EA025263B1 - Dilutant-adjuvant for dry living vaccine against trichophytosis of bovine cattle - Google Patents

Abstract

The invention is related to microbiology, in particular, to micology, namely, to veterinary micology, and can be used as a dilutant-adjuvant for dry vaccine, in particular, against trichophytosis of bovine cattle. The invention proposes a dilutant-adjuvant for dry living vaccine against trichophytosis of bovine cattle comprising an active ingredient with immunomodulating activity, additive agents and, if necessary, an excipient. As an active ingredient, the dilutant-adjuvant contains a substance of recombinant bovine γ-interferon in an amount ensuring antiviral activity not lower than 1×10IU per 100 ml of water suspension containing additive agents. As additive agents, the dilutant-adjuvant contains aminoacids including L-arginine, L-cystine, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophane, L-tyrosine, L-valine, vitamins including calcium pantothenate, chloride choline, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, inorganic salts including sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium dihydrogenphosphate, baking soda, organic salts including humates, sugars including glucose, and purified water with a definite proportion of the components. The proposed dilutant-adjuvant provides higher immunological potency of dry living vaccine against trichophytosis of bovine cattle.

Description

The invention relates to microbiology, in particular to mycology, namely to veterinary mycology, and can be used as an adjuvant diluent for a dry vaccine, in particular against cattle trichophytosis.

Various vaccines have been developed for vaccinating cattle against trichophytosis. For their introduction into the body, physiological solvents or special physiologically acceptable diluents are necessary. The goal of vaccination is to create an effective immunity in an animal (including humans) that provides long-term protection against infections. Most modern vaccines are developed based on specific antigens, in contrast to fully inactivated or attenuated pathogens. Antigens of microorganisms, tumor cells, etc. introduced into the body of the animal in the form of purified proteins. But such molecular vaccines may in some cases have weak immunogenicity, and adjuvants must be added to enhance the immune response from the animal’s immune system. Adjuvants have been used in vaccines to enhance the immune response for over 80 years. An immunological adjuvant is understood to mean substances of various chemical nature that act non-specifically and increase a specific immune response. The development of adjuvants and adjuvant systems has gone from the first empirical experiments to the creation of targeted systems, which is mainly due to successes in the study of the immune system and the improvement of analytical, chemical and immunological techniques [1]. For dry vaccines, adjuvants are usually used in a mixture / solution with a physiological solvent or a special physiologically acceptable diluent.

It is known from the prior art that both simple and complex multicomponent systems, including adjuvants, are used as a diluent for dry vaccines. Among such systems, mention may be made in particular of peptone, phosphate buffer solution and Trilon B [2], peptone and potassium phosphate buffer solution [3], sodium thiosulfate solution [4], saline solution IaC1 and aluminum hydroxide [5], sucrose, disubstituted potassium phosphate, pH indicator phenol red and distilled water [6], as well as glycerin, agar-agar and sodium chloride; cattle milk and blood serum; food sugar and glycerin and others.

The closest technical solution to the proposed adjuvant diluent is a diluent for dry live culture of fungi trichophytone or microsporum or dry vaccine against trichophytosis or microsporia, which contains as an active substance with immunomodulating activity a 1% solution of lipopolysaccharide peptide fraction of the E-antigen bacteria of the genus Eusebys 8a1toe11a, sodium chloride and yeast extract and water as excipients of sodium [7]. The specified diluent provides 100% immune protection of animals against dermatophytosis only with resuspension in it of 80 million fungal microconidia.

The objective of the invention is to develop a diluent adjuvant for a dry live vaccine, which would provide an increase in the immunogenicity of a dry live vaccine against trichophytosis in cattle.

The problem is solved by the claimed diluent adjuvant for dry live vaccine against trichophytosis in cattle containing the active substance with immunomodulating activity and excipients. The problem is solved due to the fact that the diluent adjuvant as an active substance contains a bovine recombinant γ-interferon substance in an amount providing antiviral activity of at least 1 x 10 ⁶ IU per 100 ml of an aqueous suspension containing excipients, while containing excipients amino acids, including b-arginine, b-cystine, b-glutamine, b-histidine, b-isoleucine, b-leucine, b-lysine, b-methionine, b-phenylalanine, b-threonine, b-tryptophan, b-tyrosine, b -valine, vitamins, including feces pantothenate l, choline chloride, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, inorganic salts, including sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium phosphoric acid monosubstituted, drinking soda, humic acid and glucose salts, glucose purified in the following ratio of components, wt.%:

- 1,025263 salts of humic acid glucose

B-arginine

B-cystine

E-glutamip

B-histidine

B-isoleucine

[.-leucine

B-lysine

B-methionine

B-phenylalanine

B-threonine

B-flufan

E-tyrosine

E-valine calcium pantothenate choline chloride folic acid nicotinamide pyridoxal hydrochloride riboflavin thiamine sodium hydrochloride potassium chloride calcium chloride magnesium chloride (M ^ Cb '6H ₂ O) sodium phosphate mono-amine (Ν3Η ₂ ΡΟ ₄ ' H ₂ O) soda

0.0015-0.0035

3.0-5.0

0.005-0.011

0.0015-0.003

0,0) 5-0,03

0.002-0.004

0.003-0.006

0.003-0.006

0.003-0.006

0.001-0.0015

0.002-0.004

0.0025-0.005

0.0005-0.0011

0.002-0.004

0.0025-0.005

0.00006-0.0001

0.00006-0.0001

0.00006-0.0001

0.00006-0.0001

0.00006-0.0001

0.000005-0.00001

0.001-0.002

0.5-0.8

0.02-0.04

0.01-0.02

0.01-0.02

0.01-0.015

0.1-0.22 purified water the rest.

The inventive diluent adjuvant is obtained by resuspension in purified water containing bovine recombinant γ-interferon substance with an antiviral activity of at least 1 × 10 ⁴ IU / cm ³ , amino acids b-arginine, b-cystine, b-glutamine, b-histidine, b-isoleucine , B-leucine, lysine, b-methionine, b-phenylalanine, b-threonine, b-tryptophan, b-tyrosine, b-valine; vitamins calcium pantothenate, choline chloride, folic acid, nicotinamide, pyridoxal HC1, riboflavin, thiamine HC1; sodium salts of chloride, potassium chloride, calcium chloride, magnesium chloride, sodium phosphoric acid monosubstituted, drinking soda, salts of humic acid and glucose.

Due to the presence in the inventive diluent adjuvant of specially selected various biologically active substances providing a wide spectrum of activity, one can even say, synergistically, the effectiveness (immunogenicity) of the vaccine is increased, and the risks of side effects are also reduced. In this case, the active substance with immunomodulating activity in combination with the other biologically active substances indicated above contributes to a significant increase in the immunogenicity of the inventive diluent adjuvant. Recombinant bovine interferon is a species-specific drug that exhibits antiviral and immunostimulating activity in calves and adult cattle. The effect of the drug is determined by the total effect of the exogenous protein directly on the cells affected by the virus, by the rapid induction of the endogenous interferon system, cellular and humoral immunity. Recombinant bovine interferon acts as an inducer of lysozyme and bactericidal activity of blood serum (BASK). It has anti-inflammatory effect. Increases the resistance of the animal organism to the effects of DNA and RNA containing viruses and pathogenic microorganisms. The protein of interferon after intramuscular or subcutaneous administration is well absorbed, reaching a therapeutic concentration in the blood after 6 hours. The immunostimulating activity of interferon reaches its maximum value after 12 hours and lasts for the next 24-30 hours. Traditionally, the drug is used for prophylactic and therapeutic purposes for gastrointestinal intestinal and acute respiratory diseases of viral and mixed (bacterial-viral) etiology in calves and adult cattle. It is indicated for use in case of a threat of the spread of such viral diseases as parainfluenza-3, infectious rhinotracheitis, cattle viral diarrhea, adeno-, respiratory syncytial viruses and other DNA or RNA viruses. As an immunostimulant, the drug is used for immunodeficiency in animals caused by adverse conditions of feeding, feeding, etc. In the claimed diluent adjuvant, it contributes to a significant increase in the immunogenicity of the vaccine.

Further, the advantages and advantages of the claimed diluent adjuvant will be illustrated by some possible, but not limiting examples of its implementation, as well as comparative examples (diluents and diluents adjuvants that are not included in the scope of claims of this invention).

Examples

Example 1 (prototype).

The diluent for dry living culture, trichophytone or microsporum or dry vaccine against trichophytosis or microsporia, was prepared by mixing a 1% solution of the lipopolysaccharide peptide fraction of the O-antigen of bacteria of the genus Exceisia or 8a1toie11a, sodium chloride, yeast extract and water in the following ratio of components, wt.%:

1% solution of the lipopolysaccharide peptide fraction of the O-antigen of bacteria of the genus Exskeg'mMa or 8a1tope11a 10-20 sodium chloride 0.5-0.9 yeast extract 0.1-1.0 water the rest.

The live dry vaccine against trichophytosis was resuspended in a diluent to a content of 12 million spores / cm ³ . The vaccine resuspended in this solvent protected calves from trichophytosis by administering twice as many 40 million spores (a total of 80 million spores) of trichophyton.

Example 2 (according to the invention).

An adjuvant for a dry live vaccine against cattle trichophytosis was prepared by resuspending the recombinant bovine γ-interferon substance in an amount providing antiviral activity of at least 1 χ 10 ⁶ IU per 100 ml of aqueous (in purified water) suspension containing amino acids, vitamins, salts of humic acid, glucose, in the following ratio of components, wt.%:

- 3 025263

humic acid salts 0.0015 glucose 3.0 B-arginine 0.005 B-cystine 0.0015 B-glutamine 0,0 (5 [.-histidine 0.002 B-isoleucine 0.003 1.-leucine 0.001 E-lysine 0.001 E-methionine 0.001 E-phenylalanine 0.002 E-threonine 0.0025 E-tryptophan 0,0005 E-tyrosine 0.002 E-valine 0.0025 calcium pantothenate 0.00006 choline chloride 0.00006 folic acid 0.00006 nicotinamide 0.00006 pyridoxal HC1 0.00006 riboflavin 0.000005 thiamine HC1 0.001 sodium chloride (NaCl) 0.5 potassium chloride (COP!) 0.02 calcium chloride (CaCb) 0.01 magnesium chloride (Μ ^ ΟΙ? 6H; O) 0.01 monosubstituted sodium phosphate (MAEEO ,, · N ₂ O) 0.01. drinking soda (ΝπΙ ΙΕΌι) about,! purified water rest.

The live dry vaccine against cattle trichophytosis was resuspended in the above solvent adjuvant to a content of 12 million spores / cm ³ . The cattle trichophytosis vaccine resuspended in this adjuvant solvent protected calves from the disease by trichophytosis with a single immunization at a dose of 40 million trichophyton spores.

Example 3 (according to the invention).

An adjuvant for a dry live vaccine against cattle trichophytosis was prepared by resuspending the recombinant bovine γ-interferon substance in an amount providing antiviral activity of at least 1 χ 10 ⁶ IU per 100 ml of aqueous (in purified water) suspension containing amino acids, vitamins, salts of humic acid, glucose, in the following ratio of components, wt.%:

- 4 025263 salts of humic acid glucose

B-arginine

B-cystine

B-glutamine

B-histidine

B-isoleucine

B-leucine

B-lysine

B-methionine

B-phenyl alanine

B-threonine

B-tryptophan

B-tyrosine

L-valine calcium pantothenate Choline chloride Folic acid Nicotinamide pyridoxal, riboflavin, thiamine HC1 HC1 sodium chloride (Να € 1) potassium chloride (KC1), calcium chloride (CaCl), magnesium chloride (M§S1 ₃ .6H ₂ O), sodium phosphate monobasic <Kang ₂ RO ₄ · Н ₂ О) drinking soda (KANSO ;;)

0.0035

5,0

0.011

0.003

0,03

0.004

0.006

0.006

0.006

0.0015

0.004

0.005

0.0011

0.004

0.005

0.0001

0.0001

0.0001

0.0001

0.0001

0.00001

0.002

0.8

0.04

0.02

0.02

0.015

0.22 purified water the rest.

The live dry vaccine against cattle trichophytosis was resuspended in the above solvent adjuvant to a content of 12 million spores / cm ³ . The cattle trichophytosis vaccine resuspended in this adjuvant solvent protected calves from the disease by trichophytosis with a single immunization at a dose of 40 million trichophyton spores.

Example 4 (comparative).

The adjuvant diluent for the dry live vaccine against cattle trichophytosis was prepared analogously to Example 2, but calcium pantothenate, choline chloride, folic acid, nicotinamide, pyridoxal HC1, riboflavin, thiamine HC1 were excluded from the diluent adjuvant.

The live dry vaccine against cattle trichophytosis, resuspended in this adjuvant solvent, protected calves from the disease by trichophytosis with a single immunization in

- 5,025263 dose of 49-51 million spores of trichophytone.

Example 5 (comparative).

The diluent adjuvant for the live cattle trichophytosis vaccine was prepared analogously to Example 3, but sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium phosphate monosubstituted, and baking soda were excluded from the diluent adjuvant.

The live dry vaccine against cattle trichophytosis resuspended in this adjuvant solvent protected calves from the disease with trichophytosis during a single immunization at a dose of 50-51 million trichophyton spores.

Example 6 (comparative).

An adjuvant diluent for a live cattle trichophytosis vaccine was prepared analogously to Example 3, but humic acid salts were excluded from the adjuvant.

The live dry vaccine against cattle trichophytosis resuspended in this adjuvant solvent protected calves from the disease by trichophytosis during a single immunization at a dose of 52-54 million trichophyton spores.

Example 7 (comparative).

The adjuvant diluent for dry livestock trichophytosis vaccine was prepared analogously to Example 3, but glucose was excluded from the adjuvant diluent.

The live dry vaccine against cattle trichophytosis, resuspended in this adjuvant solvent, protected calves from trichophytosis with a single immunization at a dose of 51-52 million trichophyton spores.

Example 8 (comparative).

The adjuvant diluent for dry livestock trichophytosis vaccine was prepared analogously to Example 3, but amino acids were excluded from the adjuvant diluent: b-arginine, b-cystine, b-glutamine, b-histidine, b-isoleucine, b-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine.

The live dry vaccine against cattle trichophytosis resuspended in this adjuvant solvent protected calves from the disease with trichophytosis during a single immunization at a dose of 53-55 million trichophyton spores.

Example 9 (comparative).

The adjuvant diluent for the live cattle trichophytosis vaccine was prepared analogously to Example 3, but the substance of the bovine recombinant γ-interferon was excluded from the composition of the diluent adjuvant.

The live dry vaccine against cattle trichophytosis resuspended in this adjuvant solvent protected calves from the disease by trichophytosis upon double immunization at a dose of 62-64 million trichophyton spores.

From the presented materials it can be seen that the exclusion of vitamins (example 4), inorganic salts (example 5), amino acids (example 8), glucose (example 7) or humic acid salts (example 6) from the composition of the adjuvant diluent reduces the immunogenicity of the dry vaccine live against trichophytosis of cattle by 23-38%.

The presence of bovine recombinant γ-interferon substance in the diluent-adjuvant has a significant effect on the immunogenicity of the live dry vaccine against cattle trichophytosis. So the immunogenicity of the vaccine resuspended in a diluent adjuvant containing the substance of the bovine recombinant γ-interferon (examples 2 and 3) was 55-60% higher than without it (example 9) and required two doses of the vaccine with an interval of 14 days.

Thus, in order to ensure high viability of the spores and their immunogenicity, it is advisable to resuspend the live dry vaccine against cattle trichophytosis in the diluent adjuvant prepared according to the recipe indicated in examples 2 and 3.

Information sources

1. Adjuvants as an important component of vaccines. Electronic version of the journal Biopreparations, No. 3 (43) 2011. [Electronic resource] March 21, 2014. Access mode: 11ir: // \ y \ y \ u. Yurgsraga1utada / 1ps.sg / gsu1S ^ / 40_01 /

2. §I 1490739 A1, 1994.

3. KI 2115431 C1, 1998.

4. ΒΥ 6670 C1, 2004.

5. KI 2093178 C1, 1997.

6. KI 2179035 C1, 2002.

7. ΒΥ 12706 C1, 2009.

Claims (1)

CLAIM Adjuvant thinner for dry live cattle trichophytosis vaccine containing active substance with immunomodulating activity and excipients, - 6 025263 characterized in that the active substance contains a bovine recombinant γ-interferon substance in an amount that provides at least 1 x 10 ⁶ IU per 100 ml of an aqueous suspension of excipients with antiviral activity, while it contains amino acids, including B-arginine, b-cystine, b-glutamine, b-histidine, b-isoleucine, b-leucine, b-lysine, b-methionine, b-phenylalanine, b-threonine, b-tryptophan, b-tyrosine, b-valine, vitamins, including calcium pantothenate, choline chloride, folic acid, nor cotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, inorganic salts, including sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium phosphoric disubstituted, drinking soda, humic acid salts, glucose and purified water in the following ratio, wt. %: salts of humic acid glucose B-arginine B-cystine B-glutamine B-histidine B-isoleucine B-leucine B-lysine B-methionine B-phenylalanine B-threonine B-tryptophan B-tyrosine B-valine calcium pantothenate choline chloride folic acid nicotinamide pyridoxal hydrochloride riboflavin thiamine sodium hydrochloride potassium chloride calcium chloride magnesium chloride (MgCl ₂ 6H ₂ O) sodium phosphate monosubstituted (19aH ₂ PO ₄ · N ₂ O) soda 0.0015-0.0035 3.0-5.0 0.005-0.011 0.0015-0.003 0.015-0.03 0.002-0.004 0.003-0.006 0.003-0.006 0.003-0.006 0.001-0.0015 0.002-0.004 0.0025-0.005 0.0005-0.0011 0.002-0.004 0.0025-0.005 0.00006-0.0001 0.00006-0.0001 0.00006-0.0001 0.00006-0.0001 0.00006-0.0001 0.000005-0.00001 0.001-0.002 0.5-0.8 0.02-0.04 0.01-0.02 0.01-0.02 0.01-0.015 0.1-0.22 rest.