Recombinant plasmid dna ptbi-hbsag containing chimeric gene tbi-hbsag under the control of the promoter p7.5k of vaccinia virus and recombinant strain of vaccinia virus, causing immune response against hiv and hepatitis b human animals

 

(57) Abstract:

The invention relates to biotechnology, particularly genetic engineering. The invention is a recombinant strain of vaccinia virus and recombinant plasmid DNA pTBI-HBsAg. Recombinant plasmid DNA encodes nine immunogenic epitopes of two major structural proteins of human immunodeficiency virus type-1. Recombinant strain of vaccinia virus, modified plasmid pTBI-HBsAg, causes T - and b-cell immune response against HBsAg and HIV-1 vaccinated animals they. The strain can be used as the basis for multivalent vaccines against HIV-1, hepatitis b virus human and orthopoxviruses. 1 C.p. f-crystals, 3 ill., 3 table.

The invention relates to biotechnology and, in particular, to genetic engineering and is a recombinant strain of vaccinia virus, causing the synthesis of structural protein of hepatitis b virus human (HBsAg), in which the pre-S2 region is replaced by an artificial polypeptide encoding nine immunogenic epitopes of two major structural proteins of human immunodeficiency virus type 1 (HIV-1) infected cells, and T - and b-cell immune response against HBsAg and HIV-1 in th world. Treatment and prevention of hepatitis b is of great importance for medicine, as it is probably the most common culprit of chronic liver diseases, including hepatocellular carcinoma in humans [1].

The human immunodeficiency virus is increasingly spreading epidemically dangerous disease, 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 Europe was considered an area with low levels of HIV infection in the population. After 1995, when outbreaks of HIV infection have occurred first in Ukraine, and then in Belarus and in the Russian Federation, these countries have been characterized as having the highest relative increase in the number of cases of HIV infection [2].

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

In modern literature describes several vectors, allowing expressions, adenoviruses, papillomaviruses and papovaviruses have a number of disadvantages: low speed reproduction in cell culture, small capacity relative to the size of the embedded genetic sequences, inability to multiply in the body or those of other animals.

Good progress in the development of recombinant vaccines have been achieved with the use of vaccinia virus (BOB). BOB is widely used for the expression of foreign proteins, presenting them to the immune system and determine the role of individual antigen in the immune response [3, 4]. Were constructed recombinant BOB, separately expressing individual proteins of HIV-1: gpl60, gpl20, tat and reverse transcriptase [5-9]. The obtained recombinant WWII were used for immunization of laboratory animals and identify proteins of HIV-1, can cause the induction of neutralizing antibodies. In addition, it gave the opportunity to assess the relative contribution and interaction of humoral and cellular component of immunity, because BOB is an effective inducer of T-and b-cell immune response, i.e. actually performs the role of adjuvant. The results of these studies confirmed the th induction of humoral and cellular immune response, what is crucial for the development of protective immunity and, therefore, to create a vaccine.

However, in none of the published studies using recombinant BOB failed to induce full protective immune response against HIV infection. Apparently this is due to the low immunogenicity of individual viral proteins. For full induction of the immune response system, apparently, it is necessary to present antigenic determinants of multiple viral proteins and more immunogenic than that of these determinants in the natural viral proteins.

A productive approach to overcome the difficulties encountered is the creation of virus-like supramolecular protein structures on the surface which repeatedly presents certain antigenic determinants. It is necessary the presence of carrier protein, which is under certain conditions capable of self-Assembly of virus-like particles. A suitable candidate for such a protein carrier is surface birinyi protein of hepatitis b virus human HBsAg (called S), which during the synthesis in eukaryotic cells forms vicecommodore is in the synthesis in eukaryotic cells forms a supramolecular structure, on the surface, which repeatedly presents the sequence peS2. Such particles induce when vaccination is even more pronounced immune response against hepatitis b compared with S-particles [11].

Known recombinant plasmid DNA and recombinant BOB, expressing the peptide is 15 amino acid residues from the structure of gp120 of HIV-1 (peptide P18IIIB) as part of a chimeric protein of hemagglutinin of influenza virus N-1) [12, prototype] . Immunofluorescence studies with antisera against H1N1 influenza virus peptide P18IIIB allow to identify chimeric protein is intracellular, but the peptide P18IIIB not detected on the outer surface of infected cells. These recombinants determined mainly cytotoxic specificity of T-lymphocytes CD8+to P18IIIB and influenza virus; observed stimulation of production of specific antibodies against it, but not against P18IIIB. However, these T cells are able to lyse cells infected with HIV-1.

The disadvantage of the above above mentioned approach is synthesized recombinant BOB chimeric protein is not secreted from infected cells and does not form supramolecular structures, which potentially can be vysokogo generation is the use of synthetic polypeptides, bearing protective T and b epitopes. To improve the efficiency of synthetic vaccines use techniques such as repetition of a protective epitope, the connection in one chain of several b-epitopes, proshivku to the b-epitope T-determinants, the expression of the epitope in the composition of a protein carrier. It is assumed that the vaccine is effective in heterogeneous HLA antigens of the human population, must contain at least a few T-determinants and several In-determinants [13].

In Russia developed a universal approach to the design of synthetic vaccines in the form of proteins with optimal composition of the epitope of an infectious agent with a pre-defined tertiary structure. In this approach, implemented design 4-helical protein candidate vaccines against HIV-1 [14, 15]. This protein is named after TBI.

Protein is the immunogen (TBI) includes the following T-cell epitopes of HIV-1: 105-117 HEDIISLWDQSLK, 421-437 KQIINMQEVGKAMYA, 827-841 DRVIEVVQGAYRAIR of ENV, 291-305 EPFRDYVDRFYKTLR of GAG (strain MN). In the protein are present 5 different neutralizing b-epitopes: 255-265, 309-317, 730-744 of ENV and 92-109, 351-361 of the GAG. Protein TBI - candidate vaccines against HIV - meets the basic requirements of an effective vaccine (multivalently conformation is creating a strain of recombinant vaccinia virus, performing expression in vivo artificial gene encoding the major experimentally localized epitopes of the HIV virus that can cause the induction of humoral and cellular immune response against HIV-1, comprising a chimeric protein, a protein carrier which is HBsAg, with the ability to self-Assembly into highly immunogenic virus-like particles.

The problem is solved by constructing recombinant plasmid DNA p TBI-HBsAg size 6322 p. N., bearing chimeric HBsAg gene, in which instead of the pre-S2 region is a sequence encoding a gene of artificial protein TBI, under the control of the promoter of the protein 7.5 To vaccinia virus, and consists of (Fig.1-3):

- ClaI-BamHI vector fragment of DNA plasmids p7.5S113 size 4647 p. N. , providing breeding and reproduction of hybrid plasmids in cells of the bacterium E. coli, as well as recombination integration and expression in mammalian cells of foreign genes in the gene composition timedancing of vaccinia virus;

- Bg1II- > PST synthetic oligonucleotide size 8 p. N., providing the correct dock gene TBI with the site of translation initiation, in sequence, a vector plasmid having the structure


- Kpn2I-BgIII synthetic DNA fragment size 17 p. N., linking genes to proteins TBI and S in the right frame broadcast that provides a synthesis of the chimeric protein TBI-HBsAg, having the structure

< / BR>
- BamHI-ClaI fragment of the genome of hepatitis b virus encoding surface virginny protein S (HBsAg), size 1226 p. N.;

To obtain the target recombinant strain of vaccinia virus, which ensure expression in vivo artificial gene TBI, encoding the major antigenic epitopes of HIV-1 comprising chimeric HBsAg described plasmid R TBI-HBsAg is inserted into the gene timedancing commercial strain LIVP of vaccinia virus under the control of the promoter 7.5 K. the resulting recombinant strain of WWII marked VR18, contains in its genome, in comparison with the original strain LIVP, additional sequence consisting of:

fragment length 284 N. p., comprising the promoter region of the gene of the protein TO 7.5;

fragment length 1675 N. p., containing the gene for HBsAg, in which instead of the pre-S2 region is the sequence of a gene of artificial protein TBI.

The nucleotide sequence of the embedded fragment shown in Fig. 1.

The resulting recombinant strain of vaccinia virus VR18 across obichnogo representative of the family of orthopoxviruses. The strain virions have a size of 200300 nm, characteristic biketours the form and according to electron microscopy does not differ from the original strain LIVP.

Physiological and biochemical characteristics and cultural properties of the strain.

DNA strain VR18 has a length of about 190 T. p. N., HindIII-restriction analysis of this viral DNA instead of HindIII-K fragment of the DNA of strain LIVP (5008 p. N.) revealed a fragment of a larger molecular weight (6967 p. N.), according to DNA-DNA blot hybridization of sequences of genes HBsAg and TBI.

When the reproduction VR18 on developing chicken embryos character defeats at chorioallantois shells is the same as when reproduction of the original strain LIVP. In addition, the strain VR18 not differ from the original in productivity in the monolayer transplantable cell lines CV-1 and N-D-. Unlike strain LIVP recombinant VR18 has the phenotype TC-unable to reproduce on transplantable cell line N-D-in the presence of 25 μg/ml of bromosuccinimide.

Pathogenicity for animals.

The study of the properties of strain VR18 on laboratory animals showed that the standard tests for toxicity, safety, necrotic activity of the ski properties of strain VR18.

Strain VR18 vicecommodore, it causes humoral (or antibody-based test) and T-cell immune responses against HBsAg and HIV immunization of mice BALB/c mice at a dose of 1106PFU/animal. Antibodies detect enzyme-linked immunosorbent assay, and cellular immune response in reaction besttransport of splenocytes.

The main difference of strain VR18 from LIIT is the ability to synthesis of a chimeric protein TBI-HBsAg infected cultures of mammalian cells, sensitive to BOB (for example, CV-1). The display expression is carried out using the method enzyme immunoassay.

The strain described recombinant VR18 deposited in the Collection of cultures of microorganisms of the State research center of Virology and biotechnology "Vector" for number V-312 06.10.2000,

Study of the expression of a chimeric protein TBI-HBsAg was conducted in animal cells CV-1 and N-D-when the infection in their recombinant BOB VR18 obtained from the above components. The nucleotide sequence integrated in the genome BOB chimeric gene TBI-HBsAg was confirmed by sequencing. In addition, a study was made of T - and b - cell immune response in mice of BALB/c mice immunized with recombinant BOB VR1 HBsAg reached 28-27 ng 24 hours, calculated per million cells CV-1 or H-D (multiplicity of infection of 0.1 PFU/cell; time of cultivation to 48 hours; see table. 1).

The determination of the number of TBI-protein was performed by the method competitive enzyme immunoassay using mouse serum against TBI-protein of bacterial origin. Products TBI-protein reached 11 and 5.8 ng ng 24 hours, calculated per million cells CV-1 and H-143 TC, respectively (multiplicity of infection of 0.1 PFU/cell; culturing for 48 hours; see table. 1).

Attempts to detect HBsAg in infected cell cultures as part of a chimeric protein using commercial test systems were unsuccessful. Regardless of the type of cells, the multiplicity of infection and the incubation time of HBsAg test system were not found. This suggests that the antigenic determinant "and" HBsAg, which used a test system, comprising chimeric protein shielded built-in instead of the pre-S2 region synthetic TBI-protein. We can assume that when vaccination of animals such recombinant vaccine will be somewhat reduced humoral immune response to HBsAg and raised on TBI-protein. Experiments on immunization of mice of BALB/c confirmed this assumption (see table. 2). In animals immunized with a preparation of virus 113 (drug recombi, immunized recombinant BOB VR18, antibodies are formed primarily to the antigen (HIV) in all periods of observation and slightly to HBsAg, which is observed only at 35 days.

Thus, the invention consists in the fact that the constructed recombinant BOB, expressing the chimeric protein of HBsAg in which the pre-S2 region is replaced by a synthetic protein TBI containing nine T - and b-cell epitopes of the major structural proteins of the virus HIV-1. The HBsAg molecules can assemble into virus-like particles with high immunogenicity, in which the pre-S2 region exposed on the surface. When replacing the specified area on the synthetic gene of the protein of candidate vaccine against AIDS/HIV chimeric protein on the surface of virus-like particles exhibited molecules TBI, which allows to obtain a high level of immune response. The latter was demonstrated in experiments on animals.

The invention is illustrated by the following drawings:

Fig. 1. The nucleotide sequence of the synthetic gene of the chimeric protein TBI-HBsAg. Initiation and termination triplets broadcast chimeric protein framed. The marked regions of genes TBI, HBsAg and joining them linker (the last 2. Recombinant plasmid DNA RT-HDsAg and the scheme of its design.

Fig.3. The scheme of coupling of the nucleotide coding sequences of proteins TBI and HBsAg between itself and the site of initiation of translation vector plasmids p7.5S113 using synthetic oligonucleotides (shown in bold). Single-stranded sticky ends formed after hydrolysis of the corresponding restrictase whose names are listed at the top or bottom of the sequence, indicated by broken lines.

The invention is illustrated by the following examples.

Example 1. Construction of recombinant plasmid DNA R18.

To construct the target plasmid p TBI-HBsAg using plasmid p7,5S113 [16], which is obtained based on the vector pVAR15 [17] by mounting HBsAg gene under the control of a promoter P7.5K. As the source of the gene TBI using plasmid pUCSII [15]. 30 μg of the indicated plasmids were cleaved joint hydrolysis of restrictase > PST and CRP, the obtained DNA fragment encoding TBI, isolated by electrophoresis on agarose gel, followed by elution on a dialysis membrane and are ligated under standard conditions hydrolyzed with the restriction enzyme BamHI and treated with phosphatase-p7 plasmid,5S113 and added si is coli JM103 and clones, grown on medium containing ampicillin, isolated plasmid DNA and subjected to restriction analysis with enzymes > PST, EcoRI, Xhol, BamHI. The compliance of a nucleotide sequence insertion in selected recombinant plasmids theoretical test method of Maxima-Gilbert (Fig.1). The resulting plasmid designated R TBI-HBsAg.

Example 2. The preparation of recombinant vaccinia virus VR18.

Culture vessels with monolayer cells of green monkey kidney CV-1 infected by vaccinia virus strain LIVP with a plurality of 0.05-0.1 PFU/cell. The adsorption of the virus on the cells is carried out for 1 h at 37oSince, then remove neadsorbirovanne virus and cells washed twice in culture medium, DMEM(m) that does not contain serum. For plasmid DNA P18 (0.05 mg/ml, 25 μl) add an equal volume lipofectin (0.2 mg/ml) and the mixture is left for 15 min at room temperature for formation of liposomes. The formed liposomes diluted with 1 ml of medium, DMEM(m) without serum and transfer to the prepared cells. After 16 h incubation at 37oWith the cells add 4 ml of medium, DMEM(m) containing 2% fetal serum and 80 u/ml of gentamicin and incubation continued for 24 h Cells after t is - under agar coating containing 25 μg/ml of 5-bromosuccinimide. After 48 h incubation at 37oC in an atmosphere containing 5% CO2cause the second floor, containing 0.01% dye neutral red. Staining of monolayer cells for visualization of viral plaques carried out for 3 h at 37oC in an atmosphere containing 5% CO2. Viral clones from individual plaques pokasivaut in 24-hole tablets and analyzed using DNA-DNA dot-hybridization for the presence of alien sequence. Positive hybridization clones re-clone and preparative develop the culture of cells CV-1.

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

The monolayer cells CV-1 or H-D-in the wells of 6-well plates infect recombinant viruses ospowiki 113 (contains the gene for HBsAg and pre-S2 region) and VR18 (contains a chimeric gene TBI-HBsAg) with a plurality of 0.1 or 0.01 PFU/cell. Infected cells incubated for 24 or 48 hours at 37oC in an atmosphere containing 5% CO2. In all experiments on cell cultures using the culture medium of DMEM(m) containing 2% fetal serum and 100 µg/ml penicillin and streptomycin. Posttribulationism at 600g for 10 minutes Supernatant used to determine the amount of HBsAg and TBI - protein immunoassay methods.

The determination of the number of HBsAg in samples carried out by the method of enzyme immunoassay calibration made by dilution of the drug with known HBsAg concentration using a commercial test system (JSC "Vector-best", p. Koltsovo). The determination of the number of TBI-protein in the samples carried out by the method competitive enzyme immunoassay using mouse serum against TBI-protein of bacterial origin.

The results of determination of titers of recombinant viruses and levels expresii HBsAg and TBI-protein in cell cultures of CV-1 and N-D-presented in table 1. As can be seen, the outputs of both recombinant viruses comparable to cells CV-1 and N-D-and vary in 2-10 times depending on the multiplicity of infection and the incubation time of the cells after infection. The levels of expression for recombinant HBsAg virus 113 and TBI protein for recombinant virus VR18 correlate well with the titles of the respective viruses. Products HBsAg reaches 28-27 ng for 24 h in terms of millions of cells CV-1 or H-D-(multiplicity of infection of 0.1 PFU/cell; time kultivirovanie (multiplicity of infection of 0.1 PFU/cell; time of cultivation - 48 h).

Attempts to detect HBsAg in the composition of the chimeric protein using commercial test systems were unsuccessful. Apparently, antigenic determinant "and" HBsAg, on which the test system, comprising chimeric protein shielded built-TBI-protein. Based on these data, we assume that when vaccination of animals with recombinant virus VR18 will be somewhat reduced humoral immune response to HBsAg and raised on TBI-protein. Subsequent experiments by immunization of mice of BALB/c confirmed this assumption (see table. 2).

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

The researchers used mouse strain BALB/c (Galati H-2b) weight 12 grams in the amount of 30 goals, which were kept on a standard diet.

The study of the immunogenic properties of the recombinant viruses 113 and VR18 were conducted at 0, 21 and 35 days after immunization, the animals were divided into 2 groups). Immunization was performed subcutaneously in the root of the tail (0.1 ml) dose 10 BATTLE. On these terms was studied indicators of humoral immunity in the reaction enzyme immunoassay using strips adsorbed antigens of HIV and HBsAg in the s, in table 2, in animals immunized with a preparation of virus 113, are formed antibodies only to HBsAg. In contrast, animals immunized with a preparation of virus VR18, antibodies are formed primarily to the HIV antigen (in all periods of observation) and slightly to HBsAg, which is observed only at 35 days.

Indicators of cellular immunity was studied in the reaction of besttransport of splenocytes. For carrying out this reaction from the spleens of the animals of the experimental groups distinguish cells that are washed twice in the cultural medium (RPMI medium 1640 with 2 mM L-glucamine, 510-5M 2-mercaptoethanol, 80 mg/l gentamicin, 5% and heated at 56oWith fetal serum). After that, the concentration of splenocytes lead up to 2 million/ml and used for the reaction of besttransport in 96-well plates. In wells add 2105cells in a volume of 100 μl. As the antigens used:

the lysate of HIV virus at a concentration of 2 μg/ml;

protein TBI at a concentration of 2 μg/ml;

- an inactivated antigen of mink enteritis virus at a concentration of 2 µg/ml.

As mitogens use concanavalin A (Sigma) at a concentration of 10 μg/ml lipopolysaccharide from E. coli (serotype 055:B5, Sigma) at a concentration of 25 mn 4 wells.

Splenocytes are stimulated by antigens, cultured for 96 h, and stimulated mitogen - 78 h For 18 h before the end of the cultivation add3H-thymidine at a concentration of 2 µci per well. At the end of the term culturing cells frozen, thawed and collect on "Cell-Hawester" ("Flow" UK). The expense of radioactivity is carried out in toluene scintillator counter "Mark III". The proliferative activity of splenocytes estimate absolute numbers of stimulated and unstimulated of splenocytes in the dynamics of supervision, and the stimulation index, defined as the ratio of accounts of stimulated splenocytes to the account of unstimulated splenocytes.

As follows from the results presented in table. 3, in animals immunized with virus 113, 21 and 35 days after immunization there is a marked proliferation of splenocytes in vitro upon stimulation with HBsAg protein, the proliferative response to stimulation proteins of the HIV virus and mink enteritis is missing. This is a confirmation of the specific proliferation of HBsAg and indicates the presence of the gene of this protein in the preparation of the virus 113. In animals immunized with a drug designed virus VR18, throughout dynamics nab is and HBsAg. This value significantly exceeds the same indicator for intact animals (0 day). The specificity of response of splenocytes of this group was confirmed by the absence of pronounced proliferation upon stimulation with antigen mink enteritis.

Thus, the new recombinant strain of vaccinia virus expressing the chimeric protein of HBsAg in which the pre-S2 region is replaced by a synthetic protein TBI containing protective T - and b-cell epitopes of human immunodeficiency virus type 1, and causing an immune response against HIV and hepatitis b in the body of animals. The obtained recombinant virus can be used as a candidate for creation on its basis of polyvalent vaccines against HIV-1, hepatitis b virus human and orthopoxviruses. It is expected that obtained on the basis of this approach, the vaccine will be characterized by high immunogenicity simultaneously against three of the above viral infectious diseases, and against HIV-1, affinity conformations In-epitopes in the vaccine and viral antigen efficiency in heterogeneous HLA antigens of the human population, the stimulation of the response to different In-epitopes, no unwanted epitopes of proteins Varmint and adaptability of receipt.

References

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2. Kozlov, A. P. // Russian journal of HIV/AIDS and related problems, 2000, T. 4, 1, pp. 11-14.

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1. Recombinant plasmid DNA RT-HBsAg encoding chimeric gene TBI-HBsAg, with a molecular weight of 4.2 MDA and size 6322 p. N., containing ClaI-BamHI vector fragment of DNA plasmids p7.5S113 size 4647 p. N. with nucleotide sequence of a gene timedancing of vaccinia virus with in-built P7 promoter.5 vaccinia virus; BglII- > PST sinclairii, located in the sequence vector plasmid having the structure 5'-GATCTGCA-3'; > PST -Kpn21 DNA fragment size 432 p. N., encoding an artificial polypeptide TBI, immunogenic HIV-1; CRP-synthetic BglII DNA fragment size 17 p. N., linking genes to proteins TBI and S in the right frame broadcast, having the structure

< / BR>
BamHI-ClaI fragment of the genome of hepatitis b virus encoding surface virginny protein S (HBsAg), size 1226 p. N.; genetic markers: bla-gene ampicillinresistant - gene-lactamase, which determines resistance to ampicillin in transformation of E. coli cells; TC - interrupted gene timedancing of vaccinia virus, causing the LC phenotype when the recombination insertions in the genome TC of vaccinia virus; unique restriction sites: HindIII - position 1; Cloned - 1303; Kpn21 - 1453; XhoI - 1462.

2. The recombinant strain of vaccinia virus, deposited in the Institute of ECR number V-312, producing chimeric protein TBI-HBsAg.

 

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