RU2192277C1 - Strain of bacterium salmonella enteriditis e-23 bmc 120 for immunization against human immunodeficiency virus - Google Patents

Abstract

FIELD: immunology, virology, microbiology. SUBSTANCE: strain Salmonella enteriditis E-23 BMC 120 is obtained from the strain Salmonella enteriditis. Using method of genetic transformation plasmid pBMC 120 is inserted into the parent strain that provides synthesis of protein gp120 of human immunodeficiency virus. Strain Salmonella enteriditis E-23 BMC 120 shows high ability to induce the expressed and effective immunization against human immunodeficiency virus. Strain is used for development of immunity against the human immunodeficiency virus. EFFECT: valuable properties of strain. 3 tbl, 3 ex

Description

 The invention relates to immunology and can be used to create immunity against the human immunodeficiency virus.

 A known bacterial strain Salmonella typhimurium SL 7207, see "Induction of mucosal and systematic responses against human immunodeficiency virus type 1 glycoprotein 120 in mice after oral immunization with a single dose of a Salmonella-HIV vector", Shaoguuang Wu, DBPascual, GKLewis , DM Hone, 1997, AIDS Research and Human Retroviruses, vol. 13, no 14, pp. 1187-1193 (a copy of the link is attached). The strain was administered to mice per os. As a result, a pronounced immune response to the human immunodeficiency virus gp 120 viral protein was induced in mice; Salmonella cells secrete this viral protein into the periplasm of bacterial cells; a proliferative response of splenocytes of immunized mice is observed on the 14th day after per os immunization; mainly Th 1 and Th 2 responses were induced, and the induction of IgA-secreting cells in lamina propria and in mesenteric lymph nodes was also noted.

 However, it should be noted that in this analogue, a significant drawback is that the protein synthesized by the bacterial cell does not glycosylate, as a result of which it does not cause a full-fledged immune response.

Also known is a bacterial strain Salmonella typhimurium SL 7207 (aro A his G 46) containing eukaryotic vectors expressing two truncated proteins (Act A and listeriolysin) of the pathogenic bacterium Listeria monocytogenes, see "Oral somatic transgene vaccination using attenuated S.typhimurium", A Darji, CAGusman, B. Girstel, P. Wachholz, KN Timmis, J. Wehland, T. Chakraborty, S. Weiss, 1997, Cell vol. 91, pp. 765-775 (a copy of the link is attached). Mice were immunized per os. The formation of cytotoxic lymphocytes, T-helper cells and antibodies to these proteins was observed in animals thus immunized, as well as their immunity to intravenous administration of Listeria monocytogenes at a dose of 10 • LD ₅₀ .

 The disadvantage of this strain is the lack of ability to elicit an immune response against human immunodeficiency virus.

 A known bacterial strain Salmonella typhimurium SL 3621, see "Potential live vaccines for HIV", M.S. Burnett, N. Wang, M. Hofmann, G. B. Kitto, Vaccine (2001) vol. 19, pp. 735-742 (a copy of the link is attached). These bacteria synthesize the human immunodeficiency virus Pol protein as a fusion protein, Pol-OmpA. Mice were immunized per os. After immunization of mice with cells of these bacteria, the appearance of specific immunoglobulin A to the target protein in fecal extracts, the appearance of a proliferative cell response, and the induction of spleen cytotoxic lymphocytes to cells representing polymerase fragments are observed.

 This technical solution was adopted as a propotype of the present invention.

 Its disadvantage is the fact that this strain does not induce antibodies to the surface protein gp 120 of the human immunodeficiency virus. As a result of this, antibodies that react with the surface of viral particles do not arise, which leads to insufficient immunization of the body with the human immunodeficiency virus.

 The objective of the present invention is to obtain a strain of bacteria that can cause effective immunization of the body against the human immunodeficiency virus.

 To solve this problem, a bacterial strain Salmonella enteriditis E-23 BMC120 is proposed, which has a high ability to cause a pronounced and effective immunization to the human immunodeficiency virus.

 The bacterial strain Salmonella enteriditis E-23 BMC120 obtained from a strain of Salmonella enteriditis E-23 (cya), which is a modification of the wild-type strain Salmonella enteriditis 1791, isolated from natural sources.

 The Salmonella enteriditis E-23 BMC120 strain was obtained by introducing the plasmid pBMS-120 into the Salmonella enteriditis E-23 strain, which synthesizes the human immunodeficiency virus gp 120 protein in mammalian cells and also carries the ampicillin resistance gene; the result is a strain of Salmonella enteriditis E-23 BMC120.

 This strain was deposited in the Collection of microorganisms of the All-Russian Research Institute of Agricultural Microbiology under the number VNIISKHM 423-D of July 16, 2001. The identification of the strain was carried out according to the "Identifier of bacteria Bergi"; M., 1997.

Cultural and morphological characteristics of the strain:
Morphology: gram-negative rods, medium size, randomly arranged.

Cultural properties: grows on meat-peptone agar, Davis glucose-minimum agar, MacConkey, Levin, Endo, Kaufman environments, small-sized S-colonies after 18 hours of growth at 37 ^o C. In meat-peptone broth, gall broth, liquid glucose-minimal medium A (Davis) - diffuse opacification after 18 hours of growth. In a citrate-minimal environment (Davis) - lack of growth.

 Antibiotic resistance: on all of the above media grows in the presence of ampicillin (100 μg / ml).

Antigenic structure of the strain:
O _9.12 H _{G, M}
Biochemical properties of the strain:
It produces hydrogen sulfide, has lysine decarboxidase, ornithine decarboxylase, arginine dehydrogenase activity, does not produce indole, does not thin the gelatin, does not hydrolyze urea. Does not recycle citrate on Simmons agar.

 Ferments glucose, sorbitol, mannitol, arabinose, dulcite, trehalose. It does not utilize sucrose, lactose, maltose, inositol, adonite, esculin, within 48-72 hours it utilizes ramnose.

 Virulence of the strain.

Laboratory mice are white, mongrel, 12-14 gr. LD ₅₀ = 10 ⁷ CFU (colony forming units) after intraperitoneal infection.

 The sensitivity of strains to bacteriophages.

Sensitive to bacteriophage P ₂₂ .

 Plasmid profile.

 A plasmid with a molecular weight of 55 kD and plasmid rVMS-120 with a molecular weight of 4.8 mD, which expresses the synthesis of human immunodeficiency virus gpl20 protein in eukaryotic cells.

 Example 1

A liquid nutrient medium was prepared: Peptone 10 g / L, Yeast extract 5 g / L, sodium chloride 10 g / L, to which ampicillin was added to a final concentration of 100 μg / ml after autoclaving. 100 ml of the above medium was placed in a 1 L flask, after which they were inoculated with material from one colony grown on an agarized solid nutrient medium of the same composition. The flask was incubated in a shaker at a temperature of 37 ^o C and 250 rpm for 18 hours, after which the number of bacteria in one milliliter was determined, which turned out to be equal to 2 • 10 ⁸ .

Bacteria were concentrated 10 times by centrifugation, after which 1 x 10 ⁸ cells / mouse were introduced per os to BALB / C mice. After 14 days, the immunization of mice was repeated, after another 7 days the animals were killed, splenocytes were isolated, which were mixed with syngene target cells expressing the surface protein of the human immunodeficiency virus. After co-incubation for 5 hours, the decay of target cells was determined in a known manner. The results are presented in table 1.

 Thus, immunization of mice with Salmonella enteriditis E23 strain BMS120 results in the appearance of splenocytes lysing mouse cells expressing the human immunodeficiency virus env protein.

 Example 2

For immunization, BALB / C mice weighing 12 g were used. Animals were orally or perrectally injected with a suspension of recombinant Salmonella cells containing a plasmid, 10 ⁹ living cells for each mouse for one immunization. Animals were immunized twice: on day 0 and on the 14th day after the first immunization. As a negative control, the initial Salmonella typhimurium E-23 strain was used, which did not have the ability to elicit an immune response to the human immunodeficiency virus. Assessment of the proliferative cellular response of splenocytes of animals was carried out by a known method and expressed as a proliferation index. The results are presented in table 2.

 (Salmonella enteriditis E-23 is a control strain that does not have the ability to elicit an immune response to the human immunodeficiency virus).

 As can be seen from table 2, splenocytes of animals immunized with Salmonella enteriditis E-23 BMC120, rectally or orally, retain the ability to proliferate by adding a lysate of human immunodeficiency virus up to 12 weeks after the first immunization.

 Example 3

BALB / C laboratory mice weighing 14-16 grams were immunized orally and perrectally with a suspension of Salmonella enteriditis E-23 BMC120 cells and a control strain of Salmonella enteriditis E-23, 10 ⁸ cells per mouse, after which the titers were determined by the dates indicated in the table antibodies to purified human immunodeficiency virus lysate in the blood serum of immunized animals. The results are presented in table 3.

 As can be seen from the table, immunization of Salmonella enteriditis E-23 BMC120 mice causes the formation of antibodies to the human immunodeficiency virus.

Claims (1)

 The bacterial strain Salmonella enteriditis, deposited at the All-Russian Research Institute of Agricultural Microbiology under the number "VNIISCHM 423-D" for immunization against human immunodeficiency virus.

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