Agent enhancing immunogenic property of tetanus anatoxin

FIELD: medicine, immunology.

SUBSTANCE: invention proposes an agent enhancing the immunogenic properties of tetanus anatoxin (adjuvant). Invention proposes the vegetable triterpenic compound miliacin as an agent enhancing immunogenic properties of tetanus anatoxin. Agent enhances the immune response value in its applying as a vaccine preparation of tetanus anatoxin. The agent miliacin elicits its stimulating effect for both the first and repeated administration of vaccine that allows suggesting its possible applying for prophylactic vaccinations with tetanus anatoxin. Taking into account the high tolerance of miliacin in the broad range of its doses it is suggested its practical applying as an agent promoting to the enhanced formation of vaccinal immunity in prophylactic vaccinations with tetanus anatoxin.

EFFECT: valuable medicinal properties of agent.

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The invention relates to medicine, namely to epidemiology, and may find use in vaccination.

Currently in medicine widely used substances are able to stimulate nonspecific immune response when using vaccination preparations. Such substances are called adjuvants.

To the category of immunological adjuvants usually are substances added to vaccines to enhance the immune response, in order to obtain a larger number of antibodies, to reduce the amount of antigen required for immunization and reduce the number of injections of antigen (Immunological adjuvants. Report of a who scientific group. Geneva, 1978, p.7). Among them are aluminum compounds (including phosphate and aluminium hydroxide), emulsified oil-water adjuvants (a mixture of water with mineral oil - incomplete beta-blockers; a mixture of water with peanut butter).

However, even the use of adjuvants in some cases does not allow a full immune response and to generate reliable protection. The reason for this can be various circumstances, including the inhibition of the ability of the immune system to fully respond to antigenic stimuli under the influence of environmental factors (E. Belyaev, 1996). Toxic substances such as carbon monoxide, hydrogen sulfide, sulfur dioxide, hydrocarbons the odes have a pronounced immunosuppressive action (Goryachev CENTURIES, Dotsenko SCI, Parfenov, L. F., Melnikova I.K., 1988; Boyko V.I., 1990; Petrov R.V., Khaitov R.M P.M., Ordovsky IV, Bulova A.V., 1993). The toxicants may cause disruption of normal regulatory mechanisms and genetic factors affecting the maturation of the immune system, metabolism of xenobiotics, leading to altered postnatal metabolism and lack of immunity. Particularly susceptible to such adverse effects children, have formed disorders can persist in a latent state until puberty (D.B. Roberts, J.R. Chapman, 1981).

Bwitalski and Sgemv (1991) presented the materials that convincingly demonstrate the negative impact of pollution on the efficacy of the DPT vaccine. DTP vaccine contains tetanus and diphtheria toxoids to create immunity against tetanus and diphtheria, as well as killed pertussis bacteria, used as the third vaccination component to create immunity against pertussis. All components of the vaccine, adsorbed on aluminium hydroxide, which it (the vaccine) adjuvant. Analyzing possible link between immunological effectiveness of this vaccine and environmental pollution, the authors found increasing vulnerability of the population, i.e. insufficient the level of the immune response after vaccination, in direct dependence on ecological trouble. In particular, in East Kazakhstan region, where the dominant environmental pollutants are compounds of metallic, chlorine, oxides of nitrogen and carbon, the rate of exposure of the population (according to the titer of antibodies to the diphtheria toxoid) in the contaminated zone, compared to a net area increased: in preschool children with 11,8±1,6% to 22.0±1,2%; students with 0,7±0,4% to 19.3±1,6%; adults from 37.1±3,0% to 57.2±1.4 percent. A similar direction of changes observed in relation to the exposure of the population from tetanus, which is determined by the titer of antibodies to the tetanus toxoid (component of the DPT vaccine). In Zhambyl region, where the leading pollutants are compounds of phosphorus and fluorine, the increase of insecurity among residents of contaminated areas compared to net amounted to: in preschool children from 0 to 3.8±1,1%; students with 0,7±0,7% to 7.1±1,3%; adults from 19.7±3.4 to 38,8±2,6%. The data obtained showed stratification under the influence of pollution of the human population on the intensity of the immune response to toxoids.

Currently, the number of nonspecific substances capable of increasing the magnitude of the immune response include triterpenoids (triterpenes). This is one of the groups of chemicals related to terpenoid (or isoprenoid) connections. The latter are of the Wallpaper the most numerous class of low molecular weight compounds, molecules are built from branched C5units (Mlynar, 1979; Goodwin, Mercer, 1986). They are combined into one class on biogenetic grounds, since all of these compounds are formed from the product recovery 3-hydroxy-3-methylglutaric acid (avalonbay acid). A large part of the known terpenoids refers to the substance of specialized metabolism in plants, where they are involved in signaling processes, protection against phytopathogens and so Many antibiotics, phytoalexin, repellents have a terpenoid structure (Vaisesika, 1998). Among the terpenoids allocate connection monoterpenes (C10P16); sesquiterpenes (C15H24), diterpenes (C20H32), triterpenes (C30,H48) (B.A., Pasechnick, 1998). The last, at present, are the subject of deep study, in connection with the found they have different biological properties that are used in medicine.

Clear information about the immunotropic properties of triterpenoids were obtained in vivo studies characterizing features of the formation of the immune response in terms of their application (S.Behboudi, Vmage, .Ronnberg, 1995). It is experimentally shown that triterpenoids soap tree are expressed adjuvant activity (Century Ronnberg et al., 1995). Exploring this activity to the antigen the WMD complex GP-340 - recombinant virus, Epstein-Barr, .Dotsika et al. (1997) found that without changing the nature of the immune response in mice, triterpenoids contributed to the increased production of specific antibodies (Ig G1and Ig G) in animals and stimulated antigenspecific lymphocyte proliferation. Similar results were obtained when testing the same triterpenoids with envelope antigen of influenza virus on human volunteers who were not only dropped, but nutricionista vaccination (Vmage et al., 1998). The inclusion in the system of triterpenoids as adjuvants allowed to get a better response at a lower dose of antigen, and intranasal use, as well as through the mucous genital tract - stimulated induction and secretion of Ig A. as another positive point associated with the use of such vaccines, the authors call the lack of local reactions of the mucous in the place of its introduction.

The novelty of the invention is identified property plant triterpenoid-Milyutina as immunostimulant in the application of tetanus toxoid.

The essential difference is that when vaccination tetanus-toxoid used Miliutin.

The proposed 3-β-methoxy-Δ18-oleane (Milutin) belongs to the group of natural cyclic triterpenoids (see chemical structure below), siderits the millet in the oil and is a white substance with a melting point 285-286° C. It is optically active, insoluble in water, slightly soluble in ethanol, diethyl ether, acetone, soluble in chloroform. Miliutin is well tolerated in the dose range from 2 to 1000 mg/kg LD50this connection is greater than 1000 mg/kg (Leelan et al., 1991), indicating that it has no toxic properties.

The chemical structure of Pyh triterpenoids - Milyutina. 3-β-methoxy-Δ18-oleane (Milutin)

Previously we have investigated experimentally the influence of Milyutina on the peculiarities of formation of the humoral immune response of mice after immunization with sheep red blood cells (EB) under different schemes introduction triterpenoids (Averilla, Tveeprom, Bahramov, 2000, 2002).

The experiments were conducted according to the scheme presented in table. 1. Just was performed three experimental series, each consisted of three groups of animals. The first one (experienced) was a mouse immunized with sheep erythrocytes (5×108after preliminary intraperitoneal administration of Milyutina (2 mg/kg): tenfold in 10 days (series I), three times in three days (series II) and three times with an interval between doses in three days (series III). Appropriate controls for each experimental group served as animals, immunity is consistent with sheep red blood cells after pretreatment with solvent (control II), and mouse immunized DL without prior introduction

Milyutina or solvent (control I). Immunization of animals (5×108in all series was carried out in 11 days from the beginning of the experiment. The intensity of the humoral immune response was evaluated according to the indicators of relative and absolute content of antibody productive cells (ATOC) in the spleen of mice (CBA×C57B16F1 on the 5th day after immunization.

Data on the effect of a tenfold introduction of Milyutina on the primary immune response are presented in table 2.

As can be seen from the table, a tenfold introduction of solvent (physiologic solution with tween-21 at a final concentration of 1.6×10-7mol/kg (control II) had no effect on spleen weight and the content of splenocytes compared with mice immunized DL without any prior exposure (control I). The animals of this group were decreased (by 20.7%) relative level of ATOC/million, resulting in a decrease in their total content: ATOC/spleen (21.2%) compared with only immunized mice (control I).

Doing Milyutina (experience) contributed to the weight gain of the spleen and an increase in the content of splenocytes compared with the control animals first and second the RUPE. Moreover, compared with the animals of the first group (KI) these differences (respectively, 15.2% 19.3%) were statistically significant. As for the ability to the formation of ATOC in the experimental mice, as in animals treated with the solvent (C) it was reduced compared to immunized mice group KI. However, this decrease in the relative number of atok, first, was less pronounced (17.5 percent instead to 28.05%), and secondly, was compensated by the growth of the total number of splenocytes. In the end, the absolute content of ATOC the experimental animals did not differ significantly from the figure recorded at only immunized animals (KI), a compared to immunized mice exposed to solvent, was higher by 26%.

Study of the effects of daily administration of Milyutina for three days has not discovered any meaningful impact triterpenoids nor on the weight of the spleen, nor on the number of splenocytes in the body (table 3).

Not installed and also changes in the ability of animals to the formation of humoral immune response. Relative and absolute content of ATOC in mice of the experimental group were in the range of values defined as in animals exposed before immunization (same pattern) to effect solution of the indicator (KII), and only premirovany mice (KI). It is possible that the lack of immune response to the introduction of Milyutina due to the specifics of this series of experiment, where there was a significant time period (8 days) between the cessation triterpenoids and immunization of animals.

A different picture was characteristic of the studied indicators at three times the interval introduction of Milyutina (table 4).

As shown in this experimental series, as in both previous, the solvent (C) had no effect neither on the weight of the spleen, nor on the number of splenocytes. His three-time interval introduction did not lead to the disruption of the formation of the immune response: the relative and absolute content of atok mice in this group did not differ from the parameters determined in animals subjected only to immunization (KI).

In contrast, the introduction of Milyutina (experience) has led to an increase in the spleen weight and the content of splenocytes compared with animals treated with the solvent (KII), and mice of the first control group (KI). Noteworthy that the intensity of this increase was even more pronounced than in series with a tenfold introduction triterpenoids. So, if the last increment of mass of the spleen and the number of splenocytes compared to non-treated immunized mice (KI) was accordingly, 15,2% and 19.3%, while three-time interval the introduction of Milyutina of 39.8% and 30.3%. It is essential that the increase in the number of spleen cells in this series, initiated by the interval introduction triterpenoids, was not accompanied by inhibition of the formation of ATOC. And their relative content in animals treated before immunization Miliutin (306±24,6), even slightly exceeded the level determined in mice immunized without any prior exposure (250±14,2). The consequence of these shifts, the experimental animals showed significant increase in the absolute number of antibody productive cells. The increase of this indicator, the total capacity of the immune response relative to the control groups of mice (KI and KII), amounted, respectively, and 39.9% and 37.1%. The results obtained indicate that in this scheme the use of triterpenoids, he has a clear stimulatory effect on the humoral immune response, and that the basis for such stimulation is increased pool of splenocytes and increased formation of ATOC.

As shown by subsequent observations (table. 5), the ability of Milyutina to increased formation of antibody productive cells, most clearly revealed when carrying out a similar series of experiments using to immunize more than the bottom of the first dose of sheep erythrocytes (5× 107).

As can be seen from the table, in this case, along with the increase in the number of splenocytes in mice of the experimental group recorded a marked increase in the relative level of ATOC, which surpasses as only immunized mice (KI), and in animals immunized against the background of the preliminary injection of solvent (C). It resulted in the most significant increase in total number of antitelomerase, formed with respect to KI and K, respectively 50,3% and 48.3%.

Thus, the obtained results allow to conclude that the impact of Milyutina on humoral immune response depends on the schema of its application and may be accompanied by the absence of any effect (three times daily administration), inhibition of the formation of ATOC (tenfold daily administration) and stimulation of the immune response (three-time interval introduction). Defining mechanisms of this stimulation is an increase of total pooled splenocytes and increased formation of antitelomerase. The fact that such increased formation of atok depends on the size of the antigenic load and is most pronounced when using doses of EB, part 5×107may be related to the fact that under these experimental conditions, non-specific effect is their triterpenoids on the formation of antitelomerase not “overlap” of their specific antigenic induction at higher dose of antigen.

The influence of Milyutina on the immunogenic properties of tetanus toxoid

Description of the experiment

For immunization of animals (mice (CBA×C57Bl6)F1) was used enriched tetanus liquid toxoid (AU), adsorbed on aluminium hydroxide Perm scientific production Association BIOMED (series 411-12, control room 445, shelf life of up to 08.2005).

In contrast to sheep red blood cells, which corpuscular antigen, i.e. consisting of suspended particles, tetanus toxoid is soluble antigen. Secondly, the sheep red blood cells are the classic antigen in laboratory practice in experiments on animals. Tetanus toxoid is a classic antigen used in humans. Thirdly, sheep red blood cells are used to study the immune response. Tetanus toxoid is used to create a vaccination immunity, i.e. represents a prophylactic for the prevention of tetanus.

Animals were immunotherapies subcutaneously in the region of the anterior subscapularis limbs, one dose of 0.5 ml, containing 10 EU tetanus toxoid.

Used 2 groups of animals:

Group I - control. Before immunization received 3-fold, interval solvent (twin-21) at a dose of 1.6·10-7mol/khamassi animal.

Group II - experience. Before immunization received 3-fold, the interval Miliutin at a dose of 2 mg/kg of weight of animal.

Immunization with toxoid was performed on the 11th day from the beginning of the experiment.

The slaughter of animals in both groups was carried out after 14 and 28 days after immunization.

To assess the impact of Milyutina on the formation of secondary immune response part of the mice experimental and control groups at 28 days after primary immunization was re-subjected to immunization tetanus-toxoid in the same dose. Assessment of response to repeated immunization of these animals was carried out after 14 days.

The number of animals to assess the immune response during primary immunization at every period in the experimental and the control group consisted of 15 mice. When re-immunization - 20 mice. Animals were scored by decapitation and collected the blood then get whey.

Evaluation of the immune response was performed by determining the titer of antibodies micromethods RNA using erythrocyte diagnosticum tetanus antigen liquid (kit # 1). Manufacturer: JSC “Biomed” them. Immunicon (activity 1:1280; series 73; date of manufacture 12.02).

The results are presented in table 6 reflect the average values of decimal logarithms of antibody titers detected in the control (recip is our solvent) and experimental (treated Miliutin) groups of animals at different times after the primary immunization (14 and 28 day) and 14 days after the second immunization.

Table 6

Logarithm (lg) antibody titers in the serum of mice (CBA×C57l6)F1] at various times after immunization with tetanus-toxoid prior to the introduction of solvent (control) and Milyutina (experience).
 Group I (control)Group II (experiment)P
day 141,33±0,13


28 day1,2±0,09





As follows from the presented data, immunization of mice tetanus-toxoid prior administration of Milyutina, provided the formation of a more pronounced immune response, compared to control animals immunized with a tetanus toxoid after the introduction of solvent. These differences began to emerge already at 14 days after immunization and reached significance (confidence) for 28 days. Thus it is essential that if the control animals by the deadline registered a decreasing tendency to the icesta antibodies the animals of the experimental group was noted for their growth.

Repeated immunization of mice (secondary immune response) was accompanied by the highest increase in the level of antibodies in the serum of animals as experimental and control groups. However, noteworthy that the experimental animals he reached higher values than in the control.

Thus, the obtained data indicate that 3 times, interval (3 days) introduction of Milyutina results in a more intense immune response after immunization of animals tetanus-toxoid. This stimulating effect of Milyutina manifested in response, both the original and the re-introduction of the vaccine, which allows to make a conclusion about the prospects of its use in prophylactic vaccination tetanus-toxoid.

Proceeding from the above, given the high tolerance of Milyutina (Leelan et al., 1991) in a wide range of doses, it is assumed possible its practical application as a means of contributing to the formation of vaccination immunity in preventive vaccination tetanus-toxoid.


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The use of Milyutina as a means of improving immunogene properties of tetanus toxoid.


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FIELD: medicine, immunology.

SUBSTANCE: invention proposes an agent enhancing the immunogenic properties of tetanus anatoxin (adjuvant). Invention proposes the vegetable triterpenic compound miliacin as an agent enhancing immunogenic properties of tetanus anatoxin. Agent enhances the immune response value in its applying as a vaccine preparation of tetanus anatoxin. The agent miliacin elicits its stimulating effect for both the first and repeated administration of vaccine that allows suggesting its possible applying for prophylactic vaccinations with tetanus anatoxin. Taking into account the high tolerance of miliacin in the broad range of its doses it is suggested its practical applying as an agent promoting to the enhanced formation of vaccinal immunity in prophylactic vaccinations with tetanus anatoxin.

EFFECT: valuable medicinal properties of agent.

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