RU2571944C1 - Broad-spectrum m2 protein ectodomain avian influenza type a vaccine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: vaccine contains M2 protein ectodomain, a molecular adjuvant and a buffer solution taken in effective amounts. The M2 protein ectodomain is expressed by recombinant pseudoadenovirus particles based on a human adenovirus genome of serotype five in an individual's body in the form of a fusion protein consisting of four sequential M2 protein ectodomains of avian influenza type A and TLR 5 activator - flagellin Salmonella enterica serovar Dublin. As a molecular adjuvant, it contains TLR-4 activator - a herbal acid peptidoglycane having a molecular weight from 1000 to 40000 kDa.

EFFECT: invention is non-toxic and may be used for clinical applications in a human individual.

3 cl, 15 dwg, 5 tbl, 9 ex

Description

Technical field

The claimed invention relates to medicine and healthcare, in particular the prevention of influenza. A vaccine is proposed for the prevention of influenza A caused by various avian influenza A viruses.

State of the art

Influenza is a highly contagious viral disease of mammals (including humans) and birds. Although most avian influenza viruses do not cause disease in humans, some are of particular interest to the medical and scientific communities, as they are capable of infecting people and have pandemic potential. According to the World Health Organization, such viruses include the subtype of H5N1 avian influenza viruses, which is currently circulating among poultry and has caused human cases and death since 1997. Other subtypes of avian influenza viruses, including H7N7 and H9N2, have also infected people, including those with a fatal outcome.

Therefore, the problem of creating highly immunogenic and safe influenza vaccines that provide immunity that can protect a person immediately from various variants of the bird flu virus A is urgent, with the development of modern biotechnology, interest in creating new vaccines that can induce broad protective immunity has renewed.

Immunization with modern influenza vaccines is a scientifically proven effective way of mass prevention of influenza. Modern vaccines against influenza A virus cause the formation of mainly strain-specific antibodies to the surface glycoproteins of the influenza virus: hemagglutinin and neuraminidase. However, influenza A viruses are able to constantly modify their structure as a result of genetic changes. Antigenic variations are the result of molecular changes in surface proteins - hemagglutinin and / or neuraminidase. Such genetic variability contributes to a change in the antigenic structure so significantly that the specific immunity developed for the vaccine received may not be effective. Thus, vaccines based on one of the surface antigens of the influenza virus are characterized by a narrow specificity of action, which is a significant drawback.

One of the most promising influenza A antigens for creating broad-spectrum vaccines is the M2 protein, which forms the ion channel. M2 is one of the three proteins of the influenza A virus expressed on the surface of the virion, and, unlike hemagglutinin and neuraminidase, it is highly conserved. This makes the M2e protein a good candidate for the development of broad-spectrum vaccines. The ectodomain of the influenza A virus protein M2 protein is a small (24 a.a.) protein, and phylogenetic analysis shows that influenza A virus proteins of different subtypes that are circulating in the human population or among birds are similar in amino acid composition. Thus, it is possible to create consensus ("averaged") sequences of M2e protein, allowing you to "block" or most strains of human influenza viruses or avian influenza viruses.

It is known that the use of various conservative influenza antigens to create modern genetic vaccines is also promising. Such antigens can be M1 and M2 influenza A virus proteins, influenza A virus nucleoprotein NP and even PB2 polymerase protein. Since the variability of these proteins from strain to strain is much lower than that of influenza glycoproteins, it is possible to create vaccines based on them with a wide spectrum of action that protect against various subtypes of influenza A. Currently, new recombinant vaccines based on the ectodomain of M2 protein are being actively developed. the flu virus.

A disadvantage of conservative influenza antigens is their low immunogenicity. To increase immunogenicity, the use of adjuvants is required, including molecular adjuvants or inducers of the innate immune response.

Most of the solutions known from the prior art are based on the use of expression systems based on E. coli.

One of the analogues of the present invention is a vaccine against infection caused by avian influenza virus, comprising as an active agent virus-like particles based on the hepatitis B virus antigen, bearing on its surface ectodomain polypeptides M2 of the avian influenza virus protein and expressed in E. coli with further purification . The vaccine showed high immunogenicity, as well as resistance after infection with a strain of avian influenza virus homologous to M2e peptide in laboratory animals, which makes it possible to consider it as a candidate for a universal vaccine against avian influenza virus type A. (RF Patent No. 2358981). The disadvantage of such a design of a recombinant protein molecule is the presence of one copy of the ectodomain of the M2 protein of the bird influenza virus, which underestimates the immunogenic potential of the drug, in addition, the vaccine is not intended for the prevention of bird flu in humans and only protects the bird virus strain homologous to M2e (A / Duck / Potsdam 1402-6 / 1986 (H5N2)) after double or triple immunization.

A study is also known on the intramuscular immunization of mice with the recombinant fusion protein 4xM2e.HSP70c, consisting of successive repeats of the M2e protein and the HSP70 protein of Mycobacterium tuberculosis, obtained in E. coli, upon infection of animals with a lethal dose of influenza A viruses H1N1, H3N2 or H9N2 significantly weight loss, a decrease in the titer of the virus in the lungs and a less pronounced manifestation of the symptoms of the disease (Ebrahimi S., et. al. In contrast to conventional inactivated influenza vaccines, 4xM2e. HSP70c fusion protein fully protected mice against lethal dose of H1, H3 and H9 influenza A isolates circulating in Iran, Virology 430 (2012) 63-72). In fusion protein, including the HSP70 protein of Mycobacterium tuberculosis, a significant drawback may be sensitization of the body with the subsequent inability to conduct diagnostic allergic studies for tuberculosis due to false positive results.

Thus, there is a need to select more suitable adjuvants for inclusion in the fusion protein. Studies are known to increase the immunogenicity of the candidate subunit vaccine based on the M2 protein of the influenza A virus using flagellin, a ligand for Toll-like receptor 5 (TLR5). TLRs activate the innate immunity system and largely determine the development of adaptive immunity, the use of this mechanism is a modern approach to creating vaccines. Fusion proteins derived from E. coli production, consisting of flagellin and influenza antigens, were more effective at immunization than just influenza antigens [Huleatt JW, et.al., Potent immunogenicity and efficacy of a universal influenza vaccine candidate relative a recombinant fusion protein linking influenza M2e to the TLR5 ligand flagellin, Vaccine, 2008].

The closest solution to the invention is a composition containing a recombinant fusion protein of 4 M2e and a TLR5 ligand - flagellin selected from the group: S. typhimurium fljB / STF2, E. coli flagellin fliC, S. muenchen flagellin fliC, designed to stimulate an immune response in a subject. This decision was made as a prototype (Application WO 2006/069262). The fusion protein presented in this application is essentially recombinant expressed in vitro in cell culture (Drosophila Dmel-2 cells is transduced with a plasmid carrying the corresponding fusion protein gene). To obtain various pharmaceutical compositions, the recombinant fusion proteins thus obtained must be recovered from the culture fluid and thoroughly purified in a multi-step way. As a result, the subject is administered a drug whose active ingredient is a ready-made recombinant fusion protein. However, the prototype has several disadvantages. The production of recombinant proteins in vitro in large quantities is a significant biotechnological problem - it is difficult to scale the process of cultivation of cell cultures and the subsequent multi-stage purification of the accumulated target proteins. As a result, the process of obtaining recombinant proteins is long, laborious and very expensive. In addition, pharmaceutical compositions containing ready-made proteins have known drawbacks - they have short elimination periods, are weakly immunogenic as vaccines and require two to three administrations, and also have an increased allergenic potential.

Therefore, the search for new industrially applicable technological solutions for the creation of cheaper, effective and safe influenza vaccines based on the conserved proteins of the avian influenza A virus is relevant.

Disclosure and inventions

An object of the invention was the creation of a broad-spectrum influenza vaccine based on recombinant pseudo-adenovirus particles expressing the ectodomain genes of influenza A virus M2 protein, with a molecular adjuvant that can effectively protect a person from infection with type A avian influenza viruses.

The problem is solved due to the fact that a wide-spectrum influenza vaccine was created against bird flu A based on the M2 protein ectodomain, while the M2 protein ectodomain is expressed by recombinant pseudo-adenoviral particles directly in the subject's body as a fusion protein consisting of four sequentially located four ectodomains of the M2 protein avian influenza A virus and TLR5 activator flagellin Salmonella enterica serovar dublin, in addition, the vaccine additionally contains a molecular adjuvant and a buffered solution taken in eff active quantities.

The claimed influenza vaccine contains the dose:

- recombinant pseudo-adenovirus particles expressing the fusion protein gene -10 ⁶ -10 ⁹ act.ed .;

- molecular adjuvant - 20-100 units;

- pharmaceutically acceptable buffer - up to 0.5 ml.

In the claimed influenza vaccine, the nucleotide sequence of the fusion protein gene, which consists of four consecutive ectodomains of the M2 protein and TLR5 activator flagellin Salmonella enterica serovar Dublin, is represented by SEQ ID NO: 1. Moreover, the nucleotide sequence of the fusion protein gene SEQ ID NO: 1 encodes the amino acid sequence of the fusion protein SEQ ID NO: 2.

As a molecular adjuvant, the claimed vaccine contains an activator TLR-4, namely, acidic peptidoglycan of plant origin with a molecular weight of from 1000 to 40,000 kDa.

One of the most effective approaches to creating safe and effective new generation vaccines today is the use of genetic vaccines, including those based on recombinant adenoviral vectors. When such vaccines are introduced into the body, genetic material enters the cells of the body and the genes of the target pathogen proteins are expressed in them. As a result, antigens of the corresponding pathogens are recognized by the immune system, which leads to the induction of both a humoral and a cellular immune response. To date, the most promising and often used to create genetic vaccines are recombinant pseudo adenovirus particles (RPANs), created on the basis of human adenovirus of the fifth serotype. RPAN-based vaccines have several advantages:

- RPANs are replicatively defective and not capable of causing disease. The safety of human adenoviruses of the fifth serotype with deleted E1 and E3 regions of the genome is confirmed by a number of clinical trials of various vaccine and therapeutic drugs based on them [Van Kampen K.R. et.al., Vaccine. 2005];

- do not require multiple additional introductions, since the expression of the target antigen (fusion protein) occurs directly in vivo in the body of the immunized subject;

- At present, fast and flexible technologies for obtaining RPAN have been developed, which allow the implementation of large-scale production of various candidate vaccines based on them on one technological line, without its re-equipment and changes in the regulations;

- RPANs allow for intranasal immunization and, as a result, induce the formation of a mucosal immune response;

- in comparison with recombinant proteins obtained in eukaryotic culture, they are cheaper and highly immunogenic, since a small dose of RPAN allows significant amounts of protein to be produced in the body.

The above properties make RPAN a good technological platform for creating a wide range of vaccines against various pathogens.

In the vaccine according to the invention, another way to increase the immunogenicity of the vaccine is implemented - the introduction of another activator of the innate immune response - acid peptidoglycan with a molecular weight of 1000-40000 kDa. This is a biologically active saccharide isolated from plant materials and is an effective adjuvant. The action of the drug consists in the activation of Toll-like receptor 4 (TLR4), a significant increase in the effectiveness of antigen-specific and nonspecific reactions of the immune system, in strengthening the immune defense mechanisms against infections. Based on the current level of knowledge, the stimulation of innate immunity initiates a specific immune response. A study of TLR revealed a link between innate and acquired immunity. The relationship between innate and acquired immunity is carried out through dendritic cells, specialized phagocytes concentrated in the spleen, lymph nodes and skin. Dendritic cells, being antigen-presenting cells, are responsible for stimulating immunocompetent cells. They express a high level of co-stimulatory molecules necessary for the activation of T-lymphocytes, which is the beginning of a specific immunity.

Brief Description of the Figures

In FIG. 1 shows the nucleotide sequence of the generated fusion protein gene (SEQ ID NO: 1).

The figure 2 presents the amino acid sequence (SEQ ID NO: 2) of the generated fusion protein encoded by the gene of SEQ ID NO: 1.

The figure 3 presents a diagram of the RPAN genome carrying a genetic construct expressing the fusion protein gene containing the genes of the ectodomains M2 protein and flagellin (SEQ ID NO: 1).

The components of the structure are depicted:

CMV - promoter of human cytomegalovirus,

LP - the gene for the leader peptide of secreted alkaline phosphatase SEAP,

M2e is the consensus nucleotide sequence of the ectodomain of the M2 protein of influenza A virus (in four successive repeats),

LN - linker,

flagellin - conserved N and C-terminal domains of the flagellin protein Salmonella enterica serovar Dublin,

pA - SV40 polyadenylation signal (Simian vacuolating virus 40),

Ad5 - genomic part of human adenovirus 5 serotype,

ΔE1 is a deletion of the E1 region of the adenoviral genome.

The figure 4 presents the results of PCR analysis of DNA of a recombinant pseudo-adenovirus particle based on serotype 5 human adenovirus (RPAN) with a fusion protein gene insert (SEQ ID NO: 1).

A positive result is a dark strip on the gel track.

On the tracks, depending on the number displayed:

1, 5, 9 - molecular weight marker (10,000 bp);

2, 6, 10 - negative control;

3 - DNA RPAN, PCR with primers complementary to the ectodomain gene M2 of the influenza virus protein and flagellin gene;

4 - positive control, PCR with primers complementary to the ectodomain gene M2 of the influenza virus protein and flagellin gene (pShuttle-CMV-M4-FI);

7 - DNA RPAN, PCR with primers complementary to the fifth gene serotype of the human adenovirus hexon;

8 - positive control, PCR with primers complementary to the fifth serotype human adenovirus hexon gene (pAd-Easy);

11 - DNA RPAN, PCR with primers complementary to the E1 region of the genome of the human adenovirus of the fifth serotype;

12 — positive control, PCR with primers complementary to the E1 region of the fifth serotype of the human adenovirus genome (wild type adenovirus genome).

The figure 5 shows the levels of specific antibodies to M2e of the influenza A H5N2 virus in the blood serum of mice immunized intranasally:

Group 1 - RPAN in a dose of 10 ⁸ active units without adjuvant;

Group 2 - RPAN in a dose of 10 ⁸ active units + 10 units of adjuvant;

Group 3 - RPAN in a dose of 10 ⁸ active units + 20 units of adjuvant;

Group 4 - RPAN in a dose of 10 ⁸ active units + 100 units of adjuvant;

Group 5 - RPAN in a dose of 10 ⁸ active units + 110 units of adjuvant;

6 group - negative control, 0.9% NaCl.

Figure 6 shows the optical density diagrams of solutions of lysed cells expressing Toll-like receptor 4 (HEK293-hTLR4) after adding acid peptidoglycan (CNG) to them in various concentrations, as well as positive and negative controls.

The columns shown are:

1 - acid peptidoglycan at a concentration of 15 μg / ml

2 - acid peptidoglycan at a concentration of 5 μg / ml

3 - acid peptidoglycan at a concentration of 1.67 μg / ml

4 - acid peptidoglycan at a concentration of 566 ng / ml

5 - acid peptidoglycan at a concentration of 185 ng / ml

6 - acid peptidoglycan at a concentration of 61.7 ng / ml

7 - acid peptidoglycan at a concentration of 20.6 ng / ml

8 - lipopolysaccharide (positive control) 10 μg / ml

9 - lipopolysaccharide (positive control) 1 μg / ml

10 - Negative control (K-) - HEK293-hTLRnull.

The figure 7 shows the results of determining the level of lymphoproliferation in the cell culture of the lymph nodes of immunized mice in response to the addition of antigen.

Columns:

1 - the level of lymphoproliferation in the group receiving the vaccine without subsequent stimulation of the cell culture by the antigen;

2 - the level of lymphoproliferation in the group receiving the vaccine, followed by stimulation of the cell culture with the antigen;

3 - the level of lymphoproliferation in the group receiving phosphate buffer without subsequent stimulation of the cell culture with an antigen (negative control);

4 - the level of lymphoproliferation in the group receiving phosphate buffer followed by stimulation of the cell culture with antigen (negative control);

5 - the level of lymphoproliferation in the group receiving RPAN without a transgene, without subsequent stimulation of the cell culture with an antigen (negative control);

6 - the level of lymphoproliferation in the group receiving RPAN without a transgene, followed by stimulation of the cell culture with an antigen (negative control);

The figure 8 presents the survival curves of mice receiving the vaccine and the control substance infected with 50 LD ₅₀ influenza virus A / Mallard duck / Pennsylvania / 10218/84 (H5N2).

The curves represent data for the groups that were entered:

1 - 10 ⁹ active units RPAN + 100 IU adjuvant

2 - 10 ⁹ active units RPAN + 20 units adjuvant

3 - 10 ⁶ active units RPAN + 20 units adjuvant

4 - 10 ⁶ active units RPAN + 100 IU adjuvant

5 - control substance (buffer)

Figure 9 depicts weight loss curves of vaccine and control mice infected with 50 LD ₅₀ of A / Mallard duck / Pennsylvania / 10218/84 (H5N2) influenza virus.

The curves represent data for the groups that were entered:

1 - 10 ⁹ active units RPAN + 100 IU adjuvant

2 - 10 ⁹ active units RPAN + 20 units adjuvant

3 - 10 ⁶ active units RPAN + 20 units adjuvant

4 - 10 ⁶ active units RPAN + 100 IU adjuvant

5 - control substance (buffer)

The figure 10 shows the survival curves of mice during the observation period of 14 days after infection with 50 LD ₅₀ of influenza A virus H5N2.

1 - survival curve of mice that received the vaccine before infection.

2 - survival curve of mice treated with buffer solution (negative control).

The figure 11 shows the curves of the weight loss (total in%) of mice during the observation period 14 days after infection with 50 LD ₅₀ of influenza A virus H5N2.

1 - weight loss curve of mice that received the vaccine before infection.

2 - weight drop curve of mice treated with buffer solution (negative control).

The figure 12 shows the survival curves of mice during the observation period of 14 days after infection with 50 LD ₅₀ of influenza A virus H2N3.

1 - survival curve of mice that received the vaccine before infection.

2 - survival curve of mice treated with buffer solution (negative control).

The figure 13 shows the curves of the weight loss (total in%) of mice during the observation period 14 days after infection with 50 LD ₅₀ of influenza A virus H2N3.

1 - weight loss curve of mice that received the vaccine before infection.

2 - weight drop curve of mice treated with buffer solution (negative control).

The figure 14 shows the survival curves of mice during the observation period of 14 days after infection with 50 LD ₅₀ of influenza A H1N1 virus.

1 - survival curve of mice that received the vaccine before infection.

2 - survival curve of mice treated with buffer solution (negative control).

The figure 15 shows the curves of the weight loss (total in%) of mice during the observation period 14 days after infection with 50 LD ₅₀ of the influenza A H1N1 virus.

1 - weight loss curve of mice that received the vaccine before infection.

2 - weight drop curve of mice treated with buffer solution (negative control).

Examples of the claimed invention

Example 1. The creation of a nucleotide sequence encoding a fusion protein consisting of four ectodomains of the M2 protein and the ligand for the Toll-like receptor 5, flagellin.

Example 2. Construction of a recombinant pseudo-adenovirus particle expressing the gene of the developed fusion protein, consisting of four ectodomains of the M2 protein and the ligand for the Toll-like receptor 5, flagellin.

Example 3. The selection of an effective molecular adjuvant capable of activating innate immunity.

Example 4. The selection of the effective composition of the vaccine

Example 5. Obtaining a vaccine

Example 6. Evaluation of the T-cell response to the vaccine

Example 7. Evaluation of the effectiveness of the vaccine

Example 8. The study of the breadth of action of influenza vaccine

Example 9. Preclinical studies of vaccine safety.

Example 1

This example shows a method for creating the nucleotide sequence of a fusion protein consisting of four ectodomains of the M2 protein and the ligand for the Toll-like receptor 5, flagellin, necessary for further incorporation into recombinant pseudo-adenovirus particles.

The source nucleotide and amino acid sequences for creating the target gene were taken from the official public source NCBI, GenBank, USA (www.ncbi.nlm.nih.gov). Work with sequences was carried out using the AliBee computer program - Multiple Alignment.

First, a unique M2e protein sequence was created; for this, the nucleotide sequences of the M2e gene of a protein of several tens of avian influenza A virus strains were selected and aligned, and a consensus (“averaged”) version was compiled. Further, the amino acid cysteine at positions 17 and 19 of the M2e gene was replaced by serine in order to prevent the protein from collecting in the tetramer and having the ability to secrete.

To increase the immunogenicity of the M2e protein of avian influenza virus, the ligand of the Toll-like receptor 5 flagellin was selected and a flagellin gene sequence was created based on Salmonella enterica serovar dublin (GenBank No. AAA27081), which is represented by conserved N and C-terminal domains, the central hypervariable region was excluded , and at the position of 176-402 amino acids, a linker linking the N and C-terminal domains was inserted.

To create the final genetic construct encoding the fusion protein (fusion protein) (SEQ ID NO: 1), the genes of the four ectodomains of the M2 protein (M2e) with serines at positions 17 and 19, the linker, and the flagellin gene Salmonella enterica serovar dublin were sequentially arranged. For efficient extracellular expression, secreted alkaline phosphatase leader peptide (LP) was attached to the N-terminus of the fusion protein. The nucleotide sequence of the fusion protein gene SEQ ID NO: 1 encoded the amino acid sequence of SEQ ID NO: 2.

Table 1 shows the nucleotide and amino acid sequences of the components of the expressed fusion protein gene construct containing the leader peptide, four M2e proteins, and flagellin linked to them by a linker.

The obtained unique nucleotide sequence of the fusion protein gene was synthesized by a well-known chemical method and ligated into a well-known auxiliary plasmid for further cloning.

Example 2

This example shows the preparation of recombinant pseudo-adenoviral particles expressing a fusion protein consisting of four ectodomains of the M2 protein and the ligand for the Toll-like receptor 5, flagellin.

At this stage, a recombinant pseudo-adenovirus particle based on the 5th serotype human adenovirus genome was obtained containing an insert of the fusion protein gene SEQ ID NO: 1 created in Example 1.

All of the cloning work described below was carried out using well-known standard techniques described in manuals for specialists in this field.

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 regionl 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 E1 region impaired, and the auxiliary plasmid pACCMVpLpA (Roth MG, Methods in cell biology, No. 43, p. 175 - Me odes 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, pp. 25129-25134 - Adenovirus-mediated transfer of muscle glycogen phosphorylase gene to hepatocytes changes the regulation of glycogen metabolism). Previously, SEQ ID NO: 1 was transferred from the auxiliary plasmid to the shuttle plasmid pACCMVpLpA, and as a result, pACCMVpLpA-M4FL was obtained with an expression cassette with the target transgene (fusion protein gene) flanked by sections of the adenovirus genome that are involved in further recombination. Homologous recombination was performed in HEK293 cells after cotransfection with plasmids pJM17 and pACCMVpLpA-M4FL. Since the 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 prevented the propagation of wild-type adenoviruses. 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.

Thus, after recombination, an RPAN was obtained with an expression cassette in the region of the E1 deletion of the genome containing the human cytomegalovirus (CMV) promoter, the gene for the leader peptide of secreted alkaline phosphatase SEAP (LP), the fusion protein gene (including four sequential repeats of the unique M2e sequence , linker (LN), flagellin gene encoding the conserved N and C-terminal domains of flagellin protein Salmonella enterica serovar dublin), SV40 polyadenylation signal (Simian vacuolating virus 40) - pA.

Scheme expressing cassettes included in the genome of the obtained RPAN, shown in figure 3.

A preparation containing recombinant pseudo-adenovirus particles was prepared by the following method. A few days after transfection, plaques formed on HEK293 cell culture, they were taken with a Pasteur pipette, the resulting material was propagated on HEK293 cells to obtain a titer of 10 ⁸ act.ed / ml. Then chromatographic purification was carried out, as a result, a preparation with a titer of 2 × 10 ⁹ act.ed./ml was obtained.

The activity of RPAN preparations hereinafter was evaluated by the standard titration method on a culture of sensitive HEK293 cells in plaque formation reactions.

To confirm the construction of a recombinant pseudo-adenovirus particle according to the invention based on human adenovirus 5 serotype (RPAN) with insertion of the fusion protein gene (SEQ ID NO: 1), PCR (polymerase chain reaction) was carried out according to a known standard method. For PCR, pairs of primers were used that were complementary to: serotype 5 human adenovirus hexon gene; on the E1 region (which should be absent in the created recombinant pseudo-adenovirus particles); on the fusion protein gene. According to PCR results, it was concluded that the DNA sequence of the recombinant pseudo-adenovirus particle based on human adenovirus 5 serotype was detected, the fusion protein gene (SEQ ID NO: 1) was detected, the E1 region that should be deleted from the recombinant pseudo-adenovirus particles was not detected (see Fig. 4).

Thus, we obtained a recombinant pseudo-adenovirus (E1 region of adenovirus) particle based on human adenovirus 5 serotype (there is a human adenovirus hexon 5 serotype) with an insertion of the fusion protein gene consisting of four ectodomains of the M2 protein and a ligand for Toll-like receptor 5 - flagellin (the fusion protein gene is present - SEQ ID NO: 1), which indicates the compliance of this design with the requirements of the invention claimed.

The ability of engineered recombinant pseudo-adenoviral particles to express the fusion protein gene and immunogenicity, i.e. their functional activity.

In the study, Balb / c mice (females) were used, 7-8 weeks (weighing 16-18 g), in each experimental group there were 10 animals.

The experiment was carried out in mice after intranasal administration of 50 μl of the RPAN preparation obtained earlier in 2 example (10 ⁸ active units in total). Biological material (blood) was taken from animals before administration, as well as after administration of the candidate vaccine on day 28. In blood serum titers of specific antibodies of the IgG isotype to M2e protein consensus for avian influenza viruses were determined in ELISA.

ELISA was performed using the artificially synthesized M2e peptide, consensus for avian influenza viruses, conjugated to a KLH carrier (cochlear lymphocytic hemocyanin).

Enzyme-linked immunosorbent assay was performed by the conventional method. The M2e peptide was immobilized on 96-well plates with high sorption ability (Greiner, Germany) at a concentration of 3 μg / ml (in carbonate buffer, pH 9.5-9.6), kept overnight at 4 ° C. The tablets were treated with blocking buffer (0.01 M PBS, pH 7.2-7.4 with 5% fetal calf serum) for 1 hour at room temperature, washed 3 times with PBS with tween.

100 μl of 2-fold dilutions of serum in blocking buffer were added to the plate wells, incubated for 1 hour at room temperature. To determine specific antibodies of the IgG isotype, rabbit monoclonal anti-mouse IgG (Invitrogen, USA) at a dilution of 1: 1000 labeled with horseradish peroxidase was used as a conjugate. Tetramethyl benzidine (TMB) (Invitrogen, USA) was used as a substrate. The reaction was taken into account at a wavelength of 450 nm. The titer was taken as the largest dilution of serum, which gives an optical density of at least 2 times more than the serum taken before immunization, in the same dilution.

On the 28th day after immunization, the average titers of serum IgG in mice immunized with RPAN supplemented with a fusion protein insert were significantly higher in the control groups. The geometric mean titer was 8444.851, respectively (p <0.05). In animals treated with phosphate buffer and control RPAN, titers were less than 100. Table 2 presents the results of determining the immunogenicity of the preparation RPAN with an insert of fusion protein

Thus, the obtained results indicate the ability of RPAN to express fusion protein and show its immunogenicity.

It was concluded that the constructed recombinant pseudo-adenovirus particle based on human adenovirus 5 serotype, containing an insert of the fusion protein gene consisting of four ectodomains of the M2 protein and the ligand for the Toll-like receptor 5, flagellin, produces fusion protein in the body of laboratory animals. The presence of specific activity - immunogenicity in the RPAN preparation means the possibility of using an influenza vaccine as an active substance.

Example 3

Selection of an effective molecular adjuvant capable of activating innate immunity.

In this example, the question of the selection of a molecular adjuvant that can enhance the immunogenicity of RPAN with the insertion of the fusion protein according to the invention and retains its activity in the composition of the drug is considered.

Important components necessary to increase the immunogenicity of a vaccine are adjuvants. The ability of adjuvants to increase the immunogenicity of the vaccine antigen when used together is realized through interaction with pattern-recognizing receptors, the activation of which leads to additional stimulation of immune responses.

In our experiments, we discovered such a molecular adjuvant - acidic peptidoglycan, enhancing the immunogenicity of RPAN with the transgene insert of the invention.

To confirm the ability of acidic peptidoglycan to enhance the immunogenicity of RPAN with an insert of fusion protein, an experiment was conducted with increasing dosages of adjuvant. A total of 6 groups of 10 mice were formed. Mice were once intranasally immunized with 50 μl of the drug containing:

Group 1 - RPAN in a dose of 10 ⁸ active units without adjuvant;

Group 2 - RPAN in a dose of 10 ⁸ active units + 10 units of adjuvant;

Group 3 - RPAN in a dose of 10 ⁸ active units + 20 units of adjuvant;

Group 4 - RPAN in a dose of 10 ⁸ active units + 100 units of adjuvant;

Group 5 - RPAN in a dose of 10 ⁸ active units + 110 units of adjuvant;

6 group - control, 0.9% NaCl.

Three weeks after immunization, animals were bled to determine the level of antibodies to avian influenza virus A / Mallard duck / PA / 10218/84 (H5N2) in blood serum. The level of specific antibodies to avian influenza A virus H5N2 was determined using ELISA, according to the method described in 2 example (Fig. 5).

The titer of antibodies to M2e of avian influenza virus in the blood serum of mice receiving only RPAN at a dose of 10 ⁸ act.ed. without adjuvant, 4.2 log2; adding 10 IU of adjuvant increased to 6.0 log2, adding 20 IU of adjuvant increased to 7.8 log2, adding 100 IU of adjuvant increased to 8.2 log2, adding 110 IU of adjuvant 8.2 log2 remained . In the control - a negative result.

The results obtained showed a significant increase in the immunogenicity of RPAN with an insertion of fusion protein with simultaneous intranasal administration with the inventive molecular adjuvant, which allows them to be used together to create highly immunogenic compositions.

The ability to react acid peptidoglycan with Toll-like receptors, in other words, the ability to activate innate immunity, was determined. The ability of a molecular adjuvant to specifically bind to human Toll-like (TLR) receptors was evaluated in vitro by a method well known to those skilled in the art using a set of eukaryotic cell lines based on the human embryonic renal epithelial cell line (HEK293) expressing various types of Toll-like receptors . The beta-galactosidase gene under the NF-kB dependent promoter is included in the genome of these cells. Thus, the activation of the transcription factor NF-kB as a result of the interaction of the ligand with its specific Toll-like receptor leads to the expression of beta-galactosidase by cells. The activity of beta-galactosidase after adding a molecular adjuvant to the cells was determined by adding a substrate for this enzyme, o-nitrophenyl-β-D-galactopyranoside. The level of β-galactosidase activity was determined spectrophotometrically (414 nm) by the conversion of o-nitrophenyl-β-D-galactopyranoside.

The ability of the molecular adjuvant to activate the following Toll receptors was studied: TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9. For the experiment, hTLR2 / CD14, HEK293-hTLR4 / CD14-MD2, HEK293-hTLR5, HEK293-hTLR7, HEK293-hTLR8, HEK293-hTLR9 cells were used; cells that did not carry Toll-like receptors (HEK29) served as a negative control.

Of all the listed Toll receptors, the molecular adjuvant activated Toll-like receptor 4 (TLR4) (Figure 6).

Based on the results obtained, it was concluded that the studied acid peptidoglycan from all of the above receptors is capable of causing activation of the Toll-like receptor 4 (TLR4) of innate immunity in vitro at a concentration of from 61.7 ng / ml to 15 μg / ml.

Thus, as a result of the studies, it was found that an acid peptidoglycan from all of the above receptors is capable of causing TLR4 activation, which allows it to be considered as a molecular adjuvant that activates innate immunity, and to use it to increase the efficacy of RPAN vaccine preparations.

To assess the stability of the molecular adjuvant in the vaccine, it was stored for 6 months (accelerated aging at 25 ° C) and the adjuvant activity was checked every 3 months using cells expressing Toll-like receptor 4 and carrying the beta-galactosidase gene in its genome under the NF-kB dependent promoter.

The composition of the vaccine was adjusted with buffer to a dose volume of 0.5 ml. The buffer solution can be any that supports the viability of recombinant pseudo-adenovirus particles and does not affect the stability of the molecular adjuvant (e.g. 10 mM TrisHCl, 75 mM NaCl, 1 mM MgCl ₂ , 5% sucrose, 0.05% polysorbate 80, 0.5% ethanol , 100 μm EDTA, pH 8.0). Table 3 presents the results of evaluating the stability of the molecular adjuvant in the vaccine.

In addition, the stability of pH, color, transparency of the vaccine during storage by accelerated aging was shown.

This experiment shows the absence of the effect of RPAN and molecular adjuvant when formulated jointly on each other's activity, which indicates the stability of the developed vaccine.

Thus, it was concluded that acidic peptidoglycan showed the properties of a molecular adjuvant for RPAN with an fusion protein insert of the invention capable of activating TLR4, which means innate immunity. Adjuvant is stable in the composition of the vaccine and does not affect the stability of RPAN.

Thus, the task of developing an effective vaccine composition was accomplished by adding a molecular adjuvant and a buffer solution to recombinant pseudo-adenovirus particles.

Example 4

Selection of the effective composition of the vaccine

It was carried out according to the results of a preclinical study of the immunogenicity of various vaccine compositions.

A single vaccination of 50 μl was carried out. The experimental and control groups were 10 mice of the Balb / c line. On day 42 after a single immunization, blood was taken from mice from which serum was obtained. Evaluation of the level of antibodies to the M2e protein of the influenza A H5N2 virus was performed using enzyme-linked immunosorbent assay (ELISA). A chemically synthesized M2e peptide of avian influenza virus was used as an antigen. For analysis, a 96-well, highly binding, flat-bottomed plate was sensitized with antigen at a concentration of 50 μg / ml in 20 mM potassium phosphate buffer pH 8.0. Sensitization was carried out at + 4 ° C for 12 hours. At the end of sensitization, the plate was washed from unbound antigen with a working buffer (20 mM potassium phosphate buffer pH 8.0 with 0.05% Tween 20). Then, two dilutions of the studied blood serum of laboratory mice were introduced into the wells of the tablet. At the end of the incubation (1 hour at + 37 ° C on a shaker), the plate was washed and antibodies to mouse IgG conjugated with peroxidase were added at a dilution of 1: 10000 in the working buffer. After incubation, the plate was washed and a TMB indicator mixture was used to manifest the reaction. Then the reaction was stopped with 4M H ₂ SO ₄ and the optical density of the colored product was determined at a wavelength of 450 nm.

The results of the analysis are presented in table 4. The data are presented in the form of a geometric mean titer (CHT) for each group of mice.

Table 4 presents the results of the immunogenicity of the various vaccine formulations.

Thus, the immunogenicity of the obtained vaccine was shown in effective doses of 10 ⁶ -10 ⁹ act.ed., as in combination with 20 units. adjuvant, and in combination with 100 units. adjuvant, with a single immunization schedule. Doses with the content of 10 ⁵ act.ed. RPANs did not show high immunogenicity, but with 10 ¹⁰ act.ed. RPAN - showed immunogenicity, significantly comparable with 10 ⁹ act.ed. RPAN, which makes their use not economical. The most optimal combination of immunogenicity and economic benefit was recognized composition containing: 10 ⁸ act.ed. RPAN in combination with 20 units of adjuvant.

Example 5

Getting the vaccine

The developed technology should ensure the production of vaccines of the following quality:

- dose of 10 ⁶ -10 ⁹ active units (active units) of RPAN, (recombinant pseudo-adenovirus particles);

- adjuvant content of 20-100 units;

- dose volume of 0.5 ml (adjusted with a pharmaceutically acceptable buffer solution).

Getting this vaccine takes place in several stages.

For this, the cell suspension obtained in Example No. 2 containing recombinant pseudo-adenovirus particles with a titer of 10 ⁸ PIECES per ml was used to further increase the titers of recombinant pseudo-adenovirus particles and to prepare a finished vaccine with a given content of recombinant pseudo-adenovirus particles.

Thus, to produce the necessary titers of recombinant pseudo-adenovirus particles, a wave bioreactor with 4500 ml of a suspension of a permissive cell culture 293 was seeded with a 500 ml cell suspension containing recombinant pseudo-adenovirus particles with a titer of 10 ⁸ U activity per ml.

Cultivated to build recombinant pseudo-adenovirus particles inside the cells and achieve their activity of 2 × 10 ⁸ IU / ml, approximately within 48 hours. Upon reaching the required content, the cell mass was fed for purification, which consisted of several stages:

1. Conducted deposition of cell mass by centrifugation. The suspension arriving for cleaning had at least 3.3 × 10 ¹¹ particles per 5 l. Centrifugation was carried out at a regime of 6000 g for 15 min, while the liquid supernatant was discharged, and the remaining solid part containing cells and recombinant pseudo-adenovirus particles was fed to further purification steps.

2. The extraction of recombinant pseudo-adenovirus particles from the cell culture was carried out by destroying the cells with four times thawing and thawing. A buffer solution was prepared with a pH of 8.0: 5 mM Tris HCl, 0.075 MNaCl, 1 mM MgCl ₂ , 5% sucrose, 1% polysorbate 80. The precipitate obtained in the previous step was resuspended in 70 ml of buffer (content ratio × 71). The volume of the solution was 80 ml.

Freezing was carried out for 2 hours in liquid nitrogen, thawed in a water bath (at + 37 ° C), avoiding overheating.

3. To facilitate further removal of genomic cell DNA, an additional nuclease treatment was performed. For this, benzonase was added to a concentration in the solution of 150 U / ml and put on gentle stirring using a magnetic stirrer for 3 hours at room temperature (21-23 ° C).

4. Separation of recombinant pseudo-adenovirus particles from the destroyed cells was carried out by centrifugation at 9000 g for 10 min. A supernatant containing recombinant pseudo-adenovirus particles was selected.

5. Further purification was carried out by ultrafiltration. For this, the obtained supernatant was diluted with buffer (50 mM TrisHCl pH 7.5, 1 M NaCl, 2 mM MgCl ₂ , 5% sucrose, pH 7.5) to a volume of at least 200 ml, and stirred using a magnetic stirrer. During the filtration process, the volume of the circulating solution (retentate) was constantly adjusted to the initial (200 ml).

6. Further purification was carried out by anion exchange chromatography.

The retentate was applied to a column (AxiChrom 70/300 with a volume of 400 ml) containing the Q Sepharose virus licenced anion exchange sorbent. Recombinant pseudo-adenovirus particles are adsorbed on the column, while impurities are not adsorbed, but washed with buffer A. After removal of impurities, recombinant pseudo-adenovirus particles were desorbed by washing with buffer B. Chromatography conditions: flow 193 ml / min, buffer A (40 mM TrisHCl, 0.27 M NaCl, 2 mM MgCl ₂ , 5% Sucrose, 0.1% Polysorbate 80, pH 7.5), conductivity ~ 28-30 mS / cm; buffer B (40 mM TrisHCl, 0.5 M NaCl, 2 mM MgCl ₂ , 5% sucrose, 0.1% polysorbate 80, pH 7.5) conductivity ~ 50 mS / cm. The eluate in a volume of 200 ml was sent to the next stage.

7. Size exclusion chromatography

The eluate obtained in the previous step was applied to a column (AxiChrom 100/300 with a volume of 800 ml) containing the Q Sepharose 4 FastFlow sorbent. High-molecular substances that are not included in the pores of the sorbent were eluted with the first peak (these include recombinant pseudo-adenovirus particles), impurities were eluted after the peak of non-replicating nanoparticles exited. Chromatography conditions: stream 130 ml / min, buffer (10 mM TrisHCl, 75 mM NaCl, 1 mM MgCl ₂ , 5% sucrose, 0.05% polysorbate 80, pH 8.0).

Ethanol was added to the obtained eluate (80 ml) to a concentration of 0.5% and ethylenediaminetetraacetic acid (EDTA) to a concentration of 100 μM was sent to the next stage.

8. Normal filtering

To sterilize the resulting preparation, filtration was performed through a filter system with a pore size of 22 μM.

After sterilization, the resulting volume of the preparation is diluted with a sterile pharmaceutically acceptable buffer solution (for example, 10 mM TrisHCl, 75 mM NaCl, 1 mM MgCl ₂ , 5% sucrose, 0.05% polysorbate 80, 0.5% ethanol, 100 μm EDTA, pH 8.0 ) to obtain the active component, taking into account the required final activity 10 ⁶ -10 ⁹ act.ed. recombinant pseudo-adenovirus particles with the insertion of the fusion protein gene per dose, then add a sterile solution of molecular adjuvant with an initial concentration of 1000 PIECES / ml, in the amount necessary to prepare the required concentration of 20-100 PIECES per dose. The resulting volume of the drug is poured into 0.5 ml vials per dose.

Thus, the task of preparing the vaccine according to the invention is completed. It is possible to obtain a vaccine containing:

- recombinant pseudo-adenoviral particles expressing the fusion protein gene, which consists of four consecutive ectodomains of the M2 protein (M2e) and TLR 5 activator flagellin Salmonella enterica serovar dublin - 10 ⁶ -10 ⁹ act.ed .;

- molecular adjuvant, TLR 4 activator, acid peptidoglycan - 20-100 U;

- pharmaceutically acceptable buffer solution - up to 0.5 ml.

Example 6

Evaluation of the T - cell response to the vaccine

This example shows the ability of a vaccine to stimulate cellular immunity, as it is involved in the regulation of a specific immune response.

The ability to stimulate cellular immunity for a modern vaccine is a prerequisite. Modern scientific evidence suggests the need for activation of T-lymphocytes for the formation of specific immunity. Depending on the presence of certain markers on their surface, they perform different functions, the main ones being the induction of immune responses by T-helpers - CD4.

To assess cellular immunity resulting from the intranasal immunization of mice with a vaccine, lymphoproliferative analysis of immunocompetent cells of immunized mice after stimulation with antigen was performed. As control groups were used mice that received RPAN without transgene and phosphate buffer. The immunized mice were euthanized with ether, after which sternal and deep cervical lymph nodes were taken from them, from which a culture of immunocompetent cells was obtained. The resulting cell culture was stimulated by the addition of antigen (sonicated A H5N2 avian influenza virus virus) at a concentration of 10 μg per 300,000 cells. After incubation with the antigen, the level of lymphocyte proliferation was determined by the intensity of 3H-thymidine incorporation into the DNA of cultured cells, which was determined in a special counter, comparing the results by the number of pulses per minute (cf.). The results of the analysis are shown in figure 7.

As a result of H3-thymidine analysis, it was demonstrated that the T-lymphocytes of the mice immunized with the vaccine developed after the addition of the antigen actively included labeled thymidine, which indicates their proliferation in response to stimulation with the antigen. In the control groups, this effect was not detected.

It was concluded that the assessment of the level of T-cell response in animals immunized with the vaccine according to the invention showed the presence of a pronounced cellular response, which corresponds to modern requirements for vaccines.

Example 7

Evaluation of vaccine efficacy

The experiment showed the ability of a vaccine to protect against lethal doses of the influenza virus.

At 42 days after immunization, mice were intranasally infected with 50 LD ₅₀ of the influenza virus A / Mallard duck / Pennsylvania / 10218/84 (H5N2) adapted for mice. In the experimental groups and the control group there were 10 mice. Within 14 days after infection, animals were monitored, mortality and weight change were recorded.

The experimental data are presented in figures 8 and 9.

As a result of the experiment, it was shown that the vaccine is protective, i.e. protects laboratory animals from flu. Mice treated with a vaccine containing a dosage range from the minimum (10 ⁶ active units of RPAN, 20 units of adjuvant) to the maximum (10 ⁹ active units of RPAN, 100 units of adjuvant) were protected from infection with the influenza virus. Mice receiving 10 ⁹ act.ed. RPANs, regardless of the amount of adjuvant, were fully protected. Mice receiving 10 ⁶ act.ed. RPAN in combination with both 20 PIECES and 100 PIECES of adjuvant, slightly lost weight, but there were no deaths among these groups. The control group that received the buffer completely died on day 10.

Example 8

A study of the breadth of action of the influenza vaccine

This example shows the breadth of the spectrum of action of the developed vaccine in terms of protection against various strains of influenza A virus of subtypes H5, H2 and H1. Several epidemiologically relevant subtypes of avian influenza A virus were used for infection.

At the first stage, the mice were given a single intranasal vaccine. 42 days after immunization, mice were intranasally infected with ₅₀ LD ₅₀ of influenza virus A / Mallard duck / Pennsylvania / 10218/84 (H5N2), influenza virus A / BlackDuck / NewJersey / 1580/78 (H2N3) and influenza virus A / California / 04 / 09 / (H1N1) adapted for mice. The influenza viruses A / Mallard duck / Pennsylvania / 10218/84 (H5N2) and A / BlackDuck / NewJersey / 1580/78 (H2N3) are avian and the influenza A / California / 04/09 / (H1N1) virus contains the “bird” gene M2e. Within 14 days after infection, animals were monitored, mortality and weight change were recorded. The vaccine contained the optimal composition: RPAN 10 ⁸ and adjuvant 20 PIECES in 0.5 ml. The experimental design is presented in table 5.

As a result of the experiment, it was shown that mice receiving a single intranasal vaccine were 100% protected against infection with the avian influenza virus A / Mallard duck / Pennsylvania / 10218/84 (H5N2). The mice receiving the vaccine lost little weight, but there were no deaths among them. The control group completely died on day 8 after infection. The results of the experiment are presented in figures 10 and 11.

As a result of the experiment, it was shown that mice that received a single intranasal drug were 80% protected against infection with the avian influenza virus A / BlackDuck / NewJersey / 1580/78 (H2N3). In this case, the mice receiving the drug, slightly lost weight. The control group completely died 10 days after infection. The experimental data are presented in figures 12 and 13.

As a result of the experiment, it was shown that mice receiving a single intranasal drug were protected against infection with the influenza virus A / California / 04/09 / (H1N1). Mice receiving the vaccine showed a survival rate of 90%, lost up to 13% of the weight. Moreover, the control group completely died on the 8th day after infection. The experimental data are presented in figures 14 and 15.

Thus, it was shown that intranasal single immunization with a drug containing RPAN in combination with a molecular adjuvant effectively (80-100% efficiency) protects against lethal doses of influenza A viruses of birds of various subtypes, which indicates a wide spectrum of action of the developed vaccine.

Example 9

Preclinical Vaccine Safety Studies

According to current regulatory and methodological documentation, all vaccines designed for clinical use should be safe.

The vaccine has passed the required range of preclinical safety studies, which included:

- studies of acute toxicity;

- studies of chronic toxicity;

- studies of reproductive toxicity;

- studies of immunotoxicity and allergenic properties;

- studies of mutagenic activity.

A preclinical study of acute toxicity of the vaccine did not reveal any results that hinder clinical trials for medical use; preclinical studies of the "chronic" toxicity of the vaccine did not reveal results that hinder clinical trials of the vaccine in adults when it is used in the declared finished dosage form at a dose of 0.5 ml per person; preclinical studies of reproductive toxicity did not reveal negative effects that impede the clinical study of the vaccine; an assessment of the allergic effect and immunotoxicity did not reveal a negative effect, the stimulating effect of the drug on the humoral immunity was established, and the vaccine was recommended for clinical trials; when studying mutagenic properties in the Ames test and micronucleus test, there was no undesirable mutagenic activity.

Thus, the vaccine developed according to the invention meets the safety requirements - does not have toxicity and reactogenicity, meets the requirements of hazard class 4 "low-hazard substances".

Industrial applicability

All these examples confirm the industrial applicability of the claimed vaccine.

The developed vaccine was recognized as a candidate drug for clinical use as a broad-spectrum influenza vaccine against bird flu A in humans.

Claims (3)

1. A broad-spectrum influenza vaccine against human influenza A of a person based on the M2 protein ectodomain, characterized in that the M2 protein ectodomain is expressed by recombinant pseudo-adenovirus particles based on the fifth serotype human adenovirus genome directly in the subject's body in the form of a fusion protein consisting of sequentially arranged fusion protein four ectodomains of avian influenza A virus protein M2 protein and TLR 5 activator flagellin Salmonella enterica serovar dublin, in addition, the vaccine additionally contains molecular adju ant and buffer solution, taken in effective amounts, the nucleotide sequence of the fusion protein gene, which consists of four consecutive ectodomains of the M2 protein and TLR 5 activator flagellin Salmonella enterica serovar dublin, is represented by SEQ ID NO: 1, and as a molecular adjuvant it contains the activator TLR-4 - an acidic peptidoglycan of plant origin with a molecular weight of from 1000 to 40,000 kDa. 2. The influenza vaccine according to claim 1, characterized in that it contains a dose:
- recombinant pseudo-adenovirus particles expressing the fusion protein gene, - 10 ⁶ -10 ⁹ act.ed.,
- molecular adjuvant - 20-100 units,
- pharmaceutically acceptable buffer - up to 0.5 ml. 3. The influenza vaccine according to claim 3, characterized in that the nucleotide sequence of the fusion protein gene SEQ ID NO: 1 encodes the amino acid sequence of the fusion protein SEQ ID NO: 2.

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