RU2348427C2 - Adjuvant composition for injection tissue helminthiasis vaccines - Google Patents

Abstract

FIELD: medicine; bioengineering.

SUBSTANCE: composition contains mineral oil, Tween 80, L- arginine, RNA of Saccharomyces cerevisiae culture and physiologic saline in ratio as follows: mineral oil - 100 ml, Tween 80 - 2 ml, L-arginine - 5 g, RNA of Saccharomyces cerevisiae culture - 0.5 g, physiological solution to final volume 150 ml.

EFFECT: combination of biological and chemical components of the composition with immunodominant antigens of tissue helminths within veterinary preparation provides lasting expressed immunity in target animals.

2 tbl, 6 ex

Description

The invention relates to veterinary medicine, in particular to the creation of veterinary preparations for the immunoprophylaxis of dangerous anthropozoonoses (trichinosis, echinococcosis) of domestic and agricultural animals, and can also be used in medicine in the manufacture of antiparasitic vaccines.

Introduction

At present, vaccination has established itself as the most promising tool in the fight against tissue helminthiases (larval hydatidoses, trichinosis) of animals, while the main goal of immunizing animals with parasitic antigens is to achieve a balanced immune response similar to that induced by natural infection (4-9 )

Our studies on the protective properties of immunodominant tissue helminth antigens (Echinococcus granulosus, Echinococcus multilocularis and Trichinella spiralis) showed that immunization with only immunogenic proteins of these parasites does not create sufficient immunity in vaccinated laboratory animals. The data obtained are comparable with numerous foreign publications proving that it is possible to optimize the immunological mechanisms when vaccinating animals with parasitic antigens by selecting appropriate adjuvants. As the latter, various compounds are currently used that act in a nonspecific manner, enhancing the immune response to a specific antigen (11).

Such vaccination complexes can cause the earliest, most pronounced and long-term immunological reactions that provide a high level of protection of the animal's body from invasion by tissue helminths.

It is known that the components of the cell walls of certain microorganisms and their nucleic acids possess immunoadjuvant properties, i.e. perceived by the immune system as a non-specific signal that enhances the immune response. The best known is Freund's complete adjuvant (PAF), which is used to immunize only laboratory animals and is an oily suspension of killed Mycobacterium tuberculosis cells with a lipophilic emulsion agent (10% Arlacel A); before the introduction of the animal, this drug is emulsified in an aqueous solution of antigen (Freind L. et al., 1937). The effect of the adjuvant on the immune response is mainly due to the ability to retain and even accumulate antigen in the place where it interacts with lymphocytes (“depot effect”), and the ability of killed mycobacteria to cause the synthesis of cytokines that regulate lymphocytic functions. Cytokines have been shown to act as effective adjuvants only in the case of direct binding to a specific antigen. However, a significant drawback of PAF is the high viscosity of the drug, its strong reactogenicity, which is manifested in the formation of abscesses and extensive granulomas at the injection site, and therefore this adjuvant is not used in practical veterinary medicine.

Currently, among the means of immunocorrection from among natural preparations, the leading place in the breadth of the spectrum of biological activity is occupied by nucleic acid preparations and the products of their enzymatic degradation. A typical representative of this group is the sodium salt of RNA isolated from the simplest eukaryotic organisms - Saccharomycis cerevisiae. The active biological component of this substance are nucleotides. Being a natural component of the animal organism, the sodium salt of yeast RNA does not have species specificity, has a wide range of biological activity, and is free from side effects. Clinical trials have shown that this drug accelerates the regeneration processes, stimulates natural resistance factors, migration and cooperation of T and B lymphocytes, and phagocytic activity of neutrophils. RNA stimulates factors of both innate and acquired immunity, activating the proliferation of T and B lymphocytes. The main pharmacological property of nucleic acids is the stimulation of leukopoiesis, the processes of regeneration and repair, the functional activity of almost all cells of the immune system. Drugs of this group stimulate the functional activity of neutrophils and macrophages, increasing their ability to eliminate foreign elements (e.g. bacteria and parasites), increase anti-infection resistance to infection by various pathogens, probably due to the stimulation of phagocytosis, increase the functional activity of T-helpers and T-killers, B cell proliferation and antibody synthesis. The immunomodulating properties of RNA have been established in connection with the pronounced induction of interferon (IFγ), which contributes to an increase in the process of the immune response of a subpopulation of high affinity antibodies in the blood serum of immunized animals. Interferon is known to increase the ability of macrophages to produce highly active oxygen metabolites and nitric oxide.

Being the most important signaling molecule and intracellular mediator, nitric oxide instantly passes into neighboring and distant cells by simple diffusion, performing intercellular regulation in almost all body tissues.

It has been shown that endogenous synthesis of nitric oxide by macrophages, neutrophils, and vascular endothelium is one of the mechanisms for protecting the host organism from tissue helminths by increasing the cytotoxic and cytostatic properties of the cellular immunity unit.

Actually, nitric oxide (NO) performs numerous mediator and signaling functions in the macroorganism, and peroxynitrite, which is formed as a result of the interaction of NO with the products of oxygen reduction in the foci of inflammation and stimulation of the phagocytic activity of macrophages, has a toxic effect on helminths and protozoa.

To stimulate the functional activity of macrophages, neutrophils, vascular endothelial cells during the synthesis of nitric oxide, the presence of a sufficient amount of the amino acid L-arginine in the animal body is necessary.

The aim of the invention was the creation of an optimal adjuvant, the combination of biological and chemical components of which with immunodominant antigens of tissue helminths in a veterinary preparation would provide long-term intense immunity in vaccinated animals.

The essence of the invention consists in the development of a technology for the production and use of a multicomponent water-in-oil emulsion, the use of which significantly increases the protective effect when immunizing animals with tissue helminth antigens.

The closest prototype of the proposed innovation can be considered the adjuvants used by foreign researchers (Lightowlers MW, Rickard MD, Heath DD, Woollard DJ) when testing the protective properties of vaccines for tissue helminthiases (trichinosis, echinococcosis): complete Freund's adjuvant (PAF), incomplete adjuvant without mycobacteria) and glucoside saponin (Heath DD, Lawrence SB, 1996; Heath DD, Lightowlers MW, 1997; Woollard DJ, Heath DD, Lightowlers MW, 2000; Gamble HK, 1985).

High immunostimulating properties have a complete Freund's adjuvant (PAF), the main components of which are mineral oil, a lipophilic emulsifier and killed mycobacteria. The disadvantages of the prototype - vaccine complexes with PAF - are the high viscosity of the drugs that complicate their administration, and a strong local reaction, expressed in the formation of abscesses and granulomas. The high cost of Freund's complete adjuvant (manufactured by Difco) and the above problems limit the use of this substance in veterinary medicine. Used by foreign scientists as an adjuvant in combination with the recombinant antigens of Echinococcus EG-95 glucoside saponin (8) is currently unavailable in the Russian Federation. In this regard, it seems very relevant to search for the optimal combination of inexpensive domestic components for an adjuvant composition in the production of a vaccine against tissue helminthiases (trichinosis, larval hydatidoses).

Our work is characterized in that the composition of the presented adjuvant composition used an original combination of several immunogenic agents of both biological and chemical nature.

The value of the invention lies in the fact that the immunogenic effect of the developed adjuvant composition in combination with immunodominant antigens of tissue helminths manifests itself in the form of 3 main processes:

1 - in changing the state of the introduced parasitic antigens (their aggregation with the components of the adjuvant), which contributes to the interaction with sensitive cells of the animal’s immune system, facilitating phagocytosis.

2 - in the deposition of helminth antigens, their prolonged circulation in the body and in slowing down the hydrolysis of antigenic molecules by tissue enzymes through the use of finely dispersed oil base and the Tween-80 hydrophilic emulsifier.

3 - in additional stimulation of auxiliary components of immunogenesis, including enhancing the macrophage reaction, the synthesis of nitric oxide, stimulation of T and B lymphocytes, cytokines, globulins, antibodies associated with the introduction of eukaryotic ribonucleic acid (RNA) and amino acid L- into the adjuvant composition arginine. Small granulomas in immunized animals at the site of application serve as an indicator of the adjuvant effect of the vaccine preparation and its morphological correlation.

Many immunoadjuvants stimulate the formation of antibodies, but only a few are able to increase their affinity, and consequently, their effectiveness, as well as activate the cellular component of immunity. Since the strength of humoral and cellular immunity depends on these properties, the use of RNA, the amino acid L-arginine, a hydrophilic emulsifier and mineral oil in the adjuvant composition in combination with immunosominant antigens of parasites allows a prolonged protective effect of the vaccine preparation against tissue helminthiases.

The method of obtaining adjuvant oil-water emulsion consists of the following steps:

1. Obtaining preparative quantities of RNA from a culture of Saccharomycis cerevisiae and its purification.

2. Qualitative and quantitative analysis of isolated RNA.

3. The technology of water-in-oil emulsions for adjuvant composition.

4. The technology of using the presented adjuvant for the preparation of a vaccination complex with immunodominant antigens of tissue helminths (Echinococcus granulosus, Echinococcus multilocularis, Trichinella spiralis).

In view of the difficulties in obtaining a commercial preparation of Na RNA of appropriate quality, the preparation of preparative amounts of RNA from the Saccharomycis cerevisiae culture and its purification were carried out by conventional methods according to the recommendations of Sambrook J., Fritch E.F., Maniatis T., 1989.

Qualitative and quantitative analysis of eukaryotic RNA used in the adjuvant composition was carried out according to the “Method for the determination of Na RNA in vaccine and anthelmintic preparations” previously developed by the authors, approved by the section “Invasive Animal Diseases” of the Department of Veterinary Medicine of the Russian Academy of Agricultural Sciences on May 27, 2006 (2).

The technology for producing a water-in-oil emulsion for an adjuvant composition includes several stages.

1. Mineral oil (Markol brand - 52, in the absence of replacement Oleum Vaselini is allowed) in an amount of 50 ml (50% by volume) is poured into Erlenmeyer flasks with a volume of at least 250 ml and autoclaved for 1 hour at a pressure of 0.5 atm.

2. In a 50 ml volumetric flask, place 30-40 ml of sterile physiological saline and successively dissolve 5 grams of the amino acid L-arginine and 500 mg (0.0025%) of freeze-dried RNA in it. A 2 ml (2%) hydrophilic emulsifier of Tween-80 is gradually added to the resulting mixture of biological components and gently mixed to prevent foaming, after which the volume is adjusted to 50 ml with a solution of 0.9% NaCl. Then, the resulting mixture of reagents in a sterile box is passed through a bacterial membrane with a pore diameter of 200-240 μm in a nitrocellulose filter.

3. Add 50 ml of the above reagent mixture to the Erlenmeyer flask with sterilized mineral oil and place it on a shaker with a magnetic stirrer (or laboratory shaker) at a temperature not exceeding 4-6 ° C and 120-150 rpm for 2 hours to obtain water -oil emulsion.

4. The technology of using the obtained adjuvant for the preparation of a vaccination complex with immunodominant antigens of tissue helminths (Echinococcus granulosus, Echinococcus multilocularis, Trichinella spiralis).

For the preparation of an immunizing complex, immunodominant antigens of tissue helminths are used (RF patent No. 2234539 from 08/20/04 and RF patent No. 2287342 from 10/20/06), dissolved in sterile physiological solution. Protein concentration is preliminarily measured by the Lowry method and should be at least 350-500 μg / ml in the final preparation.

Re-emulsification is carried out for 4-6 hours at a temperature of 2-4 ° C with vigorous stirring, after combining 50 ml of dissolved antigen and 150 ml of the adjuvant composition.

The protective properties of immunopreparations were studied in several types of laboratory animals with double subcutaneous administration with an interval of 21-30 days and test infection of immunized and control animals with invasive material 14 days after the last immunization.

The obtained biological preparations were tested for their immunogenicity using traditional commercial adjuvants as a control: Freund's complete and incomplete adjuvants and immunofan, while the amount of specific antigen administered to animals of each group was exactly the same.

Examples of specific performance

Example 1

A comparative analysis of the immunogenic properties of the vaccination complex based on Trichinella immunodominant antigens with a molecular weight of 29-63 kDa in combination with various adjuvants was studied on white outbred mice weighing 18-20 g. Animals in the amount of 50 animals were divided into 5 groups, 10 animals each. Immunization was performed twice, subcutaneously, with an interval of 21 days. The volume of the administered drug was 0.25 ml, while the total amount of the administered antigen for animals of all groups was 100 μg.

Group 1 animals were immunized with Trichinella excretory-secretory antigens emulsified with an equal volume of PAF (125 μl of antigen and 125 μl of Freund's complete adjuvant).

Animals of the 2nd group were immunized only with Trichinella antigens, the volume of the administered drug was replenished with sterile saline.

Animals of the 3rd group were immunized with Trichinella antigens emulsified in an equal volume of the adjuvant presented in this application.

Animals of the 4th group were injected with Trichinella antigens mixed with an equal volume of 0.005% immunophan (a commercial drug recommended for increasing titers and the duration of circulation of specific antibodies when co-vaccinated with antigens).

Animals of the 5th group were control and were injected with sterile saline in a timely manner.

14 days after the last immunization, all mice were infected per.os with Trichinella invasive larvae in the amount of 100 larvae per mouse. Slaughter of experimental animals was carried out on 63 days after their infection. The results of the experiment showed that all animals of the control (5th) group were intensively infected with trichinosis, the number of helminths in the average sample of mincemeat after digestion in artificial gastric juice (IHL) was 1785 larvae / g.

The number of larvae in the average sample of bone-muscle mincemeat in animals of the 1st and 3rd groups after digestion in IHL was almost the same and amounted to 26.4 and 28.2 larvae / g, respectively. Thus, the protection effect was 98.1%. Animals immunized with Trichinella excretory-secretory antigens only (group 2) were significantly more infected (586 larvae / g, respectively, the protective effect was 60.5%), and in animals of group 4 the invasion intensity was 379 larvae / g ( 74.5%), which indicates an insufficient protective effect when using these drugs.

Example 2

The study of the dynamics of antibody production during immunization of rabbits with recombinant antigens of Echinococcus EgFB with various adjuvants.

To determine the optimal combination of adjuvant substances in the EgFB biological product that mutually stimulate the development of humoral and cellular immunity, 4 experimental groups of animals were formed. At the beginning of the experiment, serum samples were taken from each of 12 rabbits. During the experiments, the titer of specific antibodies in immunized animals in the IGF was taken into account minus the optical density of these samples, which served as a negative control specifically for each rabbit. Animals of all groups were immunized subcutaneously once in a dose of 600 μg of EgFB antigens, dissolved in 1 ml of physiological saline with an equal volume of one of the 4 studied adjuvant compositions.

Echinococcus EgFB antigens with 1 ml of a 0.05% immunofan solution (synthetic hexapeptide C ₃₆ H ₆₁ O ₁₀ N ₁₂ ), the combined use of which with vaccines dramatically increases titers and the duration of circulation of specific antibodies, were administered to the 1st group of rabbits.

In group 2 rabbits, T. spiralis excretory-secretory antigen at a dose of 476 μg per protein was used as adjuvant. The total volume of the vaccinating composition was 2 ml.

The 3rd group of rabbits was given a vaccination composition with Echinococcus EgFB antigen in a volume of 2 ml, and the 4-component adjuvant emulsion presented in this application for the invention was used as an adjuvant.

The 4th group of rabbits, which is the basic control, was given the same amount of EgFB antigen (600 μg) emulsified with PAP. The volume of vaccine composition administered was 2 ml.

The dynamics of the titer of specific antibodies was determined in the IGF on 7, 14, 21, 30, 90 and 120 days after completion of immunization.

Studies have shown that in once-immunized animals, specific antibodies against EgFB echinococcus antigens begin to be detected from 7 days after vaccination, reach their maximum values by 21-30 days, then a period of decrease in their level sets in, and the dynamics and intensity of this process vary significantly depending on the composition of the adjuvant used.

Of the tested compositions, the best performance in IGF was in the EgFB antigen in combination with PAF (manufactured in the USA). High titers of specific antibodies are recorded in immunized animals for a long time, but the high cost of PAFs, the formation of large granulomas and abscesses in animals at the injection site limit the use of this adjuvant in vaccine production in veterinary medicine.

A multicomponent water-in-oil emulsion, the formulation and preparation technology of which is given in this application for the invention, can serve as a substitute for PAP in the biological preparation EgFB.

At the injection sites of this drug, only small granulomas are formed that resolve within a few weeks.

Specific antibodies in the blood serum of immunized animals of the 3rd and 4th groups were recorded during the entire observation period in the diagnostic titer, which indicates the presence of prolonged immunity against invasion (drawing a, b). Taking into account the immunobiological properties of the adjuvant presented and the availability of all its components (domestic production), we suggest using it in combination with various parasitic antigens for the prevention of helminthiases.

Example 3

Studies were performed on 62 white mongrel mice weighing 18-20 grams, divided into 3 groups.

The first group was immunized subcutaneously twice with an interval of 21 days with a preparation consisting of an excretory-secretory antigen (50 μg of protein in 0.125 ml by volume) and an equal amount of the adjuvant presented (125 ml).

The second group of mice was immunized subcutaneously with one adjuvant composition (without antigen) in the same dose twice with an interval of 21 days.

The third group was twice injected with sterile saline according to the same scheme.

After 14 days after the last immunization, all three groups of mice were infected with Trichinella invasive larvae at a dose of 200 larvae per animal. Accounting for the results of the experiment 60 days after infection is presented in table 1.

The above calculations show that the level of protection during experimental infection of T.spiralis in mice vaccinated with a drug containing antigen and adjuvant was 94.25%.

Example 4

The studies were carried out on 16 young rabbits of the breed “Soviet Chinchilla”, divided into two equivalent groups - experimental and control. The experimental group of animals was twice (with an interval of 20 days) subcutaneously immunized with a drug consisting of equal volumes (0.25 ml) of Freund's complete adjuvant and T. secretion secretory antigen (protein dose of 250 μg).

The control group of rabbits was injected at the same time with sterile saline. After 14 days after the last immunization, both groups were infected with invasive larvae of Trichinella at a dose of 4 thousand per animal.

Slaughter of experimental animals was carried out 60 days after infection with Trichinella. All muscle tissue was separated from the carcasses of rabbits and ground in an electric meat grinder (each separately), thoroughly mixed, and proteolysis of three aliquots of minced meat from each carcass in artificial gastric juice was carried out. The results of the experiment are presented in table 2.

The above calculations also show that the level of protection during experimental infection of T. spiralis in rabbits vaccinated twice with this drug was 95.5%.

The experiments showed a high immunogenicity of the drug based on the Trichinella excretory-secretory antigen with the adjuvant presented for two subcutaneous administration to laboratory animals, the protection effect ranged from 94 to 98%.

Example 5

Analysis of the protective properties of the adjuvant composition in alveolar hydatidosis of white outbred rats.

Studies were performed on 24 white mongrel rats weighing 180-200 g, divided into 4 groups. Immunization was carried out twice, subcutaneously, in a volume of 0.5 ml, with an interval of 21 days.

The first group of rats was injected with the recombinant echinococcus EgF antigen (240 mg protein) in combination with the adjuvant presented.

The second group of animals was administered one EgF antigen in the same dose.

A third group of rats was immunized with one adjuvant, and a fourth group of control animals was injected with sterile saline.

Twenty-one days after the last immunization, animals of all groups were infected with 600-800 specimens of Echinococcus multilocularis protoscolexes and acephalocysts.

Slaughter of animals and taking into account the results of the experiment was performed 6 months after infection. An autopsy showed that all rats of the 4th group were infected with alveolar hydatidosis, with cysts as large as 2-3 cm in diameter, mature protoscolexes were detected by microscopy, while 5 animals of the 1st group were free from invasion. In one rat, 2 parasite larvocysts with a diameter of about 1 mm surrounded by a purulent capsule were found in the liver. In 4 animals of the 2nd and 3rd groups, small parasite larvocysts were found (compared with those in rats of the 1st group), mainly without germ elements, which indicates an insufficient protective effect when the adjuvant composition and antigen are used separately . The combined use of these two substances provides reliable protection against infection with alveolar hydatidosis.

Example 6

The immunogenic properties of the vaccination complex, consisting of the recombinant EgF antigen and presented in the application for the invention of an adjuvant, were studied on 25 sheep of the Grozny breed in the Dzheyrakhsky district of the Republic of Ingushetia. The animals were divided into two groups. The first, in the amount of 20 sheep, twice, with an interval of 30 days, was immunized subcutaneously with an emulsion consisting of equal volumes of the recombinant echinococcus EgF antigen (15 ml of protein was contained in 2 ml of the preparation) and the adjuvant composition. The second group, consisting of 5 goals, remained intact. Two weeks after the second immunization, all animals were infected with activated oncospheres E.granulosus, isolated from the intestines of wild carnivorous forests of the North Caucasus (jackal and 3 foxes) infected with echinococcosis, in an amount of 1000 specimens per sheep. Sheep blood samples were taken at 45 and 60 days after infection with larval hydatidosis. The level of specific antibodies was evaluated by the enzyme immunoassay using the ELISA-echinococcus-IgG-strip test system (we filed an application for the invention, “Method for the intravital diagnosis of larval sheep hydatidoses,” No. 137821 of October 26, 2006). Statistical data processing was performed according to the method of Fisher and student, 1957; the reliability was p <0.05. The titers of specific antibodies to echinococcus antigens in the group on average 45 and 60 days after infection were: for immunized sheep 1: 12800, for infected sheep 1: 100, 1: 200, which indicates high immunogenic properties of the tested vaccination complex.

Information sources

1. Odoevskaya I.M. Analysis of titers of specific antibodies during immunization of animals with the biological product EgFB with various adjuvants. // J. Medical parasitology and parasitic diseases. - M .: - 2006. - No. 4. - S. - 13-16.

2. Odoevskaya I.M., Aseev V.V., Benediktov I.I. and others. "Methodology for the determination of Na RNA in vaccine and anthelmintic drugs." // M .: - Proceedings of VIGIS. - 2007. - T.43 - S.286-297.

3. Odoevskaya I.M., Plieva A.M., Benediktov I.I. Laboratory regulations for the conduct of IGF with larval sheep hydatidosis. // Proceedings of VIGIS. - M. - 2007. - T. 43 - S. - 307-317.

4. Gamble H.R. Trichinella spiralis: Immunization of Mice Using Monoclonal Antibody Affinity-Isolated Antigens. Exp.Parasitology-1985. V. 59. - P.398-404.

5. Heath D.D., Lawrence S.B. // Antigen polipeptides of Echinococcus granulosus oncospheres and definition of protective molecules. Parasite Immunol., 1996, V.18. - P.347-357.

6. Heath D.D., Lightowlers M.W. // Vaccination on Hydatidology-State of the Art. Arch. Int. Hidatid. - 1997. - V.32. - P.209-214.

7. Lightowlers M.W., Jensen O.E., Femandez J.A., Iriarte D.J., Woollard D.J., Jenkins D.J., Heath D.D. Vaccination trials in Australia fhd Argentina confirm the effectiveness of the EG95 hydatid vaccine in sheep // Int. J. Parasitol. - 1999. - V.29. - P.531-534.

8. Lightowlers M.W., A. Flisser, Gauci C.G., Heath D.D., Jensen O., Roife R. Vaccination against cysticercosis and hydatid disease // Parasitol. Today. - 2000. - V.16. - P.191-196.

9. Lightowlers M.W., Lawrence S.B., Gauci C.G., Young J., Ralston M.J., Maas D., Health D.D. Vaccination against hydatidosis using a defined recombinant antigen // Parasite Immunol. - 1996. - V.18. - P.457-462.

10. Sambrook J., Fritch E.F., Maniatis T. Extration and purification of RNA. // In: Molecular Cloning. A laboratory manual. Second edition. Cold Spring Harbor Laboratory Press. - 1989. - P. - 730-737.

11. Woollard D.J., Heaht D.D., Lightowlers M.W. Assessment of protective immune responses against hydatid disease in sheep by immunization with synthetic peptide antigens // Parasitology - 2000. - V.121. - N2. - P.145-153.

Table 1 Analysis of the protective properties of the adjuvant composition in trichinosis of white outbred mice Group No. The number of mice in the group Immunizing agent Immunizing dose (μg protein) The frequency of immunization The interval between immunizations (days) Dose of infection (number of larvae) The average number of larvae in 1 g of muscle tissue after digestion in I.Zh.S. one thirty E / s antigen T.spiralis (1: 1) fifty 2 21 200 149.5 2 12 Adjuvant + saline (1: 1) - 2 21 200 829.7 3 twenty Sterile saline - 2 21 200 2578 ((2578-149.5): 2578) × 100% = 94.25

Table 2 Analysis of the protective properties of the adjuvant composition in experimental rabbit trichinosis Group No. Immunizing agent (composition, volume) Protein Immunizing Dose (mg) The frequency of immunization The interval between immunizations (days) Dose of infection (number of larvae) The average number of larvae in 1 g of muscle tissue one E / Cc antigen + adjuvant (1: 1) 500 2 twenty 4000 71.8 2 Sterile saline - 2 twenty 4000 1596.3 ((1596.3-71.8): 1596.3) × 100% = 95.5%

Claims (1)

An adjuvant composition for injectable vaccines against tissue helminthiasis, consisting of an emulsified mixture of biological and chemical components, characterized in that it contains mineral oil, Tween-80, L-arginine, RNA from the Saccharomyces cerevisiae culture and physiological saline in the following ratio of components:
Mineral oil 100 ml Twin 80 2 ml L-arginine 5 g RNA from Saccharomyces Culture cerevisiae 0.5 g Saline to a final volume of 150 ml.