RU2507258C1 - Recombinant pseudo adenoviral particle based on human being adenovirus genome of 5-th serotype for induction of specific immunity to influenza virus a of h3n2 subtype and method of its use - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use. Provided particle contains expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus, haemagglutinin gene of A/Perth/16/2009(H3N2) strain with pre-optimised for expression in human being cells nucleotide sequence presented in SEQ ID NO:2. The specified haemagglutinin gene of influenza virus of A/Perth/16/2009(H3N2) strain is cloned in expressing cassette containing polyadenylation signal SV40 under control of cytomegalovirus promoter. Presented invention may be used for induction of specific immunity to influenza virus A of H3N2 subtype during injection in efficient quantity.

EFFECT: providing intense expression of recombinant haemagglutinin of the specified influenza virus.

6 cl, 9 dwg, 1 tbl, 4 ex

Description

Technical field

The invention relates to the field of biotechnology and relates to recombinant vectors that can be used in the pharmaceutical industry for the production of vaccine preparations, in particular for influenza vaccines.

Prior development

Vaccination of human influenza is the most important link in the implementation of anti-epidemic measures to reduce the incidence rate among people during seasonal outbreaks of the disease. Vaccines based on the so-called pandemic influenza virus strains are especially important. Influenza pandemic subtypes can spread globally, often with a high mortality rate for affected people. Epidemic monitoring of the circulation of influenza viruses is carried out by WHO (World Health Organization), which announces the strains recommended for inclusion in seasonal influenza vaccines in certain territories (Resource available: www.who.int).

The manufacture of a vaccine requires the presence of highly immunogenic antigens in its composition. As a rule, attenuation or inactivation of a strain in the production of classical vaccines reduces its immunogenicity. Therefore, at the present stage of development of biotechnology, the production of genetic vaccines is much more technologically and immunobiologically effective.

To date, all the antigens that make up the vaccines can be divided into two main groups according to the production method - native and recombinant, the latter, due to the development of genetic engineering and biotechnology, begin to displace antigens obtained by classical methods. To obtain antigens by classical methods, it is necessary to initially have a strain of the influenza virus isolated from patients with influenza.

The basis of any vaccine preparation are various pathogen antigens, which necessarily have immunogenic properties. The main specific immunogen of the influenza virus is its hemagglutinin, which is taken into account when developing immunobiological preparations against influenza.

Thus, a vaccine strain of influenza virus B / 60 / Brisbane / 08/83 is known for the production of a live influenza intranasal vaccine for adults and children (RF Patent No. 2,442,517). This strain is live and attenuated by a reassortment method with a donor strain that is harmless to humans, which deprived it of pathogenicity, but retained the immunogenicity of its hemagglutinin. The strain propagates in chicken embryos. As you can see, the strain is live attenuated, that is, it can be reversed to its original pathogenic form, it requires the use of chicken embryos for cultivation, which is uneconomical, inefficient, reassortment in other influenza viruses is possible if they infect embryos, in addition, the residual amounts of chicken protein in drugs can cause allergic reactions.

Also known from the classic options is the vaccine strain of influenza virus A / 8 / Perth / 16/2009 (H3N2) for the production of inactivated influenza vaccine (RF Patent No. 2458124). This strain is pathogenic and requires special care when handling it in the laboratory and in production, as well as, during the production process, an inactivation step is required to put the virus into a non-living state, which, however, dramatically reduces immunogenicity.

To solve the problems associated with native strains of various microorganisms intended for the production of vaccines, the direction for the use of safe vectors expressing the genes of pathogen antigens is promising (Abdulhaqq SA, Weiner DB DNA Vaccines: developing new strategies to enhance immune responses, 2008, No. 42, p. .219-232 - Vaccines: developing new strategies to enhance the immune response); (Zaia J.A., The status of gene vectors for the treatment of diabetes, Cell Biochem. Biophys, 2007, No. 48 (2-3), pp. 183-90. Status of genetic vectors for the treatment of diabetes mellitus). The vectors constructed on the basis of human adenovirus are recognized as one of the safest for the body (Van Kampen KR et al., Safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans, Vaccine, 2005, No. 23 (8), p. 1029 -36 - Safety and immunogenicity of adenovector nasal and cutaneous influenza vaccines in humans).

Recombinant adenoviral vectors have the following useful characteristics: they are not pathogenic, since the pathogenicity regions are removed from their genome, they can transduce both dividing and postmitotic cells, the adenovirus DNA remains in an extrachromosomal form, it can reproduce only in special cell lines in vitro , are eliminated from the body within 4-5 weeks, induce both a cellular and a humoral immune response, the process of obtaining a new recombinant adenovirus takes only a few n del, they provide a high level of target gene expression in the target cell.

Adenoviral vector expressing recombinant influenza hemagglutinins is known today (Patent Application WO No. 2008/157419). This design is selected by the author for the prototype, as the closest to the invention (prototype).

Due to the fact that at the priority date of the application, nothing was known about the strain of the influenza virus A / Perth / 16/2009 (H3N2), since this pathogen was first isolated and characterized during the influenza epidemic in 2009, the nucleotide sequence of its hemagglutinin gene could not be used to construct recombinant adenoviral particles. Accordingly, the applicants were not able to create an adenoviral construct with the characterized cultural and morphological properties, expression level, immunogenic and protective properties. This is a significant drawback of the claimed design, since it cannot be used to induce specific immunity to influenza viruses of the H3N2 subtype, in particular to strain A / Perth / 16/2009 (H3N2), which prevents the creation of influenza vaccines based on it.

Disclosure of invention

The technical task of the invention is aimed at creating a recombinant pseudo-adenovirus particle based on the genome of human adenovirus of the 5th serotype, producing recombinant influenza hemagglutinin, capable of providing enhanced expression of recombinant influenza virus hemagglutinin A / Perth / 16/2009 (H3N2) for use specific immunity to influenza A virus subtype H3N2.

The technical problem is solved due to the fact that they create a recombinant pseudo-adenovirus particle based on the genome of human adenovirus 5 serotype, containing an expression cassette with the insertion of the influenza hemagglutinin gene, while the strain A / Perth / 16/2009 (H3N2) virus hemagglutinin gene is used a hemagglutinin gene with a nucleotide sequence previously optimized for expression in human cells, providing increased expression of the influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2). Moreover, the optimized nucleotide sequence of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) is SEQ ID NO: 2, and the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) with the optimized nucleotide sequence is cloned into an expression cassette under the control of the promoter and contains a polyadenylation signal, the promoter being the cytomegalovirus promoter, and the polyadenylation signal being SV40. The expression cassette in this technical solution is located in the region of the E1 deletion of the genome of the human adenovirus 5 serotype. A method of using a recombinant pseudo adenovirus particle based on the genotype 5 human adenovirus genotype producing hemagglutinin A / Perth / 16/2009 (H3N2,) influenza virus is to administer the generated recombinant pseudo adenovirus particles in an effective amount to induce specific immunity to influenza A virus subtype H3N2.

List of figures

Figure 1 - presents:

SEQ ID NO: 1 — non-optimized nucleotide sequence of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2);

SEQ ID NO: 2 — nucleotide sequence of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) optimized for expression in human cells;

Protein is the amino acid sequence encoded by the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Figure 2 is a diagram of an expression cassette of a recombinant pseudo-adenovirus particle with the hemagglutinin gene of influenza virus strain A / Perth / 16/2009 (H3N2).

Figure 3 - shows an electrophoregram of PCR analysis of DNA of a recombinant pseudo-adenovirus particle based on human serotype 5 adenovirus with an optimized influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) (SEQ ID NO: 2).

Figure 4 - shows the results of an immunoblot analysis. Lanes indicate the result of an analysis with a lysate of HEK293 cell lines.

Figure 5 - presents a chart, the columns of which show the level of anti-hemagglutinating antibodies, defined in rtga, to influenza virus A / Perth / 16/2009 (H3N2).

Figure 6 - shows the survival curves of immunized mice after infection with a lethal dose of influenza virus strain A / Perth / 16/2009 (H3N2).

7 - presents the non-optimized nucleotide sequence of the hemagglutinin gene A / Perth / 16/2009 (H3N2).

On Fig - presents the optimized nucleotide sequence of the hemagglutinin gene A / Perth / 16/2009 (H3N2).

Figure 9 - shows the amino acid sequence of the hemagglutinin gene A / Perth / 16/2009 (H3N2).

It is well known that the genetic code is degenerate, that is, one amino acid can be encoded from one to six different triplets (codons) from different nucleotides, which, as a rule, differ only in the last nucleotide (N. Dubinin, General genetics, M., 1970, p.204-207). Each such codon corresponds to a single tRNA, which in the process of protein synthesis delivers the corresponding amino acid to the ribosome. However, in different organisms, the set of tRNAs differs, with the preferred use of one of several codons encoding the same amino acid. Data on the frequency of use of codons in various organisms is publicly available (for example, the resource www.kazusa.or.jp).

Based on the biological characteristics of protein transcription in organisms described above, the author decided to replace individual nucleotides in the codons in the nucleotide sequence of the hemagglutinin gene that do not entail replacing the amino acid itself, taking into account the preference for using tRNA codons in humans, that is, to use the hemagglutinin gene with previously optimized for expression in human cells with a nucleotide sequence, which ultimately led to increased expression of the target gene upon administration vector with the insertion of the obtained hemagglutinin gene into the human body.

To solve the technical problem is necessary:

1) to optimize the nucleotide sequence of the influenza virus hemagglutinin gene A / Perth / 16/2009 (H3N2) to increase the expression of this gene in human cells;

2) to construct a recombinant pseudo-adenovirus particle with a target transgene by embedding the obtained optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2) in human adenovirus 5 serotype;

3) show the cultural - morphological features of the obtained recombinant pseudo-adenovirus particles;

4) show improved expression of the optimized hemagglutinin gene in vitro;

5) show the improved immunogenicity and protective properties of recombinant pseudo-adenovirus particles obtained according to the claimed invention based on human adenovirus 5 serotype for influenza A / Perth / 16/2009 (H3N2) virus, and accordingly, justify their use for the induction of specific immunity to subtype influenza A virus H3N2.

The examples below confirm the solution of the technical problem.

Examples of carrying out the invention

Example 1

This example shows the progress of constructing a recombinant pseudo-adenovirus particle based on the genotype 5 human adenovirus genome with the insert of the optimized influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2).

At the first stage, to improve the expression of the transgene by the recombinant pseudo-adenovirus particle, the sequence of the hemagglutinin gene of the influenza virus strain A / Perth / I 6/2009 (H3N2), selected on the basis of WHO recommendations (taken from the official public source NCBI, GenBank, USA, No. GQ293081.1; whose data are available: www.ncbi.nlm.nih.gov), based on well-known information about the degeneracy of the genetic code and the frequency of use of certain types of codons in humans, has been previously optimized for expression in human cells.

To do this, in the nucleotide sequence of the influenza virus hemagglutinin gene of strain A / Perth / I 6/2009 (H3N2), codons that were “inconvenient” for human tRNA were replaced so that the amino acid sequence did not change. In the selection we used well-known data on the frequency of occurrence of codons in humans (http://www.kazusa.or.jp/codon/). For example, the amino acid leucine (L) is encoded by six codons - UUA, UUG, CUA, CUC, CUG, CUU, however, in humans, CUC (20%) and CUG (40%) are most often used, therefore they were replaced by all the remaining four codons, in the appropriate proportion (for CUC - 24.7% and CUG - 56.4%).

The same was done with all other codons, not singularly encoding one amino acid.

The figure 1 presents:

SEQ ID NO: 1 — non-optimized nucleotide sequence of the hemagglutinin gene of influenza virus strain A / Perth / 16/2009 (H3N2);

SEQ ID NO: 2. - the nucleotide sequence of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) optimized for expression in human cells;

Protein is the amino acid sequence of the hemagglutinin gene of influenza A / Perth / 16/2009 (H3N2) encoded by the nucleotide sequences of SEQ ID NO:

1 and SEQ ID NO: 2.

The numbers above the sequences indicate the nucleotide number in the sequence.

Conducted with the aim of optimization, the replacement of nucleotides in the nucleotide sequence of SEQ ID NO: 1 of the non-optimized influenza A / Perth / 16/2009 (H3N2) hemagglutinin gene with nucleotides in the nucleotide sequence of SEQ ID NO: 2 of the optimized influenza A / Perth / 16 hemagglutinin gene / 2009 (H3N2) darkened with gray rectangles.

The amino acid letter is located under the three nucleotides of the two compared nucleotide sequences (SEQ ID NO: 1 and SEQ ID NO: 2) with which it is encoded.

All letter designations of nucleotides and amino acids are standard and well-known.

Thus, the non-optimized and optimized nucleotide sequences of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) shown in FIG. 1 consist of 1701 nucleotides pairs, are located under each other and compared (on top is the sequence of the non-optimized hemagglutinin gene, under it the optimized sequence ) In total, both nucleotide sequences encode 566 amino acids (the amino acid sequence is displayed under the name "protein"), both of which are the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 encode the same amino acid sequence of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2), that is, the hemagglutinin gene of the above strain of influenza virus produced from an optimized nucleotide sequence does not differ from the native one, which is what is required when optimizing the nucleotide lnosti.

Thus, the lower (SEQ ID NO: 2) nucleotide sequence is a unique nucleotide sequence of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) optimized for expression in human cells.

The obtained unique optimized cDNA sequence of the human influenza virus hemagglutinin gene was synthesized by a well-known chemical method and ligated into a well-known auxiliary plasmid for further cloning. Thus, the synthesized nucleotide sequence contained optimized codons that did not affect the amino acid composition of hemagglutinin, but significantly improved its expression in human cells.

At the next stage, a recombinant pseudo-adenovirus particle based on the 5th serotype human adenovirus genome was obtained containing an insert of an optimized sequence of the influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) - SEQ ID NO: 2.

All of the cloning work described below was carried out using well-known techniques described in Sambrook J. et al., Molecular cloning: a laboratory manual, 3rd ed., Russell, 2001, vol. 1, 2, 3 — Molecular cloning — laboratory manual.

The construction of a recombinant pseudo-adenovirus particle based on the fifth serotype human adenovirus genome (size 70-80 nm) was carried out by homologous recombination between the well-known plasmid pJM17 (McGrory WJ et al., A simple technique for the rescue of early regioni mutations into infectious human adenovirus type 5 , Virology, V. 163, No. 2, 1988 - A simple technique for removing early region 1 in human infectious adenovirus type 5) containing the genomic part of human adenovirus 5 serotype with an E1-impaired region and an auxiliary plasmid pACCMVpLpA (Roth MG, Methods in cell biology, No. 43, p.175 - M methods in cell biology.), (Go 'mez-Foix A. et al., Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism, J. Biol. Chem., 1992, No. 267 ( 35), 15, p.25129-25134 - Adenovirus-mediated transfer of muscle glycogen phosphorylase gene to hepatocytes changes the regulation of glycogen metabolism). Previously, the optimized influenza A / Perth / 16/2009 (H3N2) hemagglutinin gene from an auxiliary plasmid was cloned into the shuttle plasmid pACCMVpLpA, and pACCMVpLpA was obtained with an expression cassette with the target transgene (the hemagglutinin gene that is involved in the flank recombination. Homologous recombination was performed in HEK293 cells (for example, No. 300192, CLS, Germany) after cotransfection with plasmids pJM17 and pACCMVpLpA. Since plasmid pJM17 contained the bacterial replication initiation site (Ori) and the ampicillin resistance gene within the E1 region of the genomic part of the adenovirus, this plasmid could not be packed into adenovirus virions, and thus the propagation of wild-type adenoviruses was prevented. After recombination, Ori and the ampicillin resistance gene disappeared, being replaced by a cassette with the target transgene. Recombinant DNAs were packaged in a capsid of virions, resulting in the formation of recombinant pseudo-adenovirus particles that could not multiply in non-permissive cell cultures, including ordinary human cells, due to the absence of E1 in the genome of recombinant pseudo-adenovirus particles.

Figure 2. Scheme of the expression cassette of a recombinant pseudo-adenovirus particle with a gene

fourteen

hemagglutinin of influenza virus strain A / Perth / 16/2009 (H3N2). Pictured:

1 - adenoviral nucleotide sequence (-);

2 - cytomegalovirus promoter (→);

3 - transgene ( _--- );

4 - SV40 polyadenylation signal (Simian vacuolating virus 40) (………).

To confirm compliance with the claimed properties of the created design of a recombinant pseudo-adenovirus particle based on human adenovirus 5 serotype with the insert of the optimized influenza hemagglutinin gene of type A strain A / Perth / 16/2009 (H3N2) (SEQ ID NO: 2), the following PCR (polymerase chain) was carried out reaction) according to a known standard method.

We checked the following properties of the created structure:

1. The authenticity of the recombinant pseudo-adenovirus particle, that is, the presence of the genome of the human adenovirus 5 serotype. To do this, PCR was performed on the adenovirus part (DNA sequence encoding human adenovirus 5 serotype hexon).

A pair of primers was used to confirm the presence of the recombinant adenovirus genome (hexon gene).

ADH12-reverse

5'-CTCAAAAGTCATGTCTAGCGC-3 '

ADHH-forward

S'-AACTTCCAGCCCATGAGCCG-3 '

2. The absence of pathogenicity, that is, the inability to cause adenovirus disease in humans, the E1 region is deleted in the resulting recombinant pseudo-adenovirus particles, in contrast to the wild-type adenovirus that can cause the disease. To confirm the absence of the E1 region, PCR was performed.

Used a pair of primers on the E1 region, which should be absent in the created recombinant pseudo-adenovirus particles.

KD25-Forward

5'-CGCGGGAAAACTGAATAAGAGGA-3 '

wtE1 R-Reverse

5'-ATGTCGGGCGTCTCAGG-3 '

3. The presence of an insert optimized hemagglutinin gene (SEQ ID NO: 2). For this, PCR for influenza hemagglutinin was used with primers for the target influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), optimized for expression in human cells.

H3opt - forward

5 '- CGT GCA CCA TCC CGG AAC AG - 3';

H3opt-reverse

5 '- TAG AGT TTG TCG AAC TGC TC - 3'

The results of PCR with the above primers are presented on the same electrophoregram (Figure 3).

Figure 3. shows an electrophoregram of PCR analysis of DNA of a recombinant pseudo-adenovirus particle based on serotype 5 human adenovirus with an optimized gene for the hemagglutinin gene of influenza strain A / Perth / 16/2009 (H3N2) (SEQ ID NO: 2).

Light horizontal stripes on the track - a positive result of the reaction, the absence of stripes on the track - a negative result.

Tracks: 1, 2, 3 - negative control, adenovirus DNA and positive control, respectively, with transgen primers. Tracks 4, 5, 6 - negative control, adenovirus DNA and positive control, respectively, with primers for human adenovirus hexon of the fifth serotype.

Tracks 7, 8, 9 - negative control, adenovirus DNA and positive control, respectively, with primers on the E1 region of human adenovirus of the fifth serotype.

M - molecular weight marker Gene Ruler ™ DNA Ladder Mix, Fermentas. The analysis was performed on a 0.8% agarose gel.

Thus, according to the results presented in figure 3, the obtained recombinant pseudo-adenovirus particle was analyzed by PCR using pairs of primers complementary to the corresponding target transgene (SEQ ID NO: 2), the human adenovirus hexon gene of the fifth serotype, as well as the E1 adenovirus region for control the possible presence of replicatively competent viral particles (“wild-type” adenovirus revertants).

The results of PCR analysis depicted in the figure show that the E1 region that should be deleted in recombinant pseudo-adenovirus particles was not detected (negative result on track 8), the DNA sequence encoding human adenovirus hexon 5 serotype was detected (positive result on track 5) , gene for the optimized influenza virus hemagglutinin gene A / Perth / 16/2009 (H3N2) (SEQ ID NO: 2) - detected (positive on track 2).

According to PCR results, it was concluded that a recombinant pseudo-adenovirus (E1 region of adenovirus is absent) particle based on human adenovirus 5 serotype (there is a human serum adenovirus 5 serotype) with an optimized gene for the hemagglutinin gene of influenza strain A / Perth / 16/2009 (H3N2) is present the optimized hemagglutinin gene - SEQ ID NO: 2), which indicates the compliance of this design with the requirements claimed according to the invention.

Example 2

This example describes the cultural and morphological features of the constructed recombinant pseudo-adenovirus particles based on the human serotype 5 adenovirus genome with the insert of the optimized influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2), SEQ ID NO: 2.

All applied cultural methods were well known (Freshni R. et al. Culture of animal cells. Methods, M., Mir, 1989, p. 333).

Recombinant pseudo-adenovirus particles are collected in cells of the HEK293 line (human embryonic kidney - kidneys of a human embryo) (for example, No. 300192, CLS, Germany). The genome of this type of cell contains an E1 region, which allows recombinant pseudo-adenovirus particles with similar regions removed to collect and multiply in cells of this line. Particle assembly is accompanied by cell lysis, from the moment of transduction to the time of cell lysis and obtaining a new generation of recombinant pseudo-adenovirus particles with the insertion of the influenza virus hemagglutinin gene, 48 hours pass.

For the growth of HEK293 cells in an adhesion culture, it is necessary to use DMEM medium (for example, Invitrogen, No. 52100-047, USA) containing 25 mM glucose, 4 mM D-glutamine and 10% fetal bovine serum, a special incubator with a temperature of + 37 ° C and 5% CO ₂ .

The number of recombinant pseudo-adenovirus particles capable of transducing (activity) HEK293 cells was determined using the standard plaque titration method, resulting in a particle titer expressed in plaque forming units per ml (PFU / ml).

To assess the activity of a monolayer of HEK293 cells with a confluency of 50-70%, they were infected with a suspension of infected cells, in the amount of 10 PFU of the virus per culture cup with a diameter of 15 cm. After two days, the infected cells were removed, concentrated by low-speed centrifugation, suspended in phosphate buffer (pH 7.2- 7.4) and destroyed by 3-fold freeze-thaw. The resulting suspension was clarified by centrifugation at 2000 rpm for 10 min at + 4 ° C and titrated by plaque.

Thus, the activity of recombinant pseudo-adenovirus particles was established from 5 × 10 ⁷ to 3 × 10 ⁸ PFU / ml (plaque-forming units per ml of HEK293 culture).

Example 3

The ability of the recombinant pseudo-adenoviral particles to enhance the expression of the optimized hemagglutinin gene of the influenza virus strain A / Perth / 16/2009 (H3N2) was determined in vitro by standard immunoblotting (western blot) using monoclonal antibodies to hemagglutinin.

For this, HEK293 cells were transduced with recombinant pseudo-adenovirus particles with an optimized hemagglutinin gene insert, as well as recombinant pseudo-adenovirus particles with translation-optimized codons and recombinant pseudo-adenovirus particles having a target gene that is not related to hemagglutinins. One day after transduction, the cells of all samples were washed 2 times with phosphate-buffered saline, pH = 7.4, and lysed in an application buffer containing sodium dodecyl sulfate and dithiothreitol. The samples were heated for 7 min at + 95 ° С and cooled to room temperature. Next, the obtained lysates of control and transduced cells were subjected to polyacryamide gel electrophoresis under denaturing conditions.

As a positive control, purified hemagglutinin of influenza A virus was used (Sion Biological Inc., Cat. No. 40043-V08H). After carrying out the phoresis, the proteins from the gel were transferred to a special membrane for immunoblot analysis. Immunoblot analysis was performed according to a standard scheme using antibodies to influenza hemagglutinin A / Perth / 16/2009 (H3N2) (Sion Biological Inc., cat. No. SEK11056-1). The results of the immunoblot analysis are shown in figure 4.

The figure 4 shows the results of an immunoblot analysis. Lanes represent an analysis with a lysate of HEK293 cell lines:

1 - positive control (purified hemagglutinin of influenza A virus (Sion Biological Inc., cat. No. 40043-V08H);

2 - negative control (recombinant pseudo-adenovirus particles that do not produce hemagglutinin);

3 - recombinant pseudo-adenovirus particles with the insert of the optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 2;

4 - recombinant pseudo-adenovirus particles with the insertion of the non-optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 1.

Thus, in figure 4, the staining of the detection band on lane 1 (positive control) and lane 3 (in the lysate of 293 cell cells transduced with recombinant pseudo-adenovirus particles with an insert of the optimized hemagglutinin gene, SEQ ID NO:

2. In the lysate of cells transduced with recombinant hemagglutinin pseudo-adenovirus particles with non-optimized codons, SEQ ID NO: 1 in lane 4, a very weak band was observed, indicating a very low expression level, in the case of a negative control in lane 2, there was no staining.

The obtained results of the immunoblot analysis allow us to conclude that the expression of recombinant pseudo-adenoviral particles of the optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2) compared to the non-optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), which corresponds to the solution of the task of the invention to create a recombinant pseudo-adenovirus particle based on the genome of human adenovirus 5 serotype, intensively producing hemagglutinin of influenza virus A / Perth / 16/2009 (H3N2), SEQ ID NO : 2.

Example 4

This example describes a method of using recombinant pseudo-adenovirus particles based on the genotype 5 human adenovirus genome producing hemagglutinin of influenza A / Perth / 16/2009 (H3N2) virus by administering them in an effective amount to induce specific immunity to influenza A virus of the H3N2 subtype.

To confirm the method of use, mice were immunized. The best immunogenicity and protective properties of the created recombinant pseudo-adenovirus particles were obtained with a dose of 2 × 10 ⁷ PFU / mouse, with no side effects at the time of administration and in the long term.

For immunization of laboratory mice with recombinant pseudo-adenovirus particles, the animals were divided into groups of 10 individuals and under light ether anesthesia, they were intranasally immunized once with recombinant pseudo-adenovirus particles with an optimized gene for the hemagglutinin gene of the influenza virus strain A / Perth / 16/2009 (H3N2): , at a dose of 2 × 10 ⁷ PFU / mouse. As control groups of mice were used that received recombinant pseudo-adenovirus particles that did not carry a transgene and physiological saline NaCl. The comparison drug was recombinant pseudo-adenovirus particles with an insert of the non-optimized influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2), SEQ ID NO: 1.

Blood collection for serum analysis was carried out three weeks after immunization.

The immunogenicity of recombinant pseudo-adenovirus particles with the insertion of the hemagglutinin gene was determined by the level of anti-hemagglutinating antibodies in blood serum in standard RTGA (hemagglutination inhibition reaction) (Table 1 and figure 5).

Table 1 shows the titers of specific antibodies to the influenza A / Perth / 16/2009 (H3N2) virus in blood serum on day 21 after immunization of mice.

Table 1 Name of the administered drug Antihemagglutinating antibodies in rtga (geometric mean titer) Recombinant pseudo-adenovirus particles with an optimized influenza hemagglutinin gene insert A / Perth / 16/2009 (H3N2), SEQ ID NO: 2 3429.77 ± 863.5 Recombinant pseudo-adenoviral particles with the insertion of the gene for hemagglutinin HA3 (not optimized) influenza virus A / Perth / 16/2009 (H3N2), SEQ ID NO: 1 203.44 ± 61.8 Recombinant pseudo-adenovirus particles without transgene insertion <8 Control substance (physiological solution of NaCl) <8

According to table 1, it is seen that the immunogenicity of recombinant pseudo-adenovirus particles with the insert of the optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 2 significantly exceeds the immunogenicity of the recombinant pseudo-adenovirus particles with the insert of the non-optimized gene of the hemagglutinin hemagglutinin A virus / 16/2009 (H3N2), SEQ ID NO: 1. Control substances were non-immunogenic.

The figure 5 presents a chart, the columns of which show the level of anti-hemagglutinating antibodies defined in rtga to the influenza virus A / Perth / 16/2009 (H3N2), while the immunization was carried out with the following substances:

1 - recombinant pseudo-adenoviral particles with the insert of the optimized gene for hemagglutinin of the influenza virus A / Perth / 16/2009 (H3N2), SEQ ID NO: 2;

2 - recombinant pseudo-adenovirus particles with the insertion of the not optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 1;

3 - recombinant pseudo-adenovirus particles without transgene insertion;

4 - physiological NaCl solution.

The results of the diagram in Figure 5 show a significant increase in the level of anti-hemagglutinating antibodies when immunized with recombinant pseudo-adenovirus particles with the insert of the optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 2, compared with recombinant pseudo-adenovirus particles with no hemagglutinin gene of the same strain, SEQ ID NO: 1, as well as control substances (anti-hemagglutinating antibody titers were absent).

Also, three weeks after immunization, to study the protective properties of the created recombinant pseudo-adenovirus particles, a lethal infection was simulated by infecting laboratory mice with a high lethal dose of the influenza virus strain A / Perth / 16/2009 (H3N2) intranasally under light ether anesthesia. Observation of animals was carried out for 16 days after infection.

The figure 6 presents the survival curves of immunized mice after infection with a lethal dose of influenza virus A / Perth / 16/2009 (H3N2). The immunization was carried out:

1 (◆) - recombinant pseudo-adenovirus particles with the insert of the optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 2;

2 (■) - recombinant pseudo-adenovirus particles with the insertion of the not optimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 1;

3 (▲) - recombinant pseudo-adenovirus particles without transgene insertion;

- физиологический раствор NaCl.four

- physiological solution of NaCl.

The data presented in figure 6 indicate the presence of protective properties of recombinant pseudo-adenovirus particles with the insert of the optimized gene for hemagglutinin of the influenza virus A / Perth / 16/2009 (H3N2), SEQ ID NO: 2, since none of the immunized mice when infected with a lethal dose of the virus A / Perth / 16/2009 (H3N2) did not die (100% survival). In the case of immunization with recombinant pseudo-adenovirus particles with the insertion of an unoptimized influenza hemagglutinin gene A / Perth / 16/2009 (H3N2), SEQ ID NO: 1, and without the insertion of the transgene, the protective properties were much weaker, the survival rate was 32% and 9%, respectively . At the same time, in the control group receiving saline, the death of 100% of mice was observed within 12 days.

Thus, according to the results of the experiments, it was concluded that the created recombinant pseudo-adenovirus particles with the insert of the optimized gene for hemagglutinin of the influenza virus strain A / Perth / 16/2009 (H3N2), SEQ ID NO: 2, producing hemagglutinin of the influenza virus strain A / Perth / 16/2009 (H3N2), have enhanced immunogenic and protective properties against a specific strain of the causative agent of the influenza virus, which allows them to be used to induce specific immunity to the influenza A virus subtype of H3N2 in the body.

Output. Based on the research results presented in the examples and tables, it was concluded that the technical task of the invention was completed. A recombinant pseudo-adenovirus particle with the insertion of an optimized influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2), with an activity of 5 × 10 ⁷ to 3 × 10 ⁸ PFU / ml and increased expression level of the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2), enhanced immunogenic and protective properties, which allows it to be used to induce specific immunity to influenza A virus subtype H3N2 in the body, which also confirms the industrial applicability of the claimed invention.

Claims (6)

1. Recombinant pseudo-adenovirus particle based on the human adenovirus genome 5 serotype for the induction of specific immunity to influenza A virus subtype H3N2 containing an expression cassette with an insertion of the influenza hemagglutinin gene,
characterized in that
the influenza virus hemagglutinin gene was used the influenza virus hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) SEQ ID NO: 2 with the nucleotide sequence previously optimized for expression in human cells, providing increased expression of the influenza A / Perth / hemagglutinin gene 16/2009 (H3N2). 2. The recombinant pseudo-adenovirus particle according to claim 1, characterized in that the influenza hemagglutinin gene of strain A / Perth / 16/2009 (H3N2) with an optimized nucleotide sequence is cloned into an expression cassette under the control of a promoter and containing a polyadenylation signal. 3. The recombinant pseudo-adenovirus particle according to claim 2, characterized in that the promoter is a cytomegalovirus promoter. 4. The recombinant pseudo-adenovirus particle according to claim 2, characterized in that the polyadenylation signal is SV40. 5. The recombinant pseudo-adenovirus particle according to claim 1, characterized in that the expression cassette is located in the E1 deletion region of the human adenovirus genome 5 serotype. 6. The method for using the recombinant pseudo-adenovirus particle according to claim 1 based on the human adenovirus genome 5 serotype producing hemagglutinin of influenza A / Perth / 16/2009 (H3N2) virus by administering to the individual in an effective amount to induce specific immunity to influenza A virus subtype H3N2.

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