WO2016130047A1

WO2016130047A1 - Immunobiological drug and method for using same for inducing specific immunity against the ebola virus

Info

Publication number: WO2016130047A1
Application number: PCT/RU2016/000065
Authority: WO
Inventors: Денис Юрьевич ЛОГУНОВ; Максим Михайлович ШМАРОВ; Ирина Леонидовна ТУТЫХИНА; Ольга Вадимовна ЗУБКОВА; Дмитрий Викторович ЩЕБЛЯКОВ; Андрей Александрович ЛЫСЕНКО; Дмитрий Николаевич ЩЕРБИНИН; Инна Вадимовна ДОЛЖИКОВА; Алина Шахмировна ДЖАРУЛЛАЕВА; Наталья Михайловна АРТЕМИЧЕВА; Дарья Андреевна БУРМИСТРОВА; Амир Ильдарович ТУХВАТУЛИН; Владимир Борисович ПАНТЮХОВ; Светлана Ивановна СЫРОМЯТНИКОВА; Ирина Викторовна ШАТОХИНА; Сергей Владимирович БОРИСЕВИЧ; Борис Савельевич НАРОДИЦКИЙ; Александр Леонидович ГИНЦБУРГ
Priority date: 2015-02-13
Filing date: 2016-02-12
Publication date: 2016-08-18
Also published as: RU2578159C1

Abstract

The invention relates to immunology and virology, and can be used as a specific prophylactic drug against diseases caused by the Ebola virus. An immunobiological drug has been developed, comprising a recombinant human adenovirus of serotype 5, which comprises an expressing cartridge with an insert of the GP gene of the Ebola virus, wherein the GP gene of the Ebola virus which is used is the GP gene of the Ebola virus /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 additionally comprising a recombinant human adenovirus of serotype 5, which comprises an expressing cartridge with an insert of the NP gene of the Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 SEQ ID NO: 5 which enhances the immunogenic properties of the drug developed. The immunobiological drug additionally comprises hyaluronidase. A method has also been developed for using the immunobiological drug for inducing specific immunity to the Ebola virus by said immunobiological drug being introduced in an effective quantity.

Description

Immunobiological agent and method of its use for the induction of specific immunity against the virus

Ebola

Technical field

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

The prior 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 the 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 WO2012050 93 in which the use of liposomes comprising protein antigens of the Ebola virus is contemplated.

The invention according to patent CA2768801 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 US2013323243, 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.

US Patent Application No. 20060269572 A1 discloses an agent characterized in that it contains two recombinant human adenoviruses of serotype 5, where the first adenovirus contains an expression cassette with an Ebola virus GP gene insert and the second adenovirus contains an expression cassette with an Ebola virus NP gene insertion. A method for immunizing an individual using such an agent is also described.

However, when using unmodified nucleotide sequences, immunogenicity is reduced compared to proteins obtained using the modified nucleotide sequence. As a result, an agent with low specific immunogenicity is obtained.

A solution is known according to US20100047282 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 induction. an immune response directed against a 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 date of national priority of application US20100047282 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 pathogen was first detected 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 and morphological properties, expression level, immunogenic properties. This is a significant drawback of this design, since 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.

An object of the present invention is to provide an immunobiological agent for efficiently inducing an immune response against an Ebola virus / H.sapiens-wt / SLE / 2014 / Makona-G3735.1 GenBank ID KM233056. s 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 expression cassette with N gene insert P Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056 SEQ ID NO: 5 to ensure the enhancement of the immunogenic properties of the developed tool. 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.

Description of the sequence listing

SEQ ID NO: 1 is the nucleotide sequence of GPNQ1, 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. SEQ ID NO: 2 is the GPN ° 2 nucleotide sequence, 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.

SEQ ID NO: 3 is the nucleotide sequence of GPN ° 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 the 887 position and replacing the codon GAG to codon GAC.

SEQ ID NO: 4 is the nucleotide sequence of GPN ° 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 the 887 position and replacing 2 codons AAA, to AAG codons

SEQ ID NO: 5 - nucleotide sequence of the NP gene of the Ebola virus /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056

SEQ ID NO: 6 - Ebola virus GP nucleotide sequence /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056

Description of figures

In FIG. one

presents the results of determining the expression of GP and NP proteins in HEK293 cells, after adding to them recombinant adenoviruses (Ad-GPN21, Ad-GPN22, Ad-GPN23, Ad-GPN ° 4 and Ad-NP, respectively).

Track 1 to 7 - data on GP protein expression are presented, where

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

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

3 - cell lysate to which Ad-wt was added

4 - cell lysate to which Ad-GPN ° 1 was added

5 - cell lysate to which Ad-GPN22 was added

6 - cell lysate to which Ad-GPN23 was added

7 - cell lysate to which Ad-GPN24 was added

From track 8 to 10 - data on NP protein expression 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. 2 presents the results of lymphoproliferative analysis

The ordinate axis - the number of proliferating cells,%

The abscissa axis - various groups of animals that were introduced

1. phosphate buffer (100 μl)

2. Ad-null 2 ^* 10 ⁸ PFU / mouse

3. Ad-NP 10 ⁸ PFU / mouse, Ad-GPN21 10 ⁸ PFU / mouse

4. Ad-NP 10 ⁸ PFU / mouse, Ad-GPN22 10 ⁸ PFU / mouse, 5. Ad-NP Yu OE / mouse, Ad-GPN23, 10 ^a PFU / mouse

6. Ad-NP 10 ⁸ PFU / mouse, Ad-GPNs4 10 ⁸ PFU / mouse

In FIG. 3 presents the results of an analysis of the ability of hyaluronidase to increase the ability of adenoviruses to penetrate mammalian cells.

Y-axis - luminescence, quanta / second

The abscissa axis - various groups of animals that were introduced

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 HUME / mouse hyaluronidase

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

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

5. Hyaluronidase UME / mouse

6. Hyaluronidase 25ME / mouse

7. Hyaluronidase 50ME / 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. Using both antigens at the same time will achieve 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 5 adenovirus 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: GPN21, GPN ^Q 2, GPNQ3, GPNQ4.

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

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 GPNQ1, GPN ^Q 2, GPN ° 3, GPN24 - 10 ⁷ up 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. In this case, a modified sequence was used as the GP gene, chosen as one of 4 options: GPN21, GPN ° 2, GPN ° 3, GPN ° 4.

The GPN21 sequence was obtained from the Ebola virus GP gene sequence /H.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 are generally available (for example, the resource www.kazusa.or.jp). The use of a nucleotide sequence optimized in this way will ultimately lead to increased expression of the target gene in human cells.

The GPN ° 2 sequence was obtained from the Ebola virus GP gene sequence /H.sapiens-wt / SLE / 2014 / Makona-G3735.1 GenBank ID KM233056 by adding adenine at the 887 position. It is known (J. Lee, E. Saphire, Ebolavirus glycoprotein structure and mechanism of entry, Future Virol., 2009; 4 (6), C.621-635) that a hairpin and L-polymerase are formed in the GP gene at this point Ebola virus stops, and a “short” secreted form of GP is formed. 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 a modified GPN22 sequence is used, a transmembrane highly immunogenic protein will be expressed.

The GPN ° 3 sequence was obtained from the Ebola virus GP gene sequence /H.sapiens-wt / SLE / 2014 / Makona-G3735.1 GenBank ID KM233056 by adding adenine at 887 position, which is necessary for expression of the transmembrane high 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 GPN ^Q 4 sequence was obtained from the Ebola virus GP gene sequence /H.sapiens-wt / SLE / 2014 / Makona-G3735.1 GenBank ID KM233056 by adding adenine at the 887 position, which is necessary for expression of the transmembrane highly 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.

Six plasmid constructs carrying various variants of the GP gene and the Ebola virus NP gene were obtained: pShuttle-NP, pShuttle-GPNsl, pShuttle-GPN ° 2, pShuttle-GPN ^Q 3, pShuttle-GPNs4, pShuttle-GPwt (control, contains unmodified Ebola virus GP gene /H.sapiens- wt / SLE / 2014 / Makona-G3735.1 GenBank ID KM233056. All Ebola antigen sequences 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-GPNel, pShuttle-GPN ^Q 2, pShuttle-GPN ^Q 3, pShuttle-GPN24, pShuttle-GPwt) were processed with Pad and Bstll07l endonucleases for extraction expression cassettes 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 has also been treated with Ras endonucleases! and Bstll07l. The hydrolysis products were ligated. The resulting DNA contained in the ligase mixture was packaged in phage heads and competent DH5a cells were transduced with them. Transformed cells were selected on LB agar medium with the antibiotic ampicillin. Plasmid DNA was isolated by boiling

Thus, as a result, cosmid constructs containing the genome of the recombinant human adenovirus of the fifth serotype with expression cassettes, which include the NP gene (cosmid cAd5-NP), the GPN21 gene (cosmid cAd5-GPNs1), GPN22 (cosmid cAd5-GPN22) , GPN23 (cosmid cAd5-GPN23), GPN24 (cosmid cAd5-GPN24). At the next stage, to obtain mini-preparations of recombinant adenoviruses, plasmid constructs carrying expression cassettes with the Ebola virus genes as part of the full-size recombinant adenovirus genomes were hydrolyzed by restriction endonucleases Rac! and Swal to remove the plasmid portion and transfected into eukaryotic HEK293 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-GPNsl, Ad-GPNQ2, Ad-GPNs3, Ad-GPNs4 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 expression of NP and GP proteins in cells transfected with Ad-NP, Ad-GPN21, Ad-GPNs2, Ad-GPNs3, Ad-GPN ^Q 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% C0 ₂ . Cells were placed on 30 mm ² Petri culture dishes and incubated for one day until 70% confluency was achieved. Next, the studied preparations of adenoviruses (Ad-NP, Ad-GPN ° 1, Ad-GPN ^Q 2, Ad-GPNs3, Ad-GPNs4) and control preparations of adenovirus (Ad-null - recombinant adenovirus without inserts, Ad- GPwt recombinant adenovirus containing non-optimized GP sequence) at a rate of 100 PFU / cell or phosphate buffer as a negative control. After a day, the expression of Ebola virus 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.

Then electrophoresis was carried out under denaturing conditions in a 10% SDS-polyacrylamide gel SPRINT NEXT GEL® 10% (Amresco) in the NEXT GEL® Running Buffer, 20X (Amresco) using the Mini-PROTEAN® Tetra Cell (Bio-Rad) 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.

The results of immunoblotting are presented in figure 1.

Track 1 to 7 - data on GP protein expression are presented, where:

1 - cell lysate, to which was added phosphate-buffered saline, 2 - cell lysate, to which Ad-null was added,

3 - cell lysate to which Ad-wt was added

4 - cell lysate to which Ad-GPN ° 1 was added

5 - cell lysate to which Ad-GPN ^Q 2 was added

6 - cell lysate to which Ad-GPN ° 3 was added

7 - cell lysate to which Ad-GPN ° 4 was added

From track 8 to 10 - data on NP protein expression 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 obtained data, the expression of the target NP protein was detected in the cells to which Ad-NP was added 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 against NP and GP after immunization of animals with recombinant adenoviruses Ad-NP, Ad-GPN ° 1, Ad-GPNQ2, Ad-GPN ° 3, Ad-GPN ° 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, selected from 4 options: GPN21, GPN22, GPN23, GPN24 and a recombinant adenovirus containing the Ebola virus NP gene /H.sapiens-wt/SLE/2014/Makona-G3735.1 GenBank ID KM233056.

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

1) Ad-NP 10 ⁷ PFU / mouse, Ad-GPNs1 10 ⁷ PFU / mouse

2) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN21 10 ⁸ PFU / mouse

3) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN21 10 ⁹ PFU / mouse

4) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN22 10 ⁷ PFU / mouse

5) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN22 10 ⁸ PFU / mouse

6) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN22 10 ⁹ PFU / mouse

7) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN23 10 ⁷ PFU / mouse

8) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN23 10 ⁸ PFU / mouse

9) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN ° 3 10 ⁹ PFU / mouse

10) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN24 10 ⁷ PFU / mouse

) Ad-NP 10 ⁷ PFU / mouse, Ad-GPN24 10 ⁸ PFU / mouse 12) Ad- -NP 10 ⁷ PFU / mouse, Ad-GPN ^Q 4 10 ⁹ PFU / mouse

13) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPN ^Q 1 10 ⁷ PFU / mouse

14) Ad- ^■ NP 10 ⁸ PFU / mouse, Ad-GPNs1 10 ⁸ PFU / mouse

15) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPNs1 10 ⁹ PFU / mouse

16) Ad- ^■ NP 10 ⁸ PFU / mouse, Ad-GPNQ2 10 ⁷ PFU / mouse

17) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPN ° 2 10 ⁸ PFU / mouse

18) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPNQ2 10 ⁹ PFU / mouse

19) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPN ^Q 3 10 ⁷ PFU / mouse

20) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPN ^Q 3 10 ⁸ PFU / mouse

21) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPN ^Q 3 10 ⁹ PFU / mouse

22) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPN ° 4 10 ⁷ PFU / mouse

23) Ad- ^■ NP 10 ⁸ PFU / mouse, Ad-GPNs4 10 ⁸ PFU / mouse

24) Ad- -NP 10 ⁸ PFU / mouse, Ad-GPN ^Q 4 10 ⁹ PFU / mouse

25) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPNs1 10 ⁷ PFU / mouse

26) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPNe1 10 ⁸ PFU / mouse

27) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPNQ1 10 ⁹ PFU / mouse

28) Ad- -NP 10 ⁹ PFU / mouse, Ad-GPNe2 10 ⁷ PFU / mouse

29) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPN ° 2 10 ⁸ PFU / mouse

30) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPN ^Q 2 10 ⁹ PFU / mouse

31) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPN ° 3 10 ⁷ PFU / mouse

32) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPN ^Q 3 10 ⁸ PFU / mouse

33) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPNs3 10 ⁹ PFU / mouse

34) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPNQ4 10 ⁷ PFU / mouse

35) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPNe4 10 ⁸ PFU / mouse

36) Ad- ^■ NP 10 ⁹ PFU / mouse, Ad-GPN ° 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-GPNs1 10 ⁷ PFU / mouse 41) phosphate buffered saline, Ad-GPN ° 1 10 ⁸ PFU / mouse

42) phosphate buffered saline, Ad-GPN ° 1 10 ⁹ PFU / mouse

43) phosphate buffered saline, Ad-GPNs2 10 ⁷ PFU / mouse

44) phosphate buffered saline, Ad-GPNs2 10 ⁸ PFU / mouse

45) phosphate buffered saline, Ad-GPNs2 10 ⁹ PFU / mouse

46) phosphate buffered saline, Ad-GPN ° 3 10 ⁷ PFU / mouse

47) phosphate buffered saline, Ad-GPNs3 10 ⁸ PFU / mouse

48) phosphate buffered saline, Ad-GPNs3 10 ⁹ PFU / mouse

49) phosphate buffered saline, Ad-GPN ° 4 10 ⁷ PFU / mouse

50) phosphate buffered saline, Ad-GPNs4 10 ⁸ PFU / mouse

51) phosphate buffered saline, Ad-GPNs4 10 ^e 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 recombinant adenovirus vectors of a heterosubtipic immune response against influenza A virus, Biologics, 2011, 1 (41); Scherbinin D.N. et al., Induction of the immune response to bacillus anthracis with intranasal the introduction of a recombinant adenovirus expressing a protective antigen fused to the Fc fragment of the IGG2a antibody, Acta Naturae, 2014, 1 (20), v. 6, C.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), C.9693-9703.). Thus, the doses of recombinant adenoviruses 10 ⁷ - 10 ⁹ PFU / dose were selected as the most optimal for immunization of animals, however, as a mid-level 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 at a wavelength of 450 nm was measured using a spectrophotometer. Antibody titer was determined 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.

Table 1 - The titer of antibodies to the protein GP in the blood serum of mice

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.

Table 2 - The titer of antibodies to protein NP in the blood serum of mice

As can be seen from the results of the experiment, the developed immunobiological agent, including recombinant human adenovirus of serotype 5, 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 .. and 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, wherein the amount of each of the adenoviruses varies from October ⁷ PFU / dose to 10 ⁹ PFU / dose eff su- stimulate humoral immune responses against Ebola virus antigens in the entire range of selected doses.

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 that forms in labeled cells is retained by these cells throughout development, as well as during division. Label transmitted to daughter cells and never passes to neighboring cells in a population. CFSE allows you to track cell division, because when dividing the concentration of the label 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-GPNQ1 10 ⁸ PFU / mouse

4. Ad-NP 10 ⁸ PFU / mouse, Ad-GPN ° 2 10 ⁸ PFU / mouse,

5. Ad-NP 10 ⁸ PFU / mouse, Ad-GPNQ3, 10 ⁸ PFU / mouse

6. Ad-NP 10 ⁸ PFU / mouse, Ad-GPN ° 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 later, after immunization, the spleen was taken from 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), C: 2049-2056. ) and analyzed by flow cytometry. The results are presented in the form of a histogram (FIG. 2). As can be seen from the results of the experiment, the developed immunobiological agent, including recombinant human adenovirus of serotype 5, 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. and 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, effectively stimulate the proliferation of lymphocytes.

Example 5

The aim of this experiment was to compare the immunogenicity of the recombinant proteins GPNsl, GPN22, GPN ^Q 3, GPN ^Q 4 obtained by introducing a modified GP gene sequence (selected from SEQ ID N2I, SEQ ID N22, SEQ ID N23, SEQ ID N24, respectively) with the immunogenicity of the GPN26 protein obtained by introducing into the producer cells an unmodified GP gene sequence (SEQ ID N26). Proteins were isolated from producer cells and purified using chromatography methods.

Protein immunogenicity was evaluated by the formation of specific antibodies after double immunization of BALB / c mice (dose of 10 μg / mouse) with an interval of 3 weeks. One week after the last immunization, animals were bled. Next, blood tubes were kept for 30 min in an incubator at a temperature of 37 ^° C and then serum was taken. The titer of specific antibodies was determined using enzyme immunoassay. 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 at a wavelength of 450 nm was measured using a spectrophotometer. The antibody titer was determined as the last dilution in which the optical density of the solution was significantly higher than in the negative control group (intact animals). The results are presented in table 3. Table 3 - The titer of specific antibodies to the protein GP in the blood serum of mice

As shown in table 3, immunization with all proteins led to the formation of specific antibodies. Moreover, the highest titers of antibodies to GP were found during immunization of animals with modified proteins GPNsl, GPNs2, GPN ^Q 3, GPN ^Q 4 (compared with the unmodified GPN26 protein).

Thus, the results presented in this example show that the modification of the nucleotide sequence of the GP gene leads to an increase in the immunogenicity of the protein itself, and not due to an increase in its expression (since all proteins were taken in equivalent amounts). Example 6

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 enters, 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.

The experiment used laboratory mice of the BALB / c line of females weighing 18 g. 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 HUME / mouse hyaluronidase

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

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

5) Hyaluronidase UME / mouse

6) Hyaluronidase 25ME / mouse

7) Hyaluronidase 50ME / 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). Next, the samples were centrifuged at 12000 xd 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 presented in Fig.Z.

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 to which only adenovirus was administered. 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 (N ^Q 5,6,7), there were no differences in comparison with intact animals.

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

Example 7

Estimated dosage forms of an immunobiological agent

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. zo The developed immunobiological agent can be used for intramuscular, subcutaneous and inhalation administration and can be presented in various dosage forms: a solution for subcutaneous / intramuscular administration, powder or solution for inhalation, in the form of a lyophilizate.

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 non-toxic to humans and containing all the necessary components ensuring the viability of the adenoviral vector and enzyme activity can act 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.

Thus, in comparison with analogues and prototype, the claimed invention used the technology of modification of the nucleotide sequence

(optimization) for its more efficient expression in a heterologous organism by replacing rare codons with optimal ones for a given heterologous organism. This technology is based on the use of the “redundancy” characteristics of the genetic code. In the process of modifying the nucleotide sequence, a codon encoding a specific amino acid in the amino acid sequence, but having a small amount of the corresponding tRNA in the producer cell, as a result of which reduces the total expression level of the protein product, is replaced by another codon encoding the exact same amino acid, but having in the cell produce more of the corresponding tRNA. The result of this modification of the nucleotide sequence is a noticeable increase the expression of the protein product in this case without changing the amino acid sequence of the expression product.

However, in the developed immunobiological agent, the authors carried out an original modification of the nucleotide sequence, the purpose of which is not so much to change some codons to others coding for the same amino acids for more efficient expression of the expression product, but to change the nucleotide sequence to change the amino acid sequence. As a result of the experiments performed by the authors in the examples, the amino acid sequences of the Ebola virus were changed and those that, due to their altered structure, were found to have significantly higher specific immunogenicity compared to the unchanged variant.

This effect is confirmed in Example 5. This example shows that when using equal amounts of recombinant proteins for animal vaccination, proteins obtained using modified nucleotide sequences have significantly greater immunogenicity compared to proteins obtained using an unchanged nucleotide sequence.

This proves that in addition to increasing the level of expression, modified nucleotide sequences allow significant

ss increase the immunogenicity of the claimed immunobiological agent.

Thus, increasing the level of expression and obtaining the effect of significantly increasing the immunogenicity of the expression products of the nucleotide sequences used in the inventive tool proves the fulfillment of the technical task of the present invention - the creation of 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 authors experimentally selected nucleotide sequences that ensure the effective action of the claimed immunobiological agent, the creation of which is not obvious to a mid-level specialist without carrying out the corresponding experimental work.

Industrial applicability

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

Claims

Claim

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.