the 750,000 daltons, or equal to or greater than 1,000,000 daltons, or equal to or more than 1200000 daltons, or equal to or more than 1500000 daltons, or equal or more than 2000000 Dalton.

In one aspect of particular interest, the invention relates to a polynucleotide adjuvant composition comprising polyribosome-polyribosylribitol acid (PIC), Cana is izin and calcium, moreover, the composition includes an adjuvant molecules, are heterogeneous in molecular size, with a coefficient of deposition in Svedbergs (sedbergh) (S) approximately 6,43S to 24,0S.

In related embodiments, the implementation of the molecules of the polynucleotide adjuvant composition are heterogeneous in molecular size and molecular size of approximately 12,8S to 24,03S, or from about 6,43S to 18,31S, or from about 12,8S to 18,31S, or from about 12,8S to 30,31S, or from about 12,8S to 41,54S, or from about 13,5S to 18,31S, or from about 13,5S to 24,03S, or from about 16,14S 22,12S, or from about 22,12S to 26,6S, or from about 26,6S 30,31S, or from about 30,31S to 3,55S, or from about 33,55S to 36,45S, or from about 36,45S to 39,1S, or approximately 39,1S to 41,54S, or from about 41,54S to 43,83S, or from about 43,83S to 45,95S.

In other related embodiments, the implementation polyribosome-polyribosylribitol acid (PIC), kanamycin and calcium are the average rate of deposition is greater than 9 or more than 12 or more than 13,5 or more than 15, or greater than 16, or more than 17 or greater than 18, or greater than 19, or greater than 20, or greater than 21 or more than 22 or more than 25, or greater than 30.

In some embodiments implementing the invention relates to a polynucleotide adjuvant composition comprising polyribosome-polyribosylribitol sour is the (PIC) kanamycin and calcium, and may be preferably from the composition were excluded molecules, in particular, to the extent that such excluded molecules had no significant immunogenic effect, and eliminated the molecules have a molecular weight of about or less than 30,000 daltons, about or less than 40,000 daltons, about or less than 50,000 daltons, about or less than 60,000 daltons, about or less than 70000 daltons, about or less than 80,000 daltons, about or less than 90000 daltons, about or less than 100,000 daltons, about or less than 150000 daltons, about or less than 200,000 daltons, about or less than 300,000 daltons, about or less than 350,000 daltons, about or less than 400000 daltons, about or less than 450000 daltons, about or less than 500000 daltons, about or less than of 600,000 daltons, about or less than the 700,000 daltons, about or less than 800000 daltons, about or less than 900000 daltons, about or less than 1,000,000 daltons.

In some embodiments implementing the invention relates to a polynucleotide adjuvant composition comprising polyribosome-polyribosylribitol acid (PIC), kanamycin and calcium, and may be preferably from the composition were excluded molecules, in particular, to the extent that such is skruchennye molecules had no significant immunogenic effect, moreover, the excluded molecules have a molecular size of about or less than 4,49S, about or less than 5,12S, about or less than 5,67S, about or less than 6,16S, about or less than 6,6S, about or less than 7,02S, about or less than 7,4S, about or less than 7,77S, about or less than 9,34S, about or less than 10,64S, about or less than 11,78S, about or less than 12,8S, about or less than 13,73S, about or less than 14,59S, about or less than 15,39S, about or less than 16,14S, about or less than 17,54S, about or less than 18,81S, about or less than 19,99S, about or less than 21,09S, about or less than 22,12S.

In one aspect of particular interest, the invention relates to immunogenic compositions for enhancing the antigenicity of antigenic compounds, comprising the polynucleotide adjuvant composition.

In related embodiments, the implementation of the immunogenic composition includes a polynucleotide adjuvant and antigen.

In related embodiments, the implementation of the source of antigen is a human antigen, the antigen of the animal other than human, plant antigen, a bacterial antigen, a fungal antigen, a viral antigen, a parasitic antigen, or a cancer antigen.

In related embodiments, the implementation of the immunogenic composition includes the polynucleotide adjuvant composition and the rabies antigen.

In certain embodiments of the implementation of the antigens can be purified from natural source, synthesized by solid phase synthesis or can be obtained by recombinant genetics. The antigen may include protein fragment comprising one or more immunogenic regions of the molecule. Antigens may also be provided by whole cells or microorganisms (e.g., whole viral particles), which may be live, attenuated or truncated, or killed.

In other embodiments, the implementation of antigens include one or more antigens from infectious agents, plant antigen, carcinoembryonic antigen, antigenic agents, and other human antigen, such as for the development of autoimmune diseases. In other embodiments, the implementation of antigens include one or more infectious agents from any virus, bacteria, mycobacteria, fungi and parasites.

Polynucleotide adjuvant composition of the present invention can also be used to enhance the immune response against antigens produced by the use of DNA vaccines. The DNA sequence in these vaccines encoding the antigen can be either "naked"or contained in the delivery system, such as liposomes.

In some embodiments, the implementation of the rabies antigen in the bran of the vaccine, human diploid cells (HDCV), or cleared of rabies vaccine, inactivated renal hamster cells (HKC-IPRV), or untreated of rabies vaccine, inactivated renal hamster cells (HKC-ICRV), or purified vaccine against rabies from vero cells (PVRV), or purified chicken embryo cells (RSES), or purified vaccine duck embryo (PDEV), or purified rabies antigen, inactivated renal hamster cells (HKC-IPRA), or untreated rabies antigen, inactivated renal hamster cells (HKC-ICRA).

In one aspect of particular interest, the invention relates to immunogenic compositions for enhancing the antigenicity of antigenic compounds, comprising the polynucleotide adjuvant composition which is capable of cause-specific antigen cell-mediated immune response.

In one aspect of particular interest, the invention relates to immunogenic compositions for enhancing the antigenicity of antigenic compounds, comprising the polynucleotide adjuvant composition which is capable of cause-specific antigen In cellular immune response.

In one aspect of particular interest, the invention provides an immunogenic composition for enhancing the antigenicity of antigenic compounds, comprising the polynucleotide adjuvant to the position, which can cause a combined specific antigen T - and b-cell immune response.

In one aspect of particular interest, the invention relates to immunogenic compositions for enhancing the antigenicity of the connection, including polynucleotide adjuvant composition and purified rabies antigen, inactivated renal hamster cells, where the presence of rabies antigen must attain a minimum amount, such as more than 1 International Unit (IU).

In related embodiments, the implementation of the immunogenic composition includes a polynucleotide adjuvant composition and purified rabies antigen, inactivated renal hamster cells, where the presence of rabies antigen must attain a minimum amount, such as more than 0.25 International Units, is more than 0.5 International Units, is more than 1.2 International Units, more than 1.4 International Units, more than 1.6 International Units, more than 1.8 International Units, is more than 2.0 International Units, is more than 2.2 International Units, is more than 2.4 International Units, more than 2.6 International Units, is more than 2.8 International Units, is more than 3,0 International Units, is more than 3.2 International Units, more than 3.4 International Units, is more than 3.6 International Units, Bo is its 3.8 International Units or more than 4,00 International Units.

In one aspect of particular interest, the invention relates to immunogenic compositions for enhancing the antigenicity of the connection, including polynucleotide adjuvant composition and purified rabies antigen, inactivated renal cells hamster, where the adjuvant and the antigen of rabies are present in a ratio of about 1:1.

In related embodiments, the implementation of the immunogenic composition includes a polynucleotide adjuvant composition and purified rabies antigen, inactivated renal cells hamster, where the adjuvant and the antigen of rabies are present in a ratio less than 1:10, about 1:9, about 1:8, about 1:7, about 1:5, about 1:4, about 1:3, about 1:2, about 2:1, about 3:1, about 4:1. about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, more than 10:1.

In one aspect of particular interest, the invention relates to adjuvant compositions or immunogenic compositions, where the immunogenic composition or adjuvant composition contained in the immunogenic compositions presented in solid or liquid form or in solution, or in suspension.

In one aspect of particular interest, the invention relates to adjuvant compositions or immunogenic compositions, including adjuvant composition, where adjuvant to notice or immunogenic composition is lyophilized.

In related embodiments implementing the invention relates to a kit including adjuvant composition and antigenic connection.

In one aspect of particular interest, the invention relates to the use of the polynucleotide adjuvant composition to obtain drugs to enhance the immunogenic response of the host.

In one aspect of particular interest, the invention relates to a method of enhancing immune responses to antigenic compound comprising an introduction to the master of the immunogenic composition to enhance the antigenicity of antigenic compounds, comprising the polynucleotide adjuvant composition.

In related embodiments, the implementation of the method of administration the master of the immunogenic composition may be performed by one selected from the group including parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, inhalation, rectal delivery, vaginal delivery, intranasal delivery, oral delivery, ocular delivery, local delivery, transdermal delivery, or intradermal delivery.

In one aspect of particular interest, the invention relates to a method of enhancing immune responses to antigenic connection involving the introduction of a master immunogen the Noah compositions for enhancing the antigenicity of antigenic compounds, comprising the polynucleotide adjuvant composition, where the host is a human.

In one aspect of particular interest, the invention relates to a method of enhancing immune responses to antigenic compound comprising an introduction to the master of the immunogenic composition to enhance the antigenicity of antigenic compounds, comprising the polynucleotide adjuvant composition, where the host is an animal.

These and other features and advantages of the invention will become apparent from the following detailed descriptions of preferred options for its implementation in connection with the accompanying drawings.

Brief description of drawings

Figure 1 shows the relative molecular mass for samples Av-PICKCa and PIKA.

Figure 2 shows that PIKA causes dose-dependent production of a specific cytokine interferon-gamma.

Detailed description of illustrative embodiments of the invention

The present invention can be more easily understood by reference to the following detailed description of certain embodiments of the invention and the examples included here.

According to the description with reference to the published descriptions of these publications are fully incorporated here by reference for a more complete description of the state of the field to which the invention relates.

Before dalnas is m the description of the present invention should be understood, that this invention is not limited to the specific options described implementation, because they, of course, may vary. It should also be understood that the terminology used here is intended solely for the purpose of describing specific embodiments and is not intended to be limiting, since the scope of the present invention will be limited only by the attached claims.

In the absence of other definitions used here is the technical and scientific terms have the same meaning, which is usually understandable to the average person skilled in the art to which this invention. Although the implementation or testing of the present invention can use any methods and materials similar or equivalent to the methods and materials described herein are now described the preferred methods and materials. All these are publications included here as a reference to disclose and describe the methods and/or materials in connection with which provides these links.

It should be noted that while in the context of no other clear indications used herein and in the attached claims of the form of the singular indefinite article, the conjunction "and" and the definite article include the corresponding plural forms. In affect, the, for example, the reference text includes many such texts, and reference to "segment" includes reference to one or more segments and cash equivalents, known to specialists in this field, etc. in Addition, it is noted that the claims may be drafted with the exception of any optional element. The actual instruction is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with reference to the elements of the claims, or using the "negative" limitation.

Definition of terms

Before outlining the details of the present invention may be useful for understanding the presentation of the definitions of several terms used in the present description.

Used herein, the term "adjuvant" refers to any substance or mixture of substances that can increase or diversify the immune response of the host to antigenic compound. In particular:

1. The term “PICKCa” generally refers to the composition of poly I:C, kanamycin and calcium, regardless of specific physical and immunogenic properties.

2. The term “Av PICKCa” refers to the shape of PICKCa, industrial used as an antiviral drug.

3. The term “PIKA” refers to the composition of izaberete the Oia, includes poly I:C, an antibiotic (e.g., kanamycin) and positive ion (e.g. calcium), where PIKA is characterized by physical characteristics (e.g., molecular weight, size and the like, as described here), so after the introduction of PIKA shows the characteristics of the adjuvant with reduced adverse side effects (e.g., reduced toxicity) relative to, for example, PICKCa and greater activity (e.g., stimulates enhanced immune response) relative to, for example, Av-PICKCa.

"Molecule containing a PIC or a compound containing PIC"refers without limitation to the PIC, which may not necessarily form complexes or otherwise combined, with at least one or both of the antibiotic (e.g., kanamycin) and positive ion (e.g., calcium)present in the composition containing a molecule containing PIC.

The term "heterogeneous"as used in the context of adjuvant compositions according to the invention, indicates that the components of the composition, for example a molecule containing the PIC, heterogeneous in relation to the physical characteristics of the molecular weight, size, or both of them.

The term "animal" includes humans and all domestic and wild animals and birds, including, without limitation, cattle, horses, cows, pigs, sheep, goats, dogs, cats, rabbits, deer, the Orok, chickens, ducks, geese, turkeys, quail and the like.

The term "antibody" includes polyclonal and monoclonal antibodies as well as fragments of such antibodies that bind antigenic compound, including Fab fragments, F(ab')2, Fd, Fv and single-chain derivative. In addition, the term "antibody" includes naturally occurring antibodies as well as unnaturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms.

Used herein, the term "antigenic compound" refers to any substance that can recognize the immune system (for example, to the related antibody, or processed in such a manner as to cause the cellular immune response in appropriate circumstances).

"Antigen" refers to a substance, including compositions in the form of vaccine and that the vaccine comprises an antigenic compound, and may include or not include adjuvant other than PIKA, which with the introduction of appropriate means (e.g., parenteral) causes an immune response, for example the formation of antibodies, including antibodies that specifically bind the antigen. 2 distinguishing characteristic antigens are immunogenic, i.e. their ability to induce an immune response in vivo, and their antigenicity, i.e. their ability to be selectively dissolved, and is known by antibodies originating from antigens.

The terms "cell-mediated immunity and cell mediated immune response" is intended to denote the immune protection provided by lymphocytes, such as the protection provided by T-cell lymphocytes, when they come into contact with their cell-victims. Cell mediated immune response usually involves the proliferation of lymphocytes. When measuring the proliferation of lymphocytes" measures the ability of lymphocytes to proliferate in response to specific antigen. Proliferation of lymphocytes is intended to describe the proliferation of b-cells, T-helper cells or cytotoxic T lymphocytes (CTL).

"An effective amount of the antigenic compound" refers to the number of antigenic compounds, which, in optional combination with an adjuvant, will cause the patient products specific immunological response to antigenic connection.

The expression "enhanced immune response" or similar means that the immune response is enhanced, improved or strengthened in the interests of the owner relative to the previous status of the immune response, for example, before the introduction of the immunogenic compositions according to the invention.

The term "humoral immunity" and "humoral immune response" refers to a form of immunity, in which molecules are antibodies p is observada in response to antigenic stimulation.

The term "immune response" refers to any reaction of the immune system of the vertebral individual on antigenic compound. Illustrative immune responses include, without limitation cellular, local and systemic humoral immunity, such as CTL response, including specific antigen induction reactions CD8+ CTL and helper T cells, including T-cell proliferative response and cytokine and cellular responses, including T-cell proliferative response and cytokine and cellular reactions, including the reaction of antibodies.

The term "call of the immune response" is used here in General to cover induction and/or augmentation of the immune response.

The term "induction of an immune response" refers to an immune response that is stimulated initiated or invoked.

The term "potentiation of the immune response" refers to pre-existing immune response, which is superior, received assistance, enhanced, increased, extended or renewed.

The term "poly I:C" or "PIC" refers to compositions containing polyribosomes and polyribosylribitol nucleic acid, which can also be referred to respectively polyinosinic acid-polycytidylic acid.

The term "immunogenic amount" refers to the number of antigenic compounds, sufficient to steam the regulation of the immune response when introduced with the composition according to the invention compared with the immune response, observed in the absence of a polynucleotide adjuvant.

The term "immunopotency number" refers to the amount of adjuvant required for the implementation of the increase in the titer of antibodies and/or cell-mediated immunity in the introduction of antigenic compound in the composition according to the invention compared with the increase mediated by antibodies and/or cell-mediated immunity observed in the absence of a polynucleotide adjuvant.

Used herein, the term "mixing" includes any method used to join components of the composition; such methods include, without limitation, the mixing, distribution, dissolution, emulsification, coagulation, suspension or other physical Association of the components of the composition.

"Pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compounds. Such salts include: (1) acid additive salts formed with inorganic acids such as hydrochloric acid, Hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acids is, cyclopentanepropionate acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamony acid, mandelic acid, methanesulfonate acid, econsultancy acid, 1,2-ethicality acid, 2-hydroxyethanesulfonic acid, benzolsulfonat acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonate acid, 4-toluensulfonate acid, camphorsulfonic acid, glucoheptonate acid, 4,4'-methylenbis-(3-hydroxy-2-EN-1-carboxylic acid); 3-phenylpropionate acid, trimethylhexane acid, tertiary Butylochka acid, louisanna acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, Mukanova acid and the like; or (2) salts formed when an acidic proton part in the parent compound or replaced by a metal ion, for example an alkali metal ion, alkali earth metal ion or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.

The term "treatment" encompasses any lakenyagiuliani the vertebrate animal in particular person, and includes: (i) preventing a disease in a patient that may be predisposed to the disease but has it not been diagnosed; (ii) inhibiting the disease, i.e. stopping the development; or (iii) facilitation of the disease, i.e. induction of reverse disease progression.

Used herein, the term "standard dosage form" refers to physically discrete units suitable as unitary dosages for individuals men and animals, each unit contains a given number of compounds of the present invention calculated in an amount sufficient to provide the desired effect, in combination with pharmaceutically/a physiologically acceptable diluent, carrier or base.

Consideration inventions

The present invention is directed to compounds and methods that can be used to enhance the immune response may be humoral and/or cell-mediated, human, animal or cell culture. In General, the composition consists of immunogenic compositions containing the adjuvant. The presence of the adjuvant enhances or modifies the immune response. Thus, humoral and/or cell-mediated immune responses are more effective in the presence of adjuvant. In addition, adjus the HT can change the quality of the immune response impact on subclasses (isotypes) of immunoglobulins and produced cytokines. Key features adjuvant represent its ability to stimulate the desired level and type of immune response without causing adverse side effects. Currently, there are only a limited number of adjuvants approved for use in people who have this combination of characteristics. Security standards for immunogenic substances and, in particular, vaccines are rigid and strictly observed. Therefore, a significant constraint to the development of a successful adjuvant is to develop a product that is potent enough to induce the appropriate immune response, at the same time without causing adverse side effects.

The preferred embodiment of the invention is a polynucleotide adjuvant, where polynucleotide is polyribosome-polyribosylribitol acid (PIC). It was shown that one PIC is an effective adjuvant, but reveals unacceptable safety profile and unstable in humans and primates. The present invention relates to compositions PIC in combination with an antibiotic and a positive ion, which increases the desired immunogenic attributes adjuvant, at the same time improving the security profile and stability.

The present invention,in addition, based on the discovery that the physical and biological characteristics of the molecules of the PIKA adjuvant composition influence the characteristics of the immune response and adverse side effects. During the research it has been unexpectedly discovered that the selection of certain characteristics of the polynucleotide adjuvant he becomes more or less potent and/or more or less toxic ways, which are further described below. Therefore, by determining the adjuvant composition from the point of view of its physical characteristics can accurately describe the attributes of the adjuvant composition, which provides a preferred immunogenic reaction and preferred profile security/stability.

The adjuvant of the present invention, for convenience referred to here PIKA adjuvant, therefore, completely determined by a combination of its chemical composition plus the fundamental physical attributes of the molecules that comprise adjuvant. Thus, some form of PIKA, which exhibits significantly superior immunogenic properties, at the same time, being safe for use in animals and humans, the best way is determined by one or more, typically, by a combination of specific attributes, including: composition, molecular weight, size of molecules and pH.

PIKA generally includes polynucleotide, Antibes is Otik and positive ion where polynucleotide can be polyribosome-polyribosylribitol acid (PIC), and the antibiotic is an aminoglycoside (e.g., kanamycin, streptomycin, tobramycin, neomycin, anthracyclin, butirosin sulfate, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, puromycin or streptozocin, and ion is a calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, or zinc.

"Aminoglycoside" antibiotics are antibiotics, whose structure contains the amino sugar that is attached to iminocyclitol ring (hexane core) glycosidic bonds. Aminoglycoside antibiotics derived from various species of Streptomyces and Micromonospora or are synthetically. For example, kanamycin is an aminoglycoside antibiotic derived from a soil bacterium Streptomyces Kanamycetics used to treat various infections, particularly infections caused by gram-negative bacteria.

The composition of PIKA is made by mixing polyinosinic acid, polycytidylic acid, antibiotics and source of the positive ion in solution of sodium chloride/phosphate buffer which has a pH from pH 6 to pH 8. Polyinosine acid and policitally acid is generally provided in a concentration of from 0.1 to 10 mg/ml, preferably from 0.5 to 5 is g/ml, and preferably, from 0.5 to 2.5 mg/ml Amount of hyperchromatic should be more than 10%, preferably more than 15%, and preferably greater than 20%. Obtaining PIC and Association with kanamycin and calcium is preferably carried out in accordance with quality standards, consistent with International norms of due process of manufacture.

In some embodiments, implementation of the present invention kanamycin in the polynucleotide adjuvant composition can be used together or substituted one or more antibiotics selected from the group including tobramycin, anthracyclines, bucerotidal, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, neomycin, puromycin, streptomycin and streptozocin. Antibiotic (e.g., kanamycin or similar) in the polynucleotide adjuvant composition according to the invention as a whole is provided in a concentration of from about 10 to 100,000 units/ml, preferably from about 100 to 100,000 units/ml, preferably from about 500 to 5000 units/ml

In some embodiments, implementation of the present invention polynucleotide adjuvant composition also includes a positive ion (cation), usually divalent cation, typically, the cation of an alkali metal. Positive ion in General is provided in the composition according to the invention as the e source of positive ions, such as salt or complex, organic or inorganic salt or complex, usually inorganic salt or an organic complex. Examples of positive ions include, but are not mandatory exceptions calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron, or zinc.

Positive ion (e.g., calcium) may be present in compositions according to the invention in a concentration in the range of from about 10 μmol to 10 mmol/ml, preferably from about 50 μmol to 5 mmol/ml, and preferably from about 100 mmol to 1 mmol/ml

As noted above, the positive ion may be in the form of any suitable salt or an organic complex, including without binding limits for chloride, fluoride, hydroxide, phosphate or sulfate. For example, when a positive ion is a calcium, ion can be in the form of calcium carbonate, calcium chloride, calcium fluoride, calcium hydroxide, calcium phosphate or calcium sulfate.

When a positive ion in the adjuvant composition according to the invention is a calcium, it can be combined or replaced by other positive ions, including cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron and zinc, and ions can be in the form of inorganic salts or organic complexes.

According to the scientists, the composition further transformed into PIKA through additional method of manufacture, which includes a selection of molecules of a certain molecular size and/or weight. Department of molecules polynucleotide with certain characteristics, using filtration, chromatography, thermal processing, centrifugal separation, electrophoresis and similar methods, which are standard methods known to experts in this field.

In some embodiments, implementation of the present invention polynucleotide adjuvant composition, in addition, determined by the physical characteristics of the molecular weight. The study unexpectedly found that there is a positive correlation between molecular weight and efficiency polynucleotide adjuvant composition. The observed level of activity immunogenic compositions containing the polynucleotide adjuvant composition, including the ability to cause the production of antibodies and cytokines, increases with molecular weight polynucleotide adjuvant composition. The molecular weight of the polynucleotide adjuvant may be determined by electrophoresis on an agarose gel as described in example 1.

As illustrated in the Examples section below, the inventor has found that a vaccine composition comprising PIKA adjuvant variety of molecular weights, showed direct the Yu correlation between molecular weight and specific protective antigen activity (see example 2). Similarly, the inventor has found that there is a direct correlation between the molecular weight of the adjuvant compositions PIKA and the ability to induce production of interferon-gamma when administered to the host in combination with rabies antigen (see example 3).

The author of the invention during testing in 1996, people in China with the use of rabies vaccine with adjuvant, including PICKCa specifications with particularly high molecular weight, in addition, found that, as has been shown, the obtained composition, surprisingly, has an unacceptable level of adverse side effects. The results of clinical trials in 1996, which have not been previously published, shown in example 4. The study of molecular weight are presented in examples 5 and 6. The test was conducted under the jurisdiction of the Chinese Administration Food and Drug. Thus, the adjuvant was not introduced to the people in controlled clinical trials, if such side effects would be expected on the basis of available at that time.

The inventor has found that adjuvant composition PIKA according to the invention in preclinical studies with molecular masses of up to 1.0×106and compositions of vaccines, including adjuvant composition PIKA with molecular mass of d is of 5.5×10 5demonstrated a wide margin of safety in tests specific toxicity (see example 7). PIKA with a maximum molecular weight of 1.2×106has been successfully used in preclinical studies (see example 3). Further research conducted by the author of the invention, showing the safety PIKA when used in combination with the connection of the antigen in the form of a vaccine (see example 8).

The results of a subsequent experiment conducted by the inventor in China in 2002, also demonstrate that the use of PIKA allows you to get a safe and effective adjuvant for administration to man. The results of this experiment, not previously published, are presented in example 9.

On the basis of the above observations, the preferred implementation of the PIKA therefore includes molecules having physical characteristics molecular weight and/or size, which provide a beneficial effect from increased activity and efficiency, at the same time providing an adequate degree of margin of safety so as not to cause any adverse side effects. The molecules present in the lower part of the interval molecular weight Av PICKCa can be effective as an antiviral composition, but are considerably less effective than the molecular is the first song PIKA when used as adjuvant in an immunogenic composition. In addition, it was shown that PIKA has a safety profile that is better than PICKCa.

Thus, one aspect of the present invention is a molecular weight PIKA compositions according to the invention.

The composition of PIKA according to the invention generally includes a collection or population of molecules, and molecules have physical characteristics, such as molecular weight and/or size that provides the desired effect of the induction of the immune response, at the same time, preferably, reducing or avoiding adverse side effects (such as effects associated with the introduction of PICKCa). In General, PIKA molecules are heterogeneous in molecular weight and/or size.

In General, and has no other special instructions, as used herein, the term "PIKA", adjuvant composition according to the invention, includes a PIC, which can form complexes with antibiotic (e.g., kanamycin) and positive ion (e.g., calcium). Molecules in PIKA are heterogeneous in molecular weight (for example, according to estimates in daltons) or size (for example, according to estimates by the ratio of the deposition).

When heterogeneous characteristics of the molecules PIKA range is used (e.g., molecular weight or size), the link is here on this range indicates the approximate upper and lower limits the s values of molecular weight and size of the PIKA molecules in the composition, but does not imply and is not intended to indicate that the composition contains a molecule PIKA, which has a molecular weight or size that are representative for each molecular weight or size within this range. Thus, for example, the range of molecular weight from about 66000 to 1200000 Dalton indicates that the composition contains molecules PIKA approximately 66000 daltons to 1200000 Dalton, but not required, that in the composition of the present molecules PIKA 88000 Dalton (although they certainly can be).

When the physical characteristics of the PIKA molecules in the composition according to the invention are defined by the range of molecular masses, PIKA molecules are heterogeneous in molecular weight, with the range of molecular weight is from about 300,000 to 660000 daltons, from about 300,000 to 1200000 daltons, from about 66000 to 660000 daltons, or from about 66000 to 1200000 Dalton.

The invention also relates to compositions which are molecules PIKA, are heterogeneous in molecular weight, with the range of molecular weight of approximately 300,000, up to 2,000,000 daltons, from about 300,000 to 4000000 daltons, from about 500000 to 1000000 daltons, from about 1000000 to 1500000 daltons, from about 1500000 to 2000000 daltons, from about 2500000 2000000 to daltons, from about to 3000000 2500000 daltons, from about 300,000 to 3500000 daltons, about or to 4000000 Dalton, about to 4000000 4500000 daltons, or from about 4500000 to 5000000 daltons. In the composition of the present PIKA molecules having a molecular weight in the upper and lower limits of these ranges, and also within these ranges.

When the physical characteristics of the PIKA molecules in the composition according to the invention is determined by the average molecular weight, PIKA molecules can have an average molecular weight equal to or greater than 150,000 daltons, equal to or greater than 250,000 daltons, equal to or greater than 350,000 daltons, equal to or more than 500000 daltons, equal to or greater than about 650,000 daltons, equal to or greater than the 750,000 daltons, equal to or greater than 1,000,000 daltons, equal to or more than 1200000 daltons, equal to or more than 1500000 daltons, or equal to or greater than 2000000 Dalton.

When the physical characteristics of the PIKA molecules in the composition according to the invention are determined by the ratio of the deposition, which is a measure of molecular weight and size, PIKA molecules may have a coefficient of deposition is greater than 9, or greater than about 12, or greater than about 13.5, or greater than 15, or greater than 16, or more than 17 or greater than 18, or greater than 19, or greater than 20, or greater than 21 or more than 22 or more than 25, or greater than 30.

In some embodiments implementing the invention relates to polynucleotide adjuvant the composition is, including polyribosome-polyribosylribitol acid (PIC), kanamycin and calcium, where the composition prevents detectable number of molecules that have a molecular weight around and below 30000 daltons, about and below 40000 daltons, about and below 50000 daltons, about and below 60000 daltons, about and below the 70,000 daltons, about and below the 80,000 daltons, about and below 90000 daltons, about and below 100,000 daltons, about and below 150000 daltons, about and below the 200,000 daltons, about and below the 250,000 daltons, about and below 300000 daltons, about and below 350000 daltons, about and below of 400,000 daltons, about and below to 450,000 daltons, about and below of 500,000 daltons, about and below of 600,000 daltons, about and below the 700,000 daltons, about and below to 800,000 daltons, about and below nearly 900,000 daltons, or about and below 1000000 daltons. In this embodiment, of particular interest is the exclusion of such molecules with low molecular weight to such an extent that such excluded molecules had no significant immunogenic effect.

The inventor has shown that PIKA, including molecules with molecular weight up to 1.0×106Dalton, safe in animal tests when specific toxicity (see example 7). PIKA, including molecules with molecular weight up to 1.2×106Dalton, safely been used in preclinical studies (see example 3). Was that the same shows that PIKA safe for use in immunogenic compositions (see example 8). This composition PIKA provides favorable effects from the point of view of efficiency. PIKA, including molecules with molecular weight up to 6.6×105Dalton, also results in an effective immune response with a wider margin of safety for use in humans and animals. The increase in molecular weight of the smallest molecules present to 6.6×105daltons, and preferably up to 3.0×105Dalton, increases the effectiveness of adjuvant without violating safety standards.

In addition, it was found that the concentration of the polynucleotide adjuvant composition can affect the molecular weight of the molecules contained in the composition. It was shown that the molecular weight PICKCa increases with the concentration of the adjuvant compositions (see example 5). The inventor observed that the increase in the concentration of polynucleotide adjuvant can lead to coalescence (or aggregation) of molecules PICKCa, leading to the formation of molecules with large molecular mass. It was shown that this process is irreversible. Thus, the subsequent dilution of the polynucleotide adjuvant composition in a suitable medium does not reduce the molecular weight of the adjuvant molecules. As was observed in example 6, when concentrated the data macromolecular form polynucleotide adjuvant composition is combined with the antigen, rabies, the result is a composition that retains its wide range of molecular weight. Vaccine against rabies, formed in this way, demonstrated adverse effects in clinical trials in humans (see example 4).

The composition of PIKA according to the invention can be provided in any physiologically acceptable buffer, which can be used here, but the preferred phosphate buffers. Other acceptable buffers, such as acetate, Tris, bicarbonate, carbonate or the like can be used as substituents phosphate buffers.

pH of the aqueous component preferably should be from 4.0 to 10.0, although it is preferable to bring the pH of the system to range from 6 to 8.5, in which the pH significantly reduces the stability of other components of the composition and is not physiologically wrong in other aspects. In some embodiments of the invention the water part immunogenic composition is a buffered saline solution. When these compositions are intended for parenteral administration, it is preferable to prepare these solutions so that toychest, i.e. osmollnosti, was essentially the same as the normal physiological fluids, to prevent swelling after injection or rapid absorption of the composition due to the different the Oh-ion concentration between composition and physiological fluids.

The number of buffered salt solution used in these compositions should be the amount needed to bring the amount of the composition to unity. That is, the number of buffered salt solution sufficient to obtain 100%, is mixed with other components to bring the volume of the composition to the desired value.

In some embodiments, the implementation of the antigens can be purified from natural source, synthesized by solid phase synthesis or can be obtained by recombinant genetics. The antigen may include protein fragment comprising one or more immunogenic regions of the molecule. The antigen may be provided by whole cells or microorganisms (e.g., whole viral particles), which may be live, attenuated, truncated or killed.

In other embodiments, the implementation of antigens include one or more agents of infectious agents, vegetable antigen, a cancer antigen, antigenic agents and other human antigen, such as for the development of autoimmune diseases. In other embodiments, the implementation of antigens include one or more infectious agents from any viruses, bacteria, mycobacteria, fungi and parasites.

Polynucleotide adjuvant composition in this image is the shadow can also be used to enhance the immune response against antigens produced by application of DNA vaccines. The DNA sequence in these vaccines encoding the antigen can be either "naked"or be contained in a delivery system, such as liposomes.

In some embodiments, the implementation of the polynucleotide adjuvant composition can be used in combination with vaccines. It does not matter whether the vaccine adjuvants or not. Included classes vaccines are vaccines against infectious diseases, anti-cancer, anti-Allergy, against autoimmune diseases and for immunocontraception.

The invention also relates to the use of the polynucleotide adjuvant according to the invention in combination with any suitable antigen rabies.

In some embodiments, the implementation of the rabies antigen can be an inactivated crude rabies antigen, such as untreated rabies antigen, inactivated renal hamster cells (HKC-ICRA), or purified rabies antigen, inactivated renal hamster cells (HKC-IPRA).

In some embodiments, the implementation of the polynucleotide adjuvant composition can be used with the vaccine against rabies. Suitable vaccines against rabies are commercially available or in research, including inactivated, subunit, recombinant and peptide vaccines, yet is how the vaccine, human diploid cells (HDCV), or purified vaccine against rabies inactivated renal hamster cells (HKC-IPRV), or untreated vaccine against rabies inactivated renal hamster cells (HKC-ICRV), or purified vaccine against rabies from vero cells (PVRV), or purified chicken embryo cells (RSES), or purified vaccine duck embryo (PDEV). However, not all of rabies vaccine to cause cell mediated immune response, which is important when immunization before and after contact with the pathogen. When the polynucleotide adjuvant composition (e.g., ISDN) is injected with a vaccine against rabies, caused by immune responses include: nonspecific reaction (for example, increased functions of macrophages), humoral responses (e.g., increased production of specific antibodies) and cell-mediated responses (e.g., production of cytokines, including interferon and interleukin-2).

In some embodiments implementing the invention relates to a kit comprising the polynucleotide adjuvant and immunogenic connection.

Immunogenic composition comprising PIKA capable of inducing a specific immune response in two ways: (i) humoral-mediated immunity, which involves the stimulation of b-cells and production of antibodies or immunoglobulins (other cells are also involved in the generation of antibody responses, n is the sample representing the antigen cells (APCs, including macrophages and helper T cells (Th1 and Th2)), and (ii) cell-mediated immunity, which generally involves T cells, including cytotoxic T lymphocytes (CTLs), although other cells are also involved in the generation of CTL responses (e.g., cells of the Th1 and/or Th2 and APCs). Ways to evaluate humoral and/or cellular response in an individual well known in the art (see examples 10, 11, 12 and 13).

In addition, the polynucleotide adjuvant composition may change the quality of the immune response impact on subclasses (isotypes) produced immunoglobulins (IgG1, IgG2, IgG3 and IgG4 human IgGs; IgG1, IgG2a, IgG2b and IgG3 for mouse IgGs), as well as their affintity.

The answer is regulated by Th1 cells in mice induces IgG1, IgG2a, IgG2b, and to a lesser extent, IgG3, and promotes cell-mediated immune response to the antigen. If the reaction of the IgG to the antigen is regulated by the cells of the Th2 type, it mainly enhance the production of IgG1 and IgA.

Tests activity NIH (National institutes of health) using the composition of the PIKA adjuvant and inactivated renal cells hamster purified rabies antigen showed that the immunogenic activity of the composition requires minimal presence of antigen (see example 14). The activity of the composition increases rapidly relative to the presence of additional antigen rabies when the house is ke 1 IU antigen. Thus, it was observed that the rate of increase activity of the composition was the largest at about 1.5 to 2.5 IU IU of rabies antigen present in the composition. Test activity NIH described in the document: Laboratory Techniques in Rabies, Edited by F.X.Meslin, M.M.Kaplan, H.Koprowski, 4thEdition, ISBN 92 4 1544 1.

Tests using the compositions of the PIKA adjuvant and inactivated renal cells hamster purified rabies antigen showed that the immunogenic activity of the composition was increased as the amount present adjuvant exceeded the number present antigen. The activity was increased when increased with respect to PIKA inaktivirovannaja renal cells hamster purified rabies antigen, preferably the ratio is greater than 3:1 (see example 15).

The invention provides methods of using polynucleotide adjuvant according to the invention with an antigen, for example, in order to cause the individual-specific antigen humoral response and/or specific cell (e.g., T-cell) response. Caused by the immune response may be a response to the antigen in the intact individual, or can be used to enhance an existing immune response (i.e., the agent re-immunization).

In some embodiments, the implementation of whom adjuvant who azizia PIKA and immunogenic composition, including PIKA adjuvant and antigenic compound can be freeze dried (lyophilized) for long-term stability and storage in solid form. The method of freeze-drying is known to specialists in this field. Dissolution immunogenic composition comprising PIKA and antigenic compound, demonstrated long-term preservation of the level of efficiency (see example 16). The immunogenic composition can be obtained in the form of tools for injection, liquid solution, suspension or emulsion. Getting preparative forms the desired immunogenic compositions generally described in the document New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., USA, 1978. Immunogenic composition of the present invention can be used in such forms as capsules, liquid solutions, emulsions or suspensions. Preferably, use any inert carrier, such as saline or saline with phosphate buffer, or any such carrier in which the compounds used in the method according to the present invention have suitable properties solubility for use in the methods of the present invention.

Immunogenic composition according to the present invention it is possible to enter the patient using a variety of methods known in this field. In some embodiments, the implementation of the immunogenic comp the stand can be delivered parenterally, injection, such as intramuscular, intraperitoneal, intravenous or subcutaneous injection, or by inhalation. In other embodiments, implementation of the immunogenic composition can be delivered rectally, vaginally, intranasally, orally, in the eye, topical, transdermal or intradermal. When the method of administration is by injection, encapsulated antigenic connection can remain in the injection site for up to 2 weeks, thus providing a depot of antigen, which will provide long-term release or pulsating release in vivo. This delivery system can provide immunogenic preparative forms the input of a single injection of antigenic compounds, which otherwise would require multiple injections to invoke an immune response.

For example, for parenteral administration in an aqueous solution the solution should be suitably buffered, and the liquid diluent first need to give isotonicity sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, and intraperitoneal administration. In this connection, sterile aqueous medium that can be used will be known to specialists in this field in the light of the present description. Examples of injection with the ed, which can be used in the present invention include a buffer with dispersing agents and/or preservatives or without them and edible oil, mineral oil, cod liver oil, squalene, mono-, di - or triglyceride, and mixtures thereof.

The exact required number of such compositions will vary from patient to patient depending on the species, age, weight and General condition of the patient, severity of disease, infection or condition, which is subjected to treatment or prevention, a particular connections in use, the method of its introduction, and similar factors. The average person skilled in the art can determine the appropriate number, using only the usual experimentation with the accounting provisions contained herein. After the initial introduction patients may receive one or more repeated immunizations through appropriate intervals.

The above description generally discloses the present invention. The following examples will help understanding of the present invention. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Depending on the circumstances or the advisability stipulate changes in form and substitution of equivalents. Although there were used specific terms, such terms prednaznachendlya use in a descriptive sense, and not for purposes of limitation.

EXAMPLES

Example 1

Determination of molecular weight of PIKA and Av PICKCa

This example illustrates how determined molecular mass for the PIKA adjuvant in comparison with the Av PICKCa.

Electrophoresis on agarose gel known to specialists in this area, so here is just the distinctive features of the present invention. The agarose gel used in the present invention has a concentration of 1.5% agarose. Molecular markers represented DNA ladder of 100 pairs 100 pairs of nucleotides up to 1000 base pairs, corresponding to the range of molecular weight between 6.6×104to 6.6×105daltons. Loading the samples consisted of 4 ál at 1 mg/ml figure 1 shows a representative picture of the samples on an agarose gel in accordance with the provisions of this paragraph. Five (5) different tested series showed a wide range of distribution of their molecular masses. The upper limits of their molecular masses ranged from 2.3×105Dalton for Av-PICKCa to 5,28×105Dalton for PIKA.

Example 2

Immune efficiency PIKA compared to Av-PICKCa

This example demonstrates the difference between the activity Av PICKCa with a maximum molecular weight of over 230,000 daltons and samples PIKA with a maximum molecular weight of up to 528000 dal is he.

3 series PIKA adjuvant with different molecular weight and a 1-series with molecules having a molecular weight corresponding to a molecular mass Av PICKCa, combined with purified rabies antigen, inaktivirovannye renal hamster cells (HKC-IPRA). The obtained composition was subjected to the test of the effectiveness of the NIH.

The NIH test is a stringent conditions and extensive comparative study between the investigational vaccine against rabies and standardized vaccine against rabies. Vaccinated mice infect a live strain of rabies virus and measure their survival. Different groups of mice administered various cultivation of rabies vaccine. Comparison of survival rates between groups of mice subjected to experimental and standardized vaccines, determines the activity of the experimental vaccine (Laboratory Techniques in Rabies, Edited by F.X.Meslin, M.M.Kaplan, H.Koprowski, 4thEdition, ISBN 92 4 1544 1).

The effectiveness of each combination vaccine was normalized against the standard non-composite / hardcore vaccines, where the effectiveness of unmerged vaccine was marked as 1, and relative efficiency was defined as the number of times that the effectiveness of combination vaccines has increased compared to non-composite / hardcore vaccine. Table 1 summarizes the result is you. As can be seen from table 1, the higher the molecular weight of the adjuvant PICKCa, the higher the efficiency increase of the titer of the vaccine against rabies.

Table 1
Influence of molecular weight on the activity of rabies vaccine
The type of adjuvantAntigenSample # The upper limit of the molecular weight of the adjuvantED50Activity (IU/ml)
PIKAHKC-IPRA200003045,28×1052,105,00
PIKAHKC-IPRA20000907to 4.62×1052,003,98
PIKAHKC-IPRA990202of 3.96×1051,983,80
Av-PICKCaHKC-IPRA000703of 2.30×105 1,883,00
HKC-IPRAVaccine control1,401,00

Example 3

Comparison of interferon production between PIKA and Av PICKCa

The example demonstrates the difference between the ability to induce production of interferon between samples Av-PICKCa with a maximum molecular weight of over 230,000 daltons and samples PIKA with a maximum molecular weight 1200000 Dalton.

2 series PIKA with upper limits of molecular weight of 1.2×106Dalton and 4.6×105Dalton was compared with a series Av-PICKCa with an upper limit of molecular weight of 2.3×105daltons.

The composition of PIKA and Av PICKCa combined with purified rabies antigen, inaktivirovannye renal hamster cells (HKC-IPRA). The compositions were injected with subcutaneously to mice. After 2 h each mouse was determined by the presence of interferon. General procedure for measurement of interferon-known specialists in this field. Briefly, each well of 96-well tablets made L929 cells in a volume of 0.15 ml/well with approximately 30,000 cells. Three (3) days, when the cells were grown to confluence in the wells was added to serum samples (0.1 ml/well) at a dilution of sera from 1:20 to 1:640. For each diluted sample used in the 3 hole. The wells were incubated overnight at 37°C. serum Samples were washed away. Particles of vesicular stomatitis virus VSV was used to detect the production of interferon. Table 2 shows the production of interferon induced by mixtures. As can be seen from table 2, the higher molecular weight samples PIKA, the better were induced production of interferon.

Table 2
The relationship between molecular weight and the production of interferon
Type adjuvant-TASeriesThe upper limit of the molecular weight in daltonsHKC-IPRA
Series, No.
The ratio PIKA:HKC-IPRAThe titer of interferon production
PIKA200106011,20×105200012054:1868,6
PIKA200009-7to 4.62×105200012054:1530,6
Av-PICKCa000703of 2.30×105 200012054:146,4

Example 4

A clinical trial in humans, 1996 (with toxic side effects)

This example demonstrates that adjuvant PICKCa in combination with vaccine generates unacceptable levels of side effects when administered to humans.

The aim of the study was to evaluate the safety and immune response of the vaccine against rabies, including adjuvant PICKCa in a concentration 11,95 mg/ml and a molar mass 69700 (it should be noted that in this case, the molar mass is not equivalent to the daltons, see example 5) and untreated rabies antigen, inactivated renal hamster cells (HKC-ICRA). Results and conclusions the above clinical trials have not been previously published.

40 patients who participated in the trial were divided into 2 groups of 20 people. Each group received five (5) doses of 2 ml, administered intramuscularly in the 1st, 3rd, 7th and 30th days. One group received the rabies antigen with adjuvant PICKCa, and the other group received the rabies antigen with adjuvant “alum”.

For security purposes, controlled body temperature, local and systemic symptoms after 24 h, 48 h and 72 h after each injection. When observation revealed the following side effects.

Side effectGroupThe number of volunteersThe number of side effect
LocalPICKCa plus HKC-FOSI206
“Alum” plus HKC-ICRA×5202
SystemPICKCa plus HKC-FOSI204
“Alum” plus HKC-ICRA×5200

Systemic side effects include: fever (1), rash (2), pain (2), lymph node (1), laryngeal edema (1). Local side effects include: redness at the injection site (6).

Subsequent studies conducted by the inventor, determined the relationship of the observed side effects with molecular weight molecules in Freund (see examples 5 and 6).

Example 5

The relationship between the concentration PICKCa and its molecular weight

This example demonstrates that increasing the concentration of adjuvant PICKa results in compositions with increased molecular weight.

PICKCa can be obtained in various concentrations. It has been hypothesized that PICKCa as complex polymers can exist in different forms when it is received in various concentrations. For this purpose used the scattering of laser light. The scattering of laser light has been widely used to determine the weighted average molecular weight (Mw) and the radius of gyration (Rg). Instruments are commercially available, and the way well-known experts in this field. Table 4 shows that the observed molecular mass PICKCa on the scattering of laser light is correlated with its concentration.

Table 4
The observed molecular mass of the scattering of laser light
Concentration PICKCa (mg/ml)Weighted average molar mass
11,956,97×104
2,007,30×103
1,002,00×103

Example 6

The relationship between pre-concentration PICKCa and molecular mass vaccine

This example demonstrates the correlation between the increased molecular weight of the adjuvant PICKC and the final molecular weight of the composition, which includes adjuvant PICKCa and untreated rabies antigen, inactivated renal cells hamster.

It was also assumed that the concentration PICKCa before the merger will affect the antigens in vaccines. Samples PICKCa was combined with the crude antigen rabies, inaktivirovannye renal cells hamster. With this purpose used the scattering of laser light. The scattering of laser light has been widely used to determine the weighted average molecular weight (Mw) and the radius of gyration (Rg). Instruments are commercially available, and the way well-known experts in this field. Table 5 shows that increasing concentrations PICKCa before unification led to the increase of Mw vaccine against rabies.

Table 5
The relationship between the concentration PICKCa and molecular weight before merging and Mw vaccines against rabies
Concentration PICKCa (mg/ml)Weighted average molar massThe radius of rotation
11,9529,6×104is 17.2×102
4,0022,2×10415,0×102
2,0013,8×10411,8×102
1,00the ceiling of 5.60×104at 7.55×102
1,00of 5.29×104of 6.50×102

Example 7

Test toxicity PIKA

This example demonstrates the safety features of the PIKA adjuvant, when there is a restriction on the maximum molecular weight.

The toxicity test was performed in accordance with the provisions of the National Drug Standard of China (WSI-XG-050-2000). Briefly, five (5) mice weighing approximately 18-22 g intravenously were injected with 0.5 ml/mouse of a solution of sodium chloride containing 0.3 mg PIKA adjuvant having an upper limit of molecular weight from about to about 525000 1000000 daltons. Mice after injection was observed for 7 days and weighed at the end of the observation period. Table 6 summarizes the results, which showed that the molecular mass of the PIKA adjuvant can reach up to 1.0×106Dalton without obvious toxicity.

Table 6
Test the toxicity of the PIKA adjuvant
SeriesIn the of rhny the limit of the range of molecular weight (daltons) Body weight of mice before test (g)The number entered into the tail veinThe condition of the mice at the end of the testBody weight of mice after testNote
20000304the 5.25×10518-190.5 ml/mouseHealthy23-36Satisfactory
200101035,20×10518-190.5 ml/mouseHealthy22-25Satisfactory
200108165,20×10518-190.5 ml/mouseHealthy23-25Satisfactory
200105111,00×10618-200.5 ml/mouseHealthy24-26Satisfactory

Example 8

The toxicity study PIKA in the vaccine composition

The purpose of this experiment is to validate the safety of the PIKA adjuvant.

PIKA adjuvant (molecular weight of 66,000 daltons to 660000 daltons) was combined with purified antigen, inaktivirovannye renal hamster cells (HKC-IPRA) in the ratio PIKA:HKC-IPRA 4:1.

Vaccine composition of PIKA and HKC-IPRA was compared with a commercially available inactivated purified rabies vaccination (IPRV), which included adjuvant “alum”.

Mice were administered five (5) doses of vaccine compositions in the 0-th day, 3rd day, 7th day, 14th day and 28th day. Put the dose was equivalent to a dose of approximately 300 times the dose for an adult in the normal scheme of immunization of humans against rabies.

The results of observations of the toxicity presented below in table 7.

Table 7
Security surveillance after vaccination against rabies
Preparative formEffectDay 0Day 3Day 7Day 14Day 28
HKC-IPRA plus PIKAAll the rgiya 0/200/200/200/202/20
HKC-IPRA plus PIKADeath0/200/200/200/200/20
IPRV (including "Alum")Allergy0/200/200/205/20-
IPRV (including "Alum")Death0/200/200/202/207/20
Key: (Observed frequency)/(the Total number)

The conclusion is that the combination of ISDN/HKC-IPRA safer than commercially available IPRV.

Example 9

The safe use of PIKA adjuvant in humans

In 2002, five (5) volunteers were immunized with a composition of PIKA (molecular weight from 66000 to 660000 daltons) and purified rabies antigen, inactivated renal hamster cells (HKC-IPRA). Volunteers in the-Odile vaccine composition in the 0-th day, 3rd day, 7th day, 14th day and 30th day.

None of the patients after each vaccination were observed local or systemic side effects.

The activity of the vaccine was measured using the standard NIH test, with the results presented in table 8 below.

Table 8
Activity data of rabies vaccine
DayED50Neutralizing antibody IU/ml
000
141,9>1,84
452,355,17

The results indicate that the vaccine composition of PIKA and HKC-IPRA induces a specific immune response and causes the production of protective neutralizing antibodies.

Example 10

Test after exposure to antigen (cell-mediated immunity)

Test after exposure to antigen is the most certain proof that the vaccine is able to eradicate pathogens from the body of the host after infection. As such, it indicates cell-oposed the cell immune response, induced by the vaccine.

In test after exposure to antigen, the mice were injected with wild strain of rabies virus and subsequently infected: purified rabies antigen, inaktivirovannye renal hamster cells (HKC-IPRA), in combination with the PIKA adjuvant (range molecular weight of from 1.65×105to 1.2×106daltons), or HKC-IPRA in combination with the adjuvant aluminum hydroxide (alum), a commercially available purified vaccine against rabies from vero cells (PVRV), or phosphate buffer solution (PBS). The results convincingly show that the PIKA adjuvant improved survival rates, see table 9.

9.1 Mortality after treatment

Table 9
Test antigenic stimulation after contact with antigen
GroupPredefined dosage, giving 80% mortalityPredefined dose giving 50% mortality
PIKA plus HKC-IPRA2/200/20
Alum plus HKC-IPRA10/209/20
PVRV16/203/20
Control (BPS)15/2014/20

9.2. Survival after treatment

GroupPredefined dosage, giving 80% mortalityPredefined dose giving 50% mortality
PIKA plus HKC-IPRA90,00%100,00%
Alum plus HKC-IPRA50,00%55,00%
PVRV20,00%85,00%
Control (BPS)25,00%30,00%

Mice infected by subcutaneous injection of live rabies virus, treated with vaccine after 6 h, 1 day, 2 days and 3 days after infection.

Example 11

The production of interferon-gamma in antigen-specific cell-mediated immune response

The production of interferon-gamma is a measure of the activity of cell-mediated immunity.

In this experiment, blood samples were taken from the two included in the test patients and two control individuals. Patients on brovelli were vaccinated with the vaccine against rabies PIKA, which contained PIKA (range of molecular weight from 66000 to 660000 daltons) and purified rabies antigen, inactivated renal hamster cells (HKC-IPRA) in 2.5 g to obtain blood samples.

The results in figure 2 illustrate the significant difference of interferon-gamma produced two included in the test patients when compared with control individuals.

Isolated monocytes from blood samples incubated with the same HKC-IPRA, which was used in the original test. After three days of incubation were collected cell-free supernatant and interferon-gamma in the supernatant liquids were analyzed by cytokine-specific ELISPOT (enzyme-linked immunosorbent spot assay). Observed dose-dependent effect.

Conclusions from the above observations the following:

vaccine against rabies, which included the PIKA adjuvant according to the invention, is able to induce production of interferon-gamma and, consequently, to induce cell-mediated immune response;

reaction of the interferon-gamma is specific (i.e., in contrast to the nonspecific reaction, the reaction was directed at the antigen rabies). If the reaction of interferon-gamma was nonspecific, there would be changes in the level of production of interferon-gamma in the blood of vaccinated patients with increasing concentrations of the Antiga the aqueous stimulator.

Example 12

The effectiveness test PIKA

The purpose of this experiment is to demonstrate the ability PIKA induce production of interferon-gamma and interleukin 12 (IL-12).

Samples of splenocytes from normal healthy mice were incubated for 3 days in the presence of PIKA (range of molecular weight from 66000 to 660000) in a clean environment. At the end of this period the level of cytokines in the supernatant liquids were analyzed by ELISA tests that are specific for IL-12(p40) and interferon-gamma. The results of the experiment are presented below in table 10.

Table 10
Test production of the cytokine in vitro
PIKA, ug/mlInterferon-gamma, PCG/mlIL-12P40, PCG/ml
030
0,42391
22298
1030134
50179186
100559 N/A
2501340N/A

Conclusion from the above experiment is that PIKA causes dose-dependent production of interferon-gamma and IL-12 cytokines and, thus, induces cell-mediated immune response.

In another experiment four (4) mice were injected with 500 μg/mouse PIKA (range of molecular weight from 66000 to 660000) by intraperitoneal injection. A solution of phosphate buffer was used as negative control test. After 5 h after injection took a blood sample and received the serum. The levels of cytokines in serum were analyzed by ELISA tests that are specific for IL-12(p40) and interferon-gamma. The results of the experiment are presented below in table 11.

Table 11
Test production of the cytokine in vivo
GroupInterferon-gamma, PCG/mlIL-12P40, PCG/ml
PBS42
PIKA41040

Conclusion from the above experiment is that PIKA is effective in stimulating cleto is about-mediated immune response.

Example 13

Use PIKA with inaktivirovannye purified rabies antigen from cells vero

The purpose of this experiment is to evaluate the effectiveness of PIKA in combination with inactivated purified rabies vaccination from vero cells (PVRV).

PIKA with a range of molecular weight from 66000 to 660000 Dalton was combined with PVRV for the formation of rabies vaccine. The NIH test was used to evaluate the activity of the obtained vaccine composition. The results are presented below in table 12.

Table 12
The results of the test for NIH PIKA purified and inactivated vaccines against rabies from vero cells
Vaccine compositionAntigenAdjuvantActivity (IU/ml)
PVRV0,021 IU/mlnot rated0,46
PVRV plus PIKA0,021 IU/mlPIKA3,68

The conclusion is that PIKA increases the activity of inactivated purified vaccine against rabies from vero cells.

Example 14

Dose is ntigen

This experiment demonstrates the need for a minimum quantity of purified rabies antigen, inactivated renal hamster cells (HKC-IPRA)that is present in the composition, together with the adjuvant (range of molecular weight from 66000 to 660000 daltons) to run significantly increased the level of specific immune response.

Increased the number of rabies antigen was added to a constant volume of 0.1 mg PIKA adjuvant. Activity was measured using the standard test activity of rabies vaccine NIH. After the projected initial increase in activity distinct and sharp increase was observed before the activity has levelled off, as expected, adding antigen (see table 13).

Table 13
The activity of the vaccine with increasing amounts of purified rabies antigen, inactivated renal cells hamster
HKC-IPRA (ME)HKC-IPRA plus 0.1 mg PIKA (IU)A marginal increase in activity
0,250,42-
0,511,433,88
1,012,732,60
1,406,078,56
2,0719,7820,46
2,9121,381,90

A marginal increase in activity represents an increase of activity of the vaccine HKC-IPRA/PIKA observed when adding 1 IU present HKC-IPRA.

The conclusion is that the minimum presence of antigen is necessary before significant induction of the immune response. In addition, excess antigen excess starting point causes only a marginal surplus refund activity.

Example 15

The ratio of antigen and adjuvant

This experiment shows the optimal mixture of purified rabies antigen, inactivated renal hamster cells (HKC-IPRA) and PIKA adjuvant (range of molecular weight from 66000 to 660000 daltons).

Different amounts of the antigen was mixed with different amounts of the adjuvant with PBS, added to ensure a consistent total. The activity received vaccines was determined using the test activity NIH. The data presented in table 14.

Consideration of the results to binyavanga test indicates what is the optimal combination vaccine is against PIKA to the antigen in the range of at least 3:1.

Example 16

Storage in dried form PIKA and PIKA in combination with a vaccine against rabies

This example demonstrates that PIKA stable in lyophilized form.

The technology of freeze-drying has been used for long-term storage of vaccines against rabies up to three years. The inventor sought to test also did would be a beneficial effect storage in dried form PIKA (with a range of molecular weight from 66000 to 660000 daltons) and vaccines against rabies, containing PIKA. The following composition was used for the test storage in dried form: (i) unfrozen PIKA, added to the dissolved purified rabies antigen, inaktivirovannaja renal hamster cells (HKC-IPRA), (ii) dissolved lyophilized composition PIKA plus HKC-IPRA iii) dissolved lyophilized, commercially available vaccine against rabies (without adding PIKA), and (iv) unfrozen, commercially available vaccine against rabies. Table 15 shows that dried PIKA and the rabies vaccine, containing PIKA, were ideal for long-term storage of vaccines against rabies.

Table 15
The impact of storage in dried form on the activity of vaccines against rabies
SampleED50Relative activityIU/ml
i) freeze-dried vaccine against rabies, divorced PIKA2,892,3415,71
ii) dissolved in PBS lyophilized vaccine against rabies, containing PIKA3,003,00on 20, 23
iii) dissolved in PBS lyophilized vaccine against rabies, commercially available1,850,211,43
iv) standard, non-refrigerated vaccine against rabies2,521,006,70

Although the present invention has been described with reference to specific embodiments of, it should be understood that these embodiments of are illustrative and that the invention is not limited to them. Alternative ways to implement this is part II of the invention will become apparent to the average of the experts in this field, belongs to the present invention. It is believed that such alternative implementation is included in the nature and scope of the present invention. Accordingly, the scope of the present invention described in the attached claims and confirmed the previous description.

1. Polynucleotide adjuvant composition comprising polyribosome-polyribosylribitol acid (PIC), an antibiotic and a positive ion, and the composition comprises molecules of the polynucleotide adjuvant composition, heterogeneous, at least on molecular weight or size, where the range of molecular weight is from about 66000 to 1200000 and where the size is in the range of molecular size from about 6.4 to 24,0 of Svedberg.

2. Polynucleotide adjuvant composition according to claim 1, in which the range of molecular weight is from about 300,000 to 1200000 Yes or range of molecular size is approximately 12.8 to 24.0 of Svedberg.

3. Polynucleotide adjuvant composition according to claim 1, in which the range of molecular weight is from about 66000 to 660000 Yes or range of molecular size from about 6.4 to 18.3 Swedberg.

4. Polynucleotide adjuvant composition according to claim 1, in which the range of molecular weight is from about 300,000 to 660000 Yes or range of molecular size which is approximately 12.8 to 18.3 Swedberg.

5. Polynucleotide adjuvant composition that includes:
polyribosome-polyribosylribitol acid (PIC), an antibiotic and a positive ion, and the polynucleotide adjuvant composition has an average molecular weight equal to or more than 150000 Yes or medium-sized molecules, equal to or greater than the size 9.3 Swedberg.

6. Polynucleotide adjuvant composition according to claim 5, in which the average molecular weight equal to or greater than 250,000 Yes or average size equal to or greater than 11,8 of Svedberg.

7. Polynucleotide adjuvant composition according to claim 5, in which the average molecular weight equal to or greater than 350,000 Yes, or the average size of the molecules is equal to or greater than 15,3 of Svedberg.

8. Polynucleotide adjuvant composition according to any one of claims 1 to 7, in which the antibiotic is a kanamycin, neomycin, anthracyclin, butirosin sulfate, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, puromycin, streptomycin or streptozocin.

9. Polynucleotide adjuvant composition according to any one of claims 1 to 7, in which the antibiotic is a kanamycin, neomycin, anthracyclin, butirosin sulfate, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, puromycin, streptomycin or streptozocin, and the positive ion is a calcium, cadmium, lithium, magnesium, cerium, who ESI, chromium, cobalt, deuterium, gallium, iodine, iron, or zinc, and where the positive ion presented in the form of inorganic salts or organic complex.

10. Polynucleotide adjuvant composition according to any one of claims 1 to 7, in which the antibiotic is a kanamycin, neomycin, anthracyclin, butirosin sulfate, gentamicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, puromycin, streptomycin or streptozocin, and a source of positive ion is a calcium chloride, calcium carbonate, calcium fluoride, calcium hydroxide, calcium phosphate, or calcium sulfate.

11. Polynucleotide adjuvant composition according to any one of claims 1 to 7, in which the antibiotic is a kanamycin sulfate, and the positive ion is provided by calcium chloride.

12. The set includes a polynucleotide adjuvant composition according to any one of claims 1 to 7, and antigenic compound, in which the antibiotic is a kanamycin sulfate, and the positive ion is provided by calcium chloride.

13. Immunogenic composition comprising the polynucleotide adjuvant composition according to any one of claims 1 to 7, and the antigen, in which the antibiotic is a kanamycin sulfate, and the positive ion is provided by calcium chloride.

14. Immunogenic composition comprising the polynucleotide adjuvant composition according to any one of claims 1 to 7 and the antigen, in which the second antibiotic is a kanamycin sulfate, a positive ion is provided by calcium chloride, and the antigen is a human antigen, the antigen of the animal, besides humans, plant antigen, a bacterial antigen, a fungal antigen, a viral antigen, a parasitic antigen, or a cancer antigen.

15. Immunogenic composition comprising the polynucleotide adjuvant composition according to any one of claims 1 to 7 and the antigen, in which the antibiotic is a kanamycin sulfate, positive ion is provided by calcium chloride, and the antigen is an antigen of rabies.

16. Immunogenic composition according to item 15, in which the antibiotic is a kanamycin sulfate, positive ion is provided by calcium chloride, and the antigen is an inactivated purified rabies antigen.

17. Immunogenic composition comprising the polynucleotide adjuvant composition according to any one of claims 1 to 7 and the antigen, in which the antibiotic is a kanamycin sulfate, positive ion is provided by calcium chloride, and in which the polynucleotide adjuvant composition is capable of causing an amplified combined specific humoral and/or cell mediated immune response.

18. Immunogenic composition comprising the polynucleotide adjuvant composition according to any one of claims 1 to 7 and the antigen, in which the antibiotic is a kanamycin su is that, a positive ion is provided by calcium chloride, and in which at least one of the adjuvant composition or immunogenic composition is provided in solid form or in liquid form, where the liquid form is a solution or suspension.

19. Immunogenic composition comprising the polynucleotide adjuvant composition according to any one of claims 1 to 7 and the antigen, in which the antibiotic is a kanamycin sulfate, positive ion is provided by calcium chloride, and in which at least one of the adjuvant composition or immunogenic composition is lyophilized.

20. The use of polynucleotide adjuvant composition according to any one of claims 1 to 11, to obtain drugs to enhance the immune response to antigenic connection to the patient.

21. The application of claim 20, where this composition is obtained in a form suitable for parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, inhalation, rectal delivery, vaginal delivery, intranasal delivery, oral delivery, ocular delivery, local delivery, transdermal delivery, or intradermal delivery.

22. The use according to any one of p or 21, where the immunogenic composition suitable for enhancing the immune response in humans.

23. The use of any the C p or 21, where the immunogenic composition suitable for enhancing an immune response in an animal.

24. The method of obtaining immunogenic composition according to any one of PP-19, including the Association of the antigen with the polynucleotide adjuvant composition according to any one of claims 1 to 11.

25. The method according to paragraph 24, where the antibiotic is a kanamycin sulfate, and the positive ion is provided by calcium chloride.



 

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SUBSTANCE: one version of the proposed method involves passing sugar-containing liquid, particularly diffusion juice, together with a gas stream through a closed aeration chamber, addition of lime to the aerated sugar-containing liquid and then passing carbon dioxide. According to another version of the method, sugar-containing liquid is passed through a closed vacuum chamber and then through a closed aeration chamber, lime is added to the treated sugar-containing liquid, after which carbon dioxide is passed through the sugar-containing liquid.

EFFECT: reduced chromaticity of sugar-containing liquid.

5 cl, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: biotechnologies.

SUBSTANCE: plants are transformed with application of nucleic-acid constructs, which contain the first nucleotide sequence that codes γ-sein, or its fragment, which is able to direct and retain protein in endoplasmic reticulum of plant cell, the second sequence of nucleic acid, which codes aminoacid sequence, which is specifically split by ferment or chemical compounds, and the third sequence of nucleic acid, which codes target peptide or protein.

EFFECT: transformation of plant by such constructs makes it possible to produce fused proteins accumulated in endoplasmic reticulum of cells in the form of protein bodies, from which target proteins may be extracted, in particular calcitonin.

47 cl, 19 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: plants are transformed with application of nucleic-acid constructs, which contain the first nucleotide sequence that codes γ-sein, or its fragment, which is able to direct and retain protein in endoplasmic reticulum of plant cell, the second sequence of nucleic acid, which codes aminoacid sequence, which is specifically split by ferment or chemical compounds, and the third sequence of nucleic acid, which codes target peptide or protein.

EFFECT: transformation of plant by such constructs makes it possible to produce fused proteins accumulated in endoplasmic reticulum of cells in the form of protein bodies, from which target proteins may be extracted, in particular calcitonin.

47 cl, 19 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: plants are transformed with application of nucleic-acid constructs, which contain the first nucleotide sequence that codes γ-sein, or its fragment, which is able to direct and retain protein in endoplasmic reticulum of plant cell, the second sequence of nucleic acid, which codes aminoacid sequence, which is specifically split by ferment or chemical compounds, and the third sequence of nucleic acid, which codes target peptide or protein.

EFFECT: transformation of plant by such constructs makes it possible to produce fused proteins accumulated in endoplasmic reticulum of cells in the form of protein bodies, from which target proteins may be extracted, in particular calcitonin.

47 cl, 19 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to production of new hybrid polypeptide GST-CFP10 by microbiological synthesis with properties of species-specific protein-antigen CFP10 Mycobacterium tuberculosis, which may be used for early species-specific diagnostics of tuberculosis infection. Recombinant plasmid DNA pTB232 has been constructed, which codes hybrid polypeptide GST-CFP10 with properties of mycobacterial antigen CFP10, with average molecular weight (m.w.) 3.4 MDa and having size of 5257 p.n. Recombinant strain of bacteria E. coli BL21/pTB232 contains recombinant plasmid DNA pTB232, is producer of hybrid polypeptide GST-CFP 10 with properties of mycobacterial antigen CFP10 and is deposited in KM GNC VB "Vector" under number B-1027. Recombinant polypeptide GST-CFP10, produced with strain of bacteria E. coli BL21/pTB232, contains as protein-carrier N-end polypeptide fragment glutathione S-transferase S.j. (226 a.o. with m.w. of 26.3 kDa), joined via end site of thrombin hydrolysis (LVPRGS) with C-end polypeptide fragment of antigen CFP10 (100 a.o. with m.w. of 10.8 kDa) and has complete aminoacid sequence with length of 326 a.o. and m.w. of 37.1 kDa, given in text of description.

EFFECT: use of invention provides for the possibility to produce target highly pure hybrid polypeptide GST-CFP10 in preparative amounts with preservation of immunogenic properties of the latter.

3 cl, 4 dwg, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is described method for preparing a polymorphous region of gene PAR1 which can contain T to C replacement in position 3090 and/or A to C replacement in position 3329 of the polynucleotide sequence of wild type gene (NM_001992) with applying a pair of specific primers, and also the method for observing said region prepared of a DNA-containing biological sample for the presence or absence of the specified replacements. There are offered complete sets of the components application of which provides both amplification of the polymorphous region of gene PAR1 under the invention, and if needed, further analysis for genetic modifications in positions 3090 and/or 3329.

EFFECT: higher accuracy of estimating risk of cardiovascular diseases.

7 cl, 17 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method of invention is based on double-stage multiplex PCR with production of fluorescently labeled fragments of DNA with further hybridisation of these fragments on microchip that contains set of specific discriminating oligonucleotides with a certain nucleotide sequence. Definition of tuberculosis mycobacteria resistance to rifampicin and isoniazid is carried out by detection of dot nucleotide substitutions in DNA of microorganism. Invention is also related to set of primers, biochip and set of oligonucleotide probes used in method realisation.

EFFECT: invention makes it possible to perform analysis directly from clinical sample, to simultaneously detect several mutations, to reduce prime cost of analysis and to reduce time of its performance.

9 cl, 6 dwg, 2 tbl, 10 ex

FIELD: food industry.

SUBSTANCE: strain Streptococcus thermophilus which produces lactic acid is described. Sequence of nucleic acids made of the strain producing polysaccharides are also described as well as food or pharmaceutical composition and milk product containing such strain.

EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention is related to the field of medicine and is related to treatment of proliferative diseases with application of antisense oligomer IAP and chemotherapeutical preparation. Substance of invention includes method for treatment of patient suffering from proliferative disease with application of antisense oligomer SEQ ID NO: 151 or its pharmaceutically acceptable salt and chemotherapeutical preparation.

EFFECT: invention advantage consists in improved efficiency of treatment.

28 cl, 15 ex, 9 tbl, 25 dwg

FIELD: chemistry.

SUBSTANCE: method of producing cyclopropyl-condensed inhibitors of dipeptidyl peptidase IV involves using BOC-protected amine with structural formula (3) , obtained through reductive amination of acid with formula (1) by treating the said acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified concentrate of phenyl alanine dehydrogenase and formate dehydrogenase (PDH/FDH) enzymes and without separation - by treating the obtained amine of formula (2) with ditertbutyl dicarbonate, obtaining BOC-protected amine.

EFFECT: cutting on costs.

13 cl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.

EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.

5 cl, 2 ex, 2 tbl, 4 dwg

FIELD: biology.

SUBSTANCE: invention refers to molecular biology. There developed is the device and methods of acceleration of biological processes wherein there involved are charged objects, which is stimulated with an electric field, and in particular including DNA-target revealed in a biological sample. There also revealed is a reaction cell with dielectric surface, and electric field is created by means of separation induction of charges in dielectric material when the potential is being supplied to the electrode being in contact with dielectric material.

EFFECT: invention can be used for analysing biological samples and revealing DNA target.

25 cl, 11 dwg

FIELD: chemistry.

SUBSTANCE: invention concerns method of obtaining natural and modified deoxy- and ribooligonucleotide 5'-triphosphate salts, involving monophosphation of initial reagent, protected natural or modified oligonucleotide of deoxy- or ribo- range in the form of 0.1-0.5 M solution, in pyridine by double or triple excess of phosphor oxychloride for 10-15 minutes; further processing of activated oligonucleotide derivative by tenfold to fifteenfold excess of pyrophosphate bis-tributylammonium salt solution in acetonitryl and 20-fold excess of tert-amine; reacting mix maturing for 15-30 minutes, followed by degradation of intermediary oligonucleotide trimetaphosphate derivative by triethylammonium bicarbonate buffer, purification of target product by reverse-phase chromatography (RPC), removal of protection groups from functional oligonucleotide groups, and second purification of target product by RPC. Target product output after purification comprises 45-95%. Purity grade of obtained compounds by the data of high-precision electronic liquid chromatography and nuclear magnetic resonance spectrography is over 95%.

EFFECT: obtaining compounds applicable in biological molecular and genetic engineering research.

2 cl, 1 dwg, 2 tbl, 7 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention refers to genetic engineering specifically to production of muteins of placental growth factor 1st type (PLGF-1), and can be used in medicine. Monomeric human mutein PLGF-1 includes substitution or removal in polypeptide sequence of false protein of one cysteine rest (Cys) residue in position 142 of polypeptide sequence of precursor protein thus preventing multimerisation of this monomeric form.

EFFECT: reception chemically stable muteins PLGF-1 and for diagnostics and treatment of oncological and not oncological pathologies.

44 cl, 7 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present invention concerns to immunostimulating phosphorothioate CpG-oligonucleotides which can be used for stimulation of the immune response.

EFFECT: obtaining of CpG-oligonucleotides.

49 cl, 76 fig, 19 tbl, 34 ex

FIELD: medicine.

SUBSTANCE: according to invention protein NMB 1870 represents effective antigen used for immune response for meningococcus, and it is expressed among all meningococcal serogroup. Forty two various chains NMB 1870 are identified, and they are grouped by three versions. Serum produced for this version is bactericidal in the same group of version, but is inactive for strains expressing the other two other versions, i.e. intra-version, rather than inter-version cross-protection is available. Therefore this invention provides maximum cross-strain efficiency using combinations of various versions NMB 1870. Invention discovers method of mammal immune response induction providing application of compositions on based on various versions NMB 1870. Besides invention presents nucleic acid coding specified versions of protein NMB 1870 (associated chains are specified in chain list). Compositions based on protein NMB 1870 have immunogenic properties for infection, induce formation of bactericidal antibodies, cause immunity for meningococcus and not only for serogroup B meningococcus.

EFFECT: higher efficiency.

32 cl, 37 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a group of novel chemical compounds pharmacologically acceptable salts thereof having formula , where A represents COOH; B represents H; n equals 0; V represents -CH2-, a single bond; W represents a 5-7-member heteroaromatic group with one heteroatom selected from N, O, S which can optionally be substituted with 1-3 substitutes selected from a group of substitutes A, when V represents a -CH2-group, where if V represents a single bond, W represents a bicyclic condensed a ring -member heterocyclic group with one heteroatom selected from O, S, which can optionally be substituted with 1-3 substitutes selected from a group of substitutes A; X represents a 5-7-member heteroaromatic group with one O atom and one or two N atoms, which can optionally be substituted with 1-3 substitutes selected from a group of substitutes A; Y represents C6-C10 aryl which can optionally be substituted with 1-3 substitutes selected from a group of substitutes A, a 5-7-member heteroatomatic group with one S atom which can optionally be substituted with 1-3 substitutes selected from a group of substitutes A; Z represents C1-C8 alkyl, C3-C7 cycloalkyl which can optionally be substituted with 1-5 substitutes selected from a group of substitutes A; C6-C10 aryl which can optionally be substituted with 1-5 substitutes selected from a group of substitutes A; C6-C10 aryloxy which can optionally be substituted with 1-5 substitutes selected from a group of substitutes A, or C1-C12 aralkyl which can optionally be substituted with 1-5 substitutes selected from a group of substitutes A; group of substitutes A represents halogen, C1-C6 alkyl, halogen C1-C6 alkyl, C1-C6 alkoxy.

EFFECT: compounds exhibit inhibitory activity towards HvGR which enables their use to prepare a pharmaceutical composition used in therapy for autoimmune diseases.

33 cl, 6 tbl, 30 ex

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