RU2194075C2 - Recombinant plasmid dna ptbi-hbsag comprising chimeric gene tbi-hbsag under control of smallpox vaccine promoter p7,5k and strain of recombinant smallpox vaccine inducing immune response against hiv and human hepatitis b in animal body - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, molecular biology, virology, medicine. SUBSTANCE: invention presents strain of recombinant smallpox vaccine and recombinant plasmid DNA pTBI-HBsAg. Recombinant plasmid DNA encodes nine immunogenic epitopes of two main structural proteins of human immunodeficiency virus of type-1. Recombinant strain of smallpox vaccine modified with plasmid pTBI-HBsAg induces T- and B-cellular immune response against HBsAg and HIV-1 in animals vaccinated by its. Strain can be used as a base for mixed vaccine against HIV-1 and human hepatitis B virus and orthopoxviruses. EFFECT: valuable antiviral properties of strain. 3 dwg, 3 tbl, 3 ex

Description

 The invention relates to biotechnology and, in particular, to genetic engineering and is a strain of recombinant vaccinia virus, which causes the synthesis of a structural protein of hepatitis B virus (HBsAg), in which the pre-S2 region is replaced by an artificial polypeptide encoding nine immunogenic epitopes of two main structural proteins of human immunodeficiency virus type 1 (HIV-1) in infected cells, and the T- and B-cell immune response against HBsAg and HIV-1 in vaccinated laboratory animals.

 Human hepatitis B virus is widespread throughout the world. The treatment and prevention of hepatitis B is of great importance for medicine, since it is apparently the most common culprit in chronic liver diseases, including hepatocellular carcinoma in humans [1].

 The human immunodeficiency virus causes an ever-spreading epidemically dangerous disease, the methods of treatment and prevention of which are not yet available and it often ends in death. Until 1995, the territory of the former Soviet Union and Eastern European countries was considered a zone with a low level of HIV infection in the population. After 1995, when outbreaks of HIV infection occurred first in Ukraine, and then in Belarus and the Russian Federation, these countries began to be characterized as having the highest relative increase in the number of HIV infections [2].

 Creating effective vaccines against these viral infections is an urgent task. One approach to solving this problem is to use the genomes of certain viruses as vectors for the expression of protective antigens of pathogenic viruses in mammalian cells.

 Several vectors have been described in the current literature that allow the expression of foreign genes in eukaryotic cells. The vectors created to date based on retroviruses, adenoviruses, papillomaviruses, and papovaviruses have a number of significant drawbacks: the low rate of reproduction in cell culture, the small capacity relative to the size of the inserted genetic sequences, the inability to reproduce in the body of certain animals.

 Some progress in the development of recombinant vaccines has been achieved using the vaccinia virus (BOB). BOB is widely used to express foreign proteins, present them to the immune system and determine the role of an individual antigen in the immune response [3, 4]. Recombinant BOBs expressing individually the individual HIV-1 proteins: gpl60, gpl20, tat and reverse transcriptase were constructed [5–9]. The resulting recombinant BOB were used to immunize laboratory animals and identify HIV-1 proteins that can induce the neutralization of antibodies. In addition, this made it possible to evaluate the relative contribution and interaction of the humoral and cellular components of immunity, since BOB is an effective inducer of both T- and B-cell immune responses, i.e., it actually plays the role of an adjuvant. The results of the studies indicate that recombinant BOB expressing immunologically significant HIV-1 antigens are capable of inducing a humoral and cellular immune response, which is crucial for the development of protective immunity and, therefore, for creating a vaccine.

 However, in none of the published works using recombinant BOB it was not possible to induce a full-fledged protective immune response against HIV infection. This is apparently due to the low immunogenicity of individual viral proteins. To induce a full-fledged response to the immune system, it seems necessary to present the antigenic determinants of several viral proteins simultaneously and in a more immunogenic form than the one that these determinants have in natural viral proteins.

 A productive approach to overcoming the difficulties that arise is the creation of supramolecular virus-like protein structures, on the surface of which various antigenic determinants are repeatedly presented. In this case, it is necessary to have a carrier protein, which under certain conditions is capable of self-assembly of virus-like particles. A suitable candidate for the role of such a carrier protein is the surface virion protein of human hepatitis B virus HBsAg (also called S), which, when synthesized in eukaryotic cells, forms highly immunogenic spherical particles with a diameter of about 22 nm (S-particles) [10]. One of the forms of the surface protein called npeS2-S, when synthesized in eukaryotic cells, forms supramolecular structures, on the surface of which the sequence of preS2 is repeatedly represented. During vaccination, such particles induce an even more pronounced immune response against hepatitis B in comparison with S-particles [11].

Known recombinant plasmid DNA and recombinant BOB expressing a peptide of size 15 amino acid residues from the gp120 HIV-1 (peptide P18IIIB) in the composition of the chimeric protein hemagglutinin of influenza virus N-1 (HA) [12, prototype]. Immunofluorescence studies with anti-H1N1 antisera of the influenza virus and P18IIIB peptide reveal intracellular chimeric protein, but the P18IIIB peptide is not found on the outer surface of infected cells. These recombinants mainly determine the cytotoxic specificity of CD8 ⁺ T-lymphocytes to P18IIIB and HA of influenza virus; there is a stimulation of the production of specific antibodies against HA, but not against P18IIIB. However, such T-lymphocytes are able to lyse cells infected with HIV-1.

 The disadvantage of the implemented approach considered above is that the chimeric protein synthesized by recombinant BOB does not secrete from infected cells and does not form supramolecular formations, which can potentially be highly immunogenic.

 One of the promising directions in the development of effective and safe new generation vaccines is the use of synthetic polypeptides bearing protective T and B epitopes. To increase the effectiveness of synthetic vaccines, such techniques are used as repeated repetition of the protective epitope, combining several B-epitopes in a single chain, suturing T-determinants to the B-epitope, and expression of the epitope in a carrier protein. Moreover, it is believed that vaccines that are effective in HLA heterogeneous antigens of the human population should contain at least several T-determinants and several B-determinants [13].

 In Russia, a universal approach to the construction of synthetic vaccines in the form of proteins with the optimal composition of the epitopes of the infectious agent and with a predetermined tertiary structure has been developed. As part of this approach, the design of a 4-helical protein candidate for HIV-1 vaccines was carried out [14, 15]. This protein is called TBI.

 An immunogen protein (TBI) includes the following HIV-1 T-cell epitopes: 105-117 HEDIISLWDQSLK, 421-437 KQIINMQEVGKAMYA, 827-841 DRVIEVVQGAYRAIR from ENV, 291-305 EPFRDYVDRFYKTLR from GAG (B) strain. There are 5 different neutralizing B epitopes in the protein: 255-265, 309-317, 730-744 from ENV and 92-109, 351-361 from GAG. Protein TBI - a candidate for HIV vaccines - meets the basic requirements for effective vaccines (multivalence, conformational adequacy of B-epitopes, the absence of undesirable epitopes).

 An object of the invention is the creation of a strain of recombinant vaccinia virus that expresses in vivo an artificial gene encoding the main experimentally localized epitopes of the HIV virus that can induce the humoral and cellular immune responses against HIV-1, in the composition of the chimeric protein, the carrier protein of which is HBsAg, with the ability to self-assemble into highly immunogenic virus-like particles.

- BamHI-ClaI фрагмента генома вируса гепатита В, кодирующего поверхностный вирионный белок S (HBsAg), размером 1226 п.н.;
Для получения целевого рекомбинантного штамма вируса осповакцины, обеспечивающего экспрессию in vivo исскуственного гена TBI, кодирующего основные антигенные эпитопы ВИЧ-1 в составе химерного HBsAg, описанную плазмиду р TBI-HBsAg встраивают в ген тимидинкиназы коммерческого штамма ЛИВП вируса осповакцины под контролем промотора 7.5К. Полученный в результате рекомбинантный штамм ВОВ, обозначенный VR18, содержит в геноме, по сравнению с исходным штаммом ЛИВП, дополнительную последовательность, состоящую из:
- фрагмента длиной 284 н. п., включающего район промотора гена белка 7.5К;
- фрагмента длиной 1675 н.п., содержащего ген HBsAg, в котором вместо пре-S2-области находится последовательность гена исскуственного белка TBI.The problem is solved by constructing a recombinant plasmid DNA p TBI-HBsAg of 6322 bp in size, carrying the chimeric HBsAg gene, in which, instead of the pre-S2 region, there is a sequence encoding the gene for the artificial TBI protein under the control of the vaccine protein protein 7.5K, vaccinia virus, and consisting of (Figs. 1-3):
- ClaI-BamHI vector fragment of the plasmid p7.5S113 DNA with a size of 4647 bp providing for the selection and propagation of a hybrid plasmid in the cells of the bacterium E. coli, as well as the recombination insertion and expression of foreign genes in mammalian cells as part of the thymidine kinase gene of the vaccinia virus;
- Bg1II-PstI synthetic oligonucleotide of 8 bp in size, ensuring the correct docking of the TBI gene with the translation initiation site, located in the sequence of a vector plasmid having the structure
5'-GATCTGCA-3 ';
- PstI-Kpn2I DNA fragment 432 bp in size, encoding the artificial TBI polypeptide, immunogenic against HIV-1;
- Kpn2I-BgIII synthetic 17 bp DNA fragment connecting the genes of the TBI and S proteins in the correct translation frame, which ensures the synthesis of the TBI-HBsAg chimeric protein with the structure

- BamHI-ClaI fragment of the hepatitis B virus genome encoding surface virion protein S (HBsAg), 1226 bp;
To obtain the target recombinant strain of the vaccinia virus, which provides for the in vivo expression of the artificial TBI gene encoding the main antigenic HIV-1 epitopes in the chimeric HBsAg, the described plasmid p TBI-HBsAg is inserted into the thymidine kinase gene of the commercial Livp vaccine vaccine 7.5 vaccine control vaccine. The resulting recombinant BOB strain designated VR18 contains, in the genome, in comparison with the original strain of LIVP, an additional sequence consisting of:
- fragment length 284 N. p., including the region of the promoter of the 7.5K protein gene;
- a 1675 bp fragment containing the HBsAg gene, in which instead of the pre-S2 region is the gene sequence of the artificial TBI protein.

 The nucleotide sequence of the inserted fragment is shown in FIG. 1.

 The resulting strain of recombinant vaccinia virus VR18 is characterized by the following features.

 Morphological signs.

 Strain VR18 has the properties of a typical member of the orthopoxvirus family. Virions of the strain have a size of 200 • 300 nm, a characteristic briquette-like shape and according to electron microscopy do not differ from the original strain of LIVP.

 Physiological and biochemical characteristics and cultural properties of the strain.

 The DNA of strain VR18 has a length of about 190 kb, when a HindIII restriction analysis of this viral DNA replaces the HindIII-K fragment of the DNA of the LIVP strain (5008 bp), a fragment of a higher molecular weight (6967 bp) is detected , according to DNA-DNA blot hybridization containing the sequences of the HBsAg and TBI genes.

When VR18 is propagated in developing chicken embryos, the nature of the lesions on the chorioallantoic membranes is the same as when the initial strain of LIVP is multiplied. In addition, the strain VR18 does not differ from the initial one in the monolayer productivity of transplantable cell lines CV-1 and H-143TK ^- . In contrast to the LIVP strain, the recombinant VR18 possesses the TK ^- phenotype, is not capable of propagation on the transplanted cell line H-143TK ^- in the presence of 25 μg / ml bromodeoxyuridine.

 Pathogenicity for animals.

 The study of the properties of strain VR18 in laboratory animals showed that in standard tests for toxicity, safety, necrotic activity and specificity, the recombinant strain VR18 does not differ from the original strain of LIVP.

 Immunobiological properties of strain VR18.

Strain VR18 is highly immunogenic, it elicits humoral (antibody) and T-cell immune responses against HBsAg and HIV when immunizing BALB / c mice at a dose of 1 • 10 ⁶ PFU / animal. Antibodies are determined by enzyme-linked immunosorbent assay, and the cellular immune response is determined by the blast transformation of splenocytes.

 The main difference between strain VR18 and LIVP is the ability to synthesize the chimeric protein TBI-HBsAg during infection of mammalian cell cultures sensitive to BOB (for example, CV-1). Indication of expression is carried out using enzyme-linked immunosorbent assay.

 The strain of the described recombinant VR18 was deposited in the Collection of microorganism cultures of the State scientific center of virology and biotechnology "Vector" under the number V-312 from 06.10.2000

The study of the expression of the chimeric protein TBI-HBsAg was carried out in animal cells CV-1 and H-143TK ^- when they were infected with recombinant BOB VR18 obtained from the above components. The nucleotide sequence of the TBI-HBsAg chimeric gene inserted into the BOB genome was confirmed by sequencing. In addition, a T- and B-cell immune response was studied in BALB / c mice immunized with recombinant BOB VR18.

 The determination of the amount of HBsAg in the samples was carried out by enzyme immunoassay. At the same time, HBsAg production reached 28-27 ng in 24 hours in terms of CV-1 or H-143TK cells per million cells (infection multiplicity 0.1 PFU / cell; cultivation time - 48 hours; see Table 1).

 The amount of TBI protein was determined by competitive enzyme-linked immunosorbent assay using mouse serum against TBI protein of bacterial origin. Production of TBI protein reached 11 ng and 5.8 ng in 24 hours, calculated per million cells of CV-1 and H-143 TK, respectively (multiplicity of infection 0.1 PFU / cell; cultivation time - 48 hours; see table 1 )

 Attempts to detect HBsAg in infected cell cultures as part of a chimeric protein using a commercial test system were unsuccessful. Regardless of cell type, multiplicity of infection, and time of incubation, HBsAg was not detected by the test system. This suggests that the antigenic determinant “a” of HBsAg, according to which the used test system works, is screened as part of the chimeric protein by the synthetic TBI protein integrated instead of the pre-S2 region. It can be assumed that when vaccinating animals with such a recombinant vaccine, the humoral immune response to HBsAg will be slightly reduced and increased by the TBI protein. Experiments on immunization of BALB / c mice confirmed this assumption (see Table 2). In animals immunized with the virus 113 preparation (recombinant BOB preparation containing the full HBsAg gene with the pre-S2 region), only antibodies against HBsAg are formed. In contrast, in animals immunized with recombinant BOB VR18, antibodies are formed primarily against the HIV antigen (HIV) at all observation times and slightly to HBsAg, which is observed only on day 35.

 Thus, the essence of the invention lies in the fact that a recombinant BOB was constructed expressing the chimeric HBsAg protein, in which the pre-S2 region was replaced by a synthetic TBI protein containing nine T and B cell epitopes of the main structural proteins of HIV-1 virus. HBsAg molecules are able to assemble into virus-like particles with high immunogenicity, in which the pre-S2 region is exposed on the surface. When replacing this region with the synthetic gene of the candidate protein for AIDS / HIV vaccines, TBI molecules are exposed on the surface of virus-like particles in the chimeric protein, which allows to obtain a high level of immune response. The latter has been demonstrated in animal experiments.

The invention is illustrated by the following drawings:
FIG. 1. The nucleotide sequence of the artificial gene of the chimeric protein TBI-HBsAg. The initiating and terminating triplets of chimeric protein translation are circled. The regions of the TBI, HBsAg genes and their linker were marked (the sequence is shown in bold). Hydrolysis sites for certain restriction enzymes are indicated.

 FIG. 2. Recombinant plasmid DNA pTBI-HDsAg and scheme for its construction.

 Figure 3. Scheme for docking the nucleotide coding sequences of TBI and HBsAg proteins between each other and the translation initiation site of the vector plasmid p7.5S113 using synthetic oligonucleotides (in bold). Sticky single-stranded ends formed after hydrolysis by the corresponding restriction enzymes, the names of which are given above or below the sequence, are indicated by broken lines.

 The invention is illustrated by the following examples.

 Example 1. Construction of recombinant plasmid DNA p18.

 To construct the target plasmid p TBI-HBsAg, the plasmid p7,5S113 [16], which was obtained on the basis of the pVAR15 vector [17] by inserting the HBsAg gene under the control of the P7.5K promoter, is used. As the source of the TBI gene, plasmid pUCSII is used [15]. 30 μg of the indicated plasmid was digested with co-hydrolysis with restriction enzymes PstI and Kpn21; the obtained DNA fragment encoding TBI was isolated by agarose gel electrophoresis followed by elution on a dialysis membrane and ligated under standard conditions with hydrolyzed restriction enzyme BamHI and treated with phosphatase-11 synthesized plasma nucleotide adapters (figure 2, 3). Competent cells of E. coli strain JM103 are transformed with a ligase mixture and plasmid DNA is isolated from clones grown on ampicillin medium and subjected to restriction analysis using PstI, EcoRI, Xhol, BamHI enzymes. The correspondence of the nucleotide sequence of the insert in the selected recombinant plasmid theoretical is checked by the Maxam-Gilbert method (Fig. 1). The resulting plasmid is designated p TBI-HBsAg.

 Example 2. Obtaining recombinant vaccinia virus VR18.

Culture vessels with a monolayer of green monkey monkey kidney cells CV-1 infect the smallpox vaccine virus of the LIVP strain with a multiplicity of 0.05-0.1 PFU / cell. The adsorption of the virus on the cells is carried out for 1 h at 37 ^{° C.} , then the non-adsorbed virus is removed and the cells are washed twice with serum-free DMEM culture medium (m). An equal volume of lipofectin (0.2 mg / ml) was added to p18 plasmid DNA (0.05 mg / ml, 25 μl) and the prepared mixture was left for 15 min at room temperature to form liposomes. The formed liposomes are diluted with 1 ml of serum-free DMEM (m) and transferred to prepared cells. After 16 hours of incubation at 37 ^° C, 4 ml of DMEM medium (m) containing 2% fetal serum and 80 u / ml gentamicin was added to the cells and the incubation was continued for 24 hours. Cells after transfection were frozen, thawed and the progeny were cloned on the cells transplantable culture of N-143TK ^- under an agar coating containing 25 μg / ml of 5-bromodeoxyuridine. After 48 hours of incubation at 37 ^{° C.} in an atmosphere containing 5% CO ₂ , a second coating was applied containing 0.01% neutral red dye. Staining of the cell monolayer for visualization of viral plaques is carried out for 3 hours at 37 ^o C in an atmosphere containing 5% CO ₂ . Viral clones from individual plaques are grown in 24-well plates and analyzed by dot-hybridization DNA-DNA for the presence of a foreign sequence. Hybridization-positive clones are re-cloned and preparatively produced on a CV-1 cell culture.

 Example 3. Determination of the expression level of the chimeric protein TBI-HBsAg.

A monolayer of CV-1 or H-143TK cells ^- in the wells of 6-well plates is infected with recombinant vaccinia viruses 113 (contains the HBsAg gene with the pre-S2 region) and VR18 (contains the chimeric TBI-HBsAg gene) with a multiplicity of 0.1 or 0, 01 PFU / cell. Infected cells are incubated for 24 or 48 hours at 37 ^{° C.} in an atmosphere containing 5% CO ₂ . In all experiments on cell cultures, a DMEM culture medium (m) containing 2% fetal serum and 100 μg / ml of penicillin and streptomycin was used. After incubation, the cells are destroyed by double freezing-thawing. Cell debris is pelleted by centrifugation at 600 g for 10 minutes. Supernatants are used to determine the amount of HBsAg and TBI protein by enzyme-linked immunosorbent assay.

 The determination of the amount of HBsAg in the samples is carried out by enzyme-linked immunosorbent assay according to a calibration made by diluting the HBsAg preparation with a known concentration using a commercial test system (ZAO Vector-Best, Koltsovo). The determination of the amount of TBI protein in samples is carried out by competitive enzyme-linked immunosorbent assay using mouse serum against TBI protein of bacterial origin.

The results of determining the titers of recombinant viruses and the levels of expression of HBsAg and TBI protein in cell cultures of CV-1 and H-143TK ^are presented in Table 1. As can be seen, the yields of both recombinant viruses are comparable for cells of CV-1 and H-143TK ^- and differ in 2-10 times depending on the multiplicity of infection and the time of incubation of the cells after infection. HBsAg expression levels for recombinant virus 113 and TBI protein for recombinant VR18 virus correlate well with titers of the corresponding viruses. HBsAg production reaches 28-27 ng in 24 hours in terms of a million cells CV-1 or H-143TK ^- (multiplicity of infection 0.1 PFU / cell; cultivation time - 48 hours). The production of TBI protein reaches 11 and 5.8 ng in 24 hours in terms of a million cells CV-1 and H-143TK ^, respectively (multiplicity of infection 0.1 PFU / cell; cultivation time - 48 hours).

 Attempts to detect HBsAg in the composition of the chimeric protein using a commercial test system were unsuccessful. Apparently, the antigenic determinant “a” of HBsAg, according to which the test system works, is shielded by the integrated TBI protein as part of the chimeric protein. Based on these data, we suggest that when vaccinating animals with the recombinant VR18 virus, the humoral immune response to HBsAg will be slightly reduced and increased by the TBI protein. Subsequent experiments on immunization of BALB / c mice confirmed this assumption (see Table 2).

 Example 4. The study of the immunogenic properties of the recombinant virus VR18.

 In the experiment, BALB / c mice (halotype H-2b) were used, weighing 12 grams in an amount of 30 animals, which were contained in a standard diet.

 The study of the immunogenic properties of the preparations of recombinant viruses 113 and VR18 was carried out on 0, 21 and 35 days after immunization (animals were divided into 2 groups). Immunization was carried out subcutaneously in the root of the tail (0.1 ml) with a dose of 10 PFU. For the indicated periods, indicators of humoral immunity were studied in an enzyme-linked immunosorbent assay using strips with sorbed HIV and HBsAg antigens and a species-specific conjugate of rabbit immunoglobulins against mouse IgG from Sigma.

 As can be seen from the data shown in table 2, in animals immunized with the preparation of the virus 113, antibodies only to HBsAg are formed. In contrast, in animals immunized with the VR18 virus antibody, antibodies are formed primarily against the HIV antigen (at all observation times) and slightly against HBsAg, which is observed only on day 35.

Indicators of cellular immunity were studied in the reaction of blasttransformation of splenocytes. To carry out this reaction, cells are separated from the spleens of animals of the experimental groups, which are washed twice in the culture medium (RPMI 1640 medium with 2 mM L-glutamine, 5 • 10 ^-5 M 2-mercaptoethanol, 80 mg / l gentamicin, 5% heated at 56 ^o With fetal serum). After that, the concentration of splenocytes was adjusted to 2 million / ml and used for the blast transformation reaction in 96-well plates. 2 x 10 ⁵ cells are added to the wells in a volume of 100 μl. As antigens use:
- lysate of the HIV virus at a concentration of 2 μg / ml;
- TBI protein at a concentration of 2 μg / ml;
- inactivated antigen of mink enteritis virus at a concentration of 2 μg / ml.

 Concanavalin A (Sigma) at a concentration of 10 μg / ml and E. coli lipopolysaccharide (serotype 055: B5, Sigma) at a concentration of 25 μg / ml were used as mitogens. Antigens and mitogens are added in a volume of 100 μl to the cell wells, using 4 wells for each antigen or mitogen.

Splenocytes stimulated by antigens are cultured for 96 hours, and stimulated by mitogens - 78 hours. 18 H before the end of cultivation, add ³ H-thymidine at a concentration of 2 μCi per well. At the end of the cultivation period, the cells are frozen, thawed and harvested on a Cell-Hawester (Flow UK). The radioactivity count is carried out in a toluene scintillator on a "Mark III" counter. The proliferative activity of splenocytes is estimated by the absolute numbers of stimulated and unstimulated splenocytes in the dynamics of observation and the stimulation index, defined as the ratio of the count of stimulated splenocytes to the count of unstimulated splenocytes.

 As follows from the results presented in table. 3, in animals immunized with virus 113, on day 21 and 35 after immunization, marked proliferation of splenocytes in vitro was observed upon stimulation with the HBsAg protein, and there was no proliferative response to the stimulation by HIV and mink enteritis virus. This confirms the specific proliferation of HBsAg and indicates the presence of a gene of this protein in the preparation of virus 113. In animals immunized with the preparation of the engineered virus VR18, proliferative activity of splenocytes in response to stimulation as HIV proteins is observed throughout the observation dynamics - 21 and 35 days and HBsAg. This indicator significantly exceeds the same indicator in intact animals (0 day). The specificity of the reaction of splenocytes of this group is confirmed by the absence of pronounced proliferation during stimulation of the mink enteritis with antigen.

 Thus, a new recombinant vaccinia virus strain was obtained expressing the chimeric HBsAg protein, in which the pre-S2 region was replaced by a synthetic TBI protein containing protective T and B cell epitopes of type 1 human immunodeficiency virus and causing an immune response against HIV viruses and hepatitis B in animals. The resulting recombinant virus can be used as a candidate for the creation on its basis of a multivalent vaccine against HIV-1, human hepatitis B virus and orthopoxviruses. It is expected that the vaccine obtained on the basis of this approach will be characterized by high immunogenicity against the three above-mentioned viral infectious diseases simultaneously, and with respect to HIV-1, the proximity of B-conformation conformations in the vaccine and viral antigen, and efficiency in the human population heterogeneous for HLA antigens , stimulation of the response to different B-epitopes, the absence of undesirable epitopes of HIV-1 proteins, as well as the ability to optimize protective properties by changing the composition and location of T- and B-determin m and processability receipt.

List of references
1. Szmuness W. // Prog. Med. Virol., 1978, v.24, p.40-69.

 2. Kozlov A.P. // Russian Journal of HIV / AIDS and Related Problems, 2000, v. 4, 1, pp. 11-14.

 3. Moss B., and Flexner C. // Annu. Rev. Immunol., 1987, v. 5, p. 305.

 4. Moss B. // Seminars in Virology, 1992, v. 3, p. 277.

 5. Falkner F.G., et al. // Virology, 1988, v. 164, p. 450.

 6. Flexner C., et al. // Virology, 1988, v. 166, p. 339.

 7. Takahashi H., et al. // Proc. Natl. Acad Sci. USA, 1988, v. 85, p. 3105.

 8. Walker B. D., et al. // Science, 1988, v. 240, p. 64.

 9. Willey R. L., et al. // J. Virol., 1988, v. 62, p. 139.

 10. Heerman K.H., et al. // J. Virol., 1984, v. 52, p. 396.

 11. Prange R., et al. // J. Gen. Virol., 1995, v. 76, p. 2131.

 12. Chiba M. // Arch. Virol., 1999, v. 144, p. 1469.

 13. Berzofsky J. A., et al. // Curr. Top Microbiol. Immunol., 1986, v. 130, p.13.

 14. Eroshkin A.M., et al. // Protein Eng., 1993, v. 6, p. 997.

 15. Eroshkin A.M., et al. // Protein Eng., 1995, v. 8, 2, p. 167.

 16. Belyaev A.S. et al. // Dokl. USSR Academy of Sciences, 1990, v. 314, p. 488.

 17. Fodor I.I. et al. // Biotechnology, 1987, vol. 3, p. 302.

Claims (2)

1. Recombinant plasmid DNA pTBI-HBsAg encoding the chimeric TBI-HBsAg gene with a molecular weight of 4.2 Mda and a size of 6322 bp containing ClaI-BamHI vector fragment of plasmid p7.5S113 DNA size 4647 p. N. with the nucleotide sequence of the vaccinia virus thymidine kinase gene with the p7.5 vaccinia virus promoter integrated therein; BglII-PstI synthetic oligonucleotide 8 bp providing the correct docking of the TBI gene with the translation initiation site located in the sequence of the vector plasmid having the structure 5'-GATCTGCA-3 '; PstI-Kpn21 DNA fragment 432 bp encoding an artificial TBI polypeptide immunogenic against HIV-1; Kpn21-BglII synthetic DNA fragment of 17 bp connecting the genes of the TBI and S proteins in the correct translation frame, having the structure

BamHI-ClaI fragment of the hepatitis B virus genome encoding surface virion protein S (HBsAg), size 1226 bp ; genetic markers: bla-gene of ampicillin resistance - β-lactamase gene, which determines ampicillin resistance during transformation of E. coli cells; TK - the interrupted thymidine kinase gene of smallpox vaccine virus, causing the TK phenotype during recombination insertion into the genome of the smallpox vaccine virus; unique restriction sites: HindIII - position 1; KpnI - 1303; Kpn21 - 1453; XhoI - 1462.  2. The strain of recombinant vaccinia virus, deposited in the research institute of CMC under the number V-312, is the producer of the chimeric protein TBI-HBsAg.

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