Suppository for immunoprophylaxis of viral infections

FIELD: microbiology, biotechnology, medicine, in particular production of living vaccines against viral infections.

SUBSTANCE: invention relates to living vaccines for rectal administration against viral infections based on attenuated recombinant Salmonella strains bearing protective viral antigens. Suppository for immunoprophylaxis of viral infections contains (per one 2 g suppository, mass %): cell slurry of attenuated recombinant Salmonella strains transformed with pGEX-2T-TBI, pcDNA-TCI, or pKHBc bearing gene of protective viral antigens, mixed with suppository base in amount of 106-109 living cells 1 %; fatty base 94 %; and emulsifier T2 5 %. As fatty solid cooking oil (94 %); or hydrolyzed cotton oil (94 %); or mixture of cooking oil (60 %), Cocoa fat (24 %), and solid wax (10 %).

EFFECT: agent for inducing of humoral and cell immune response to respective viral antigen.

8 tbl, 11 ex, 3 dwg

 

The invention relates to Microbiology, biotechnology and medicine, specifically to the creation of methods for genetic engineering of live vaccines against viral infections based on attenuated recombinant Salmonella strains carrying protective viral antigens, in the form of suppositories.

Currently in the world there is a rapid spread of a number of viral infections, developing into an epidemic; it is AIDS caused by human immunodeficiency virus (HIV), hepatitis b, hepatitis C, etc. All of these pathogens are highly contagious, virulent and possess antigenic variability.

As in the Arsenal of medicine today, there is not enough effective ways to treat this kind of viral infectious diseases, the only measure to prevent the development of infections is mass vaccination of the population.

The main drawback of existing vaccines is that they can only induce systemic immunity. It is obvious that effective antiviral vaccine should provide both systemic and local mucosal immunity in the field "input gate" infection.

Among the promising new developments in viral vaccine in recent preference for the creation of mucosal vaccines, i.e. drugs, injected mucous membranes. Such vaccines have several advantages in the EU ETS: an introduction without a syringe, for example oral or intranasal; ensures the induction of not only the system, but local immunity, which is very important for sexually transmitted diseases. The main problems creation of mucosal vaccines based on inactivated antigens related to the fact that individual viral proteins not penetrate into the cells of the mucous membrane or degraded in the stomach or intestines.

One of the most promising vectors for mucosal delivery of vaccines are not pathogenic for humans attenuated strains of Salmonella [1, 2].

Interest in living cells of Salmonella as a carrier immunogenic epitopes of infectious agents due to the fact that attenuated strains of this bacterium is able to envirovet mucous and some time to live in the cells of lymphoid tissue of mammals. Cell-vector as a natural depot and adjuvant for antigen, is it in the most immunogenic form, increases the persistence and contributes to the accumulation of the antigen to immunocompetent cells.

The well-known series of works in which it is shown that recombinant attenuated Salmonella producing HIV antigens, induce high system (humoral and cellular) immunity and secretory immunity. An example is the recombinant attenuated Salmonella strains enteitidis E-23 VS [3] and Salmonella typhimurium T-10 Navy 160 [4], the first of which provides a fusion protein gp 120 of HIV in mammalian cells, and the second is another protein immunogen HIV gp 160. Known live HIV vaccine based on attenuated strain of Salmonella typhimurium SL 3261 for oral administration, which as immunogens carries protein reverse transcriptase transactivity protein of HIV [5]. In the described works as antigen used only one or two fragments of proteins gp 120 or gp 160 or pol, which is obviously insufficient to create an effective vaccine, because it is believed that an effective HIV vaccine should include epitopes not only of the env protein, but also gag and pol [6].

Known recombinant attenuated strain of Salmonella enteritidis E-23/pGEX-2T-TBI, and are able to produce artificial protein TBI containing nine T - and b - cell epitopes of HIV, in mammalian cells [7]. Also known recombinant attenuated strain of Salmonella typhimurium SL 7202 pGEX-TBI, and are able to produce artificial protein TBI [8].

Known recombinant attenuated strain of Salmonella enteritidis E-23/pcDNA-TCI [9], capable of producing artificial protein TCI containing multiple (more than eighty) cytotoxic T-cell epitopes of HIV-1 in mammalian cells.

All of these strains with the introduction of animals induce a pronounced specific gumor the capacity and cellular immune response to HIV and can be used to design the living DNA vaccines against HIV.

Actively developing approaches to the creation of a live vaccine against hepatitis b based on attenuated strains of Salmonella. Currently undergoing clinical testing of a vaccine based on recombinant Salmonella producing HBcAg-preS2 [10]. However, immunization of volunteers records recombinant strain of Salmonella, the answer to preS2 was only observed in one out of 13 subjects. One of the reasons for such a weak immune response is the fact that reS2-region is minor antigenic determinant, whereas the main neutralizing epitope contained in HBsAg.

Known candidate vaccine strains of bacteria, specifically designed by introducing plasmids encoding the antigens of hepatitis b virus HbsAg and HbcAg in the form of chimeric proteins in recipient attenuated Salmonella typhimurium strains T-10 and Salmonella typhimurium SL 7207 [11, 12]. After immunization of mice received oral structures and errectile (water suspension cells of recombinant strains in the form of an enema) found specific anti - HbsAg and anti-HbcAg IgG in the serum of animals and specific anti-viral IgA in the mucosa of the intestine, and also shows the formation of T-cell response to antigens of hepatitis C.

It is known that aqueous solutions, introduced into the rectum by enema, medicinal substances are absorbed very quickly. However, a lot of which of them is displayed back together with solution therefore, the total amount that absorb from the drug substance is small. The efficiency of absorption increases significantly when using suppozitornoj form. In addition, using suppozitornyj forms of medications can provide rapid and massive or progressive delivery of medicinal substance in the bloodstream [13].

The advantage of mucosal vaccines in the form of suppositories is that they can provide painless, ease of administration and to promote whiter stable and long-term induction of local immunity, because multiplying in the host organism, vaccinal strain stimulates long-term antibody production to the development of resistance at the input gate of the infection. In addition, this approach does not require expensive cleaning stage antigen.

Therefore, it seems appropriate to develop approaches to the creation of vaccines against viral infectious agents on the basis of living cells recombinant attenuated Salmonella in the form of suppositories.

There is information on the development of vaccines in the form of suppositories on the basis of inactivated microorganisms. This kind of vaccine against urogenital infections, consisting of inactivated bacteria that originate from cultures of strains uropathogenic BA is clear species Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus morganii and Streptococcus faecalis, and polietilenglikoli basics suppository, described in the patent of the Russian Federation No. 2224542 issued by the American firm Protein Express [14]. This suppozitornyj form of the vaccine promotes uniform distribution of cells of microorganisms in the pharmaceutical form and ensures the stability of the active substance for a long time.

In medical practice are created and used suppositories containing live microorganisms. It suppositories containing microorganisms normoflora person, which are used in the treatment of dysbacteriosis, inflammatory processes in the distal intestine and vagina [15, 16, 17]. Preparations suppositories contain microbial mass of live bifidobacteria or lactobacilli in the amount of 103-108cells per dose [17] or dry liofilizovannye biomass of microorganisms [16] and pharmaceutically acceptable additives. In Russia produced candles Azilect, Baydin, Lactobacterin (OJSC "Biochimmash"), which represent the microbial mass of live microorganisms, dried in an environment of cultivation with the addition of Saharsa-gelatin-dairy environment and formed the medical suppositories.

However, not described in literature preparations suppositories containing living cells of recombinant MIC the organisms, bearing protective viral antigens intended for vaccination of viral infections.

An object of the invention is to develop an effective drug for prophylaxis of viral infections (virus vaccines) based on live recombinant Salmonella strains in the form of a suppository.

The problem is solved by the development of production and composition of suppositories for rectal administration, containing live attenuated cell culture, recombinant Salmonella strains carrying genes protective viral antigens incorporated into suppozitornyj basis.

Rectal route opens the possibility of introducing into the body of drugs, collapsing in the stomach or intestines. And for attenuated Salmonella physiological environment with a pH of 6.8 to 7.2, so the environment of the rectum to the minimum extent will affect the manifestation of the biological activity of the vaccine strain.

To solve the task dynamics of the accumulation of biomass of Salmonella carrying the protective antigens of HIV and hepatitis b - Salmonella enteritidis E-23/ pGEX-2T-TBI, Salmonella enteritidis E-23/pcDNA-TCI, Salmonella typhimurium T-10/pKHBc in the fermentation process. It is shown that active cell division continues until OP=2,0. When this plasmid is stably retained in the strains. After culturing the titer of cells is of lionell in the culture fluids should not be below 10 6.

In the manufacturing process of DNA vaccines in the form of suppositories containing living cells with recombinant Salmonella strains, we studied the influence of substrate and temperature the introduction of native biomass in suppozitornyj based on cell viability. It is shown that the type of basics and nature of excipients has a significant impact on cell viability suppositories. From the data presented in Table 1, it is seen that the best cell viability provides the use of a hydrophobic (fat) basis. Based hydrophilic type no viable cells. Basics # 2 and # 6 have poor fluidity. Introduction to the framework as an excipient of Tween-80 acts detrimental to cell viability of Salmonella in suppositories. In the manufacture of suppositories there is a lack of viable cells with the introduction of cells in suppozitornyj base at a temperature of 50°C. Remains the same viability when making cells based at temperatures up to 45°C.

One dose (one suppositories weighing 2 g) contains 1·106-1·109living cells of Salmonella enteritidis E-23/pGEX-2T-TBI or Salmonella enteritidis E-23/rwma-TC1 or Salmonella typhimurium T-10/HBc.

According to studies suppositories are prepared on the basis of fats, nab the emer using cooking oil "Frying" or hydrolyzed cottonseed oil (GHM), or a mixture of solid cooking fat, cocoa butter with addition of solid paraffin oil and an emulsifier T-2 by pouring in suppozitornoj form a molten mass containing cell culture of Salmonella. The optimal temperature for introduction of biomass in suppozitornyj the basis of the temperature is 39 to 45°C.

Suppositories for prophylaxis of viral infections contain components in the following ratio to one candle by weight of 2 g, wt.%:

cell suspension of recombinant attenuated Salmonella strain carrying genes protective viral antigen, in an amount of 106-109living cells - 1%; fat basis - 94% and the emulsifier T-2 - 5%. At the same time as fat basis can be solid cooking fat (94%), or gidrolizovannogo cottonseed oil (94%), or a mixture of cooking oil "Frying" (60%), cocoa butter (24%) and solid paraffin oil (10%).

In the manufacturing process of the vaccine in the form of suppositories containing the recombinant cells of Salmonella, there is the problem of storing for a long time, standardization and stability of biomass. A necessary condition is that the cells of recombinant Salmonella must be in the active state of division, i.e. in the phase of logarithmic growth. The solution to this problem is to lyophilization biomass.

The viability of m is of croorganisms in the process of freeze-drying has a significant influence the composition of the protective environment. In the experiments we used the following protective environment: Saharsa gelatin (SG), milk-glucose (MG), sacharose gelatin with thiourea (GTL). The biomass samples of recombinant strains of Salmonella, liofilizovannye with different security environments, characterized by vitality and stability. The data are shown in Table 2.

Table 2
The viability and stability of biomass recombinant strains of Salmonella, liofilizovannyh with different security environments
The name of the biomass. Protective environment.1 month3 months6 months
ViableStabilityViableStabilityViableStability
Sal.ent. E23 DNA-TCI Environment SG4,0·109100%2,1·109100%2,0·108100%
Sal.ent. E23 DNA-TCI Environment MG4,0·109100%3,3·108100%2,7·108100%
Sal.ent. E23 DNA-TCI Media is GTL 1,8·109100%6,0·108100%5,8·108100%
Sal.ent. E23 rcns Environment SG4,3·109100%4,7·109100%4,0·109100%
Sal.ent. E23 rcns Environment MG5,3·109100%4,8·109100%3,5·109100%
Sal.ent. E23 rcns Environment GTL4,0·109100%3,5·109100%7,8·108100%

In Table 2 data show that liofilizovannye biomass recombinant Salmonella is stored without loss of cell viability and stability for 6 months, however, the process of freeze-drying is preferable to use environment SG or MG.

Moreover, it is shown that the suppositories manufactured using liofilizovannyh biomass can be stored for 6 months at 4-8 t With°, and the cells do not lose their viability and stability of the strain (see Table 3).

Table 3
A study of one hundred is innosti and viability liofilizovannyh cells of recombinant strains of Salmonella in suppositories
The name of the biomass. Protective environment.1 month3 months6 months
ViableStabilityViableStabilityViableStability
Sal.ent. E23 DNA-TCI Environment MG8,0·106100%1,0·106100%1,2·106100%
Sal.ent. E23 rcns Environment MG3,5·108100%2,7·107100%1,5·107100%

During preclinical studies in mice and Guinea pigs shows the formation of immunity against HIV infection and hepatitis b through education in animals of specific antibodies and the formation of a clone of memory cells. The maximum increase in antibody titer is achieved at 28 days and lasts for weeks after a single immunization. Two immunization held within 28 days after the first, the maximum antibody titer is logged on 42-56 days. In the pre-clinical testing performed in accordance with the Rules for state tests is s and the registration of new medical immunobiological preparations (SP 3.3.2.561-96), shows the lack of significant deviations in the condition of the vital organs, hematological and morphological indicators, the absence of allergenic activity that demonstrates the safety and tolerability of the vaccine.

The invention is illustrated in the following graphic.

Figure 1. Analysis of the systemic antibody response to purified HIV-1 sera of mice immunized with strain Salmonella enteritidis E-23/pcDNA-TCI according to ELISA.

S.enteritidis E-23/pcDNA-TCI

S.enteritidis E-23

Figure 2. Analysis of the systemic antibody response to purified protein TCI sera of mice immunized with strain Salmonella enteritidis E-23/pcDNA-TCI according to ELISA.

S.enteritidis E-23/ pcDNA-TCI

S.enteritidis E-23

Figure 3 Enzyme-linked immunosorbent assay to HBcAg sera of mice immunized with suppositories containing Salmonella typhimurium T10 rcns.

For a better understanding of the invention the following specific examples of its implementation.

Example 1. The cultivation of recombinant strains of Salmonella. The cultivation of recombinant strains of Salmonella enteritidis E-23/pGEX-2T-TBI or Salmonella enteritidis E-23/pcDNA-TCI or Salmonella typhimurium T-10/pKHBc spend on the medium S with ampicillin. Before cultivation spend the transformation of cells of Salmonella by which electroporation. For this "night" culture of Salmonella enteritidis E-23 or Salmonella typhimurium T-10 diluted in the ratio 1: 100 L-broth and pokasivaut in LB medium without ampicillin on the rocking chair at a temperature of (37±2)°C to an optical density of D550=0,2. The cell suspension is cooled in ice for 20 min and centrifuged 2 min at 6000 rpm After washing with water the precipitate cells resuspended in 40 μl of 10%glycerin solution, make a 1 µl plasmid DNA pGEX-2T-TBI or pcDNA-TCI or rcns, incubated in ice for 2 min and hold electroporation with the use of device "Ecobio LTD. The pulse time is 4 seconds, the voltage 2500 Century After electroporate in the same cuvette add 300 μl of L-broth and incubated 1 h at a temperature of (37±2)°C. the Obtained transformants plated on solid medium L - agar with ampicillin and grown overnight at a temperature of (37±2)°C. After checking for the presence of plasmids carried out clones producer strain cultivated on LB medium with ampicillin at temperature-controlled shaker at a temperature of (37±2)°With a rotation speed of 200 rpm, the Cultivation is continued for 3-4 h until the optical density D550=2,0. After culturing in the culture fluid determine the titer of cells, which should not be below 106. By centrifugation at 5000 rpm for 5 min to separate the biomass cells recombinant Salmonella that COI is lsout for the preparation of suppositories.

Example 2. Preparation of suppository Sal-HIV "B", containing living cells attenuated recombinant strain of Salmonella enteritidis E-23/pGEX-2T-TBI.

Main components of the suppository in the form of hanging (cooking oil "Frying" - 300,0±1.0 g, emulsifier T2 - 25,0±0.1 g cocoa butter - 120,0±0.1 g and paraffin - 50,0±0.1 g) quantitatively transferred into a beaker with a capacity of 1 l, which is placed in a water bath heated to 75°S, and incubated until complete melting of the mixture. Glass with molten base is placed in a mixer-homogenizer and stirred at 200 rpm until a homogeneous mass. The basis is cooled with constant stirring to a temperature of (39±1)°C. the Biomass of cells of Salmonella enteritidis E-23/pGEX-2T-TBI combined with 5 ml of sterile purified water and resuspended to obtain a homogeneous suspension which is injected into a chilled suppozitornyj basis, mixing thoroughly to obtain a homogeneous mass. Suppozitornyj was poured into the prepared forms and is cooled at a temperature of minus 15±2)°C for 60 minutes

Example 3. Preparation of suppository Sal-HIV "D"containing living cells attenuated recombinant strain of Salmonella enteritidis E-23/pcDNA-TCI.

Main components of the suppository in the form of sub-samples (solid cooking fat - 470,0±1.0 g emulsifier T2 - 25,0±0.1 g) quantitatively transferred into a glass in which estimatio 1 l, which is placed in a water bath heated to 55°S, and incubated until complete melting of the mixture. Glass with molten base is placed in a mixer-homogenizer and stirred at 200 rpm until a homogeneous mass. The basis is cooled with constant stirring to a temperature of (39±1)°C. the Biomass of cells of Salmonella enteritidis E-23/pcDNA-TCI combined with 5 ml of sterile purified water and resuspended to obtain a homogeneous suspension which is injected into a chilled suppozitornyj basis, mixing thoroughly to obtain a homogeneous mass. Suppozitornyj was poured into the prepared forms and is cooled at a temperature of minus 15±2)°C for 60 minutes

Example 4. Determination of viable cells in rectal suppositories.

One suppository weighing 2.0 g containing cells attenuated strain of Salmonella enteritidis E-23 with integrated plasmid pcDNA-TCI or pGEX-2T-TB, soften at 37°in phosphate buffer until smooth and cook 10 fold dilution of the suspension. From solutions of the 6th, 7th and 8th breeding do the sowing 100 ál of Petri-dish with L-agar containing 100 μg/ml ampicillin, and thermostatic day at 37°C. Upon expiration of the time visually counting the number of colonies on plates. The number of viable colonies of recombinant Salmonella in suppositories (X) is found by the formula:

where: X is the number of viable colonies;

N - number of colonies;

10pnumber of breeding;

Vp- volume sowing cultivation, ál; (100 μl).

The number of viable colonies in suppositories after the counting is from 106up to 109.

Example 5. Determination of the toxicity of drugs suppositories.

Suppositories weighing 100 mg, containing from 106up to 109cells of Salmonella enteritidis E-23/pGEX-2T-TBI or Salmonella enteritidis E-23/pcDNA-TCI enter not less than 5 Guinea pigs (weighing 250-350 g), once in the rectum to a depth of 0.5 to 1 cm and fixed in 2-3 minutes during the first day and for 7 days after the administration of suppositories conduct a daily visual inspection of animals, evaluating their appearance, mobility, measure body weight. During the whole observation period no animal died, visually recorded signs of intoxication and loss of body weight of the animals was not observed. Thus, the proposed product is non-toxic and harmless.

Example 6. Determination of specific activity of suppositories containing Salmonella enteritidis E-23/pcDNA-TCI

The immunization. For immunization using mice of BALB/c mice, weighing 12-15 grams, which were obtained from the vivarium of the SRC VB "Vector". The animals were kept on a standard diet.

Immunization of animals with suppositories p is avodat once, parentale (one suppository contains 108cells of S. enteritidis E-23/pcDNA-TCI). Blood sampling to produce 0, 7, 14, 21, 28, 35 the day after the introduction of Salmonella.

Enzyme-linked immunosorbent assay. The specificity of the resulting antibodies in sera of mice analyzed by ELISA. For the detection of antibodies against HIV-1 using purified inactivated HIV-1 virus and purified protein TCI. In each experiment, take blood samples from three animals. After obtaining serum samples are mixed, and then determine the titre of specific antibodies. Titer is defined as the excess of the titer of the sera of mice immunized with suppositories containing S. enteritidis E-23/pcDNA-TCI above the titer of the sera of mice immunized with the suppositories from the original strain of S.enteritidis E-23. At each point do two repetitions. The results are presented in figures 1, 2. Starting with the third week after immunization of mice with suppositories S. enteritidis E-23/pcDNA-TCI detect products of IgG to HIV-1, and antibodies production remains at a high level throughout the observation period.

The determination of the number of CTL lymphocytes on the basis of the reaction ELISPOT

When setting the ELISPOT response at the first stage are sorption anti-INF-γ MAT with a concentration of 5 μg/ml per well of 96-hole tablet ImmunoSpot M200. After incubation for 12 h at 4°With each of the wells washed twice with a solution of PBS and blocked the Ute medium RPMI 1640, containing 10% fetal bovine serum for 2 hours as cell-effectors using splenocytes of immunized animals at a concentration of 106Jr. For the stimulation of product INFγ suspension cells using protein TCI (1 μg/ml) and two peptide: N15 (DRVIEVVQGAYRAIR), N16 (KQIINMWQEVGKAMYA), to assess nonspecific products make use of the peptide of EHEC (negative control). Cells cultured in the presence of 5% CO2at 37°C for 24 h INFγ-secreting cells render using 0.5 μg/ml biotinylating anti - INF-γ antibodies and 0.25 μg/ml conjugate Avidin-HRP. Staining produced by adding a substrate for peroxidase (4-chloro-1-naphthol, diaminobenzidin phosphate). The reaction is stopped by removal of the reagents, the wells washed 3 times with distilled water. Counting the number of INFγ-producing cells is carried out using a microscope. The results of the experiments are summarized in Table 4.

Table 4

The results of the evaluation of cytotoxic T-lymphocytes of animals immunized with suppositories containing S. enteritidis E-23/pcDNA-TCI in the ELISPOT
AntigenThe period from the beginning of immunization (day) / number of INF-gamma producing cells
101421
TCI120230150143
R110220128125
P16107217129127
Of EHEC10754

Example 7. Evaluation of the neutralizing activity of sera.

Neutralizing activity of sera determined by the accounting method of inhibiting the reproduction of the virus HIV-1 by reducing the infectious titer. This is done using human lymphoblastoid cells of a human MT-4. Cells were cultured at a concentration of 3.0 to 5.0×105cells in 1 ml of RPMI medium 1640 with 10% serum of cow embryos, 100 μg/ml of gentamicin. In the reaction of virusneutralizing use strain of HIV-1899A(type X4, subtype b), obtained from the collection of strains of human immunodeficiency Institute of Virology. DI Ivanovo RAMS. The multiplicity of infection of 100 TCD50. Serum inactivate in a water bath at a temperature of 56°C for 30 min In the wells of a plastic 96-hole panel (Costar, USA) add 50 ál of the following breeding sera: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, etc. and add 50 μl of the suspension of the virus. The mixture is cuberoot for 1 h at 37° C. After incubation, make a 100 μl suspension of sensitive cells MT-4 in the amount of 90-100×103cells/well.

Panel incubated at 37°C in an atmosphere with 5% CO2and 98% humidity for 5-7 days prior accounting results. As controls use: control of virus - virus with the addition of medium without serum, "control cells" - cells in a nutrient medium, "control of cytotoxicity sera" - cells + serum. Analysis performed 5-7 days after the start of the experiment. Inhibition of virus reproduction judged by the reduction in infectious titer, which is determined by TCD50(50% tissue cytopathic dose). The data for detection of neutralizing antibody titers in the blood serum of immune animals are summarized in Table 5.

Table 5

The neutralizing activity of sera
The period from the beginning of immunization (day)
groups of animalsTiters of neutralizing antibodies
071421
Sera from mice immunized01:201:801:640
the suppositories S. Enteritidis E-231
pcDNA-TCI
Sera from mice immunized0000
the suppositories S. Enteritidis E-23

The data presented in Table 5, suggest that formed after insertion of suppositories S. enteritidis E-231 pcDNA-TCI antibodies have neutralizing activity.

Thus, suppositories containing Salmonella enteritidis E-23/pcDNA-TCI with the introduction of animals induces a pronounced specific humoral and cellular immune response to HIV-1. Proposed suppositories can be used as a living DNA vaccines against HIV-1.

Example 8. Determination of specific activity of suppositories containing Salmonella typhimurium T10 rcns.

The immunization. For immunization using mice of BALB/c mice, weighing 12-15 grams, obtained from the vivarium of the SRC VB "Vector". The animals were kept on a standard diet.

Immunization of mice conduct the introduction suppositories perfectline, twice with an interval of 3 weeks. The dose of S. typhimurium T10 rcns is 107cells on the suppository. The blood produce the via 14, 21, 28, 36 and 44 days after the first injection of Salmonella.

Enzyme-linked immunosorbent assay. The specificity of the resulting antibodies in sera of mice analyzed by ELISA. For the detection of antibodies against HBcAg using purified recombinant HBcAg. In each experiment, take samples from three animals. These samples are mixed, and then determine the titre of specific antibodies. Titer is defined as the excess of the titer of the sera of mice immunized with suppositories containing Salmonella typhimurium T10 rcns, above the titer of the sera of mice immunized suppositories with the original strain of Salmonella typhimurium T10. At each point do two repetitions. The results are presented in figure 3.

The reaction besttransport. The reaction besttransport of splenocytes to carry through the 81 day after the first injection of Salmonella according to previously described methods [18]. As a specific antigen using HBcAg, as a non - specific antigen-virus enteritis of mink. The hole in the tablet make 100 ál of each component. The antigen concentration of equal to 2 μg/ml as a mitogen use of concanavalin a (manufactured by Sigma, USA) at a concentration of 5 µg/ml Cells stimulated by antigen, incubated for 78 hours For 18 h before the end of the incubation period make3H-thymidine (St. Petersburg) in the dose of 2 µci per well. Cellular response estimate by pokazatelyakh stimulation, which characterizes the ratio of the proliferation of splenocytes induced by specific antigens to spontaneous proliferation of splenocytes (table 6). The formation of a clone of splenocytes sensitized to HBcAg, is registered in the group of animals immunized with the suppositories with S. typhimurium T10/pKHBc. The stimulation index in the group of S. typhimurium T10/pKHBc significantly exceeding that in the control group S. typhimurium T10, and stimulation of splenocytes heterologous antigen mink enteritis virus. The ability of splenocytes to respond to the stimulation of HBcAg in the 81st day of the start of immunization S. typhimurium T10/pKHBc indicates the formation of a specific memory cell.

The data suggests that suppositories containing Salmonella typhimurium T10/pKHBc, with the introduction of animals induce a pronounced specific humoral and cellular immune response to HBcAg. Proposed suppositories can be used as a live vaccine against hepatitis C.

Example 9. Preparation of suppository from freeze-dried biomass attenuated recombinant strains of Salmonella typhimurium T-10/pKHBc.

Main components of suppositories in the form of batches prepared as described in Examples 2 or 3. Glass with molten base is placed in a mixer-homogenizer and stirred at 200 rpm until a homogeneous mass. The basis is cooled with constant stirring to a temperature of (39±1)°C. the Vials (2-3 bottles) with freeze-dried biomass of Salmonella typhimurium T-10/pKHBc (titer of viable cells should be 109-1011CFU/ml) opened under aseptic conditions and triturated tablets to obtain a free flowing powder without lumps. Thus, for the first time received suppositories for prophylaxis of viral infections, containing living attenboroughii recombinant Salmonella strains carrying protective viral antigens, inducing when introducing animals expressed specific humoral and cellular immune response to the corresponding viral is tegenu, which can be used as living antiviral vaccines.

Example 10. Determination of specific activity of suppositories containing Salmonella enteriditis E23/pGEX-2T-TBI.

Recombinant strain of Salmonella enteriditis E23/pGEX-2T-TBI produces polyepitopic protein gst-TBI, consisting of b - and T-cell epitopes of HIV-1 [8].

The immunization. For immunization using mice of BALB/c mice, weighing 12-15 grams, obtained from the vivarium of the SRC VB "Vector". The animals were kept on a standard diet.

Immunization of mice conduct the introduction suppositories perfectline. The dose of Salmonella enteriditis E23/pGEX-2T-TBI is 109cells on the suppository. As a positive control animals subjected to immunization with protein gst-TBI with PAF (full beta-blockers) intramuscularly once. Blood sampling is carried out in 2, 6 and 12 weeks after injection of Salmonella.

Enzyme-linked immunosorbent assay. The specificity of the resulting antibodies in sera of mice analyzed by ELISA. For the detection of antibodies against HIV-1 using purified recombinant protein gst-TBI and the lysate of HIV-1A(type X4, subtype b), obtained from the collection of strains of human immunodeficiency Institute of Virology. Dijanoveckog RAMS. In each experiment, take samples from three animals. These samples are mixed, and then determine the titre of specific antibodies. At each point de is with two repetitions. The results are presented in table 7.

Table 6
The proliferative activity of splenocytes of mice immunized with the suppositories, carriers of recombinant Salmonella (stimulation index) on day 81 after the beginning of immunization
AntigenAntigenMitogen
The group of animalsHbcAgVirus enteritis of minkOf concanavalin
S. typhimurium T-101,00,97,4
S. typhimurium T-10/rcns3,31,07,5
Table 7

The development of HIV-specific humoral immune response in mice immunized with cells of recombinant Salmonella producing TBI
GroupWeek 2 titleWeek 6 : the titer of anti-TBI12-week titer anti-TBI
Anti-TBIAnti-HIV-1anti-TBIanti-HIV-1anti-TBIAnti-HIV-1
Protein gst-TBI1:6001:3001:36501:12001:21501:600
S. enteriditis E23/ pGEX-2T-TBI rectal1:3001:1001:14501:6001:8001:400
S. enteriditis E23 rectal (contr)1:1001:501:501:501:501:50

The reaction besttransport. The reaction besttransport of splenocytes spend 6 weeks after the introduction of Salmonella according to previously described methods [18]. As a specific antigen using gst-TBI and the lysate of HIV-1 as a non - specific antigen-virus enteritis of mink. The hole in the tablet make 10 µl of each component. The antigen concentration of equal to 2 μg/ml as a mitogen use of concanavalin a (manufactured by Sigma, USA) at a concentration of 5 µg/ml Cells stimulated by antigen, incubated for 78 hours For 18 h before the end of the incubation period make3H-thymidine (St. Petersburg) in the dose of 2 µci per well. The cellular response is evaluated in terms of stimulation index, which characterizes the ratio of the proliferation of splenocytes induced by specific antigens, to spontaneous proliferation of splenocytes (table 8). The formation of a clone of splenocytes sensitized to gst-TBI, is registered in the group of animals immunized with the suppositories with S. enteriditis E23/pGEX-2T-TBI. The stimulation index in the group of S. enteriditis E23/pGEX-2T-TBI significantly exceeding that in the control group S. typhimurium 7207 and stimulation of splenocytes heterologous antigen mink enteritis virus. The ability of splenocytes to respond to the stimulation gst-TBI 6 week from the beginning of immunization with S. enteriditis E23/pGEX-2T-TBI indicates the formation of a specific memory cell.

Table 8
Induction of T-cell response. The stimulation index
The group of animals immunized with recombinant SalmonellaThe lysate of HIV-1gst-TBI antigen mink enteritis virusConA
S. enteriditis E23/pGEX-2T - TBI rectal3,43,51,17,9
S. enteriditis E23 rectal(contr)1,11,21,27,9

Note.In each experiment took cells from 3 animals. The error in the determination of the stimulation index of not more than 0.1.

The data suggests that suppositories containing with S. enteriditis E23/pGEX-2T-TBI, with the introduction of animals induce distinct HIV-1 specific humoral and cellular immune response.

Example 11. Preparation of suppository Sal-HIV "D"containing living cells attenuated recombinant strain of Salmonella enteritidis E-23/pcDNA-TCI.

Main components of the suppository in the form of hanging (gidrolizovannogo cottonseed oil (GHM) - 425,0±1.0 g emulsifier T2 - 25,0±0.1 g) quantitatively transferred into a beaker with a capacity of 1 l, which is placed in a water bath heated to 55°S, and incubated until complete melting of the mixture. Glass with molten base is placed in a mixer-homogenizer and stirred at 200 rpm until a homogeneous mass. The basis is cooled with constant stirring to a temperature of (39±1)°C. the Biomass of cells of Salmonella enteritidis E-23/pcDNA-TCI enter into a chilled suppozitornyj basis then what s under stirring, suspended until a homogeneous suspension. Suppozitornyj was poured into the prepared forms and is cooled at a temperature of minus 15±2)°C for 60 minutes, the Number of viable colonies in suppositories after the counting is from 1.1×106up to 4×108.

Thus, for the first time received suppositories for prophylaxis of viral infections, containing living attenboroughii recombinant Salmonella strains carrying protective viral antigens, inducing when introducing animals expressed specific humoral and cellular immune response to the corresponding viral antigen that can be used as living antiviral vaccines.

LITERATURE

1. International application WO No. 98/48026, CL 12N 15/74, publ. 1998

2. International application WO No. 0014240, CL 12N 15/31, publ. 2000

3. RF patent №2192277, CL AC 39/12, publ. BI No. 31, 2002

4. RF patent №2192886, CL AC 39/19, publ. BI No. 32 of 2002

5. RF patent №2223784, CL AC 39/385, publ. BI # 4, 2004

6. Eroshkin A.M., Karginova E.A., Gileva I.P. et al. Design of four-helical protein as a candidate for HIV vaccine. // Protein engineering-1995 - 8 - p.167-173.

7. Nekrasov N.A., Ignatyev, G.M., Agafonov A.P., Il A.A. Karpenko LI Study of systemic and mucosal immune response after immunization of mice with candidate HIV-1 vaccine. // Siberian medical journal of the L. - 2004. - T. - P.60-62.

8. Karpenko LI, Ignatyev, G.M., Agafonov A.P., Poryvaev V.A., Lebedev LR, Veremail T.A., N.A. Nekrasov, Klimov N.A., Kozlov A.P., Il A.A. Design and study of the antigenic properties of the recombinant strain of Salmonella, producing protein TBI. // Virology - 2002 - N.2 - C-28.

9. Application for patent of the RF No. 2003111095/13 from 17.04.2003, "Recombinant plasmid DNA pcDNA-TCI, ensuring the expression of the synthetic gene TCI in eukaryotic cells, and recombinant attenuated strain of Salmonella enteritidis E-23/pcDNA-TCI as a candidate for constructing living DNA vaccines against HIV."

10. Nardelli-Haeftiger D., Kraehenduhl J.P, et al. Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain. Infect. Immun. - 1996 - v.64 - p.5219-5224.

11. RF patent №2216590, CL 12N 1/21, publ. BI No. 32 for 2003

12. Karpenko LI, Ignatyev, G.M., Simon E.M. and other Receipt and study of recombinant strains of Salmonella typhimurium SL 7207, producing HbcAg HbcAg-HBs. Questions Virology - 2000 - No. 2 - s.43-46.

13. Gregg M.R., Jack D.B., Smith S.R., Kendall, M.J. The pharmacokinetics ofoxprenolol following oral and rectal dosing-a comparison of delivery systems and routes of administration. // J Clin Pharm Ther. 1987 Apr; 12 (2): 91-9.

14. RF patent №2224542, CL AC 39/116, publ. BI No. 6, 2004

15. RF patent №2185842, CL AC 35/74, publ. BI No. 21 for 2002

16. RF patent №2228738, CL AC 9/02, publ. BI No. 15, 2004

17. RF patent №2073520, CL AC 35/74, publ. BI No. 5, 1997

18. Horobryh CENTURIES, Prony the AV, Kirkin AF, Sanin A.V. Methods of production of reaction besttransport in micromodification // Immunology. - 1983. - N3. - P.76-79.

1. Suppositories for prophylaxis of viral infections, containing live attenuated cell culture, recombinant Salmonella strains, transformed with pGEX-2T-TBI, pcDNA-TCI or rcns bearing genes protective viral antigens, hydrophobic suppozitornyj basis and emulsifier T2 in the following ratio of components on one candle by weight of 2 g, wt.%:

Suspension of living cells in quantities of 1·106-1·1091
Hydrophobic suppozitornyj base94
Emulsifier T25

2. Suppositories according to claim 1, characterized in that as a live attenuated cell culture recombinant Salmonella strains contain liofilizovannye biomass recombinant microorganisms.

3. Suppositories according to claim 1, characterized in that the hydrophobic suppozitornoj bases contain solid cooking fat (TKJ) or gidrolizovannogo cottonseed oil (GHM) in the following ratio, wt.%:

Suspension of living cells in quantities of 1·106-1·1911
TKJ or GHM94
Emulsifier T25

4. Suppositories according to claim 1, characterized in that the hydrophobic bases contain a mixture of cooking oil "Frying", cocoa butter and paraffin wax in the following ratio, wt.%:

Suspension of living cells in quantities of 1·106-1·101
Cooking fat "Frying"60
Cocoa butter24
Paraffin wax10
Emulsifier T25



 

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