Method for immunoprophylaxis of experimental pseudocholera with entrapped antigens burkholderia pseudomallei

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to microbiology and immunology and can be used for immunoprophylaxis of pseudocholera. The method involves antigen pretreatment to mice followed by infection and estimating level of protectivity. Antigens Burkholderia pseudomallei are chosen of superficial antigenic fractions: D, C, F, J, H. Herewith the antigen solution in amount 1.5 ml is mixed with phosphatidyl choline approximately 40 mg and cholesterol in the ratio 7:3 to produce liposomes.

EFFECT: invention allows improving survival rate of laboratory animals infected with pseudocholera owing to higher protectivity of pseudocholera antigens entrapped in liposomes.

2 cl, 4 tbl, 2 ex

The invention relates to medicine, in particular to Microbiology and immunology, and for the prophylaxis of melioidosis.

Melodos - a potentially fatal infectious disease caused by a pathogen Burkholderia pseudomallei, belonging to the group of especially dangerous pathogens. Despite many years of study of domestic and foreign scientists to date the vaccine melioidosis not developed. Quest components of future vaccines among different antigenic complexes of the cell wall, the cytoplasm or the extracellular metabolites of the pathogen melioidosis not led to the discovery vysokoproduktivnogo antigen, capable of becoming the basis for vaccine preparation. Selected by different methods antigenic melioidosis drugs, as a rule, formed a low level to protect animals from experimental infection (1).

Currently, the main immunogenic potential melioidosis vaccine greatest attention is drawn to the surface biopolymers of the pathogen involved in the implementation of virulent and pathogenic properties, but they do not have a high produktivnost for experimental animals. Thus, increasing protectively melioidosis antigens is one of the urgent problems of immunology of melioidosis, successful re the giving of which undoubtedly will precede the establishment of an effective chemical vaccines. One of the modern approaches to increase the immunogenic properties of the antigen is a method of encapsulating antigens in liposomes. A number of authors have shown the principal possibility of increased immunogenicity and protectively antigens of different pathogens (brucellosis, Pseudomonas infection, and others) when they are in liposomes (2, 3). Liposomes are formed from natural phospholipids, therefore, have no irritant or toxic effects on the body. The membrane of liposomes protects the encapsulated substance from inactivation and rapid destruction in the internal environment of the body, which creates conditions for the long-term impact included in the liposomes of the antigen to immunocompetent cells and, therefore, more pronounced stimulation of the immune response.

The closest analogue of the proposed method of immunization of experimental melioidosis is to work Piven NN. et al. (4)in which the immunization of mice 9 out of 10 selected chromatographic fractions of water-salt extract (WSE) of the pathogen melioidosis introduced in a mixture with the adjuvant's adjuvant, mortality of animals after control of infection 3-30

LD₅₀virulent culture was 70-100%.

The aim of the invention is to develop a method for the specific prevention of experimental melioidosis, call the Commissioner, to increase protectionist melioidosis antigens.

This goal is achieved by the fact that animals are subjected to immunization encapsulated in liposomes surface melioidosis complexes, and to enhance their immunogenic activities use actoprotector bromantan.

For inclusion in liposomes use of surface antigenic melioidosis fraction D, C, F, J, H, obtained from use of the pathogen melioidosis. Liposomes prepared from chromatographically pure phosphatidylcholine and cholesterol in a ratio of 7:3. The mixture of lipids (40 mg) are suspended in 2.0 ml of 2% solution of desoxycholate sodium in 0.01 M phosphate buffer, pH 7,2, then add 1.5 ml of antigen in 0,01M phosphate buffer at a concentration of 2 mg/ml of the resulting mixture is treated with ultrasound with a power of 200 W and a frequency of 20 kHz for 3 minutes, then cialiswhat 4 times in 2 hours against 200 ml of 0,01M phosphate buffer on a magnetic stirrer to remove desoxycholate sodium. The resulting liposomes are separated from neuklyuzhego antigenic material by centrifugation at 20,000 rpm for 1 h the Precipitate liposomes with incorporated antigen resuspended in 5 ml of 0,01M phosphate buffer. This technique provides a 50% inclusion melioidosis antigens in liposomes.

To determine the protective properties of the encapsulated melioidosis antigens used outbred white mice weighing 18-22, Animals subjected to immunization with a mixture dvuhsotmanatnyh antigens in a dose of 30 µg protein each, subcutaneously, twice, with an interval of 10 days. Simultaneously with primary and secondary immunization mice injected intraperitoneally immunomodulator bromantan dose of 1 mg After 14 days after the second immunization mice infect subcutaneously daily agar culture of a virulent strain of the pathogen melioidosis at doses of 3 and 30 LD₅₀. In 30 days after infection to determine the mortality rates of mice: the percentage of the fallen and life expectancy (ALE). The mortality rates in the experimental group compared with those in the control group of intact animals and make a conclusion about the level of protectively encapsulated antigens.

Example 1

In the process for obtaining immunogenic melioidosis antigens was performed fractionation of use of the pathogen melioidosis strain 111 methods of gel-chromatography and ion-exchange chromatography, which received 10 antigenic fractions: a, b, C, C₁, D, E, F, G, H, J. the fractions Obtained are heterogeneous macromolecular complexes consisting of proteins, glycoproteins or fragments of the polysaccharide. When tested immunogenic properties melioidosis fractions on white mice, it was shown that their protective activity in a stand-alone application in a mixture 1:1 with adjuvant's adjuvant was low: only 4 out of 10 agents had the ability to improve the survival odds of white mice from infection. Of these fractions, C, C₁increased survival of animals from 3-30 LD₅₀virulent melioidosis strain C-141 on 25-38%, the fraction of N - 60% of small infecting dose - 3 LD₅₀. Other fractions in varying degrees, increased the rate ALE or two infecting doses of fraction B, G, J, or from one infecting dose - fractions a, D, E, F, H (table 1).

Melioidosis faction that found protective properties in experiments on mice, was tested by us in the repeated experiments. Summary data when using different infecting doses of the pathogen melioidosis presented in table 2. As can be seen from table 2, the tested fractions C, D, C₁and H introduced animals in the mixture with the adjuvant's adjuvant, did not provide a reliably significant increase survival of animals, fraction D, C₁and N contributed only time increase ALE.

Due to low produktivnost antigenic fractions in their stand-alone application in subsequent studies have tested combinations melioidosis fractions, found some degree of protection. Combinations of antigenic fractions (each at a dose of 30 μg protein) was injected Guinea pigs, white mice with 0.2 ml subcutaneously as in the traditional form (mixed 1:1 with adjuvant-blockers), and liposomal scheme twice immunization (interval 10 day). Infection of immunized animals p is bodily through 14 days after the second immunization daily agar culture of a virulent strain of C-141 pathogen melioidosis. In 30 days after infection was determined mortality rates: the percentage of dead and ALE. The results of the experiments are shown in table 3.

As can be seen from table 3, liposomal forms of the compositions melioidosis antigens (L+J, L+F J+F) compared with adjuvant forms had higher rates of protectively for white mice. The difference in mortality rates during infection 3 LD₅₀virulent strain of the C-141 was 20-30%, while infecting 30 LD₅₀- 13-22%.

Example 2

For additional stimulation of the immune response to liposomal antigens used the immunomodulator bromantan dose of 1 mg, which was administered to the animal simultaneously with immunization (both primary and repeat) intraperitoneally in a volume of 0.5 ml of Bromantan refers to Ecoprotection adamantanol series. It is known that Ecoprotection - drugs that cause human adaptation to the adverse effects of physical factors also have an immunomodulating effect. These include, in particular, the adamantane - and imidazolidinone connection. These drugs have a broad spectrum of pharmacological activity (antitumor, antiviral, neurotropic, cardiotropic and others), many of them are the regulators of humoral and cellular processes immunogenesis (5, 6).

In table 4. reflects the results of a study in which iania of ectoprocta bromantane on the immunogenicity of the compositions encapsulated in liposomes melioidosis antigens in infected animals virulent strain 56830. As follows from the data of table 4, immunization of mice with compositions melioidosis antigens in liposomal form (L+S, J+H) increased their resistance to infection 3-30 LD₅₀the agent melioidosis strain 56830 on 25-43% compared with the control group of intact animals. Use for additional stimulation of the immune response bromantane resulted in increased survival of immunized mice by 25-30%. Immunization compositions of liposomal antigens increased the performance ALE animals, which was more pronounced when it is used to stimulate the immune response bromantane.

Summarizing the obtained results, it should be noted that protectionist compositions melioidosis antigens increases in their use for introducing animals in liposomal form compared to conventional adjuvant form by 20-30%. The inclusion in the scheme immunization with liposomal antigens of bromantane increases the survival rate of animals by another 20-30%.

The proposed method can be used in institutions for the development of means of specific prophylaxis of especially dangerous infections, to improve protectively antigenic preparations, promising to construct chemical melioidosis vaccine.

LITERATURE

1. Immunology of melioidosis / N. Tikhonov, Rybkin B.C., Zhukov, S., AND what rarawa .. // Melodos: Sat. scient. Tr. Volgograd. N.-I. pretiosum. in-TA. - Volgograd: the lower Volga book publishing house, 1995. - 119-141.

2. Minuchin BV, Brezinova NS, Tsyganenko YAKOVENKO et al. Immunogenic properties of liposomal forms toxoid Pseudomonas aeruginosa // Bulletin of the Academy of medical Sciences of the USSR. - 1990. No. 8. - P.44-47.

3. Kulikov, V.N., Kashkin C.P., Drnovska E.A. Inclusion of protective antigen Br.abortus in liposomes and immunogenic properties antigenaemia liposomes // J. of microbiol. - 1985. No. 12. - P.69-72.

4. Piven N.N., Rybkin B.C., Plekhanov N.G. et al. Immunotropnosti and immunogenicity of surface and membrane antigens of Burkholderia pseudomallei // J. of microbiol. - 2001. No. 1. - P.29-33.

5. The study of immunomodulatory activity imidazolidones polyelectrolytes / man'ko V.M., Rudnev T.B., Hajiyev R.I., etc. // Immunology. - 1990. No. 5. - P.63-66.

6. Experimental study of the immunotropic activity of new drug Kelantan / Artsimovich N.T., Fadeyeva T.A., Galushin FORCE and other // Immunology. - 1990. No. 6. - P.21-23.

1. The method of immunization of experimental melioidosis encapsulated antigens of Burkholderia pseudomallei, including the preliminary introduction of antigens to mice followed by infection and assessment of the level of protectively, characterized in that as antigens of Burkholderia pseudomallei using antigens selected from the surface antigenic fractions: D, C, F, J, H, with 1.5 ml of antigen mixed with 40 mg of phosphatidylcholine and cholesterol in a ratio of 7:3.

2. The method according to claim 1, characterized in that the mice injected with the mixture of the two encapsulated antigens, twice, with an interval of 10 days, subcutaneously, each at a dose of 30 μg protein and at the same time introducing actoprotector bromantan wew is brusino dose of 1 mg, while infecting mice virulent strain of the pathogen melioidosis dose 3-30 LD₅₀spend 14 days after the second immunization and 30 days after infection, assess the level of protectively antigens on mortality rates.