RU2578159C1 - Immunobiological agent and method for use thereof to induce specific immunity against ebola virus - Google Patents

Abstract

FIELD: medicine; virology.

SUBSTANCE: developed immuno-biological agent for inducing specific immune response to Ebola virus. Agent contains human recombinant adenovirus serotype 5 containing expressing cassette with gene insertion GP Ebola virus, and human recombinant adenovirus serotype 5 containing expressing cassette with gene insertion NP Ebola virus. GP gene of Ebola virus gene is GP Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 with modified nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and gene NP Ebola virus gene is NP Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 SEQ ID NO: 5. Immuno-biological agent additionally contains hyaluronidase. Also disclosed is method of using immuno-biological agent for inducing specific immunity to Ebola by introducing immuno-biological agent in effective amount. Presented inventions more effectively induce intensified immune response against Ebola virus in comparison with similar facilities due to features of used genetic constructs, optimising codons nucleotide sequences coding protein of Ebola virus, as well as amino acid sequences of proteins of Ebola virus.

EFFECT: presented inventions can be used in medicine as specific preventive agent for diseases caused by Ebola virus.

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Description

Technical field

The invention relates to immunology and virology and can be used as a specific prophylactic against diseases caused by the Ebola virus.

State of the art

Ebola hemorrhagic fever is currently one of the most dangerous diseases, the mortality rate from which reaches 90%. The causative agent of this disease is the Ebola virus (Ebolavirus), which belongs to the family of filoviruses. Since the discovery of this virus in 1976, it has caused several serious epidemics in West Africa, the last of which in 2014 claimed the lives of more than 4,000 people. Therefore, the development of vaccines against Ebola hemorrhagic fever is an urgent task of modern health care.

A solution is known according to patent WO 2012050193, in which the use of liposomes, including Ebola virus protein antigens, is contemplated.

The invention according to CA 2768801 provides for the use of virus-like particles, which are Ebola virus proteins expressed in mammalian cells and spontaneously assembled into virus-like structures.

A solution is known according to the patent US 2013323243, which provides for the use of a hybrid protein, some of which is represented by the antigen - glycoprotein of the Ebola virus, and the second part by the immunoglobulin segment. This fusion protein can be introduced on its own, or the gene of this protein can be inserted into a viral vector.

A solution is known according to patent US 20100047282 A1, where a recombinant adenovirus containing the Ebola virus glycoprotein gene (GP) is used as a vaccine against Ebola hemorrhagic fever. This decision is based on the fact that when an adenoviral vector enters the body, it enters the cells where the expression of the viral antigen begins. This leads to the induction of an immune response directed against the viral antigen and cells expressing it.

This technical solution as the closest to the claimed by the composition of the active substance and the method of its use is selected by the authors of the claimed invention for the prototype. Due to the fact that on the national priority date of the application US 20100047282 A1 on August 2, 2004, nothing was known about the Ebola virus strain /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, since this is the first time the pathogen was isolated and characterized in July 2014; the nucleotide sequence of its genes could not be used to construct recombinant adenoviral particles. Accordingly, the applicants were not able to create an adenoviral construct with characterized cultural-morphological properties, expression level, immunogenic properties. This is a significant drawback of this design, as it does not allow using it to obtain specific immunity to the Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056.

Disclosure of the present invention

The objective of the present invention is to provide an immunobiological agent for the effective induction of an immune response against the Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056.

The problem is solved due to the fact that an immunobiological agent has been developed, including recombinant human adenovirus 5 serotype, containing an expression cassette with an insert of the Ebola virus GP gene, while the Ebola virus gene GP /H.sapiens-wt/SLE is used as the Ebola virus GP gene / 2014/Makona-G3735.1 GenBank ID KM233056 with a modified nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and further comprising recombinant human adenovirus 5 serotype containing an expression cassette with a gene insert Ebola NP /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 SEQ ID NO: 5 providing amplification with the immunogenic properties of the developed means. The immunobiological agent further includes hyaluronidase. A method has also been developed for using an immunobiological agent to induce specific immunity to the Ebola virus by administering this immunobiological agent in an effective amount.

Brief Description of the Figures

In FIG. 1 shows the nucleotide sequence of GP No. 1, which is the Ebola virus GP gene sequence /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, modified by replacing rare codons with more common ones in humans.

In FIG. 2 shows the nucleotide sequence of GP # 2, which is the Ebola virus GP gene sequence /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, modified by adding adenine at the 887 position.

In FIG. 3 shows the nucleotide sequence of GP No. 3, which is the Ebola virus GP gene sequence /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, modified by adding adenine at 887 position and replacing the GAG codon with the GAC codon .

In FIG. 4 shows the nucleotide sequence of GP No. 4, which is the Ebola virus GP gene sequence /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, modified by adding adenine at 887 position and replacing 2 AAA codons with codons AAG.

In FIG. Figure 5 shows the unmodified nucleotide sequence of the Ebola virus NP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056.

In FIG. Figure 6 shows the unmodified nucleotide sequence of the Ebola virus / H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056.

In FIG. 7 presents the results of determining the expression of GP and NP proteins in HEK293 cells after adding recombinant adenoviruses to them (Ad-GP No. 1, Ad-GP No. 2, Ad-GP No. 3, Ad-GP No. 4 and Ad-NP, respectively).

Track 1 to 7 presents data on GP protein expression, where

1 - cell lysate to which phosphate-buffered saline was added,

2 - cell lysate, to which Ad-null was added,

3 - cell lysate to which Ad-wt was added,

4 - cell lysate to which Ad-GP No. 1 was added,

5 - cell lysate, to which was added Ad-GP No. 2,

6 - cell lysate, to which was added Ad-GP No. 3,

7 - cell lysate to which Ad-GP No. 4 was added.

From track 8 to 10, NP protein expression data are presented, where

8 - cell lysate, to which was added phosphate-buffered saline,

9 - cell lysate to which Ad-null was added,

10 - cell lysate to which Ad-NP was added.

In FIG. 8 presents the results of lymphoproliferative analysis.

The ordinate axis is the number of proliferating cells,%.

The abscissa axis - various groups of animals that were injected:

1. phosphate buffer (100 μl)

2. Ad-null 2 · 10 ⁸ PFU / mouse

3. Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 1 10 ⁸ PFU / mouse

4. Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 2 10 ⁸ PFU / mouse

5. Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 3, 10 ⁸ PFU / mouse

6. Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 4 10 ⁸ PFU / mouse

In FIG. Figure 9 presents the results of an analysis of the ability of hyaluronidase to increase the ability of adenoviruses to penetrate mammalian cells.

The ordinate axis is luminescence, quanta / second.

The abscissa axis - various groups of animals that were injected:

1. Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse

2. Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse and hyaluronidase 10 IU / mouse

3. Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse and hyaluronidase 25 IU / mouse

4. Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse and hyaluronidase 50 IU / mouse

5. Hyaluronidase 10 IU / mouse

6. Hyaluronidase 25 IU / mouse

7. Hyaluronidase 50 IU / mouse

8. Intact animals

The implementation of the invention

From literature data it is known that the most promising vaccine antigens of the Ebola virus are the glycoprotein GP, which is the surface protein of the virion, and the nuclear protein NP. The use of both antigens at the same time will allow for a more complex immune response and provide a protective effect against more strains of the Ebola virus.

The present invention is a composition consisting of recombinant human Adenovirus 5 serotype containing the Ebola virus glycoprotein (GP) gene, and recombinant human Adenovirus 5 serotype containing the Ebola virus nuclear protein (NP) gene, taken in an effective ratio. At the same time, a modified sequence was used as the GP gene, chosen as one of 4 options: GP No. 1 (Fig. 1), GP No. 2 (Fig. 2), GP No. 3 (Fig. 3), GP No. 4 (Fig. . four).

As the NP gene, the unmodified Ebola virus NP / H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 sequence was used (Fig. 5).

1 dose of the developed immunostimulating agent includes:

Recombinant human adenovirus of serotype 5, which contains the Ebola virus NP protein gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 - 10 ⁷ to 10 ⁹ plaque forming units (PFU);

Recombinant human adenovirus 5 serotype, which contains a modified Ebola virus GP gene sequence /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, selected from GP No. 1, GP No. 2, GP No. 3, GP No. 4 - 10 ⁷ to 10 ⁹ plaque forming units (PFU);

A buffer that ensures the viability of recombinant adenoviruses and is safe for administration to mammals.

To improve the penetration of recombinant adenoviruses into mammalian cells, the composition includes the enzyme hyaluronidase, which breaks down hyaluronic acid, which is part of the intercellular substance.

Example 1

Obtaining recombinant adenoviruses containing genes for antigens of the Ebola virus - nuclear protein NP and glycoprotein GP

The developed immunobiological agent is a composition consisting of recombinant human adenovirus 5 serotype containing the Ebola virus glycoprotein (GP) gene and recombinant human adenovirus 5 serotype containing the Ebola virus gene (NP) taken in an effective ratio. At the same time, a modified sequence was used as the GP gene, chosen as one of 4 options: GP No. 1 (Fig. 1), GP No. 2 (Fig. 2), GP No. 3 (Fig. 3), GP No. 4 (Fig. . four).

GP sequence No. 1 was obtained from the Ebola / H virus GP gene sequence. sapiens-wt / SLE / 2014 / Makona-G3735.1 GenBank ID KM233056 by partially replacing codons that are rarely found in human genes with codons that are most common. Data on the frequency of use of codons in various organisms is publicly available (for example, the resource www.kazusa.or.jp). The use of the nucleotide sequence optimized in this way will ultimately lead to increased expression of the target gene in human cells.

The sequence of GP No. 2 was obtained from the Ebola virus GP gene sequence /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 by adding adenine at position 887. It is known (J. Lee, Ε. Saphire. Ebolavirus glycoprotein structure and mechanism of entry. Future Virol., 2009; 4 (6), pp. 621-635) that in the GP gene a hairpin and L-polymerase of the virus are formed in this place Ebola stops and a “short” secreted form of GP forms. However, with a 20% probability, L-polymerase makes a mistake at this point by inserting an additional nucleotide, as a result of which the reading frame shifts and a transmembrane variant of GP is formed, which is considered the most immunogenic antigen of the Ebola virus. Thus, if the original sequence of the GP gene is used to construct the recombinant adenovirus, then a "short", low-immunogenic form of the protein will be expressed as a result; and if you use a modified sequence of GP No. 2, the transmembrane highly immunogenic protein will be expressed.

The sequence of GP No. 3 was obtained from the sequence of the GP gene of the Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 by adding adenine at position 887, which is necessary for expression of the transmembrane highly immunogenic protein. In addition, the GAG codon encoding glutamic acid was replaced by the GAC codon encoding aspartic acid.

This replacement leads to a decrease in the cytotoxicity of this protein in vitro while maintaining its immunogenicity.

The sequence of GP No. 4 was obtained from the sequence of the GP gene of the Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 by adding adenine at the 887 position, which is necessary for the expression of transmembrane high immunogenic protein. In addition, 2 AAA codons were replaced with AAG codons. Both of these triplets encode one amino acid - lysine. However, due to this substitution, a hairpin is not formed at this location of the nucleotide sequence, which ultimately leads to an increase in the expression of this protein in mammalian cells.

As the NP gene, the unmodified Ebola virus NP / H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 sequence was used (Fig. 5).

Six plasmid constructs carrying different variants of the GP gene and the Ebola virus NP gene were obtained: pShuttle-NP, pShuttle-GP No. 1, pShuttle-GP No. 2, pShuttle-GP No. 3, pShuttle-GP No. 4, pShuttle-GPwt (control , contains the unmodified Ebola virus GP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 (Fig. 6)). All sequences of Ebola virus antigens were chemically synthesized.

The resulting plasmid vectors were further used to obtain plasmid constructs carrying the full-sized recombinant adenovirus genomes. For this, plasmid vectors with corresponding expression cassettes containing the Ebola virus genes (pShuttle-NP, pShuttle-GP No. 1, pShuttle-GP No. 2, pShuttle-GP No. 3, pShuttle-GP No. 4, pShuttle-GPwt) were treated with endonucleases Pacl and Bstll07I to extract an expression cassette for subsequent ligation with a cosmid vector containing the complete recombinant adenovirus gene. The cAd5-EGFP construct, which we obtained earlier, was used as the cosmid vector carrying the complete genome of the recombinant adenovirus. The cAd5-EGFP cosmid was also treated with Pacl and Bstll07I endonucleases. The hydrolysis products were ligated. The resulting DNA contained in the ligase mixture was packaged in phage heads and transformed with them DH5α competent cells. Transformed cells were selected on LB agar medium with the antibiotic ampicillin. Plasmid DNA was isolated by boiling.

Thus, as a result, cosmid constructs were obtained containing the genome of the recombinant human adenovirus of the fifth serotype with expression cassettes, which include the NP gene (cosmid cAd5-NP), gene GP No. 1 (cosmid cAd5-GP No. 1), GP No. 2 (cosmid cAd5 - GP No. 2), GP No. 3 (cosmid cAd5-GP No. 3), GP No. 4 (cosmid cAd5-GP No. 4).

At the next stage, to obtain recombinant adenovirus mini-preparations, plasmid constructs carrying expression cassettes with Ebola virus genes as part of the full-size recombinant adenovirus genomes were hydrolyzed by Pacl and Swal restriction endonucleases to remove the plasmid part and transfected into HEK293 eukaryotic cells. 5 days after transfection, blind passages of material were performed to propagate recombinant adenoviruses expressing Ebola virus genes. As a result, “seed” material was obtained, which was then used to accumulate preparative amounts of recombinant adenoviruses.

Thus, as a result of the work, recombinant adenoviruses expressing Ad-NP, Ad-GP No. 1, Ad-GP No. 2, Ad-GP No. 3, Ad-GP No. 4 were obtained. According to the results of the study, it can be concluded that the selected variants of the Ebola virus antigens /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 can be integrated into the recombinant adenovirus genome. In this case, none of the variants of the genes showed pronounced toxicity, which made it possible to obtain a complete set of recombinant adenoviral vectors.

Example 2

Verification of the expression of NP and GP proteins in cells transfected with Ad-NP, Ad-GP No. 1, Ad-GP No. 2, Ad-GP No. 3, Ad-GP No. 4.

The purpose of this experiment was to verify the expression of Ebola NP and GP antigens using constructed recombinant adenoviruses. The experiment was carried out as follows.

HEK293 cells were cultured in DMEM supplemented with 10% fetal calf serum in an incubator at 37 ° C and 5% CO ₂ . Cells were placed on 30 mm ² Petri dishes and incubated for one day until 70% confluency was achieved. Next, the studied preparations of adenoviruses (Ad-NP, Ad-GP No. 1, Ad-GP No. 2, Ad-GP No. 3, Ad-GP No. 4) and control preparations of adenovirus (Ad-null, a recombinant adenovirus that does not contain inserts, Ad-GPwt - recombinant adenovirus containing an unoptimized GP sequence) at a rate of 100 PFU / cell or phosphate buffer as a negative control. After a day, the expression of Ebola antigens was checked by immunoblotting. For this, the medium was selected from the cells, the cells were washed with sterile phosphate-buffered saline, and then lysed using RIPA buffer (Pierce) according to the protocol of the manufacturer.

After cell lysis, the concentration of total protein in the samples was measured using Bradford reagent (Sigma) according to the manufacturer's instructions. An aliquot of protein-aligned samples was mixed with Laemmli Sample Buffer (Sigma) and incubated for 5 minutes at 95 ° C.

Denaturing electrophoresis was then carried out on a 10% SDS-polyacrylamide gel SPRINT NEXT GEL® 10% (Amresco) in NEXT GEL® Running Buffer, 20X (Amresco) using the Mini-PROTEAN® Tetra Cell (Bio-Rad) system for 20 minutes at 250V. After electrophoresis, proteins were transferred onto a Trans-Blot® Turbo ™ Mini Nitrocellulose Transfer Packs (Bio-Rad) nitrocellulose membrane in Tris-CAPS buffer (Bio-Rad) using the Trans-Blot® SD Semi-Dry Transfer Cell transfer system (Bio- Rad) for 15 minutes at 25V. Then blocking of a nonspecific signal was carried out. For this, the membrane was incubated in a solution of 3% skim milk in phosphate-buffered saline with 0.05% Tween20 (TFSB) for one hour at room temperature. After that, the membrane was incubated for 16 hours at 4 ° C with antibodies to the GP protein or with antibodies to the NP protein in a solution of 3% skim milk in TFSB. Next, the membrane was washed with a TFSB solution and the membrane was treated with a solution of secondary antibodies conjugated to horseradish peroxidase in a solution of 3% skim milk in TFSB at 37 ° C on a shaker for 1 hour. Then the membrane was thoroughly washed with a solution of TFSB. Further detection was performed using the Clarity ™ Western ECL Substrate kit (Bio-Rad) and developed on a Hyperfilm® ECL ™ film (Amersham). Using the same protocol, the GAPDH comparison protein was detected using Anti-GAPDH antibodies.

Immunoblot results are shown in FIG. 7.

Track 1 to 7 presents data on GP protein expression, where

1 - cell lysate to which phosphate-buffered saline was added,

2 - cell lysate, to which Ad-null was added,

3 - cell lysate to which Ad-wt was added,

4 - cell lysate to which Ad-GP No. 1 was added,

5 - cell lysate, to which was added Ad-GP No. 2,

6 - cell lysate, to which was added Ad-GP No. 3,

7 - cell lysate to which Ad-GP No. 4 was added.

From track 8 to 10, NP protein expression data are presented, where

8 - cell lysate, to which was added phosphate-buffered saline,

9 - cell lysate to which Ad-null was added,

10 - cell lysate to which Ad-NP was added.

As can be seen from the data obtained, in the cells to which Ad-NP was added, expression of the target NP protein was detected after 24 hours. In cells transduced with recombinant adenoviruses containing both the modified and unmodified GP gene, expression of the target GP protein was observed. Moreover, higher expression levels were observed in cells to which recombinant adenoviruses containing modified GP genes were added than in cells transduced with adenovirus expressing the unmodified GP gene.

Example 3

Determination of antibody titer to NP and GP after immunization of animals with recombinant adenoviruses Ad-NP, Ad-GP No. 1, Ad-GP No. 2, Ad-GP No. 3, Ad-GP No. 4

From literature data it is known that antibodies play an important role in protecting the body against the Ebola virus. The rodent model showed that the antibody titer against Ebola antigens correlates with animal survival. Therefore, in this experiment, we evaluated the titer of antibodies against NP and GP antigens of the Ebola virus 5 days after double immunization of animals with an immunobiological agent including a recombinant adenovirus containing the modified Ebola virus GP gene /H.sapiens-wt/SLE/2014/Makona-G3735. 1 GenBank ID KM233056, one of 4 options selected: GP No. 1, GP No. 2, GP No. 3, GP No. 4, and a recombinant adenovirus containing the Ebola virus NP gene /H.sapiens-wt/SLE/2014/Makona-G3735 .1 GenBank ID KM233056.

The experiment used mice of the Balb / c line, females of 18 g. All animals were divided into 52 groups of 3 animals, which were injected intramuscularly:

1) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 1 10 ⁷ PFU / mouse

2) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 1 10 ⁸ PFU / mouse

3) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 1 10 ⁹ PFU / mouse

4) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 2 10 ⁷ PFU / mouse

5) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 2 10 ⁸ PFU / mouse

6) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 2 10 ⁹ PFU / mouse

7) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 3 10 ⁷ PFU / mouse

8) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 3 10 ⁸ PFU / mouse

9) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 3 10 ⁹ PFU / mouse

10) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 4 10 ⁷ PFU / mouse

11) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 4 10 ⁸ PFU / mouse

12) Ad-NP 10 ⁷ PFU / mouse, Ad-GP No. 4 10 ⁹ PFU / mouse

13) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 1 10 ⁷ PFU / mouse

14) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 1 10 ⁸ PFU / mouse

15) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 1 10 ⁹ PFU / mouse

16) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 2 10 ⁷ PFU / mouse

17) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 2 10 ⁸ PFU / mouse

18) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 2 10 ⁹ PFU / mouse

19) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 3 10 ⁷ PFU / mouse

20) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 3 10 ⁸ PFU / mouse

21) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 3 10 ⁹ PFU / mouse

22) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 4 10 ⁷ PFU / mouse

23) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 4 10 ⁸ PFU / mouse

24) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 4 10 ⁹ PFU / mouse

25) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 1 10 ⁷ PFU / mouse

26) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 1 10 ⁸ PFU / mouse

27) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 1 10 ⁹ PFU / mouse

28) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 2 10 ⁷ PFU / mouse

29) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 2 10 ⁸ PFU / mouse

30) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 2 10 ⁹ PFU / mouse

31) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 3 10 ⁷ PFU / mouse

32) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 3 10 ⁸ PFU / mouse

33) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 3 10 ⁹ PFU / mouse

34) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 4 10 ⁷ PFU / mouse

35) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 4 10 ⁸ PFU / mouse

36) Ad-NP 10 ⁹ PFU / mouse, Ad-GP No. 4 10 ⁹ PFU / mouse

37) Ad-NP 10 ⁷ PFU / mouse, phosphate buffered saline

38) Ad-NP 10 ⁸ PFU / mouse, phosphate buffered saline

39) Ad-NP 10 ⁹ PFU / mouse, phosphate buffered saline

40) phosphate-buffered saline, Ad-GP No. 1 10 ⁷ PFU / mouse

41) phosphate-buffered saline, Ad-GP No. 1 10 ⁸ PFU / mouse

42) phosphate-buffered saline, Ad-GP No. 1 10 ⁹ PFU / mouse

43) phosphate-buffered saline, Ad-GP No. 2 10 ⁷ PFU / mouse

44) phosphate-buffered saline, Ad-GP No. 2 10 ⁸ PFU / mouse

45) phosphate-buffered saline, Ad-GP No. 2 10 ⁹ PFU / mouse

46) phosphate-buffered saline, Ad-GP No. 3 10 ⁷ PFU / mouse

47) phosphate-buffered saline, Ad-GP No. 3 10 ⁸ PFU / mouse

48) phosphate-buffered saline, Ad-GP No. 3 10 ⁹ PFU / mouse

49) phosphate-buffered saline, Ad-GP No. 4 10 ⁷ PFU / mouse

50) phosphate-buffered saline, Ad-GP No. 4 10 ⁸ PFU / mouse

51) phosphate-buffered saline, Ad-GP No. 4 10 ⁹ PFU / mouse

52) phosphate buffered saline

Doses of recombinant adenoviruses were selected based on the literature data on the use of these vectors to induce an immune response to various pathogens (Shmarov M.M. et al. Investigation of the possibility of inducing a recombinant adenoviral vector of a heterosubtypic immune response against influenza virus A. Biologics, 2011, 1 (41); Scherbinin, D.N. et al. Induction of the immune response to bacillus anthracis by intranasal administration of a recombinant adenovirus expressing a protective antigen fused to an Fc fragment of an antibody la IGG2a. Acta Naturae, 2014, 1 (20), v. 6, pp. 82-90, Kim EH et al. Intranasal adenovirus-vectored vaccine for induction of long-lasting humoral immunity-mediated broad protection against influenza in mice. J Virol. 2014, 88 (17), pp. 9693-9703).

Thus, the doses of recombinant adenoviruses 10 ⁷ -10 ⁹ PFU / dose were chosen as the most optimal for immunization of animals, however, as an average specialist understands, increasing the dose of recombinant adenoviruses will lead to an increase in the immune response to target proteins, up to toxic effects .

Three weeks later, animals were sampled from the tail vein, from which serum was then obtained. The antibody titer was determined by enzyme immunoassay.

First, the titer of antibodies to the GP protein was determined. Serum samples were diluted with a 2-fold dilution starting at a 1: 200 dilution. A total of 8 dilutions of each sample were prepared, which were added 50 μl to the wells of the tablet, with pre-adsorbed GP protein. Next, incubation was carried out for 1 hour at 37 ° C.

After incubation, the wells were washed, and then secondary antibodies against mouse immunoglobulins conjugated with biotin were added. After incubation and washing, a conjugate of streptovidin and horseradish peroxidase was added to the wells of the plate. At the final stage, after washing the wells, a TMB solution was added, which is a substrate of horseradish peroxidase and, as a result of the reaction, turns into a colored compound. The reaction was stopped after 15 minutes by the addition of sulfuric acid. Next, the optical density of the solution (OD) in each well was measured using a spectrophotometer at a wavelength of 450 nm. Antibody titer was defined as the last dilution in which the optical density of the solution was significantly higher than in the negative control group. The results are presented in table 1.

Similarly, the titer of antibodies to the NP protein was determined in the blood serum of mice immunized with an immunobiological agent. The results are presented in table 2.

As can be seen from the results of the experiment, the developed immunobiological agent, including recombinant human adenovirus 5 serotype, containing the Ebola virus GP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 with a modified nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 (FIG. 1, FIG. 2, FIG. 3, FIG. 4), and recombinant human serotype 5 adenovirus containing an expression cassette with insertion of the Ebola NP NP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, SEQ ID NO: 5 (FIG. 5), where the amount of each of the adenoviruses varies about July ¹⁰ PFU / dose to 10 ⁹ pfu / dose, to effectively stimulate humoral immune responses against Ebola virus antigens in the entire range of doses selected.

Example 4

Determination of the proportion of proliferating lymphocytes in mice after administration of the developed immunobiological agent

The lymphoproliferative test allows one of the key properties of an adaptive immune response to be evaluated - the ability of antigen-specific lymphocyte clones to rapidly proliferate and differentiate into effector cells. The most common technique for assessing lymphoproliferation is staining of cells with CFSE dye. The CFSE conjugate with proteins, which is formed in labeled cells, is retained by these cells throughout development, as well as during division. The label is passed on to daughter cells and never passes to neighboring cells in a population. CFSE allows you to track cell division, because during division, the label concentration in daughter cells decreases exactly 2 times and the fluorescence intensity, respectively, too.

In the experiment, Balb / c mice were used. All animals were divided into six groups, which were administered intramuscularly:

1) phosphate buffer (100 μl)

2) Ad-null 2 · 10 ⁸ PFU / mouse

3) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 1 10 ⁸ PFU / mouse

4) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 2 10 ⁸ PFU / mouse

5) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 3, 10 ⁸ PFU / mouse

6) Ad-NP 10 ⁸ PFU / mouse, Ad-GP No. 4 10 ⁸ PFU / mouse

Doses of the recombinant adenoviruses that make up the immunobiological agent were selected based on data from a previous experiment.

Two weeks after immunization, the spleen was taken from the animals, from which lymphocytes were then isolated by centrifugation in a ficoll-urographin gradient. Then, the isolated cells were stained with CFSE according to the procedure (Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester / Quah BJ, Warren HS, Parish CR Nat Protoc. 2007; 2 (9), pp. 2049-2056) and analyzed by flow cytometry. The results are presented in the form of a histogram (Fig. 8).

As can be seen from the results of the experiment, the developed immunobiological agent, including recombinant human adenovirus 5 serotype, containing the Ebola virus GP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 with a modified nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. (FIG. 1, FIG. 2, FIG. 3, FIG. 4), and recombinant human serotype 5 adenovirus containing an expression cassette with the insertion of the NP gene of the Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056, SEQ ID NO: 5 (Fig. 5), effectively stimulate the proliferation of lymphocytes.

Example 5

Determining the efficiency of using enzymes to improve the penetration of recombinant adenoviruses into mammalian cells in vivo

The purpose of this experiment was to determine the possibility of using hyaluronidase as part of the developed immunobiological agent to increase the efficiency of cell transduction by recombinant adenoviruses. This enzyme depolymerizes hyaluronic acid, which is part of the intercellular substance. As a result, the hyaluronic acid molecule breaks up into small fragments, which causes a thinning of the intercellular substance gel layer and, accordingly, facilitates the penetration of recombinant adenoviruses directly to the target cells.

In order to test this hypothesis, we used the recombinant Ad-luc adenovirus containing the luciferase gene. With the introduction of recombinant Ad-luc adenovirus into the body in those cells where it penetrates, luciferase expression begins. The production of the luciferase enzyme in cells is easily detected by reaction with a specific substrate, luciferin, which is accompanied by the emission of light quanta.

In the experiment, laboratory mice of the BALB / c line of females weighing 18 g were used. Animals were divided into 5 groups, which were intramuscularly injected:

1) Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse

2) Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse and hyaluronidase 10 IU / mouse

3) Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse and hyaluronidase 25 IU / mouse

4) Ad-luc (recombinant adenovirus containing the luciferase gene) 10 ⁷ PFU / mouse and hyaluronidase 50 IU / mouse

5) Hyaluronidase 10 IU / mouse

6) Hyaluronidase 25 IU / mouse

7) Hyaluronidase 50 IU / mouse

8) Intact animals

Doses of enzymes were chosen based on their solubility and the maximum volume that can be entered intramuscularly in mice. The dose of recombinant adenovirus was chosen as the lowest of those that are supposed to be used in the developed immunobiological agent. After 24 hours, the animals were euthanized and the muscle into which the preparations were injected was isolated. Muscle tissue was homogenized in a lysis buffer specifically designed to measure luciferase activity (Promega). Then, the samples were centrifuged at 12,000 × g and 100 μl of supernatant were taken onto a plate. Luciferin (E1602, Promega) was diluted according to the manufacturer's instructions and 100 μl was added to each sample. After that, luminescence was evaluated using a flat-bed spectrophotometer. The results are shown in FIG. 9.

As can be seen from the data obtained, in all groups where adenovirus was used together with hyaluronidase, higher luminescence was observed than in the group that was administered only adenovirus. Moreover, luciferase expression was observed in animal tissues, which were administered Ad-luc with hyaluronidase at a dose of 50 IU / mouse (in terms of human dose this is 672 IU). In the control groups (No. 5, 6, 7), there were no differences compared to intact animals.

Based on the results obtained, it can be concluded that hyaluronidase in doses of at least 672 IU / person provides the best penetration of adenovirus into tissues and, therefore, can be used for these purposes as part of the developed immunobiological preparation.

Example 6

Estimated dosage forms of an immunobiological agent

From literature data it is known that immunization with recombinant adenoviruses expressing various target genes works effectively with various methods of introducing vectors: subcutaneous, intramuscular, inhalation.

The hyaluronidase enzyme is approved for use by humans for the correction of various pathological conditions associated with joint diseases, for improving the absorption of radiocontrast substances, local anesthetics, etc. for intramuscular, subcutaneous and inhalation routes of administration. Thus, the developed immunobiological agent can be used for intramuscular, subcutaneous and inhalation administration and can be presented in the form of various dosage forms: solution for subcutaneous / intramuscular administration, powder or solution for inhalation, in the form of a lyophilisate.

To obtain a solution for subcutaneous / intramuscular / inhalation administration, human serotype 5 adenovirus containing the GP gene, human serotype 5 adenovirus containing the NP gene in an effective ratio and containing hyaluronidase in 1-5 ml of buffer solution are mixed. Any solution that is not toxic to humans and contains all the necessary components to ensure the viability of the adenoviral vector and enzyme activity can serve as a buffer solution. For example, phosphate buffered saline.

The lyophilized form is obtained by freeze drying a suspension containing human serotype 5 adenovirus containing the GP gene, human serotype 5 adenovirus containing the NP gene in an effective ratio and containing hyaluronidase in 1-5 ml of buffer solution. Any solution that preserves the viability of adenoviral particles and the activity of enzymes after freeze drying can be used as a buffer solution. Before use, the lyophilized preparation is diluted with sterile distilled water to a volume of 1-5 ml (it is necessary to restore the volume that was before freeze drying).

An immunobiological agent can also be presented in the form of two solutions that are in two separate bottles and mixed immediately before use. In this case, one bottle contains human serotype 5 adenovirus containing the GP gene and human serotype 5 adenovirus containing the NP gene in an effective ratio in the buffer solution, and the other bottle contains hyaluronidase, as well as buffer components. The contents of one or both vials may also be freeze-dried.

Industrial applicability

All the above examples confirm the fulfillment of the task, as well as the industrial applicability of the created invention.

Claims (3)

1. An immunobiological agent for the induction of specific immunity to the Ebola virus, comprising recombinant human adenovirus 5 serotype containing an expression cassette with an insert of the Ebola virus GP gene,
characterized in that
as the Ebola virus GP gene, the Ebola virus GP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 with a modified nucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and further comprising recombinant human adenovirus 5 serotype containing an expression cassette with an insert of the Ebola virus NP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 SEQ ID NO: 5, providing enhanced immunogenic properties of the developed product. 2. The immunobiological agent according to claim 1, characterized in that it further includes hyaluronidase. 3. A method of using an immunobiological agent to induce specific immunity to the Ebola virus by administering an immunobiological agent according to claim 1 in an effective amount.

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