Recombinant attenuated strain of salmonella enteriditis e-23/pgex-2t-tbi as a candidate for constructing a live vaccine against human immunodeficiency virus
The invention relates to biotechnology, specifically genetic engineering. Proposed recombinant attenuated strain of Salmonella enteriditis E-23/pGEX-2T-TBI, producing artificial protein TBI, consisting of T - and b-cell epitopes of HIV-1. The strain obtained by genetic transformation strain of Salmonella enteriditis E-23 (cya, CRP) plasmid pGEX-2T-TBI. With the introduction of animal recombinant strain of microorganism induces a pronounced specific humoral and cellular immune response to HIV-1. Recombinant strain can be used to construct a live vaccine against HIV-1. 5 Il., table 2.
The invention relates to biotechnology, specifically genetic engineering, and is a recombinant attenuated strain of Salmonella enteriditis E-23/pGEX-2T-TBI, and are able to produce artificial protein TBI, consisting of b - and T-cell epitopes of HIV-1. The proposed recombinant strain of microorganism is a candidate for constructing a live vaccine against HIV-1.
Salmonella have the natural ability to penetrate the epithelium of the mucous mainly through M-cells and multiply in the local intestinally lymphoid formations), which are an attractive vehicle for delivery of heterologous antigens to the mucosa of the gastrointestinal tract with subsequent involvement of the immune system [2, 3]. This approach can be used to create vaccines against viruses that may enter the body parenterally and/or through the mucosa of the genital and rectal tract.
The efficiency of the vectors on the basis of Salmonella illustrated by many experiments on animals, which used Salmonella, producing proteins of viruses, bacteria and protozoa [4, 5]. Antigens produced by strains of Salmonella, stimulate antigen-specific systemic IgG, T-helper response, CD8+ I-class or CD4+ II-class CTL response, as well as the production of secretory antibodies in the mucosa . One of the striking examples is the use of Salmonella strains expressing the gene fragment With tetanus toxsoid  and providing complete protection of mice from parenteral infection with tetanus toxin.
A number of authors described a large variation of the immune response to various antigens expressed in Salmonella. The reason for this can serve a variety of factors, including the expression level, cellular localization of the antigen, the route of administration and the scheme of vaccination .
At present, a number of different attenuated strains of Salmonella. These strains are able to envirovet lymphoid tissue of the gastrointestinal tract, but oratory expresses gp120 protein of HIV-1 [11, the prototype]. The strain was administered to mice per os. As a result, the mice were recorded a pronounced immune response to the viral protein gp120 of human immunodeficiency virus. Was observed proliferative response of splenocytes of immunized mice and was shown cytotoxic activity of splenocytes. However, it should be noted that similar significant drawback is the fact that this strain induces immunity to only one viral protein HIV-1 and not shown the formation of mucosal immunity after immunization with the indicated strain and neutralizing activity of the sera of immunized animals.
An object of the invention is the creation on the basis of the attenuated strain of Salmonella enteriditis E-23 recombinant strain of Salmonella enteriditis E-23/pGEX-2T-TBI, which provides constitutive protein immunogen TBI containing multiple epitopes immunogenic proteins of HIV-1 and causing the induction of specific neutralizing HIV-1 antibodies with the introduction of the animal, i.e. a candidate live vaccine against HIV-1.
A suitable candidate proteins immunogen is a protein TBI (T and In cell epitopes contaning immunogen). Constructed of four--helical protein on the protein sequence TBI includes four T-cell epitope and five b-cell neutralizing epitopes from proteins env and gag HIV-1. In mice and monkeys rhesus Macaque immunized with TBI, was formed as the cellular and humoral response to HIV-1. It was shown that anti-TVI antibodies had HIV-1-neutralizing activity. The results indicate the viability of this approach.
Known attenuated strain of Salmonella enteriditis E-23, (cya, CRP), which has reduced virulence . In mice, calves and monkeys, it was shown that the virulence of this strain reduced by approximately 1,000-fold compared with the parental strain, while maintaining the invasive activity. This strain on a number of basic properties can be used to solve the task, i.e. to create on its basis of a vaccine against HIV-1.
The problem is solved by genetic transformation strain of Salmonella enteriditis E-23 (cya, CRP) plasmid pGEX-2T-TBI , which provides constitutive protein synthesis TBI, and the introduction of strains of laboratory animals, causing the induction of specific anti-HIV antibodies and specific t-cell response.
The constructed strain was designated as S. enteriditis E-23/pGEX-2T-TBI and deposited under number B-884 in the collection of the Institute of CMC SRC VB "Vector".
Morphology: gram-negative rods, medium size, PAA, environments poppy-Com, Levin, Kaufman, colonies in S-form, small size after 18 hours of growth at 37°C. In mesopatamia broth, bile broth, liquid glucosaminidase environment (Davis) - diffuse opacification after 18 hours of growth. In citratemale environment (Davis) - no growth.
Antibiotic resistance: all of these environments grow in the presence of ampicillin (100 µg /ml)
Do not utilize citrate on agar Simmons, does not produce indole, not splits urea, has no phenylalaninamide activity, reaction Voges-Proskauer - negative; produces hydrogen sulfide within 48 hours, has a lysine - and intercorporate activities and argumentirovannoe activity; ferments glucose, not fermented lactose, sucrose, maltose, sorbitol, Inositol, dulcet, adonit; within 48 hours fermented rhamnose, lures, trehalose. Gelatino not thins.
Sensitivity to bacteriophages:
Sensitive to bacteriophage P22.
Plasmid pGEX-2T-TBI with a molecular mass 5382 kb. This plasmid obtained by the authors and provides protein synthesis TBI in the cells of Salmonella .
Sobro data electrophoresis in 15% SDS page.
1. The molecular mass marker
2. The lysate of cells of Salmonella enteriditis E-23/pGEX-2T-TBI.
3.Purified protein TBI
4. The lysate of cells of Salmonella enteriditis E-23, not containing plasmid.
C. Immunoblotting using sera of macaques immunized with HIV-1.
1. The lysate of cells of Salmonella enteriditis E-23/pGEX-2T-TBI.
2. The lysate of cells of Salmonella enteriditis E-23, not containing plasmid.
Analysis of the systemic antibody response to purified HIV-1 sera of mice immunized with strain of Salmonella enteriditis E-23/pGEX-2T-TBI no data ELISA.
S. enteriditis E-23/pGEX-2T-TBI
S. enteriditis E-23
Analysis of the systemic humoral response to recombinant proteins of HIV-1 sera of mice immunized with strain of Salmonella enteriditis E-23/pGEX-2T-TBI according to the IFA.
S. enteriditis E-23/pGEX-2T-TBI
S. enteriditis E-23
Assessment of mucosal immune response to purified HIV-1 sera of mice immunized with strain of Salmonella enteriditis E-23/pGEX-2T-TBI according to the IFA.
S. enteriditis E-23/pGEX-2T-TBI
S. enteriditis E-23
Assessment of mucosal immune response to recombinant proteins of HIV-1 sera of mice immunized with strain of Salmonella enteriditis E-23/pGEX-2T-TBI Dunn is his understanding of the invention the following are examples of its implementation.
Example 1. Obtaining a recombinant strain of Salmonella enteriditis E-23/pGEX-2T-TBI
Electroporation. Cell culture Salmonella enteriditis E-23 is grown in an environment YT before OP=0,5, cooled, 1 ml of the culture fluid centrifuged 2 min at 6000 rpm in the Eppendorf centrifuge. Sediment cells resuspended 800 ál of chilled, pedestrianonly water and precipitated by centrifugation for 1 min at 6000 rpm the washing Procedure is repeated 5 times. Then the precipitate resuspended in 200 ál of 10% glycerol, centrifuged 1 min at 6000 rpm and resuspended in 40 ál of 10% glycerol.
To the cell suspension add 0.5 μg of plasmid DNA. The sample is placed in a cuvette for electroporation, the pulse time is 4 seconds at a voltage of 2.5 C. For electroporation using tame the electroporator company BIORAD. Then add 300 ál of YT medium and incubated 1 hour at 37°C. Cells scatter on StI agar with ampicillin (100 µg/ml). Grown on agar with ampicillin colony known as Salmonella enteriditis E-23/pGEX-2T-TBI. All manipulations with plasmid DNA carried out according to described methods .
Cells of Salmonella enteriditis in E-23/pGEX-2T-TBI is grown as follows: a single colony of Salmonella inoculated into 50 ml of medium YT containing ampicillin (100 μg/ml), and incubated for 16 hours 3T-TBI precipitated from 100 ml of culture. The precipitate is dissolved in water and add 40 ál of lytic buffer (1M Tris-Hcl pH 8.8, 5% LTOs, 20% glycerol, 10% mercaptoethanol, of 0.02% bromophenol blue) and incubated in a boiling water bath for 5 minutes Electrophoretic separation of proteins is carried out in a 13% polyacrylamide gel in the presence of 0.1% LTOs in laemmli's method . Transfer proteins from the gel to the filter is done according to the described method  when the voltage of the electric field of 10 V/cm2within 2 hours. The filters are placed in a 3% solution of BSA in 0.01 M Tris-HCl, pH 7.5, 0.05% tween-20 for 1 hour, then incubated 1 hour with a solution of antibodies macaques immunized with HIV-1, diluted in buffer, 0.01 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% tween-20, 0.05% of NaN3, 0,001 M PMSF. The same solution is used for the subsequent cleaning of filters and for incubation with conjugate antibodies with phosphatase. The results are presented in Fig.1. In the lysate of cells of Salmonella enteriditis E-23/pGEX-2T-TBI is a protein with a molecular mass corresponding to 45 kDa, and detected antibodies to HIV-1. This demonstrates the production of the protein TBI in the cells of S. enteriditis E-23/pGEX-2T-TBI.
Example 2. The study of the ability of the target strain to induce a humoral immune response.
For immunization, cells Salmonella enteriditis E-23/pGEX-2T-TBI grown following obrazov 37°C; cells precipitated by centrifugation and resuspended in 1 ml of physiologic saline. The cell concentration is determined by titration.
The immunization. For immunization were used mouse strain 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 animals obtained strain was performed once, perfectline, the introduction of 108cells Salmonella enteriditis E-23/pGEX-2T-TBI mouse. Blood sampling was performed on 0, 14, 21, 35, 42 and 48 hours 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 inactivated HIV-1 virus and a mixture of recombinant proteins of HIV-1: gag, gp120, env1 and env2. 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 Salmonella enteriditis E-23/pGEX-2T-TBI above the titer of the sera of mice immunized with the original strain of Salmonella enteriditis E-23. At each point do two repetitions. The results are presented in Fig.2, 3. Starting with the third week after immunization of mice with cells of Salmonella enteriditis E-23/pGEX-2T-TBI obnaruzhivaemye(48 days).
The definition of a mucosal immune response. Assessment of mucosal response is performed by analyzing the titer of IgA in the intestinal mucosa of immunized animals. The selection of the intestinal mucosa of animals carried out according to the method described in . The specificity of the resulting antibodies of class As determined by ELISA using antigens purified HIV-1 virus and a mixture of recombinant proteins of HIV-1: gag, gp120, env1 and env2. The results are presented in Fig.4, 5. Products specific to HIV-1 IgA is found, starting from the third week after immunization, and reaches a maximum value of 35 days from the beginning of immunization.
Example 3. The study of the ability of the target strain to induce a cellular immune response.
The reaction besttransport. The reaction besttransport of splenocytes carried out at 0, 14, 21, 28, 35 and 48 days after the injection of Salmonella according to a previously described method . As a specific antigen using an inactivated HIV-1, as a non - specific antigen-virus enteritis of mink. The hole in the tablet make 100 ál of each component. The antigen concentration of 2 μg/ml as a mitogen use of concanavalin a (manufactured by Sigma, USA) at a concentration of 5 µg/ml Cells, stimulirovan rburg) at a 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.1). The results are shown in table. 1, indicate that immunization with cells of Salmonella enteriditis E-23/pGEX-2T-TBI causes the formation of a clone of memory cells, because the index of stimulation of cells in vitro specific HIV proteins significantly increased (P<0.01) at 14 days compared with the figures for 0 day (day of immunization). This indicator remains at a high level throughout the observation period (48 days).
Example 4. 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 3.0-5.0105cells 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 HIV-A (X4), obtained from the collection of strains of human immunodeficiency virus is otci inactivate in a water bath at a temperature of 56°C for 30 minutes 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 incubated for 1 h at 37°C. After incubation, make a 100 μl suspension of sensitive cells MT-4 in the amount of 90-100103cells/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. 2.
The data presented in the table.2 suggests that formed after introduction of the recombinant strain antibodies have neutralizing activity.
Thus, konstruirovanie humoral (like the system, and local) and cellular immune response to HIV-1. The proposed recombinant strain can be used to construct a live vaccine against HIV-1.
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Recombinant attenuated strain of Salmonella enteriditis E-23/pGEX-2T-TBI deposited in to atopy several proteins of HIV-1.