RU2095082C1 - Vaccine for helminthiasis control - Google Patents

Abstract

FIELD: biotechnology, helminthology. SUBSTANCE: vaccine is a conjugate of immunostimulating carrier with protective antigen. Protective antigen: protein isolated from connective tissues and showing hyalurosno-lyase activity, its molecular mass is 63000 Da, isoelectric point - at 5.7. Mass ratio of antigen and carrier = 1:(1-10). Vaccine can contain adjuvant, for example, polyaxidonium, muramyldipeptide, aluminium hydroxide. Vaccine is used for prophylaxis and treatment of different helminthiasis forms in human and animals. EFFECT: enhanced effectiveness of vaccine. 3 cl

Description

 The invention relates to biotechnology and can be used for the prevention and treatment of various helminthiases of animals and humans.

 Currently, the main method of combating helminths is the use of chemotherapeutic drugs of anthelmintics, such as nilverm, rintal, panapurm, etc. However, it has been established that anthelmintics are highly toxic, cause immunosuppression, repeated treatment is required, and the development of parasite resistance to the chemotherapy used is also noted. Vaccination removes the questions of multiple treatments, the resistance of parasites to the drugs used, and the safety of use.

 Known methods for producing inactivated, genetically engineered and live vaccines against helminthiases (1,2,3). The disadvantages of such vaccines are their high reactogenicity, low immunogenicity, the complexity of the production process, as well as the likelihood of infection with vaccinating material, genetically engineered low immunogenicity. In addition, vaccines are ineffective, their effect is highly specific and is directed only against a specific parasite.

 Known conjugate vaccine against helminthiases based on ecdysteroid and protein macromolecule. The disadvantage of this vaccine is its low efficiency, the use of a protein as a macromolecular carrier that causes a number of side immunopathological effects when introduced into the body (4).

где:
q (0,2-0,35)n
z (0,4-0,65)n
m (0-0,4)n
n 200-2000.The closest analogue is a vaccine for the prevention of helminthiasis containing a conjugate of the protective antigen hyaluronidase mol.m. 63 CD with an immunostimulating carrier of the general formula:

Where:
q (0.2-0.35) n
z (0.4-0.65) n
m (0-0.4) n
n 200-2000.

 In addition, the known vaccine further comprises an adjuvant, for example, aluminum hydroxide, muramyl dipeptide and / or synpole (polyoxidonium) (5).

 However, the known vaccine has undesirable side effects due to the low rate of biodegradation and excretion from the body. In addition, obtaining a drug with a predetermined structure and properties is difficult, since the synthesis is carried out under uncontrolled conditions.

 The technical result obtained by the implementation of the invention is to increase the efficiency and safety of the vaccine by reducing its toxicity, as well as the possibility of controlled synthesis of the drug with the desired structure and properties.

 The invention consists in the following.

где
R протективный антиген
n 200-2000 количество элементарных звеньев
q (0,1-0,35) количество алкилированных звеньев,
z (0,4-0,7) количество окисленных групп.The helminthiosis vaccine containing the conjugate of an immunostimulating carrier with a protective antigen contains as a protective antigen a protein isolated from the structures of connective tissues, which has hyaluronic lyase activity and is characterized by a mol.m. 63000 D, an isoelectric point of 5.7, has a mass ratio of carrier antigen 1: 1-10 and the general formula:

Where
R protective antigen
n 200-2000 the number of elementary links
q (0.1-0.35) the number of alkylated units,
z (0.4-0.7) the number of oxidized groups.

 The described protein polypeptide with a molecular weight of 63000 (P-63) is common to various forms of helminths and its amino acid composition (primary sequence) of the protective antigen has been studied and described in a well-known source (6).

 The source for the isolation of P-63 protein can be, for example, the larval forms of helminths Dictyocaulus filaria, Fasciola hepatica, Haemonchus contorius.

 Protein is isolated by grinding the original biomaterial, extraction with a solution of acetic acid (pH 4.5-4.7), separation of the extract by centrifugation, concentration of the extract by ultrafiltration, precipitation of protein ballasts, purification of the target product by chromatography and freeze drying.

 Polyoxidonium, a synthetic high molecular weight copolymer of 1,4-ethylene piperazine N-oxide and N-carboxyethyl-1,4-ethylene piperazinium bromide, is used as an immunostimulating carrier.

 The polyoxidonium structure parameters described in the invention: n is the number of elementary units, q is the number of alkylated units, z is the number of oxidized groups determine the immunobiological properties of the drug as a whole (exposure efficiency, toxicity and safety of use).

 It was found that the effectiveness of the drug increases with increasing molecular weight. The used q values (0.1-0.35) provide a sufficient degree of binding and low toxicity even at high molecular weights. The number of oxidized groups (z = 0.4-0.7) provides a reduction in toxicity and regulates the metabolic rate and excretion of the drug from the body.

 The mechanism of protection against helminthiasis, which forms in the body after immunization with the proposed vaccine, is associated with both the effect on one of the key pathogenicity factors in the early stages of helminth development and the vital processes of a mature helminth.

 Protein P-63 is a weak immunogen that does not cause a noticeable antibody response in the body. As a result of conjugation with a polyoxidonium immunostimulating carrier capable of causing a significant increase in the antibody response to the protective antigen in the conjugate, compounds are formed that cause a high antibody response against the P-63 protein in the body, which ensures high efficiency, stability and prolongation of the vaccine.

 Strengthening the antibody response and protective activity of the conjugate vaccine can be achieved by introducing additives of various natural and synthetic adjuvants in optimal immunostimulating doses.

 A conjugated vaccine is obtained by covalently binding the protective protein antigen P-63 to a high molecular weight immunostimulating carrier with the polyoxidonium azide method or the activated ester method, resulting in the formation of a covalent amide bond between the molecules of the polymer carrier and the protein antigen molecules. The described synthesis is carried out under controlled conditions and makes it possible to obtain a drug with a predetermined structure and protein-polymer ratio.

The use of the azide method requires careful observance of the reaction conditions, temperature 0-2 ^o C, pH 8.0-8.5, polymer-antigen ratio 1: 3-1: 5, reaction time 12 hours. When carrying out the reaction under strictly controlled conditions, this method conjugation allows you to completely avoid intermolecular and intramolecular crosslinking, preserve antigenic determinants and get conjugates in high yield. When using the method of activated esters, the preparation of the polymer-antigenic vaccine is carried out in two stages, the first stage is the synthesis of the polyoxidonium succinimide ester, the second stage is the conjugation reaction between the activated form of the polyoxidonium and P-63 antigen.

 Isolation of the conjugate is carried out by chromatographic method, the analysis of conjugates is carried out by electrophoresis in SDS page, fluorescence spectroscopy.

 The introduction of adjuvants in the conjugate vaccine is carried out by adding adjuvants to the conjugate vaccine in an aqueous solution at a pH of 6.5-7.45, followed by lyophilization. The amount of adjuvant added depends on its optimal dose for humans or a particular animal species. The method allows to maintain the effectiveness of the vaccine with a low content of high molecular weight carrier, which reduces the cost of production of the vaccine.

 To illustrate the invention, specific examples of the preparation of a helminth vaccine by various methods and analysis of the protective properties of the vaccine are given below.

 Example 1. Obtaining a conjugate of polyoxidonium protein P-63 using the azide conjugation method is carried out in two stages. The first stage is the preparation of polyoxidonium hydrazide.

500 mg of polyoxidonium (n = 2000; q = 0.1; z = 0.7) is dissolved in 25 ml of methyl alcohol. At a temperature of 20 ^o C add 0.2 ml (2 • 10 ^-3 M) hydrazine hydrate. The reaction is carried out for 24 hours. Methanol is distilled off on a rotary evaporator, the residue is dissolved in water and ether extraction is carried out. The product is isolated by ultrafiltration on hollow fibers (Amikon system), followed by lyophilization.

 The content of hydrazide groups is determined by the standard method of titration of primary amino groups with 2,4,6-trinitrobenzenesulfonic acid.

The second stage is the preparation of a conjugate by the condensation reaction of polyoxidonium hydrazide with protein P-63. 100 mg of polyoxidonium hydrazide is dissolved in 4 ml of 1N. HCl). The solution was cooled to 0-2 ^° C. Then, with stirring and cooling, 1.15 ml of a 3% sodium nitrite solution was added. After 15 minutes, the pH of the solution was adjusted to 8.5 by the addition of 2N. NaOH. A solution of 12 mg of P-63 protein in 10 ml of 0.05 M phosphate buffer pH 8.5 is added to the activated polyoxidonium solution. The pH of the reaction mixture was maintained at 8.5 by the addition of 2N. NaOH. The reaction continues for 12 hours with stirring and cooling (0-2 ^o C). To isolate and purify the conjugate, the reaction mixture was applied to a column (2.6 × 90) filled with P 100 biogel; a 0.05 M pH 7.5 phosphate buffer containing 0.05 M NaCl was used as an eluent. The conjugate yield is monitored using a flow spectrophotometer at 226 nm. Determination of protein content and analysis of the conjugate is carried out by fluorescence spectroscopy, electrophoresis in SDS page. 1 mg of the drug contains 0.10 mg of P-63 protein.

 Example 2. Obtaining a conjugate of polyoxidonium (molecular mass 25000, n = 200, q = 0.1, z = 0.7) with protein P 63 lead similarly to that described in example 1. The ratio of polymer to protein in the reaction mixture is 1: 5, the pH during the condensation is maintained equal to 8. 1 mg of the drug contains 0.15 mg of protein P 63.

 Example 3. Obtaining a conjugate of polyoxidonium protein P 63 by the method of activated esters is carried out in two stages.

 The first step is the preparation of a succimide polyoxidonium ester.

100 mg of polyoxidonium (n = 800; q = 0.1; z = 0.7) containing 0.30 mmol of COOH groups is suspended in 4 ml of dimethylformamide, 77.2 mg (0.30 mmol) of dicyclohexidecarbodiimide are added with stirring and 36 mg (0.30 mmol) of N-hydroxysuccinimide. The reaction is carried out with stirring and cooling (2-4 ^° C.) for 24 hours. For purification, the precipitate is washed many times with dioxane, ether and acetone, and dried in a vacuum oven. The absence of low molecular weight impurities is checked by thin-layer chromatography on Silufol plates in a 4: 1: 1 n-butyl alcohol-water-acetic acid system. The content of activated groups is determined by a standard method (pH 8.5, 0.1 M aqueous solution of NH ₃ , 259 nm, extinction coefficient 9700); the content of activated groups is 9 • 10 ^-4 moles per 1 g of polymer.

The second stage is the conjugation. 100 mg of activated polyoxidonium is dissolved in 10 ml of phosphate buffer pH 6.0 0.05 M. With stirring and cooling, a solution of 15 mg of protein P 63 in 12 ml of phosphate buffer 0.05 M pH 7.5 is added. The condensation reaction is carried out for 18 h at 0 ^o C. After the reaction, the conjugate is isolated by chromatographic method, as described in example 1. The analysis of the conjugate is carried out similarly to that described in example 1.

 1 mg of the drug contains 0.1 mg of protein P 63.

 Example 4. Obtaining a conjugate of polyoxidonium with protein P 63 is carried out similarly to that described in example 3. The ratio of polymer to protein in the reaction mixture is 1: 2.

 1 mg of the drug contains 0.5 mg of protein P 63.

 Example 5. Obtaining a complex vaccine.

 The preparation and isolation of the conjugate of polyoxidonium with protein P 63 is carried out similarly to that described in example 1. The ratio of polymer proteins in the reaction mixture is 1: 1, the content of polyoxidonium 5 mg, protein P 63 5 mg. An aqueous solution of polyoxidonium adjuvant in an amount of 40 mg is then added to the solution of the purified conjugate. The solution is lyophilized, the protein content is determined as described in example 1. 1 mg of the preparation contains 0.1 mg of protein.

Example 6. Obtaining a complex vaccine /
Obtaining a complex vaccine is carried out similarly to that described in example 5.

 The preparation and isolation of the conjugate of polyoxidonium with protein P 63 is carried out similarly to that described in example 3. As an adjuvant, polyoxidonium in an amount of 200 mg is used.

 Example 7. Obtaining a complex vaccine.

 Obtaining a complex vaccine is carried out similarly to that described in example 5.

 The preparation and isolation of the conjugate of polyoxidonium with protein P-63 is carried out similarly to that described in example 1. As an adjuvant, polyoxidonium in an amount of 200 mg is used.

 Example 8. Obtaining a complex vaccine.

 Obtaining a complex vaccine is carried out similarly to that described in example 5, as adjuvant use GMDP (derivative of muramyl dipeptide) in an amount of 10 mg.

 Example 9. Obtaining a complex vaccine. Obtaining a complex vaccine is carried out similarly to that described in example 5, aluminum hydroxide in the amount necessary to stimulate the immune response is used as an adjuvant.

 Example 10. A test of the effectiveness of the vaccine for the prevention of echinococcosis was carried out on 45 dogs. The preparations obtained according to examples 1 and 5 are administered twice with an interval of 21 days. 14 days after the second immunization, the animals are orally infected with echinococcus protoscolexes at a dose of 30,000 protoscolexes per animal. On the 30th day after infection, the animals are killed and the number of helminths in the intestine is determined. The experimental results are presented in table. one.

Assessment of the acute toxicity of the drug (LD ₅₀ ) in all cases was carried out according to the standard method described in the collection of instructions, approved by order of the USSR Ministry of Health of 13.01.83 N 31, p. 76.

 The acute toxicity of the preparations obtained according to examples 1 and 5 is 2000 mg / kg of animal weight.

 Example 11. The tests were carried out at the Ukrainian NIVS on 12 dogs. Dogs, divided into 4 groups, were administered the drug obtained as described in example 2.

 In the first group of dogs, they are immunized twice at a dose of 5 mg per animal. Dogs of the second group are vaccinated twice at a dose of 10 mg. For dogs of the third group, the first immunization is carried out at a dose of 5 mg, the second at a dose of 10 mg. The fourth group will add control.

 On the 14th day after the second immunization, the dogs are infected with echinococcus protoscolexes at a dose of 5000 per animal.

35 days after infection, the animals are killed and the number of helminths is determined. The results are as follows: 1 group 1-2 helminths per animal, 2 group 6-12, 3 group 2-3, control group 1574-2510 helminths per animal. The optimal dose of the drug was 5 mg per head, which coincides with the result of the experiment described in example 1. In this case, the acute toxicity of LD ₅₀ was 2500 mg / kg of animal weight.

Example 12. The test of the protective properties of the drug with fascioliasis is carried out on lambs 3-4 months of age. The drug, obtained as described in example 3, is administered twice intramuscularly with an interval of 21 days. During the pasture period (May-January), animals are monitored, feces are periodically examined for the presence of fasciolus eggs. At the end of the grazing period, the animals are killed and the amount of fasciol in the liver is counted. The results of the experiment are given in table. 2. LD ₅₀ 500 mg / kg.

 Example 13. For a comparative study of the protective properties of various series of the drug obtained according to examples 1, 5, 6 and 7, a test is conducted on 60 lambs of the current year of birth. One month before the pasture, the lambs are vaccinated twice in accordance with the scheme indicated in the table. 3.

 The invasion was carried out on day 14 after a secondary vaccination at a dose of 50 adolescaria per head. Then the animals were sent to a pasture, non-fragmental for fascioliasis. The results of a coprological study conducted after 10 months. revealed the presence of only individual fasciolus eggs in groups I, III, IV, V and the complete absence thereof in animals in group II. In the control group, 30-42 fasciol was determined in the liver of each animal.

 Example 14. The protective properties of Pr-1 in dactiocauliasis were tested on calves of the current year of birth. Animals were immunized twice with an interval of 21 days.

 14 days after secondary immunization, the calves were infected with 500 larvae of dictioacula larvae per animal. On day 30, the animals were sacrificed and the number of helminths in the intestine was determined. The results are presented in table. 4.

 Example 15. The protective properties of the vaccine obtained according to example 1 were carried out under production conditions for 11,000 lambs of pasture.

 45 days before the pasture, the vaccine was administered twice at a dose of 5 mg per head to the lambs of the first month of life. The interval between vaccines was 21 days.

A year after vaccination, in slaughter of 1127 lambs, only 3-4 animals were found in only 6 animals. No dictioacles, no echinococci, nor fasciol were found. The invasion rate of unvaccinated animals was 80-100%
Example 16. The protective properties of the vaccine obtained as described in example 1 were tested under production conditions for 11700 lambs of pasture.

 The vaccine was administered twice in a dose of 5 mg per head to lambs aged 20-30 days 45 days before pasture in the pasture. The interval between vaccinations was 21 days.

7 months after vaccination, when slaughtering 5000 lambs, only 1-3 animals were found 1-3 dictiocaula. No echinococci or fasciol were found. The invasion rate of unvaccinated animals was 90-100%
Example 17. The protective properties of the vaccine obtained as described in examples 8 and 9 were tested in 20 cows.

 The vaccine was administered twice at a dose of 30 mg per head with an interval between vaccinations of 21 days. 14 days after the second vaccination, the animals were infected with larvae of dictiocaula at a dose of 500 per head. After 30 days, the animals were slaughtered and the number of helminths in the intestine was determined. The results are presented in table. 5.

 Example 18. The therapeutic properties of the vaccine obtained according to example 1 were tested on 45 sheep. Before immunization, animals are infected orally with protoscolexes of efinococcus at a dose of 30,000 protoscolexes per animal. On the 30th day after infection in animals, the number of helminths in the intestine is determined. The vaccine is administered at a dose of 5 mg per head five or ten times with an interval between administrations of 3 days. 14 days after the last immunization, the animals are killed and the number of helminths in the intestine is determined. The experimental results are presented in table. 6.

 Example 19. The therapeutic properties of the vaccine obtained as described in example 1 were tested under production conditions for 500 grazing sheep.

 The vaccine was administered five times in a course dose of 50 mg per head. The interval between vaccine administrations was 3 days.

7 months after vaccination, when 3,000 sheep were slaughtered, only 2-5 dictiocauls were found in only 50 animals. No echinococci or fasciol were found. The invasion rate of unvaccinated animals was 90-100%
Sources of information taken into account:
1. Anakina Yu.G. Immunoprophylaxis of endoparasitic animal diseases. M. VNIITEIagroprom, 1990 54 p.

 2. Shishkov V.P. "Immunology and current problems of veterinary medicine. Problems of veterinary immunology." VASKHNIL, Agropromizdat, 1985 p. 3.

 3. Conto Alarcon C. et al. "Ensayo de vaccination contra babesia bovis utilizando antigenos procedentes de cultivo in vitro." Tech. pec. en Mexico, 1982, N 43, p. 43.

4. UK patent N 2037163, CL A 61 K 39/00, 1983
6. Wayne L. et al. Jnfection and Jmmunity 1989, p. 533 539.

 5. WO application 95/07100, cl. A 61 K 39/00, 1995.

Claims (3)

1. A vaccine against helminthiases containing a conjugate of an immunostimulating carrier with a protective antigen, characterized in that it is a conjugate of the General formula

where n 200 2000 the number of elementary links;
q 0.1 0.35 the number of alkylated units;
z 0.4 0.7 the number of oxidized groups;
R is a protective antigen, which is a protein isolated from the structures of connective tissues, having hyaluronic-lyase activity and is characterized by a mol.m. 63000 D, isoelectric point 5.7,
when the mass ratio of antigen and carrier 1 1 10.  2. The vaccine according to claim 1, characterized in that it further comprises an adjuvant.  3. The vaccine according to claim 2, characterized in that it contains polyoxidonium and / or muramyl dipeptide and / or aluminum hydroxide from the adjuvants.

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