RU2802753C1 - Expression vector based on human adenovirus 5 serotype, inducing cross-protective immunity to influenza a viruses of subtype h1, and a pharmaceutical composition based on it - Google Patents

Abstract

FIELD: biotechnology; immunology; medicine.

SUBSTANCE: invention relates to the field of biotechnology, immunology, medicine. An expression vector based on human adenovirus 5 serotype with deletion of E1 and E3 regions, carrying a modified hemagglutinin gene of influenza A virus of antigenically distant subtype H1, capable of inducing cross-protective immunity against infection caused by influenza A viruses of subtype H1 pathogenic to humans, without the effect of antigenic imprinting when administered to a subject as part of a pharmaceutical composition or influenza vaccine. A pharmaceutical composition based on it is also presented. The invention can be used in the pharmaceutical industry for the production of influenza vaccines.

EFFECT: invention makes it possible to create a viral vector expressing the gene of the target antigen that induces immunity to various strains of the human influenza A virus subtype H1 without the effect of antigenic imprinting, which can be used as a pharmaceutical composition for the prevention of influenza A subtype H1, and also be part of an influenza vaccine.

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Description

Field of technology

The present invention relates to the field of biotechnology, immunology, and medicine. It concerns an expression vector based on a human adenovirus with a broad-spectrum protective effect (cross-protective) against influenza A viruses of the H1 subtype, as well as a pharmaceutical composition based on it. The invention can be used in the pharmaceutical industry for the production of influenza vaccines.

Previous level of development

Influenza is a highly contagious infectious disease that is widespread throughout the world. In the structure of influenza morbidity, a significant proportion is made up of influenza A virus strains of subtype H1. The most important measure of protection against influenza infection and limiting its spread is vaccination using trivalent or quadrivalent vaccines, which necessarily include an antigenic component obtained from the strain of influenza A virus subtype H1 expected to circulate in the coming season.

The effectiveness of existing seasonal vaccines is directly dependent on the degree of correspondence between the antigenic structure of the influenza virus hemagglutinins (HA) included in the vaccine and the influenza strains circulating among the population during the current epidemic season. At the same time, the immunodominant epitopes of the influenza virus hemagglutinin are located on its globular part and antibodies to them are able to neutralize the virus, which is the basis of the protective immune response after vaccination. High antigenic variability (the so-called “antigenic drift”) of immunodominant hemagglutinin epitopes necessitates an annual update of the composition of vaccines against influenza A virus strains. However, even updated vaccines are not always effective in the next epidemiological season due to the discrepancy between the expected and arriving virus strains flu Antigenic epitopes of broad specificity, which cause the formation of cross-antibodies within the H1 subtype, are not immunodominant and therefore, when immunized with native hemagglutinin as part of inactivated vaccines, a small amount of antibodies is produced to them, which does not allow one to obtain a broad-spectrum protective immune response. The problem is widely considered in the scientific literature (Lee J., Boutz D.R., et all. Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination. // Nat Med. 2016 Dec;22(12):1456 -1464; Laursen N.S., Friesen R.H.E., Zhu X., et all. Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin. Science. 2018 Nov 2;362(6414):598-602).

In addition to the variability of influenza virus hemagglutinin, the phenomenon of “antigenic imprinting” may affect the protective effectiveness of influenza vaccines. It is assumed that the main mechanisms of this phenomenon are realized due to previously developed antibodies in response to previous vaccinations or illnesses with influenza (Zarnitsyna V.I., et all. Multi-epitope models explain how pre-existing antibodies affect the generation of broadly protective responses to influenza. PLoS Pathog 2016 Jun 23;12(6):e1005692). The phenomenon of antigenic imprinting can reduce the effectiveness of vaccination and cause adverse events, so its potential occurrence should be taken into account during the development of antigenic components. A number of publications note that the phenomenon is not observed during sequential infection of animals with antigenically distant strains of influenza virus (Gulati U., Kumari K., Wu W. et al. Amount and avidity of serum antibodies against native glycoproteins and denatured virus after repeated influenza whole-virus vaccination // Vaccine. - 2005. - Vol. 23. - P. 1414-1425; Kim J.H., Skuntzou I., Compans R. et al. // Original antigenic sin responses to influenza viruses // J. Immunol. - 2009 - Vol. 183. - P. 3294-3301).

Consequently, the main unresolved scientific and technical problem in the development of vaccines is the production of highly immunogenic antigenic components that induce broad-spectrum immunity against influenza A viruses, in particular within the H1 subtype. To provide effective prevention of seasonal influenza, vaccines must provide cross-protection not only against currently circulating influenza A virus strains, but also against those that may arise in the future as a result of virus mutations, and must not cause antigenic imprinting. Antigenic components with a broad spectrum of action against the influenza A virus of subtype H1 will not require annual replacement in the composition of influenza vaccines.

It has been shown that some influenza virus antigens have epitopes that are conserved among phylogenetically distant influenza virus strains. These antigens should serve as the basis for a broad-spectrum vaccine. It is known that conserved B-cell epitopes are located mainly in the stem domain of hemagglutinin, specific antibodies to which have recently been discovered. In this case, these antibodies react immediately with several subtypes of the influenza virus (hemagglutinins). Antibody CR6261 - with subtypes of phylogenetic group 1 (Ekiert D.C. et al. Antibody recognition of a highly conserved influenza virus epitope // Science. - 2009. - Vol. 324. - Issue 5924. - P. 246-251), antibody CR8020 - with subtypes of phylogenetic group 2 (Ekiert D.C., Wilson I.A. Broadly neutralizing antibodies against influenza virus and prospects for universal therapies // Current Opinion in Virology. - 2012. - Volume 2. - Issue 2. - April 2012. - P. 134-141 ), antibody FI6 - with subtypes of both groups (Corti D. et al. A Neutralizing Antibody Selected from Plasma Cells That Binds to Group 1 and Group 2 Influenza A Hemagglutinins // Science. - 2011. - 12 Aug 2011. - Vol. 333 . - Issue 6044. - P. 850-856). It has been shown that antibodies against the stem domain of hemagglutinin are able to prevent conformational changes in the HA structure necessary for virus penetration into the cell.

Therefore, it is promising to present HA to the immune system in a variant in which conservative sites of the stem part are immunodominant and the immunogenicity of immunodominant antigenic sites in the globular part of HA is maximally reduced. When immunized with such an HA antigen, a cross-reactive immune response is expected due to the induction of antibodies to the stem part.

Thus, the most promising antigens for creating broad-spectrum vaccines are conserved hemagglutinin epitopes. In this regard, various approaches to modifying hemagglutinin to give it new immunogenic properties are being developed.

One approach to solving the problem is to obtain only the stem part of hemagglutinin, the so-called “headless” hemagglutinins (Impagliazzo A., et al. A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.Science // 2015 - Sep 18;349(6254 ):1301-6.; Yassine H.M., et al. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection // Nat Med. 2015 Sep;21(9):1065-70).

There is a known patent that uses this approach (WO/2019/070955). Universal influenza viruses based on the truncated hemagglutinin (HA) protein of the influenza virus, which does not have a head domain (hrHA), have been disclosed. Also disclosed is a composition comprising a nanoparticle coated with the disclosed hrHA polypeptide. Also disclosed is a composition containing a virus-like particle (VLP) expressing the disclosed hrHA polypeptide on its surface.

The approach to creating “headless” hemagglutinins (WO/2019/070955) has significant drawbacks, since in practice it is extremely difficult to create them without losing the conformational fold of the protein, which causes a deterioration in the immunogenic properties of such antigens.

Another approach is the creation of chimeric hemagglutinins with exotic globular parts (Krammer F., Palese P., Steel J.. Advances in universal influenza virus vaccine design and antibody mediated therapies based on conserved regions of the hemagglutinin // Curr Top Microbiol Immunol. 2015; 386:301-21.; Hai R., et al. Influenza viruses expressing chimeric hemagglutinins: globular head and stalk domains derived from different subtypes // J Virol. 2012 May;86(10):5774-81).

This approach is implemented in technical solutions of patents US11266734B2, US20190106461A1, EP1997831.

US11266734B2 is known to disclose chimeric hemagglutinin (HA) polypeptides and their use for inducing an immune response (eg, humoral response) against influenza virus. Also provided herein are methods of obtaining antibodies to chimeric HA polypeptides from a subject.

In turn, the patent EP1997831 describes chimeric vaccine antigens against the avian influenza virus (AI). Such vaccine antigens are based on viral subunits associated with protein co-stimulatory molecules, which enhance both the cellular and humoral components of the immune response. Chimeric antigens can be produced in expression systems that ensure proper three-dimensional folding of the chimeric molecules forming the basis of the present invention.

The approach to creating chimeric hemagglutinins (US11266734B2, EP1997831) also has significant disadvantages, namely that, firstly, in the globular part there are antigenic sites C, D and E, which are quite conservative within one subtype and, therefore, when changing In the globular part, many epitopes are lost, antibodies to which are certainly protective, and secondly, the antigenic sites at the top of HA still remain immunodominant. As a result, a robust, broad-spectrum immune response to such hemagglutinins is difficult to obtain.

There is another approach to modify hemagglutinin - hyperglycosylation.

Glycosylation of antigens is a recognized strategy for viruses to evade the host immune response. Glycosylation of influenza virus hemagglutinin occurs mainly at antigenic sites, and the number of glycosylation sites becomes larger over time (Medina R.A. et al. Glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the H1N1 influenza viruses. // Sci Transl Med 2013 May 29;5(187):187ra70).

A number of publications have shown that hyperglycosylation of hemagglutinin in the globular domain leads to a significant increase in heterosubtypic antibodies (Lin S.C., et al. Broader neutralizing antibodies against H5N1 viruses using prime-boost immunization of hyperglycosylated hemagglutinin DNA and virus-like particles // PLoS One - 2012-7: e39075.; Lin S.C., et al. Glycan masking of hemagglutinin for adenovirus vector and recombinant protein immunizations elicits broadly neutralizing antibodies against H5N1 avian influenza viruses // PLoS One. 2014 Mar 26;9(3):e92822; Eggink D., Goff P.H., Palese P. Guiding the immune response against influenza virus hemagglutinin toward the conserved stalk domain by hyperglycosylation of the globular head domain // J Virol. 2014 Jan;88(1):699-704; Bajic G., Maron M.J., Adachi Y., et al. Influenza antigen engineering focuses immune responses to a subdominant but broadly protective viral epitope // Cell Host Microbe. 2019 Jun 12;25(6):827-835.e6).

For example, Eggink D. et al. in 2014, 7 mutations were introduced into the Ca1, Cb, Sa and Sb antigenic sites of the HA strain A/Puerto Rico/8/1934 H1N1 and, thus, hyperglycosylated HA (Eggink D., Goff P.H., Palese P. Guiding the immune response against influenza virus hemagglutinin toward the conserved stalk domain by hyperglycosylation of the globular head domain // J Virol. 2014 Jan;88(1):699-704). Ultimately, they showed that, unlike wild type, HA increased, firstly, the production of antibodies to the stem part of HA, and secondly, such immunization protected mice from the influenza virus subtype H9.

On the other hand, there is information about the likelihood of shielding of immunogenic epitopes in the stem part of hemagglutinin by glycosylation sites (Andrews S.F., et al. Is it possible to develop a “universal” influenza virus vaccine? Immunogenetic considerations underlying B-cell in the biology of a pan -subtype influenza a vaccine targeting the hemagglutinin stem // Cold Spring Harb Perspect Biol. 2018 Jul 2;10(7). pii: a029413).

The approach to modifying influenza virus hemagglutinin based on hyperglycosylation appears to be the most promising, since it allows precise screening of the required antigenic sites.

In general terms, the creation of broad-spectrum influenza virus antigens based on hyperglycosylation is to force the body to induce the production of antibodies to the necessary epitopes of the influenza virus hemagglutinin.

Recombinant adenovirus seems to be the most promising as a vector expressing the created modified influenza virus gene. Currently, recombinant adenoviruses, which have a number of advantages over other genetic vectors, are actively used as a vector for the expression of various target antigens of infectious disease pathogens. Such vectors are safe - the DNA of recombinant adenoviruses is not integrated into the genome of target cells and the introduced adenoviral particles are eliminated from the body within 4-5 weeks. Moreover, they are able to penetrate a wide range of both dividing and non-dividing cells, carry out highly efficient gene expression, induce a humoral and cytotoxic immune response to the expressed foreign antigen, and do not require adjuvants.

A viral vector based on the genome of human adenovirus serotype 5 is recognized as one of the most immunogenic adenoviral vectors in relation to the transgene, it is assumed that this is due to more efficient transduction of cells with the CAR receptor (Abbink P. et.al. Comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups B and D. // J. Virol. - 2007.- May;81(9):4654-63). Thus, from the point of view of expected effectiveness, the choice of human adenovirus serotype 5 for creating a vaccine is justified. (Coughlan L. Factors which contribute to the immunogenicity of non-replicating adenoviral vectored vaccines // Front. Immunol. -2020.-11:909).

Today, the world is actively developing influenza vaccines based on broad-spectrum antigens, but there are still no drugs registered for use.

Thus, to improve the effectiveness of preventive immunization against seasonal influenza, it is imperative to create new antigens with a broad-spectrum protective effect (cross-protective) against influenza A viruses of the H1 subtype, which can be used in the pharmaceutical industry for the production of influenza vaccines.

Disclosure of the invention

The technical objective of the invention is to create a viral vector expressing the gene of a target antigen that induces immunity to various strains of human influenza A virus subtype H1 without the effect of antigenic imprinting, which can be used as a pharmaceutical composition for the prevention of influenza A subtype H1, and also be part of an influenza vaccine .

The technical result consists in obtaining an expression vector based on human adenovirus serotype 5 with a deletion of the E1 and E3 regions, carrying a modified hemagglutinin gene of the influenza A virus of the antigenically distant H1 subtype, capable of inducing cross-protective immunity against infection caused by influenza A viruses of the H1 subtype pathogenic for human, without the effect of antigenic imprinting when administered to a subject as part of a pharmaceutical composition or influenza vaccine.

This technical result is achieved by creating an expression vector based on the genome of human adenovirus serotype 5, inducing cross-protective immunity to influenza A viruses of subtype H1, containing:

- the genome of human adenovirus serotype 5, with the exception of the E1 region, where one or more endogenous nucleotides are deleted with the loss of its functional activity, and the E3 region, where one or more endogenous nucleotides are deleted with the loss of its functional activity;

- an expression cassette at the site of deletion of the E1 region of the adenovirus genome, including: a promoter, a modified hemagglutinin gene of influenza A virus subtype H1, with the sequence SEQ ID NO: 1, where the gene is obtained from influenza virus strain A/swine/Illinois/A01672343/2017(H1N1 ), polyadenylation signal sequence.

In a particular embodiment of the expression vector, the genome of human adenovirus serotype 5 is represented by SEQ ID NO: 2.

In addition, in a particular embodiment of the expression vector, the expression cassette at the site of deletion of the E1 region of the adenovirus genome includes a regulated promoter, while an expression cassette is additionally placed in the expression vector at the site of deletion of the E3 region of the adenovirus genome, including: a constitutive promoter, a transcription activator gene for the regulated promoter, polyadenylation signal sequence. In this case, it is possible that the expression of the modified hemagglutinin gene of the influenza A virus subtype H1 in a eukaryotic cell is negatively regulated in the presence of a tetracycline antibiotic.

A pharmaceutical composition has also been created for protection against influenza A viruses of subtype H1, containing an effective amount of the developed expression vector and a pharmaceutically acceptable buffer solution.

In addition, the use of a pharmaceutical composition to protect a subject from influenza A viruses of subtype H1 has been developed. Including as part of an anti-influenza vector vaccine.

Description of the figures

Figure 1 schematically shows the location of single amino acid substitutions in the modified hemagglutinin swH1glyc (numbering was used relative to HA subtype H3 without taking into account the signal peptide). The diagram shows the hemagglutinin subunits HA1 and HA2, as well as the localization of the globular and stem regions of the hemagglutinin molecule.

In fig. Figure 2 shows a diagram of the expression cassettes of the created expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc, where

Ad5 is part of the adenovirus genome, Ψ is the adenovirus packaging signal;

promoter P - regulated promoter for the gene encoding the modified hemagglutinin gene swH1glyc;

target gene - modified hemagglutinin gene swH1glyc;

pA - polyadenylation signal;

promoter K - constitutive promoter for a gene, the product of which activates transcription of the regulated promoter;

activator - a gene whose product activates transcription of a regulated promoter;

ICP - adenovirus inverted terminal repeats;

ΔE1 - site of deletion of the E1 region where the cassette expressing the target gene is inserted;

ΔE3 is the site of deletion of the E3 region, where a cassette expressing a gene is inserted, the product of which activates transcription of the regulated promoter.

In fig. Figure 3 shows an electropherogram of the results of PCR analysis of an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc. The fragments (target bands) of the required size obtained during the PCR analysis confirm the creation of an expression vector based on the genome of human adenovirus serotype 5 according to the invention, since they indicate the presence of the adenoviral genome and the presence of the target transgene.

Designations:

M - molecular weight marker Thermo Scientific Gene Ruler 1 kb DNA Ladder;

Tracks 1, 2, 3 - PCR result for the adenoviral genome, where:

1 - negative control;

2 - positive control (plasmid carrying hexon of human adenovirus serotype 5);

3 - hexon of human adenovirus serotype 5 (fragment size 440 bp)

Tracks 4, 5, 6 are the result of PCR for the transgene (modified hemagglutinin gene of influenza A virus subtype H1, with the sequence SEQ ID NO: 1), where:

4 - negative control;

5 - positive control (plasmid carrying the SwH1glyc gene);

6 - SwH1glyc gene (fragment size 730 bp).

Figure 4

In fig. Figure 4 presents the results of assessing the immunogenic activity of an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc, carried out by ELISA with recombinant hemagglutinin of the heterologous influenza virus A/California/04/2009 (H1N1) in the blood sera of immunized mice. The abscissa axis is the dilution of blood serum, the ordinate axis is the optical density measured using a spectrophotometer (at a wavelength of 450 nm). The titration curves represent the following animal groups:

In the titration curves, markers indicate the average absorbance measurements by group, and the ticked lines indicate the standard deviation.

Figure 5

Figure 5 presents the results of a study of the cross-protective immune response of mice immunized with the Ad5-swH1glyc expression vector as part of a pharmaceutical composition with a single intranasal route of administration, at a dose of 10 ⁷ PFU/animal, followed by infection with the heterologous influenza virus A/Victoria/2570/19 ma(H1N1).

In the graphs, the survival curves and changes in body weight are indicated as follows:

an experimental group of mice immunized with an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition;

control group of mice that received a buffer solution.

5A. Survival of mice infected with 15 LD ₅₀ influenza virus A/Victoria/2570/19 ma (H1N1).

The y-axis is % of surviving animals.

The x-axis is days from the moment of infection.

Markers on the graph lines indicate the number of animals in the group.

The differences between survival rates in the groups of immunized mice and the control group are statistically significant (p=0.0001).

5 B. Changes in body weight of mice infected with 15 LD ₅₀ influenza virus strain A/Victoria/2570/19 ma (H1N1).

The y-axis is the change in body weight of animals in %.

The x-axis is days from the moment of infection.

The markers on the lines indicate the % change in the average body weight of animals in the group.

Figure 6

Figure 6 presents the results of a study of the cross-protective immune response of mice immunized with an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition with a single intranasal route of administration, at a dose of 10 ⁷ PFU/animal, followed by infection with a heterologous influenza virus A/California/05/09 ma (H1N1).

In the graphs, the survival curves and weight changes are indicated as follows:

- an experimental group of mice immunized with an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition;

- control group of mice that received a buffer solution.

6A. Survival of mice infected with 15 LD ₅₀ influenza A/California/05/09 ma (H1N1) virus.

The y-axis is % of surviving animals.

The x-axis is days from the moment of infection.

Markers on the graph lines indicate the number of animals in the group.

The differences between survival rates in the groups of immunized mice and the control group are statistically significant (p=0.0001).

6B. Changes in body weight of mice infected with 15 LD ₅₀ influenza virus strain A/Victoria/2570/19 ma (H1N1).

The y-axis is the change in body weight of animals in %.

The x-axis is days from the moment of infection.

The markers on the lines indicate the % change in the average body weight of animals in the group.

Figure 7

In fig. Figure 7 presents the results of a study of the cross-protective immune response of mice immunized with the Ad5-swH1glyc expression vector as part of a pharmaceutical composition with a single intranasal route of administration, at a dose of 10 ⁷ PFU/animal, followed by infection with the heterologous influenza virus A/Duck/Moscow/4970/20 ma(H1N1).

In the graphs, the survival curves and changes in body weight are indicated as follows:

- an experimental group of mice immunized with an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition;

- control group of mice that received a buffer solution.

7A. Survival of mice infected with 15 LD ₅₀ influenza virus A/Duck/Moscow/4970/20 ma (H1N1).

The y-axis is % of surviving animals.

The x-axis is days from the moment of infection.

Markers on the graph lines indicate the number of animals in the group.

The differences between survival rates in the groups of immunized mice and the control group are statistically significant (p=0.0001).

7B. Changes in body weight of mice infected with 15 LD ₅₀ influenza virus strain A/Victoria/2570/19 ma (H1N1).

The y-axis is the change in body weight of animals in %.

The x-axis is days from the moment of infection.

The markers on the lines indicate the % change in the average body weight of animals in the group.

Implementation of the invention

The novelty of this invention lies in the fact that to create an influenza vaccine for humans, the antigen of the influenza A/swine/Illinois/A01672343/2017(H1N1) virus was used, which does not circulate in the human population and is antigenically distant from influenza A viruses of subtype H1, pathogenic for humans . Thus, a person does not have pre-existing antibodies to a given antigen, which avoids the effect of antigenic imprinting. The authors performed a significant modification of the antigen, which made it possible to focus the immune response on a number of epitopes of the hemagglutinin stem part. These epitopes are conservative and have a high degree of homology with epitopes of the influenza A virus of subtype H1, which allows for a cross-protective effect.

A person of ordinary skill will understand that the immune response to an expression vector and a pharmaceutical composition based on it is primarily determined by the sequence of the target antigen. In this case, the expression cassette may include any promoter that ensures high expression of the target antigen gene (for example, the CMV promoter). At the same time, the use of a regulated promoter, which is described in the examples, provides an advantage when expanding the expression vector and allows it to be obtained with greater yield.

As an expression vector, the authors use a vector based on human adenovirus serotype 5. An expression vector was obtained from the sequence of SEQ ID NO:2 by deleting the E1 and E3 regions. In this case, in place of the E1 region, an expression cassette with a promoter, a target gene (modified hemagglutinin gene of influenza A virus subtype H1, with the sequence SEQ ID NO: 1), and a polyadenylation signal sequence is placed. A variant of the invention is possible when, in addition, instead of the E3 region, an expression cassette with a constitutive promoter, a transcription activator gene for a regulated promoter, and a polyadenylation signal sequence is placed. A preparative amount of the developed expression vector is obtained in the suspension cell line HEK293.

The resulting expression vector, as well as a pharmaceutical composition based on it, when introduced into the body of mammals, are capable of inducing an antigen-specific immune response and can be used as an independent means for the prevention of influenza A subtype H1, as well as as part of influenza vaccines.

The implementation of the invention is confirmed by the following examples, which do not limit its scope.

Example 1

Creation of a modified hemagglutinin gene sequence for influenza A viruses of subtype H1

The example describes the work on selecting the original hemagglutinin gene of the influenza A virus for genetic engineering modification, which will allow, after introducing the modified gene into the subject, to induce cross-protective immunity to influenza A viruses of subtype H1 without the effect of antigenic imprinting.

To overcome the problem of “antigenic imprinting,” antigenically distant strains of influenza A virus pathogenic for pigs were chosen as the basis for creating modified hemagglutinins of influenza A viruses, which, according to the authors, will not cause a boost of currently irrelevant pre-existing antibodies to immunodominant epitopes of human influenza A viruses. Additionally, immunodominant epitopes in the globular part of hemagglutinin were closed and the immune response was redirected to conservative epitopes of the stem part of hemagglutinin.

In order to select the initial amino acid sequence of the hemagglutinin of the influenza A virus pathogenic for pigs, a generalized (consensus) sequence of the 2017 isolates was obtained. Next, for design, the hemagglutinin sequence that is maximally homologous to the consensus sequence was taken as a basis.

As a result, hemagglutinin from the Influenza A virus A/swine/Illinois/A01672343/2017(H1N1) strain was chosen as the initial hemagglutinin for creating modified genes; the amino acid sequence number in the public NCBI Protein database is AQU12415. To carry out the modification, the corresponding nucleotide sequence of hemagglutinin A/swine/Illinois/A01672343/2017(H1N1) was taken, which is located in the public NCBI GenBank database under the number KY593239.1 (hereinafter in the text this hemagglutinin gene is designated swH1 ).

Next, to focus the immune response, the most variable and accessible immunodominant epitopes were screened on the surface of the hemagglutinin protein by introducing 2 additional glycosylation sites in the globular part of the hemagglutinin of the influenza A virus subtype H1 A/swine/Illinois/A01672343/2017(H1N1) by replacing the hemagglutinin sequence 2 amino acids. Conversely, some of the natural glycosylation sites in the stem part that mask conservative epitopes were removed by replacing 2 amino acids in the hemagglutinin sequence in order to make the epitopes more open to the immune system in order to increase their immunogenicity.

Thus, as a result of in silico work with the original nucleotide sequence of swH1, the following mutations were introduced in hemagglutinin (numbering was used relative to HA subtype H3 without taking into account the signal peptide):

1) amino acid substitution N33Q was carried out, which led to the removal of the glycosylation site in HA (33-35);

2) the amino acid substitution S291N was carried out, which led to the removal of the glycosylation site in HA (289-291);

3) an amino acid substitution was made at position T144N, which led to the addition of a glycosylation site in HA (144-146);

4) an amino acid substitution was made at position G158N, which led to the addition of a glycosylation site in HA (158-160).

As a result, glycosylation sites in the stem part were removed and glycosylation sites were added in the globular part. To increase antigen exposure, the influenza virus hemagglutinin leader peptide was replaced with the alkaline phosphatase leader peptide - SEAP. Codon optimization was also carried out for expression in mammalian cells. The resulting variant of the modified hemagglutinin gene is designated swH1glyc .

The target gene with all modifications was synthesized de novo (JSC Evrogen, Moscow).

The sequence of the modified hemagglutinin gene of the influenza A virus subtype H1 is presented in the Sequence Listing as SEQ ID NO: 1 ( swH1glyc gene).

The presence of all modifications in the hemagglutinin gene swH1glyc was confirmed by sequencing.

Thus, a modified hemagglutinin gene of influenza A virus subtype H1 was created, with the sequence SEQ ID NO: 1, where the gene is derived from influenza virus strain A/swine/Illinois/A01672343/2017(H1N1).

Example 2

Preparation of an expression vector based on the genome of human adenovirus serotype 5, with the E1 and E3 regions removed, containing a modified hemagglutinin gene of the influenza A virus subtype H1

This example describes the process of obtaining an expression vector based on the genome of human adenovirus serotype 5, containing a modified hemagglutinin gene of influenza A virus subtype H1 from example 1, by the method of homologous recombination.

The process of obtaining an expression vector based on the genome of human adenovirus serotype 5, with the E1 and E3 regions removed, containing a modified hemagglutinin gene of the influenza A virus subtype H1 consisted of several main stages, the essence of which is known to a specialist in the field, and was based on well-known methods (for example, Sambrook J. et al., Molecular cloning: a laboratory manual, 3rd ed., Russell, 2001, volume 1, 2, 3 - Molecular cloning - laboratory manual) and therefore is not discussed in detail in this example, indicating only the most basic steps :

1) Recloning of the synthesized target gene swH1glyc with SEQ ID NO: 1 from a plasmid for cloning PCR products into the plasmid pShuttle-TswH1glyc, carrying an expression cassette with a regulated promoter;

2) Construction of the plasmid pAdeno-E3/TA based on the genome of human adenovirus serotype 5, represented by SEQ ID NO: 2, with the removal of the E3 region and the insertion of an expression cassette with a gene, the product of which activates transcription of the regulated promoter;

3) Homologous recombination between the pShuttle-TswH1glyc plasmid and the pAdeno-E3/TA plasmid occurs in HEK293 (Human embryonic kidney 293) cells after transfection with plasmid DNA to obtain an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc. As a result of recombination, an expression cassette with a regulated promoter, a target gene (modified influenza A virus subtype H1 hemagglutinin gene, with the sequence SEQ ID NO: 1), and a polyadenylation signal sequence was inserted into the E1 region deletion site.

4) Obtaining a preparative amount of an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc was carried out in the HEK293 suspension cell line.

The diagram of the constructed expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc is presented in Figure 2.

The results of the PCR analysis presented in figure 3 confirm the creation of an expression vector based on the genome of human adenovirus serotype 5, carrying a modified hemagglutinin gene of influenza A virus subtype H1, with the sequence SEQ ID NO: 1. The electropherograms show that the molecular weights of the amplified DNA fragments in the preparation , containing an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc, correspond to the molecular masses of amplified DNA fragments in positive controls. Moreover, the presence of a target band with a fragment size of 440 bp. in PCR with primers AdH01/AdH02 for the hexon gene of adenovirus serotype 5 indicates the presence of DNA from the genome of human adenovirus serotype 5, and the presence of a target band with a fragment size of 730 bp. (primers SWH1glyc-forw/SW-H1TM-rev) indicates the presence of the hemagglutinin gene sequence swH1glyc.

The correctness of the specified expression vector design based on the genome of human adenovirus serotype 5 was confirmed by the results of sequencing the genome of the recombinant adenovirus.

Thus, as a result of the work, an expression vector was obtained based on the genome of human adenovirus serotype 5, with the E1 and E3 regions removed, containing in the deleted E1 region an expression cassette including a modified hemagglutinin gene of the influenza A virus subtype H1, which is under the control of a regulated promoter, the gene the activator of which is located in the expression cassette in the remote E3 region under the control of the constitutive promoter.

Example 3

Determination of the expression of the modified hemagglutinin gene of influenza A virus subtype H1 in cell culture

The example shows an experiment to study the expression of the modified hemagglutinin gene of the influenza A virus subtype H1 with SEQ ID NO: 1, created in example 2, by an expression vector based on the genome of human adenovirus serotype 5, which was carried out on a human cell culture A549 and assessed using the immunoblotting method.

The A549 cell line was transduced with an expression vector based on the genome of human adenovirus serotype 5 at a dose of 5-10 PFU per cell. An adenovirus not carrying an expression cassette (Ad-null) was used as a negative control. After 48 hours of incubation, the cell pellet was collected for further analysis by polyacrylamide gel electrophoresis, followed by immunoblotting using mouse monoclonal antibodies to hemagglutinin/HA swine influenza H1N1 (Cat: 11055-MM09, Sino Biological Inc.). Goat anti-mouse IgG H&L (HRP) (ab205719, Abcam) was used as a secondary antibody.

It was shown that the expression vector created according to the invention based on the genome of human adenovirus 5 serotype Ad5-swH1glyc ensures the production of modified hemagglutinin of influenza A virus subtype H1 in human cell culture.

Example 4

Preparation of a pharmaceutical composition based on an expression vector based on the genome of human adenovirus serotype 5 for protection against influenza A viruses of subtype H1

This example shows the process of expanding the expression vector constructed in example 2 based on the genome of human adenovirus 5 of serotype Ad5-swH1glyc in order to obtain on its basis a pharmaceutical composition to protect a subject from influenza A viruses of subtype H1.

To grow recombinant adenoviruses, a HEK 293 (human embryonic kidney) cell culture adapted for growth in suspension was used.

Quantitative measurement of recombinant adenoviruses was carried out in activity units (PFU, plaque forming units) measured in HEK 293 cell culture.

A cell suspension of the HEK293 line was infected with an expression vector based on the genome of human adenovirus serotype 5, obtained in example 2, with a multiplicity of infection of 10 PFU per cell, and was used for further expansion of a larger amount of expression vector based on the genome of human adenovirus serotype 5.

For this purpose, a 500.0 ml virus-containing liquid containing an expression vector based on the genome of human adenovirus 5 serotype from example 2 was added to a wave bioreactor with 4500.0 ml of a suspension of permissive cell culture 293HEK. To expand the expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc with a regulated promoter, were cultured for 48 hours in the presence of a tetracycline antibiotic (for example, doxycycline) with a concentration of 1-10 μg per ml of medium. The expression vector based on the genome of human adenovirus serotype 5 was then purified from the cell lysate using well-known ultrafiltration and high-performance liquid chromatography methods in several steps.

To sterilize the resulting preparation, a filter with a pore size of 0.22 μM was used and diluted with a sterile pharmaceutically acceptable buffer solution that preserves the stability of recombinant adenoviruses, for example: tris (hydroxymethyl) aminomethane - 0.606 mg; sodium chloride - 2.19 mg; sucrose - 25.0 mg; polysorbate-80 - 0.00025 ml; magnesium chloride hexahydrate - 0.1 mg; ethanol 95% - 0.0025 ml; EDTA disodium salt - 0.019 mg; water for injection - the rest, pH 7.0-8.0, with the production, based on an expression vector based on the genome of human adenovirus serotype 5, of a pharmaceutical composition containing:

- expression vector based on the genome of human adenovirus serotype 5 - 1x10 ¹¹ -5x10 ¹¹ particles/ml with an activity of at least 2x10 ⁸ PFU;

- pharmaceutically acceptable buffer solution.

Thus, as a result of the work carried out, a pharmaceutical composition was obtained based on an expression vector based on the genome of human adenovirus serotype 5 according to the invention, containing in an effective amount an expression vector based on the genome of human adenovirus serotype 5 and a pharmaceutically acceptable buffer solution.

Example 5

Immunogenic activity of an expression vector based on the genome of human adenovirus serotype 5, carrying a modified hemagglutinin gene of influenza A virus subtype H1, when administered to laboratory animals

Immunogenic activity was assessed by the ability to produce specific antibodies in response to intranasal administration of an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc to mice. The assessment was carried out in mouse blood serum using enzyme-linked immunosorbent assay (ELISA).

Balb/c mice, females, weighing 14-16 grams, were immunized once intranasally with the pharmaceutical composition from example 4, with different contents of an expression vector based on the genome of human adenovirus serotype 5 in a dose, which, if necessary, was achieved by diluting the initial concentration with a buffer solution. Animals that received a buffer solution were used as a control group. The number of mice in the experimental and control groups was 6 individuals each. Thus, the following experimental groups were formed:

Group 1) Ad5-swH1glyc, dose 10 ⁸ PFU;

Group 2) Ad5-swH1glyc, dose 10 ⁷ PFU

Group 3) Ad5-swH1glyc, dose 10 ⁶ PFU

Group 4) Buffer solution (control).

Obtaining mouse blood sera. Blood was obtained from mice 28 days after immunization into Eppendorf tubes and incubated in a thermostat for 40 minutes at 37°C to form a fibrin clot. After this, the tubes were centrifuged at 3000 rpm, 4°C, 20 minutes. Sera were collected in clean tubes, heated for 30 minutes at 57°C (to inactivate the complement system) and frozen at -20°C.

Method for performing ELISA to determine antibody titer in mouse blood serum.

The antigen for the reaction was: recombinant hemagglutinin of influenza virus A/California/04/2009 (H1N1) (Abcam, ab217662);

To perform ELISA, the wells of a 96-well plate were sensitized by adding antigens in 10 mM potassium carbonate buffer (pH = 9.6). Sensitization was carried out for 18 hours at 4°C. The plate was then washed 3 times with distillate and 3 times with TPBS (phosphate-buffered saline with 0.05% Tween 20). Next, the plate was incubated in loading buffer (TPBS with 5% ovoalbumin) at 37°C on a shaker. Then they were washed 3 times with TPBS and incubated with serum dilutions from 1/400 to 1/25600 in loading buffer for 1 hour at 37°C on a shaker. Next, they were washed again 3 times with TPBS and incubated with an anti-mouse conjugate (polyclonal horse antibodies to mouse IgG, conjugated with peroxidase, (Amersham, UK), in loading buffer for 1 hour at 37°C on a shaker (working dilution 1:5000). Then again washed 3 times with TPBS. To develop the reaction, a TMB indicator mixture was used. To stop the reaction, 4 M H ₂ SO ₄ was used. The optical density of the colored product of the peroxidase reaction was determined on an iEMS Reader MF device (Termolabsystems) at a wavelength of 450 nm. The wells served as the background. where no serum was added, but antigen and conjugate were added.

Figure 4 shows the results of an experiment to evaluate, using ELISA, the level of antibodies interacting with recombinant hemagglutinin of the influenza A virus subtype H1 in the blood sera of mice immunized with an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc in comparison with the control group, mice to which the buffer solution was injected.

As can be seen in Figure 4, in mice from the experimental group immunized with an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc, antibodies to the hemagglutinin of influenza A virus subtype H1 are detected. For group 1 (dose 10 ⁸ PFU), the limiting dilution of blood serum, in which the optical density value at A ₄₅₀ was significantly different from the control group was 1:12800 (p <0.0001), and for group 2 (dose 10 ⁷ PFU) and group 3 (dose 10 ⁶ PFU) - 1:6400 (p<0.05). The dose of the expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc for use and use in further studies was 10 ⁷ PFU per mouse. At the same time, in the control group, antibodies were detected at the background level.

Thus, the constructed expression vector based on the genome of human adenovirus serotype 5, containing a modified hemagglutinin gene of the influenza A virus subtype H1, has immunogenic activity when administered to a subject as part of a pharmaceutical composition, that is, it is capable of inducing the formation of antibodies to the influenza A virus subtype H1, which corresponds the stated purpose of the invention.

Example 6

Application of a pharmaceutical composition for the prevention of influenza A subtype H1 with assessment of cross-protective properties in laboratory animals

At this stage, work has been carried out to assess the cross-protective properties of an expression vector based on the genome of human adenovirus serotype 5 as part of a pharmaceutical composition against various heterologous strains of influenza A viruses of subtype H1. The assessment was carried out on the survival and dynamics of body weight of mice immunized with the pharmaceutical composition after they were infected with influenza viruses.

Based on the developed design of an expression vector based on the genome of human adenovirus serotype 5, immunization should stimulate the production of antibodies, including to highly conserved regions of hemagglutinins of influenza A viruses of subtype H1, therefore it was assumed that the expression vector based on the genome of human adenovirus serotype 5 as part of a pharmaceutical composition will have a wide spectrum of action (cross-protective), that is, the ability to protect against various strains of influenza A viruses of the H1N1 subtype. To demonstrate the cross-protective effect, immunized mice were infected with the following heterologous mouse-adapted strains of influenza A virus subtype H1:

1) human influenza virus A/Victoria/2570/19 ma (H1N1);

2) human influenza virus A/California/05/09 ma (H1N1);

3) avian influenza virus A/Duck/Moscow/4970/20 ma (H1N1).

To conduct the experiment, female Balb/c mice weighing 14-16 grams were used. Mice from the experimental groups were immunized once intranasally with an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition at a dose of 10 ⁷ PFU. Control mice were injected with a buffer solution. There were 10 mice in each group.

28 days after immunization, mice were infected intranasally under ether anesthesia with the above influenza viruses at a dose of 15 LD ₅₀ per animal. To prepare the necessary dilutions of influenza viruses, Hanks' medium with lactalbumin hydrolyzate and the antibiotic gentamicin (PanEco, Russia) was used.

Thus, the following experimental and control groups were formed in the experiment:

Group 1) Ad5-swH1glyc + infection A/Victoria/2570/19 ma;

Group 2) Buffer solution + infection A/Victoria/2570/19 ma;

Group 3) Ad5-swH1glyc + infection A/California/05/09 ma;

Group 4) Buffer solution + infection A/California/05/09 ma;

Group 5) Ad5-swH1glyc + infection A/Duck/Moscow/4970/20 ma;

Group 6) Buffer solution + infection A/Duck/Moscow/4970/20 ma.

The mice were observed for 14 days after infection with influenza viruses; during this period, the survival of the animals, as well as the manifestation of disease symptoms (changes in body weight), were assessed.

A computer program was used to statistically process the experimental results. Statistical processing of animal survival was carried out using the Gehan-Breslow-Wilcoxon test; differences between groups were considered significant at p <0.05.

The results of the experiment are presented in Table 1 and Figures 5, 6, 7.

Table 1. Cross-protective properties of immunity induced by an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition against influenza A viruses of subtype H1 in mice, assessed after infection with heterologous strains of influenza A virus of subtype H1 Influenza virus for infection Survival rate, % Experience Control A/Victoria/2570/19 ma (H1N1) 100% ^* 10% A/California/05/09 ma (H1N1) 100% ^* 0% A/Duck/Moscow/4970/20 ma (H1N1) 100% ^* 0%

^* - significant difference from control (p=0.0001)

From Table 1 it follows that the expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition protected mice 100% from infection with influenza A viruses of subtype H1 in all three experimental groups. At the same time, the survival rate of the control groups was 10% (for the influenza virus A/Victoria/2570/19 ma (H1N1), 0% (for the influenza viruses A/California/05/09 ma (H1N1) and A/Duck/Moscow/4970/ 20 ma (H1N1). The difference in the survival rate of the experimental and control groups for each of the influenza viruses used for infection is significant (p = 0.0001, Gehan-Breslow-Wilcoxon test). A decrease in body weight in mice of all experimental groups immunized with Ad5- swH1glyc, did not occur, while in the control groups of mice until death there was a significant drop in body weight, which reached 30%.Immunized mice in the experimental groups had no clinical symptoms of the disease, unlike control mice.

Consequently, the experiment demonstrated the protective effect of the immune response induced in mice by an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition against heterologous influenza A viruses of subtype H1: A/Victoria/2570/19 ma (H1N1) , A/California/05/09 ma (H1N1), A/Duck/Moscow/4970/20 ma (H1N1), which confirms the presence of cross-protective properties against influenza A viruses of subtype H1.

Thus, an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a pharmaceutical composition induces cross-protective immunity and can be used for the prevention of influenza A subtype H1 in a subject, which corresponds to the stated purpose of the invention.

Example 7

Application of an expression vector based on the genome of human adenovirus serotype 5 as part of an influenza vaccine with assessment of protective properties and safety

The example shows the possibility of using an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of an anti-influenza vector vaccine. The protective properties of an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of a quadrivalent influenza vector vaccine and its safety when used in laboratory animals (mice) were assessed.

The vaccine included a mixture of 4 types of expression vectors based on human adenovirus serotype 5, differing by the insertion of the influenza virus hemagglutinin gene, which were: the hemagglutinin gene of the influenza A virus subtype H1 (modified hemagglutinin gene of the influenza A virus subtype H1, with the sequence SEQ ID NO: 1- swH1glyc), hemagglutinin gene of influenza A virus subtype H3, hemagglutinin gene of influenza B virus Victoria line, and hemagglutinin gene of influenza B virus Yamagata line.

The dose of the expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc as part of the influenza vector vaccine, the dosage form of which was presented in a volume of 0.5 ml, was 1x10 ⁸ PFU.

To evaluate the protective properties of Ad5-swH1glyc and the safety of the influenza vaccine containing 4 types of expression vectors, female Balb/c mice, 14-16 grams, were used. Animals were immunized once intranasally with the vaccine in a volume of 50 μl (content of the expression vector Ad5-swH1glyc 10 ⁷ PFU). Animals that received a buffer solution were used as a control group. The number of mice in the experimental and control groups was 10 individuals each. 28 days after immunization, mice were infected intranasally under ether anesthesia with influenza A /California/07/09 (H1N1) virus at a dose of 15 LD ₅₀ per animal. Assessment of survival and loss of body weight of animals was carried out within 14 days after infection. The results of the experiment are presented in Table 2.

Table 2. Protective properties of immunity induced by an expression vector based on the genome of human adenovirus 5, serotype Ad5-swH1glyc, administered to mice as part of a quadrivalent influenza vector vaccine Group Virus used to infect mice Result Experience (Vaccine) A/California/07/09 (H1N1) Survival rate 100%, weight loss less than 3% Control (buffer solution) A/California/07/09 (H1N1) Survival rate 0%,
weight loss 20%

From Table 2 it follows that the survival rate of mice in the experimental group after infection with the influenza A virus of subtype H1 was 100%, while in the mice of the control group it was 0%. The difference between the experimental and control groups was significant (p = 0.0001, Gehan-Breslow-Wilcoxon test). The decrease in body weight of mice in the control group after infection with the influenza A virus subtype H1 until death reached 20%, while the mice in the experimental group almost did not lose body weight, and they had no clinical symptoms of influenza.

Thus, in response to the administration of a quadrivalent influenza vector vaccine containing an expression vector based on the genome of human adenovirus 5 serotype Ad5-swH1glyc, laboratory mice developed a protective immune response against influenza A virus subtype H1.

The use of the vaccine in laboratory animals was safe; no toxic effects were observed at the dose administered to mice.

As a result of the experiment, it was shown that an expression vector based on the genome of human adenovirus serotype 5 according to the invention, introduced into the influenza vector vaccine, can be used to protect a subject from infection with influenza A viruses of subtype H1.

Thus, the technical task posed, namely, the creation of a viral vector carrying an antigen that induces immunity to various strains of human influenza A virus of subtype H1 without the effect of antigenic imprinting, which can be used as a pharmaceutical composition for the prevention of influenza A subtype H1, and also be included in the composition of the influenza vaccine has been achieved, which is confirmed by the examples given.

Industrial applicability

All of the above examples confirm the ability of the developed expression vector based on human adenovirus serotype 5 to induce an immune response against the target antigen and its industrial applicability for the creation of influenza vaccines.

Claims (9)

1. An expression vector based on human adenovirus serotype 5, inducing cross-protective immunity to influenza A viruses of subtype H1, containing: - the genome of human adenovirus serotype 5, with the exception of the E1 region, where one or more endogenous nucleotides are deleted with the loss of its functional activity, and the E3 region, where one or more endogenous nucleotides are deleted with the loss of its functional activity; - an expression cassette at the site of deletion of the E1 region of the adenovirus genome, including: a promoter, a modified hemagglutinin gene of influenza A virus subtype H1, with the sequence SEQ ID NO: 1, polyadenylation signal sequence. 2. An expression vector according to claim 1, characterized in that the genome of human adenovirus serotype 5 is represented by SEQ ID NO: 2. 3. The expression vector according to claim 1, characterized in that the expression cassette at the site of deletion of the E1 region of the adenovirus genome includes a regulated promoter, while an expression cassette is additionally placed in the expression vector at the site of deletion of the E3 region of the adenovirus genome, including: a constitutive promoter, an activator gene transcription for a regulated promoter, polyadenylation signal sequence. 4. An expression vector according to claim 3, characterized in that the expression of the modified hemagglutinin gene of the influenza A virus subtype H1 in a eukaryotic cell is negatively regulated in the presence of a tetracycline antibiotic. 5. Pharmaceutical composition for protection against influenza A viruses of subtype H1, containing in an effective amount the expression vector according to any one of claims 1-4 and a pharmaceutically acceptable buffer solution. 6. Use of a pharmaceutical composition according to claim 5 to protect a subject from influenza A viruses of subtype H1. 7. Use of the pharmaceutical composition according to claim 5 as part of an anti-influenza vector vaccine.

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