RU2660562C2 - Attenuated grippose vector and mucosal universal grippose vaccine on its basis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: attenuated influenza vector induces a cross-protective response against influenza A and B viruses containing its pharmaceutical and vaccine compositions and their use. The characterized attenuated influenza vector contains the nucleotide sequences of the genes of the proteins PB2, PB1, PA, NP and M, originating from the influenza virus A/PR/8/34 (H1N1), the nucleotide sequences of the HA and N protein genes originating from the influenza A/California/7/09-like (H1N1pdm), a nucleotide sequence of a chimeric NS protein gene comprising a truncated reading frame of the 124 amino acid NS1 gene derived from the A/PR/8/34 (H1N1) virus and continued with insertion of the nucleotide sequence encoding the HA2 subunit fusion peptide From the virus gr Ipp B, and the nucleotide sequence encoding the conserved B-cell epitope of the influenza A virus nucleoprotein (TK), and the sequence of the Nep protein gene derived from the A/Singapore/1/57-like (H2N2) virus.

EFFECT: inventions can be used to prevent influenza A and B.

16 cl, 15 dwg, 4 ex

Description

Technical field

This invention relates to the field of medicine and virology, in particular, to an attenuated chimeric influenza vector and its use for the prevention of influenza as a mucosal universal influenza vaccine.

State of the art

Of all respiratory viral diseases, influenza infection is characterized by the most severe pathology and causes the greatest damage to public health and the economy. Periodically appearing new pandemic strains, to which there is no population immunity, turn the flu into a particularly dangerous infection. It is known that the Spanish flu of 1918 caused the death of 30 to 50 million people. Currently, according to the World Health Organization (WHO), during seasonal epidemics, up to 20% of the population, including 5-10% of adults and 20-30% of children, gets the flu every year worldwide (World Health Organization [Electronic resource] . URL: http://www.who.int/biologicals/vaccines/influenza/en/ (accessed March 28, 2016). Severe forms are noted in 3-5 million cases, deaths range from 250,000 to 500,000 cases. The economic losses caused by influenza and other acute respiratory viral infections (ARVIs) account for about 77% of the damage attributable to all infectious diseases. Significant losses are associated both with direct costs of treatment and rehabilitation, as well as with indirect losses of a production nature caused by a decrease in labor productivity and a reduction in the profit of enterprises. Of the total number of cases of temporary disability, influenza and SARS account for 12-14%.

The influenza virus belongs to the Ortomyxoviridae family, which includes five genera: influenza A, B, C, D ( Thogotovirus ) and Isavirus . The genomes of influenza A and B viruses are structurally similar, they consist of 8 negative polarity genomic RNA segments that encode 12 proteins and are named for a product translated from the main open reading frame: PB1, PB2, PA, HA, NP, NA, M, and NS (Chou YY, Vafabakhsh R, Doğanay S, Gao Q, Ha T, Palese P. One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis. Proc Natl Acad Sci US A. 2012 Jun 5; 109 (23): 9101-6. Doi: 10.1073 / pnas. 1206069109. Epub 2012 Apr 30). The polymerase complex PB2, PB1, PA transcribes one mRNA from each genomic fragment, which is translated into the protein of the same name. In addition, the mRNA of the genomic segments M and NS is subjected to splicing, encoding the M2 and NEP proteins in addition to the M1 and NS1 proteins, respectively. In individual strains, the PB1-F2 protein is translated from an alternative reading frame of the PB1 segment. All proteins, with the exception of NS1 and PB1-F2, are structural components of viral particles. An unstructured NS1 protein accumulates in the cytoplasm of infected cells and acts as an inhibitor of the interferon system (Krug RM. Functions of the influenza A virus NS1 protein in antiviral defense. Curr Opin Virol. 2015 Jun; 12: 1-6. Doi: 10.1016 / j.coviro .2015.01.007. Epub 2015 Jan 29. Review).

Segmentation of the genome of the influenza virus, the high mutational background of the RNA genome, as well as the presence of a natural reservoir of viral variants in birds and animals, is an inexhaustible source of new strains that are introduced into the human population. Antigenic properties of influenza virus are determined by surface glycoproteins that form spikes on the surface of virions - hemagglutinin (HA) and neuraminidase (NA). The HA molecule is responsible for the mechanisms of attachment of the virus to the sialic receptors of the cell and the fusion of the viral and cell membranes for the penetration of the genome into the cytoplasm and cell nucleus. During the reproduction of the virus, HA is cleaved (activated) by cellular proteases into two subunits - HA1 and HA2, which remain connected by a disulfide bond (Bullough PA, Hughson FM, Skehel JJ, Wiley DC. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature 1994; 371, 37-43.). The HA molecule consists of two parts: the globular, formed by the HA1 subunit, and the stem, which consists mainly of the HA2 subunit and partially HA1. The globular portion includes a receptor-binding site and five antigenic sites and is the main target for antibody formation. Antibodies that block the binding of the virus to the receptor are neutralizing. HA1 subunit is characterized by high variability. The stem part of HA is located in close proximity to the viral membrane and is characterized by weak immunogenicity. The HA2 subunit plays a major role in ensuring the fusion of the viral membrane with the endosomal and is highly conservative. In accordance with antigenic specificity, 18 HA subtypes and 11 NA subtypes are currently known for influenza A virus. The HA subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18 belong to the first group, and H3, H4, H7, H10, H14 and H15 to the second group.

It is known that the specific immunity generated after a disease or vaccination with one serotype of the influenza A virus weakly protects against infection with another serotype. Immunity to any serotype of influenza A virus does not protect against influenza B virus, and vice versa, immunization against influenza B is not effective against influenza A.

The best way to prevent the spread of influenza infection is vaccination. Existing vaccines can be divided into two types: attenuated (live, containing whole and active viruses that exhibit low pathogenicity with intranasal administration) and inactivated (containing fragments of viral particles or whole inactive viruses). Live attenuated viruses, capable of limited reproduction in the cells of the respiratory tract, cause both humoral and cellular immune responses. Therefore, live attenuated vaccines have a wider range of protection against antigenic variants of the influenza virus compared to inactivated vaccines, which, as a rule, stimulate only the humoral part of the immune system.

The basis of the unusually fast variability of the influenza virus, and hence its escape from the action of neutralizing antibodies, is based on two mechanisms: 1) the accumulation of point mutations leading to a change in the antigenic structure of surface glycoproteins (antigenic drift) and 2) reassortment of genomic segments. They lead to the emergence of new antigenic variants of the virus (antigenic shift) that can cause a pandemic.

All influenza vaccines currently in use have a low risk of immunization for the elderly and young children (Jefferson T, Rivetti A, Di Pietrantonj C, Demicheli V, Ferroni E. Vaccines for preventing influenza in healthy children. Cochrane Database Syst Rev 2012; 8, CD004879 .;

Osterholm MT, Kelley NS, Sommer A, Belongia EA. Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. Lancet Infect Dis 2011; 12, 36-44; Pfleiderer M, Trouvin JH, Brasseur D, Granstrom M, Shivji R, Mura M, Cavaleri M. Summary of knowledge gaps related to quality and efficacy of current influenza vaccines. Vaccine 2014; 32, 4586-91). In addition, these vaccines protect against circulating strains only if the epidemic virus coincides with the vaccine strain by antigenic properties. The high variability of the surface antigens of the influenza virus, HA and NA, necessitates the annual vaccination and updating of the composition of the vaccines. It should be noted that seasonal vaccines, formed on the basis of WHO recommendations, are ineffective in the case of the sudden emergence of a new pandemic strain that is radically different from all circulating variants, as happened in 2009 with the advent of the pandemic virus A / California / 7/2009 (H1N1pdm09) . Another example is the low efficacy of the 2014 seasonal vaccine component H3N2 due to the emergence of a new antigenic variant of this seropodotype as a result of antigenic drift (Skowronski DM, Chambers C, Sabaiduc S, De Serres G, Dickinson JA, Winter AL, Drews SJ, Fonseca K, Charest H, Gubbay JB, Petric M, Krajden M, Kwindt TL, Martineau C, Eshaghi A, Bastien N, Li Y. Interim estimates of 2014/15 vaccine effectiveness against influenza A (H3N2) from Canada's Sentinel Physician Surveillance Network, January 2015. Euro Surveill 2015; 20). Over the past 60 years, many variants of live and inactivated influenza vaccines have been developed that have certain advantages and disadvantages, however, none of the known drugs solves the problem of controlling the incidence of influenza due to their inability to cause cross-protective immunity to constantly evolving influenza viruses. In this regard, there is an urgent need to create a universal influenza vaccine that causes a wide cross-protective long-term immunity, able to withstand all known known circulating seropodi viruses of influenza A and B.

All known influenza vaccines - inactivated (whole-virion, split or subunit) or live (attenuated cold-adapted) - are mainly aimed at creating immunity to the globular part of HA. Unlike the variable globular part, the stem part of HA is conservative among influenza A viruses (group I and II) and B. For antibodies induced to this region of HA, several mechanisms of both direct and indirect neutralization are known. One of the mechanisms of direct neutralization is associated with the prevention of conformational changes in HA, which is necessary for the release of the fusion peptide and the subsequent integration of the endosomal and viral membranes to deliver the viral genome into the cell. The second mechanism of direct neutralization is associated with the prevention of activation cleavage of HA into HA1 and HA2 subunits using antibodies that interact with the HA site in the immediate vicinity of the cleavage site. Indirect neutralization mechanisms use antibody-dependent and complement-dependent cytotoxicity (Terajima M, Cruz J, Co MD, Lee JH, Kaur K, Wrammert J, Wilson PC, Ennis FA. Complement-dependent lysis of influenza a virus-infected cells by broadly cross-reactive human monoclonal antibodies. J Virol 2011; 85, 13463-7 .; Jegaskanda S, Weinfurter JT, Friedrich TC, Kent SJ. Antibody-dependent cellular cytotoxicity is associated with control of pandemic H1N1 influenza virus infection of macaques. J Virol 2013; 87 5512-22).

Antibodies to the HA stem region during vaccination practically do not form and are detected in small quantities only after a natural infection (Moody MA, Zhang R, Walter EB, Woods CW, Ginsburg GS, McClain MT, Denny TN, Chen X, Munshaw S, Marshall DJ, Whitesides JF, Drinker MS, Amos JD, Gurley TC, Eudailey JA, Foulger A, DeRosa KR, Parks R, Meyerhoff RR, Yu JS, Kozink DM, Barefoot BE, Ramsburg EA, Khurana S, Golding H, Vandergrift NA, Alam SM , Tomaras GD, Kepler TB, Kelsoe G, Liao HX, Haynes BF. H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination. PLoS ONE 2011; 6, e25797).

Most of the approaches currently being developed for the preparation of a universal vaccine are based on focusing the immune response to conserved regions of the flu virus proteins. Antibodies to the conserved internal proteins PB2, PB1, PA, NP, and M1 are not neutralizing, but can play an important role in eliminating the virus due to antibody-dependent cytotoxicity (ADCC).

Several examples are known of creating a universal vaccine based on the HA2 subunit. Three-fold immunization of mice with peptides representing the HA2 ectodomain (23-185 amino acid residues) or a fusion peptide (1-38 amino acid residues), in combination with KLH adjuvants ( k eyhole l impet h emocyanin) and Freund induced cross-reactive immunity, which reduces the death of animals for lethal infection with a heterologous strain (Stanekova Z, Kiraly J, Stropkovska A, Mikuskova T, Mucha V, Kostolansky F, Vareckova E. Heterosubtypic protective immunity against influenza A virus induced by fusion peptide of the hemagglutinin in comparison to ectodomain of M2 protein. Acta Virol 2011; 55, 61-7). More effective protection developed during vaccination with chimeric HA constructs. Krammer et al. showed that heterosubtypical humoral immunity in mice is induced when immunized with chimeric proteins containing globular parts of HA from different seropodotype strains and the same stem part of HA (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 2014; 386, 301-21 .; Krammer F, Hai R, Yondola M, Tan GS, Leyva-Grado VH, Ryder AB, Miller MS, Rose JK, Palese P, Garcia- Sastre A, Albrecht RA. Assessment of influenza virus hemagglutinin stalk-based immunity in ferrets. J Virol 2014; 88, 3432-42). The disadvantages of this approach include the complex immunization schedule, which includes electroporation of animals using DNA and double immunization with protein constructs intramuscularly and intranasally with poly (I: C) adjuvant.

Another example is the use of stable constructs (mini-HA) obtained by genetic engineering based on the amino acid sequence of the stem portion of the HA seropodotype H1N1 virus. Only the constructions with the highest affinity for antibodies with neutralizing activity of a wide spectrum were selected from the large collection. Immunization of mice with such constructs also protected animals from death when infected with a strain of group I - the highly pathogenic avian influenza virus seroptype H5N1 (Impagliazzo A, Milder F, Kuipers H, Wagner MV, Zhu X, Hoffman RM, van Meersbergen R, Huizingh J, Wanningen P, Verspuij J, de Man M, Ding Z, Apetri A, Kukrer B, Sneekes-Vriese E, Tomkiewicz D, Laursen NS, Lee PS, Zakrzewska A, Dekking L, Tolboom J, Tettero L, van Meerten S, Yu W, Koudstaal W, Goudsmit J, Ward AB, Meijberg W, Wilson IA, Radosevic K. A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen. Science 2015; 349, 1301-6). One-hundred-percent protection of mice from death was achieved as a result of double intramuscular immunization with purified mini-HA protein at a dose of 30 μg with Novavax Matrix-M adjuvant.

Another promising direction in the development of a universal influenza vaccine is based on the creation of self-assembled nanoparticles, which, as shown earlier, significantly increase the immunogenic properties of HA (Kanekiyo M, Wei CJ, Yassine HM, McTamney PM, Boyington JC, Whittle JR, Rao SS, Kong WP, Wang L, Nabel GJ. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 2013; 499, 102-6). Nanoparticles were added to the animals two or three times intramuscularly with the addition of a new SAS adjuvant (Sigma Adjuvant System). Despite the lack of production of neutralizing antibodies after immunization with nanoparticles, both mice and ferrets were completely protected from death when infected with the highly pathogenic avian influenza virus seroptype H5N1.

One of the modern technologies for producing a live vaccine is based on the construction of vaccine vectors in which one virus expresses antigens of another virus. Various DNA-containing viruses are used as vectors expressing influenza antigens, namely: adenovirus, herpes virus, baculovirus or poxvirus (Dudek T, Knipe DM. Replication-defective viruses as vaccines and vaccine vectors. Virology 2006; 344, 230-9 .; He F, Madhan S, Kwang J. Baculovirus vector as a delivery vehicle for influenza vaccines. Expert Rev Vaccines 2009; 8, 455-67 .; Draper SJ, Cottingham MG, Gilbert SC. Utilizing poxviral vectored vaccines for antibody induction- progress and prospects. Vaccine 2013; 31, 4223-30. Price GE, Soboleski MR, Lo CY, Misplon JA, Pappas C, Houser KV, Tumpey TM, Epstein SL. Vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent H1N1 and H5N1 influenza A viruses. Vaccine 2009; 27, 6512-21). Thus, using an adenoviral vector, it was shown that triple immunization with a plasmid (50 μg) containing sequences of conserved NP and M2 viruses of influenza A virus, followed by intranasal infection with two adenovirus vectors expressing the same proteins, completely protected mice and ferrets from death and loss weight when infected with virulent virus A / FM / 1/47 (H1N1) or a highly pathogenic strain of avian influenza virus seropodotype H5N1.

Thus, all of the above approaches of focusing the immune response against conservative influenza virus antigens confirm the fundamental possibility of creating an influenza vaccine that can protect against infection with various variants of influenza A. However, to achieve this goal, complex schemes of repeated vaccination of animals using immunological adjuvants of various nature. In addition, none of the known experimental preparations for the universal influenza vaccine provided protection against influenza B. To this, it should be added that the experimental preparations listed above require complex technological processes for the production of multicomponent vaccines associated with an unacceptably high cost of the final drug.

Thus, there remains a need for new effective attenuated influenza vectors that can be used for the prevention of influenza in the form of a universal influenza vaccine, in particular in the form of a mucosal influenza vaccine.

Disclosure of invention

The present invention relates to an attenuated influenza vector inducing a cross-protective response against influenza A and B viruses, comprising:

the nucleotide sequence of the PB2 protein gene derived from influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity to the nucleotide sequence of the PB2 protein gene;

the nucleotide sequence of the PB1 protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity with the specified nucleotide sequence of the PB1 protein gene;

the nucleotide sequence of the PA protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity to the specified sequence of the protein PA;

the nucleotide sequence of the NP protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity with the specified sequence of the NP protein;

the nucleotide sequence of the M protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity with the indicated sequence of the protein M gene gene;

the nucleotide sequence of the HA protein gene, derived from the influenza virus A / California / 7/09-like H1N1pdm), or the nucleotide sequence having at least 95% identity to the specified nucleotide sequence of the HA protein gene;

the nucleotide sequence of the NA protein gene derived from the influenza virus A / California / 7/09-like H1N1pdm), or the nucleotide sequence having at least 95% identity to the specified nucleotide sequence of the NA protein gene; and

the nucleotide sequence of the chimeric gene of the NS protein, including

the reading frame of the NS1 protein, derived from the virus A / PR / 8/34 (H1N1), where the specified reading frame is shortened and encodes the NS1 protein of 124 amino acid residues,

and a Nep protein gene sequence derived from influenza A / Singapore / 1/57-like (H2N2) virus, or

a nucleotide sequence having at least 95% identity to the indicated sequence of the chimeric NS gene;

where the indicated shortened reading frame of the NS1 protein gene is continued by the insertion of a nucleotide sequence encoding a fusion peptide of the HA2 subunit from influenza B virus and a nucleotide sequence encoding a conserved B cell epitope of influenza A virus nucleoprotein (NP).

The attenuated influenza vector of the invention induces a cross-protective response against various antigenic variants of influenza A and B virus after mucosal administration, in particular after intranasal immunization.

A preferred embodiment is an influenza vector in which the nucleotide sequence of the NS protein chimeric gene is presented in SEQ ID NO: 8.

Another object of the present invention is a pharmaceutical composition for the prevention of influenza in a subject, containing in an effective amount an attenuated influenza vector of the invention and a pharmaceutically acceptable carrier.

One embodiment of the invention is a pharmaceutical composition comprising 6-8.5 logEID 50 / ml of the attenuated influenza vector of the invention and a buffer solution containing 0-1.5 wt.% Monovalent salt, 0-5 wt.% L-carnosine, 0-5 wt.% Carbohydrate component, 0-2 wt.% Protein component, 0-2 wt.% Amino acid component and 0-10 wt.% Hydroxyethylated starch. The preferred option is a pharmaceutical composition, where the buffer solution contains 0.5-1.5 wt.% Monovalent salt, 0.01-5 wt.% L-carnosine, 1-5 wt.% Carbohydrate component, 0.1-2 wt. .% protein component, 0.01-2 mass. % amino acid component and 1-10 wt.% hydroxyethylated starch, preferably where the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose or lactose, the protein component is human recombinant albumin, casitone, lactalbumin hydrolyzate or gelatin, the amino acid component represents arginine, glycine or monosodium glutamate.

In a preferred embodiment, the subject is a mammal or a bird, in particular, the subject is a human.

The invention also relates to a flu vaccine comprising, in an effective amount, an attenuated influenza vector of the invention and a pharmaceutically acceptable carrier.

One embodiment of the invention is a vaccine containing 6-8.5 log EID 50 / ml of the attenuated influenza vector of the invention and a buffer solution containing 0-1.5 wt.% Monovalent salt, 0-5 wt.% L-carnosine, 0 -5 wt.% Carbohydrate component, 0-2 wt.% Protein component, 0-2 wt.% Amino acid component and 0-10 wt.% Hydroxyethylated starch. The preferred option is a vaccine, where the buffer solution contains 0.5-1.5 wt.% Monovalent salt, 0.01-5 wt.% L-carnosine, 1-5 wt.% Carbohydrate component, 0.1-2 wt. % protein component, 0.01-2 mass. % amino acid component and 1-10 wt.% hydroxyethylated starch, preferably where the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose or lactose, the protein component is human recombinant albumin, casitone, lactalbumin hydrolyzate or gelatin, the amino acid component represents arginine, glycine or monosodium glutamate.

The invention also relates to the use of an attenuated influenza vector of the invention or a pharmaceutical composition of the invention for the prevention of influenza in a subject.

In a preferred embodiment, the subject is a mammal or a bird, in particular, the subject is a human.

The technical result of the present invention is the preparation of a chimeric NS-based genomic fragment of an influenza vector having the properties of a universal influenza vaccine with a single mucosal administration in the absence of adjuvants. In addition, the technical result is a high growth potential of the obtained influenza vector in 10-day-old chicken embryos. Another technical result is the production of influenza vectors that have the properties of a universal influenza vaccine. Another technical result is the reduction in the cost of production of influenza vaccine due to the non-use of adjuvants.

Brief Description of the Drawings

Figure 1 shows the structure of the influenza attenuated vector. 8 fragments of the virus genome and their features are designated.

It was shown that fragments of the genome PB2, PB1, PA, Np and M are derived from the virus A / PR / 8/34 (H1N1); HA and NA surface glycoprotein genes are derived from the A / California / 7/09-like virus (H1N1pdm); The NS genomic fragment has a chimeric structure encoding two proteins: 1) an NS1 protein shortened to 124 amino acid residues, continued by inserting the sequence of the N-terminal region of the HA2 virus of influenza B and the insertion of a conserved B-cell epitope of the NP protein of influenza A virus 2) Nep protein having the sequence from a heterologous strain of influenza A virus, seropodotype H2N2 A / Singapore / 1/57 -like.

Figure 2 presents the nucleotide sequence of the genomic fragments of the vaccine vector: PB2, PB1, PA, NP, M - derived from the virus A / PR / 8/34 (H1N1); HA and NA derived from A / California / 7/09-like virus (H1N1pdm); chimeric NS (the insert in the reading frame of NS1 is shown in bold).

Figure 3 presents the results reflecting the protective properties of the vaccine vector after intranasal immunization of mice with respect to different variants of influenza A and influenza B viruses. The graphs show the% mortality in vaccinated mice after infection with the indicated subtypes of influenza A or influenza B compared with with control animals. The vaccine was administered once - 1x or twice - 2x.

 Figure 4 presents data demonstrating the protective properties of the vaccine vector after intranasal immunization of ferrets. Figure A shows the dynamics of the average value of temperature fluctuations after the control virus infection A / St. Petersburg / 224/2015 (H3N2) in vaccinated and control animals. Figure B presents the results of plating the control virus from nasal swabs of ferrets on 2.4 and 6 days after infection. The titers are expressed as the average concentration of the virus in nasal swabs, expressed as the value of 50% cytopathic doses / ml after titration on MDCK cells. The vaccine was administered once - 1x or twice - 2x.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a genetically engineered vaccine vector based on influenza A virus, which can be used to prevent influenza caused by all strains circulating in the human population, including influenza A and B. In particular, the present invention relates to attenuated influenza A virus inducing a cross-protective response against influenza A and B virus, containing a chimeric NS fragment comprising a shortened reading frame of the NS1 protein and a heterologous protein gene sequence Nep, derived from the influenza A virus subtype H2N2. Thus, the virus A subtype for the sequence encoding the truncated NS1 protein is different from the virus A subtype from which the sequence encoding the Nep protein is derived. In particular, in the vaccine vector, the shortened reading frame of the NS1 protein is derived from the H1N1 subtype influenza virus, and the heterologous sequence of the Nep protein gene is derived from the H2N2 subtype influenza virus.

In particular, the present invention relates to an attenuated influenza vector inducing a cross-protective response against influenza A and B viruses, comprising:

the nucleotide sequence of a gene encoding a PB2 protein derived from influenza virus A / PR / 8/34 (H1N1), or a nucleotide sequence having at least 95% or more (e.g. 96, 97, 98 or 99%) of the nucleotide identity PB2 protein gene sequences;

the nucleotide sequence of a gene encoding a PB1 protein derived from influenza virus A / PR / 8/34 (H1N1), or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98 or 99%) of the nucleotide identity PB1 protein gene sequences;

the nucleotide sequence of a gene encoding a PA protein derived from influenza virus A / PR / 8/34 (H1N1), or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98 or 99%) the identity of the specified sequence RA protein;

the nucleotide sequence of a gene encoding an NP protein derived from influenza virus A / PR / 8/34 (H1N1), or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98 or 99%) the identity of the specified sequence NP protein;

the nucleotide sequence of a gene encoding a protein M derived from influenza A / PR / 8/34 (H1N1) virus, or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98 or 99%) the identity of the specified sequence gene protein M;

the nucleotide sequence of a gene encoding an HA protein derived from the influenza virus A / California / 7/09-like H1N1pdm), or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98 or 99%) identity of the specified the nucleotide sequence of the HA protein gene;

the nucleotide sequence of a gene encoding an NA protein derived from the influenza virus A / California / 7/09-like H1N1pdm), or a nucleotide sequence having at least 95% or more (for example, 96, 97, 98 or 99%) identity of the specified the nucleotide sequence of the NA protein gene; and

the nucleotide sequence of the chimeric gene of the NS protein, including

the reading frame of the NS1 protein, derived from the virus A / PR / 8/34 (H1N1), where the specified reading frame is shortened and encodes the NS1 protein of 124 amino acid residues,

and a Nep protein gene sequence derived from influenza A / Singapore / 1/57-like (H2N2) virus, or

a nucleotide sequence having at least 95% or more (for example, 96, 97, 98 or 99%) the identity of the specified sequence of the chimeric NS gene;

where the indicated shortened reading frame of the NS1 protein gene is continued by the insertion of a nucleotide sequence encoding a fusion peptide of the HA2 subunit from influenza B virus and a nucleotide sequence encoding a conserved B cell epitope of influenza A virus nucleoprotein (NP).

The indicated shortened reading frame encodes an NS1 protein of 124 amino acid residues, which is extended by two glycines, an insert of the N-terminal region of the second subunit of hemagglutinin HA2 influenza B virus (23 amino acid residues) and an insertion of the sequence of the conserved B cell epitope of influenza A virus (7 amino acid residues).

The surface glycoprotein genes of this vector are derived from the influenza virus A / California / 7/09 (H1N1pdm). Genes for the internal proteins PB2, PB1, PA, NP, and M come from the influenza virus A / PR / 8/34 (H1N1). Thus, the influenza vector according to the invention is a complex genetic construct consisting of genomic sequences of various strains of the influenza virus, namely: 1) genes encoding PB2, PB1, PA, NP and M, are derived from virus A / PR / 8 / 34 (H1N1) (PB2 (Genbank accession number: AB671295), PB1 (Genbank accession number: CY033583), PA (Genbank accession number: AF389117), NP (Genbank accession number: AF389119), M (Genbank accession number: AF389121)) , 2) the genes encoding HA and NA are derived from the A / California / 7/09-like H1N1pdm virus) (HA (GenBank: KM408964.1) and (NA GenBank: KM408965.1), 3) the NS gene has a chimeric nature where the reading frame of the NS1 protein from A / P virus R / 8/34 (H1N1) was shortened to 124 amino acid residues and continued with the insertion of the sequence encoding the fusion peptide of the HA2 subunit from influenza B virus and the sequence encoding the conserved B-cell epitope of influenza A virus nucleoprotein (NP), and the reading frame of the NEP protein derived from the influenza virus subtype H2N2.

The present invention is based in particular on the fact that the authors unexpectedly discovered that a vector with the indicated structure upon intranasal immunization of mice and ferrets without the use of adjuvants protects animals from control infection not only with influenza A (H1N1) viruses, but also with influenza A viruses (H3N2) and influenza B. Therefore, the vaccine vector has the properties of a universal influenza vaccine.

The term "universal vaccine" in the context of the present invention means a vaccine capable of protecting against all known and unknown variants of the influenza virus. Conventional "seasonal vaccines" protect only against viruses similar to those viruses that make up their composition.

The term “mucosal vaccine” means that the vaccine can be administered in the cavities of the respiratory and digestive tract and applied to the mucous membranes of the mouth and nose, i.e. apply intranasally, orally, sublingually.

The influenza A / PR / 8/34 virus vector carrying the chimeric NS genomic fragment was unable to actively reproduce at 39 ° C and in the lungs of mice (attenuation phenotype), but it still reproduced to high titers of 10- daytime chicken embryos.

The pharmaceutical composition according to the present invention can be formulated as a vaccine containing in an effective amount an attenuated influenza vector according to the present invention and a pharmaceutically acceptable carrier.

The term "subject" or "animal" in the context of the present description means vertebrates that are susceptible to infection by pathogenic bacteria, viruses or protozoa, including birds (waterfowl, chickens, etc.) and representatives of various species of mammals, such as canids, cats , wolves, ferrets, rodents (rats, mice, etc.), horses, cows, sheep, goats, pigs and primates. In one embodiment, the subject is a human.

By "effective amount" is meant such an amount of a vector that, when administered to a subject, in a single or double dose, is effective for the prevention of influenza. This amount may vary depending on the patient’s health and physical condition, age, taxonomic group of the individual being vaccinated, formulation, assessment of the medical situation by the attending physician, and other important factors. It is believed that the amount can vary over a relatively wide range that a person skilled in the art can determine by standard methods. In particular, the pharmaceutical composition may contain 6-8.5 log EID 50 / ml influenza vector according to the invention.

The term "pharmaceutically acceptable carrier" in the context of the present invention means any carrier used in this field, in particular water, saline, buffer solution and the like. In one embodiment, the pharmaceutically acceptable carrier is a buffer solution containing 0-1.5 wt.% Monovalent salt, 0-5 wt.% L-carnosine, 0-5 wt.% Carbohydrate component, 0-2 wt.% protein component, 0-2 wt.% amino acid component and 0-10 wt.% hydroxyethylated starch, preferably, said buffer solution contains 0.5-1.5 wt.% monovalent salt, 0.01-5 wt.% L- carnosine, 1-5 wt.% carbohydrate component, 0.1-2 wt.% protein component, 0.01-2 wt. % amino acid component and 1-10 wt.% hydroxyethylated starch, in the most preferred embodiment, the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose or lactose, the protein component is human recombinant albumin, casitone, lactalbumin hydrolyzate or gelatin, the amino acid component is arginine, glycine or monosodium glutamate.

The present invention also relates to the use of an attenuated attenuated influenza vector or pharmaceutical composition according to the present invention for the prevention of influenza.

The introduction to the subject can be carried out by any standard method, in particular, orally, sublingually, inhalation or intranasally.

The influenza vector or pharmaceutical composition can be administered to the subject one, two or more times, single administration is preferred.

The invention is further illustrated by non-limiting embodiments.

EXAMPLES

Example 1

Obtaining influenza vector with a modified NS genomic fragment

The assembly of the recombinant virus was carried out in several stages. At the first stage, complementary DNA (cDNA) copies of 5 genomic fragments (PB2, PB1, PA, NP, M) of the influenza A / PR / 8/34 (H1N1) virus (PB2 (Genbank accession number: AB671295), PB1 (Genbank accession number: CY033583), PA (Genbank accession number: AF389117), NP (Genbank accession number: AF389119), M (Genbank accession number: AF389121)) and 2 genomic fragments (HA, NA) of A / California / 7 virus / 09-like (HA (GenBank: KM408964.1) and (NA GenBank: KM408965.1)), as well as synthesized a chimeric NS genomic fragment composed of sequences related to the H1N1 virus (NS1 gene), the H2N2 virus (Nep gene ) and sequences of two peptides from HA2 of influenza B virus and NP of influenza A virus.

In the second step, the synthesized sequences were cloned into a bidirectional vector based on the plasmid pHW2000 (Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA. 2000; 97 (11): 6108-13.). The specified plasmid vector due to the presence of Pol I and Pol II promoters provides simultaneous intracellular transcription of viral and corresponding messenger RNA during transfection of mammalian cells. Figure 1 presents the genetic scheme of the influenza vector. In FIG. 2 shows the nucleotide sequences of all 8 genomic fragments of the influenza vector.

The nucleotide sequence of the PB2 gene

1 agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactacg aaatctaatg

61 tcgcagtctc gcacccgcga gatactcaca aaaaccaccg tggaccatat ggccataatc

121 aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa atggatgatg

181 gcaatgaaat atccaattac agcagacaag aggataacgg aaatgattcc tgagagaaat

241 gagcaaggac aaactttatg gagtaaaatg aatgatgccg gatcagaccg agtgatggta

301 tcacctctgg ctgtgacatg gtggaatagg aatggaccaa taacaaatac agttcattat

361 ccaaaaatct acaaaactta ttttgaaaga gtcgaaaggc taaagcatgg aacctttggc

421 cctgtccatt ttagaaacca agtcaaaata cgtcggagag ttgacataaa tcctggtcat

481 gcagatctca gtgccaagga ggcacaggat gtaatcatgg aagttgtttt ccctaacgaa

541 gtgggagcca ggatactaac atcggaatcg caactaacga taaccaaaga gaagaaagaa

601 gaactccagg attgcaaaat ttctcctttg atggttgcat acatgttgga gagagaactg

661 gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta cattgaagtg

721 ttgcatttga ctcaaggaac atgctgggaa cagatgtata ctccaggagg ggaagtgagg

781 aatgatgatg ttgatcaaag cttgattatt gctgctagga acatagtgag aagagctgca

841 gtatcagcag atccactagc atctttattg gagatgtgcc acagcacaca gattggtgga

901 attaggatgg tagacatcct taggcagaac ccaacagaag agcaagccgt ggatatatgc

961 aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt cacatttaag

1021 agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggcaa tcttcaaaca

1081 ttgaagataa gagtgcatga gggatatgaa gagttcacaa tggttgggag aagagcaaca

1141 gccatactca gaaaagcaac caggagattg attcagctga tagtgagtgg gagagacgaa

1201 cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga ttgtatgata

1261 aaagcagtca gaggtgatct gaatttcgtc aatagggcga atcaacgatt gaatcctatg

1321 catcaacttt taagacattt tcagaaggat gcgaaagtgc tttttcaaaa ttggggagtt

1381 gaacctatcg acaatgtgat gggaatgatt gggatattgc ccgacatgac tccaagcatc

1441 gagatgtcaa tgagaggagt gagaatcagc aaaatgggtg tagatgagta ctccagcacg

1501 gagagggtag tggtgagcat tgaccgtttt ttgagaatcc gggaccaacg aggaaatgta

1561 ctactgtctc ccgaggaggt cagtgaaaca cagggaacag agaaactgac aataacttac

1621 tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgttggtcaa tacctatcaa

1681 tggatcatca gaaactggga aactgttaaa attcagtggt cccagaaccc tacaatgcta

1741 tacaataaaa tggaatttga accatttcag tctttagtac ctaaggccat tagaggccaa

1801 tacagtgggt ttgtaagaac tctgttccaa caaatgaggg atgtgcttgg gacatttgat

1861 accgcacaga taataaaact tcttcccttc gcagccgctc caccaaagca aagtagaatg

1921 cagttctcct catttactgt gaatgtgagg ggatcaggaa tgagaatact tgtaaggggc

1981 aattctcctg tattcaacta taacaaggcc acgaagagac tcacagttct cggaaaggat

2041 gctggcactt taactgaaga cccagatgaa ggcacagctg gagtggagtc cgctgttctg

2101 aggggattcc tcattctggg caaagaagac aagagatatg ggccagcact aagcatcaat

2161 gaactgagca accttgcgaa aggagagaag gctaatgtgc taattgggca aggagacgtg

2221 gtgttggtaa tgaaacggaa acgggactct agcatactta ctgacagcca gacagcgacc

2281 aaaagaattc ggatggccat caattagtgt cgaatagttt aaaaacgacc ttgtttctac

2341 t (SEQ ID NO: 1)

The nucleotide sequence of the PB1 gene

1 atggatgtca atccgacctt acttttctta aaagtgccag cacaaaatgc tataagcaca

61 actttccctt atactggaga ccctccttac agccatggga caggaacagg atacaccatg

121 gatactgtca acaggacaca tcagtactca gaaaagggaa gatggacaac aaacaccgaa

181 actggagcac cgcaactcaa cccgattgat gggccactgc cagaagacaa tgaaccaagt

241 ggttatgccc aaacagattg tgtattggag gcgatggctt tccttgagga atcccatcct

301 ggtatttttg aaaactcgtg tattgaaacg atggaggttg ttcagcaaac acgagtagac

361 aagctgacac aaggccgaca gacctatgac tggactctaa atagaaacca acctgctgca

421 acagcattgg ccaacacaat agaagtgttc agatcaaatg gcctcacggc caatgagtct

481 ggaaggctca tagacttcct taaggatgta atggagtcaa tgaacaaaga agaaatgggg

541 atcacaactc attttcagag aaagagacgg gtgagagaca atatgactaa gaaaatgata

601 acacagagaa caatgggtaa aaagaagcag agattgaaca aaaggagtta tctaattaga

661 gcattgaccc tgaacacaat gaccaaagat gctgagagag ggaagctaaa acggagagca

721 attgcaaccc cagggatgca aataaggggg tttgtatact ttgttgagac actggcaagg

781 agtatatgtg agaaacttga acaatcaggg ttgccagttg gaggcaatga gaagaaagca

841 aagttggcaa atgttgtaag gaagatgatg accaattctc aggacaccga actttctttc

901 accatcactg gagataacac caaatggaac gaaaatcaga atcctcggat gtttttggcc

961 atgatcacat atatgaccag aaatcagccc gaatggttca gaaatgttct aagtattgct

1021 ccaataatgt tctcaaacaa aatggcgaga ctgggaaaag ggtatatgtt tgagagcaag

1081 agtatgaaac ttagaactca aatacctgca gaaatgctag caagcatcga tttgaaatat

1141 ttcaatgatt caacaagaaa gaagattgaa aaaatccgac cgctcttaat agaggggact

1201 gcatcattga gccctggaat gatgatgggc atgttcaata tgttaagcac tgtattaggc

1261 gtctccatcc tgaatcttgg acaaaagaga tacaccaaga ctacttactg gtgggatggt

1321 cttcaatcct ctgacgattt tgctctgatt gtgaatgcac ccaatcatga agggattcaa

1381 gccggagtcg acaggtttta tcgaacctgt aagctacttg gaatcaatat gagcaagaaa

1441 aagtcttaca taaacagaac aggtacattt gaattcacaa gttttttcta tcgttatggg

1501 tttgttgcca atttcagcat ggagcttccc agttttgggg tgtctgggat caacgagtca

1561 gcggacatga gtattggagt tactgtcatc aaaaacaata tgataaacaa tgatcttggt

1621 ccagcaacag ctcaaatggc ccttcagttg ttcatcaaag attacaggta cacgtaccga

1681 tgccatagag gtgacacaca aatacaaacc cgaagatcat ttgaaataaa gaaactgtgg

1741 gagcaaaccc gttccaaagc tggactgctg gtctccgacg gaggcccaaa tttatacaac

1801 attagaaatc tccacattcc tgaagtctgc ctaaaatggg aattgatgga tgaggattac

1861 caggggcgtt tatgcaaccc actgaaccca tttgtcagcc ataaagaaat tgaatcaatg

1921 aacaatgcag tgatgatgcc agcacatggt ccagccaaaa acatggagta tgatgctgtt

1981 gcaacaacac actcctggat ccccaaaaga aatcgatcca tcttgaatac aagtcaaaga

2041 ggagtacttg aggatgaaca aatgtaccaa aggtgctgca atttatttga aaaattcttc

2101 cccagcagtt catacagaag accagtcggg atatccagta tggtggaggc tatggtttcc

2161 agagcccgaa ttgatgcacg gattgatttc gaatctggaa ggataaagaa agaagagttc

2221 actgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atagtgaatt

2281 tagcttgt (SEQ ID NO: 2)

The nucleotide sequence of the PA gene

1 agcgaaagca ggtactgatc caaaatggaa gattttgtgc gacaatgctt caatccgatg

61 attgtcgagc ttgcggaaaa aacaatgaaa gagtatgggg aggacctgaa aatcgaaaca

121 aacaaatttg cagcaatatg cactcacttg gaagtatgct tcatgtattc agattttcac

181 ttcatcaatg agcaaggcga gtcaataatc gtagaacttg gtgatccaaa tgcacttttg

241 aagcacagat ttgaaataat cgagggaaga gatcgcacaa tggcctggac agtagtaaac

301 agtatttgca acactacagg ggctgagaaa ccaaagtttc taccagattt gtatgattac

361 aaggagaata gattcatcga aattggagta acaaggagag aagttcacat atactatctg

421 gaaaaggcca ataaaattaa atctgagaaa acacacatcc acattttctc gttcactggg

481 gaagaaatgg ccacaaaggc agactacact ctcgatgaag aaagcagggc taggatcaaa

541 accagactat tcaccataag acaagaaatg gccagcagag gcctctggga ttcctttcgt

601 cagtccgaga gaggagaaga gacaattgaa gaaaggtttg aaatcacagg aacaatgcgc

661 aagcttgccg accaaagtct cccgccgaac ttctccagcc ttgaaaattt tagagcctat

721 gtggatggat tcgaaccgaa cggctacatt gagggcaagc tgtctcaaat gtccaaagaa

781 gtaaatgcta gaattgaacc ttttttgaaa acaacaccac gaccacttag acttccgaat

841 gggcctccct gttctcagcg gtccaaattc ctgctgatgg atgccttaaa attaagcatt

901 gaggacccaa gtcatgaagg agagggaata ccgctatatg atgcaatcaa atgcatgaga

961 acattctttg gatggaagga acccaatgtt gttaaaccac acgaaaaggg aataaatcca

1021 aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga gaatgaggag

1081 aaaattccaa agactaaaaa tatgaagaaa acaagtcagc taaagtgggc acttggtgag

1141 aacatggcac cagaaaaggt agactttgac gactgtaaag atgtaggtga tttgaagcaa

1201 tatgatagtg atgaaccaga attgaggtcg ctagcaagtt ggattcagaa tgagtttaac

1261 aaggcatgcg aactgacaga ttcaagctgg atagagctcg atgagattgg agaagatgtg

1321 gctccaattg aacacattgc aagcatgaga aggaattatt tcacatcaga ggtgtctcac

1381 tgcagagcca cagaatacat aatgaagggg gtgtacatca atactgcctt gcttaatgca

1441 tcttgtgcag caatggatga tttccaatta attccaatga taagcaagtg tagaactaag

1501 gagggaaggc gaaagaccaa cttgtatggt ttcatcataa aaggaagatc ccacttaagg

1561 aatgacaccg acgtggtaaa ctttgtgagc atggagtttt ctctcactga cccaagactt

1621 gaaccacata aatgggagaa gtactgtgtt cttgagatag gagatatgct tataagaagt

1681 gccataggcc aggtttcaag gcccatgttc ttgtatgtga gaacaaatgg aacctcaaaa

1741 attaaaatga aatggggaat ggagatgagg cgttgcctcc tccagtcact tcaacaaatt

1801 gagagtatga ttgaagctga gtcctctgtc aaagagaaag acatgaccaa agagttcttt

1861 gagaacaaat cagaaacatg gcccattgga gagtccccca aaggagtgga ggaaagttcc

1921 attgggaagg tctgcaggac tttattagca aagtcggtat tcaacagctt gtatgcatct

1981 ccacaactag aaggattttc agctgaatca agaaaactgc ttcttatcgt tcaggctctt

2041 agggacaacc ttgaacctgg gacctttgat cttggggggc tatatgaagc aattgaggag

2101 tgcctgatta atgatccctg ggttttgctt aatgcttctt ggttcaactc cttccttaca

2161 catgcattga gttagttgtg gcagtgctac tatttgctat ccatactgtc caaaaaagta

2221 ccttgtttct act (SEQ ID NO: 3)

The nucleotide sequence of the NP gene

1 agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact

61 ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt

121 tgcagggaag aacactgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct

181 gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg

241 aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa

301 catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc

361 caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata

421 caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga

481 acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact

541 aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat

601 ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctagacaaat

661 ggtgcaagcg atgagaacca ttgggactca tcctagctcc agtgctggtc tgaaaaatga

721 tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa

781 gtgatcctct cgctattgcc gcaaatatca ttgggatctt gcacttgaca ttgtggattc

841 ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc

901 cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg

961 ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt

1021 ttctact (SEQ ID NO: 4)

The nucleotide sequence of the M gene

1 agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact

61 ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt

121 tgcagggaag aacactgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct

181 gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg

241 aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa

301 catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc

361 caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata

421 caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga

481 acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact

541 aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat

601 ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctagacaaat

661 ggtgcaagcg atgagaacca ttgggactca tcctagctcc agtgctggtc tgaaaaatga

721 tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa

781 gtgatcctct cgctattgcc gcaaatatca ttgggatctt gcacttgaca ttgtggattc

841 ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc

901 cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg

961 ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt

1021 ttctact (SEQ ID NO: 5)

The nucleotide sequence of the HA gene

1 atgaaggcaa tactagtagt tctgctatat acatttgcaa ccgcaaatgc agacacatta

61 tgtataggtt atcatgcaaa caattcaaca gacactgtag acacagtact agaaaagaat

121 gtaacagtaa cacactctgt taaccttcta gaagacaagc ataacgggaa actatgcaaa

181 ctaagagggg tagccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga

241 aatccagagt gtgaatcact ctccacagca agttcatggt cctacattgt ggaaacatct

301 agttcagaca atggaacgtg ttacccagga gatttcatca attatgagga gctaagagag

361 caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaaaac aagttcatgg

421 cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcacgctgg agcaaaaagc

481 ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagccaa

541 tcctacatta atgataaagg gaaagaagtc ctcgtgctgt ggggcattca ccatccatct

601 actactgctg accaacaaag tctctatcag aatgcagatg catatgtttt tgtggggaca

661 tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcaa

721 gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa

781 gcaactggaa atctagtggt accgagatat gcattcacaa tggaaagaaa tgctggatct

841 ggtattatca tttcagatac accagtccac gattgcaata caacttgtca gacacccgag

901 ggtgctataa acaccagcct cccatttcag aatatacatc cgatcacaat tggaaaatgt

961 ccaaagtatg taaaaagcac aaaattgaga ctggccacag gattgaggaa tgtcccgtct

1021 attcaatcta gaggcctatt cggggccatt gccggcttca ttgaaggggg gtggacaggg

1081 atggtagatg gatggtacgg ttatcaccat caaaatgagc aggggtcagg atatgcagcc

1141 gacctgaaga gcacacaaaa tgccattgac aagattacta acaaagtaaa ctctgttatt

1201 gaaaagatga atacacagtt cacagcagtg ggtaaagagt tcaaccacct ggaaaaaaga

1261 atagagaatt taaataaaaa agttgatgat ggtttcctgg acatttggac ttacaatgcc

1321 gaactgttgg ttctattgga aaatgaaaga actttggact accatgattc aaatgtgaag

1381 aacttgtatg aaaaggtaag aaaccagtta aaaaacaatg ccaaggaaat tggaaacggc

1441 tgctttgaat tttaccacaa atgcgataac acgtgcatgg aaagtgtcaa aaatgggact

1501 tatgactacc caaaatactc agaggaagca aaattaaaca gagaaaaaat agatggggta

1561 aagctggaat caacaaggat ttaccagatt ttggcgatct attcaactgt cgccagttca

1621 ttggtgctgg tagtctccct gggggcaatc agcttctgga tgtgctctaa tgggtctcta

1681 cagtgtagaa tatgtattta a (SEQ ID NO: 6)

The nucleotide sequence of the NA gene

1 atgaatccaa accaaaagat aataaccatt ggttcggtct gtatgacaat tggaatggct

61 aacttaatat tacaaattgg aaacataatc tcaatatgga ttagccactc aattcaagtt

121 gggaatcaaa gtcagatcga aacatgcaat caaagcgtca ttacttatga aaacaacact

181 tgggtaaatc agacatatgt taacatcagc aacaccaact ttgctgctgg gcagccagtg

241 gtttccgtga aattagcggg caattcctct ctctgccctg ttagtggatg ggctatatac

301 agtaaagaca acagtgtaag agtcggttcc aagggggatg tgtttgtcat aagggaacca

361 ttcatatcat gctccccctt ggaatgcaga accttcttct tgactcaagg ggccttgcta

421 aatgacaaac attccaatgg aaccattaaa gacaggagcc catatcgaac cttaatgagc

481 tgtcctattg gtgaagttcc ctctccatac aactcaagat ttgagtcagt cgcttggtca

541 gcaagtgctt gtcatgatgg catcaattgg ctaacaattg gaatttctgg cccagacagt

601 ggggcagtgg ctgtgttaaa gtacaacggc ataataacag acactatcaa gagttggaga

661 aacgatatat tgagaacaca agagtctgaa tgtgcatgtg taaatggttc ttgctttacc

721 ataatgaccg atggaccaag tgatggacag gcctcataca agatcttcag aatagaaaag

781 ggaaagatag tcaaatcagt cgaaatgaat gcccctaatt atcactatga ggaatgctcc

841 tgttatcctg attctagtga aatcacatgt gtgtgcaggg ataactggca tggctcgaat

901 cgaccgtggg tgtctttcaa ccagaatctg gaatatcaga taggatacat atgcagtggg

961 attttcggag acaatccacg ccctaatgat aagacaggca gttgtggtcc agtatcgtct

1021 aatggagcaa atggagtaaa aggattttca ttcaaatacg gcaatggtgt ttggataggg

1081 agaactaaaa gcattagttc aagaaaaggt tttgagatga tttgggatcc aaatggatgg

1141 actgggacag acaataactt ctcaataaag caagatatcg taggaataaa tgagtggtca

1201 ggatatagcg ggagttttgt tcagcatcca gaactaacag ggctggattg tataagacct

1261 tgcttctggg ttgaactaat cagagggcga cccaaagaga acacaatctg gactagtggg

1321 agcagcatat ccttttgtgg tgtaaacagt gacactgtgg gttggtcttg gccagacggt

1381 gctgagttgc catttaccat tgacaagtaa (SEQ ID NO: 7)

The nucleotide sequence of the gene of the chimeric NS fragment (the insert is shown in bold)

AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGGGGCAGCACTCTTGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGCCTCTGTACCTGCGTCGCGTTACCTAACCGACATGACTCTTGAGGAAATGTCAAGGGAATGGTCCATGCTCATACCCAAGCAGAAAGTGGCAGGCCCTCTTTGTATCAGAATGGACCAGGCGATCATG GGAGGAGGTTTCTTCGGAGCTATTGCTGGTTTCTTGGAAGGAGGATGGGAAGGAATGATTGCAGGTTGGGGAGGAAGAGAGAGCCGGAACCCAGGGAATGCTTGATAATAAGCGGCCGC AGTGTGATTTTTGACCGGCTGGAGACTCTAATATTGCTAAGGGCTTTCACCGAAGAGGGAGCAATTGTTGGCGAAATTTCACCATTGCCTTCTCTTCCAGGACATACTAATGAGGATGTCAAAAATGCAATTGGGGTCCTCATCGGAGGACTTGAATGGAATGATAACACAGTTCGAGTCTCTAAAACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAGACCTCCACTCACTCCAAAACAGAAACGGAAAATGGCGAGAACAATTAGGTCAAAAGTTCGAAGAAATAAGATGGCTGATTGAAGAAGTGAGACACAAATTGAAGATAACAGAGAATAGTTTTGAGCAAATAACATTTATACAAGCCTTACAGCTACTATTTGAAGTGGAACAAGAGATAAGAACTTTCTCGTTTCAGCTTATTTAATAATAAAAAACACCCTTGTTTCTACT (SEQ ID NO: 8)

The recombinant viruses were assembled by transfection of VERO cells with eight plasmids encoding genomic unmodified fragments of the influenza virus and a chimeric NS genomic fragment using the method of electroporation of plasmid DNA (Cell Line Nucleofector® Kit V (Lonza)) in accordance with the instructions for use. After transfection, the cells were incubated in Optipro medium (Invitrogen) for 96 hours at 34 ° C with the addition of trypsin 1 μg / ml to ensure post-translational cleavage of the hemagglutinin precursor into HA1 and HA2 subunits. Vero cell viral harvest was used to infect 10-day-old chicken embryos (SPFs). Embryos were incubated for 48 hours at 34 ° C, after which allantoic fluids having a positive titer in the hemagglutination reaction were used for subsequent passages on chicken embryos. Allantoic fluids of passage 7 were purified by tangential filtration and lyophilized for storage. Immunization of animals was carried out after dissolving the lyophilisate with distilled water in an equivalent volume.

Example 2

A protective response against heterologous strains of influenza A and B virus in control mice infection

In order to determine the protective activity against heterologous antigenic variants of the influenza virus, an influenza vector of 6.8log EID ₅₀ / mouse was used to immunize mice, which was administered intranasally in a volume of 50 μl under mild anesthesia, once or twice with a period of 3 weeks. Twenty-one days after the last immunization, the animals were challenged with influenza pathogens for mice: homologous A / California / 7/09 (H1N1pdm) or heterologous A / Aichi / 2/68 (H3N2), A / Mississippi / 85/1 ( H3N2) or influenza virus B / Lee / 40 taken in a dose corresponding to 3-5 LD50, respectively.

As can be seen from Figa, the control infection of non-immune mice with the H1N1pdm virus led to their death in 90% of cases. At the same time, mice immunized once or twice with the viral preparation were reliably protected from death.

As can be seen from Figv, control infection of non-immune mice with A / Aichi / 2/68 virus (H3N2) led to their death in 100% of cases. At the same time, mice immunized once or twice with the viral preparation were significantly protected from death.

As can be seen from Figs, control infection of non-immune mice with A / Mississippi / 85/1 (H3N2) virus led to their death in 100% of cases. At the same time, mice immunized with the viral preparation twice had 100% protection.

As can be seen from Fig.3D, the control infection of non-immune mice with influenza virus / Lee / 40 led to the death of animals in 100% of cases. At the same time, mice immunized with a viral preparation twice had a significantly different protection from 60% protection.

Thus, an influenza vector carrying a chimeric NS genomic fragment showed signs of a universal influenza vaccine effective against both heterologous antigenic subtypes of influenza A virus and influenza B.

Example 3

A protective response against a heterologous strain of influenza A virus (H3N2) in control ferrets

Ferrets are the optimal WHO recommended model for studying the effectiveness of influenza vaccines and drugs. In order to determine the protective activity against the heterologous antigenic variant of the influenza virus, a 7.5 log EID ₅₀ / ferret vector was used for immunization of ferrets (9 per group), which was administered intranasally in a volume of 500 μl under mild anesthesia, once or twice with period of 3 weeks. Twenty-one days after the last immunization, the animals were challenged with the ferret pathogenic virus A / St. Petersburg / 224/2015 (H3N2). As can be seen from Figa, control infection of non-immune animals led to a rise in body temperature 2 days after infection, while vaccinated animals did not have a temperature reaction.

In order to study the effect of vaccination on the reproduction of the control virus in the respiratory tract of ferrets in animals, nasal swabs were taken on days 2,4 and 6 in order to determine the concentration of the infectious virus in them by titration of a 50% cytopathic dose in an MDCK cell culture. As can be seen from Figv, control infection of non-immune ferrets led to active reproduction of the virus without a significant reduction in titers by day 6. With a single immunization of ferrets, a significant decrease in viral titer was observed on days 4 and 6 after control infection. After double immunization, a reliable, more than 100-fold, decrease in viral titer was recorded already 2 days after infection of animals.

Thus, even a single vaccination of ferrets with the influenza vector led to the protection of animals from clinical manifestations in the form of a temperature reaction and contributed to the accelerated elimination of the control heterologous strain from the respiratory tract. Re-immunization accelerated the process of viral elimination.

Example 4

The composition of the vaccine based on influenza vector for intranasal immunization

A vaccine where the influenza vector is contained in an amount of 6.5 to 8.5 log of embryonic 50% infectious doses (EID50) / ml, and the buffer stabilizing solution contains 0.9 wt.% Sodium chloride solution, 0.5 wt.% L β-carnosine, 2.5 wt.% sucrose, 1 wt.% recombinant albumin, 1 wt.% L-arginine and 3 wt.% hydroxyethylated starch 130 / 0.4 (molecular weight 130 kDa, degree of molar substitution 0.4 )

Claims (28)

1. An attenuated influenza vector inducing a cross-protective response against influenza A and B viruses, containing: the nucleotide sequence of the PB2 protein gene derived from influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity to the nucleotide sequence of the PB2 protein gene; the nucleotide sequence of the PB1 protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity with the specified nucleotide sequence of the PB1 protein gene; the nucleotide sequence of the PA protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity to the specified sequence of the protein PA; the nucleotide sequence of the NP protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity with the specified sequence of the NP protein; the nucleotide sequence of the M protein gene derived from the influenza virus A / PR / 8/34 (H1N1), or the nucleotide sequence having at least 95% identity with the indicated sequence of the protein M gene gene; the nucleotide sequence of the HA protein gene derived from the A / California / 7/09-like influenza virus (H1N1pdm), or the nucleotide sequence having at least 95% identity to the nucleotide sequence of the HA protein gene; the nucleotide sequence of the NA protein gene derived from the A / California / 7/09-like influenza virus (H1N1pdm), or the nucleotide sequence having at least 95% identity with the nucleotide sequence of the NA protein gene; and the nucleotide sequence of the chimeric gene of the NS protein, including the reading frame of the NS1 protein, derived from the virus A / PR / 8/34 (H1N1), where the specified reading frame is shortened and encodes the NS1 protein of 124 amino acid residues, and a Nep protein gene sequence derived from influenza A / Singapore / 1/57 – like virus (H2N2), or a nucleotide sequence having at least 95% identity to the indicated sequence of the chimeric NS gene; where the indicated shortened reading frame of the NS1 protein gene is continued by the insertion of a nucleotide sequence encoding a fusion peptide of the HA2 subunit from influenza B virus and a nucleotide sequence encoding a conserved B cell epitope of influenza A virus nucleoprotein (NP). 2. The vector according to claim 1, where the nucleotide sequence of the chimeric gene of the NS protein is presented in SEQ ID NO: 8. 3. A pharmaceutical composition for the prevention of influenza A and B, containing in an effective amount an attenuated influenza vector according to claim 1 or 2 and a pharmaceutically acceptable carrier. 4. The pharmaceutical composition according to claim 3, containing 6.5-8.5 log EID 50 / ml attenuated influenza vector according to claim 1 or 2 and a buffer solution containing 0-1.5 wt.% Monovalent salt, 0-5 wt.% L-carnosine, 0-5 wt.% Carbohydrate component, 0-2 wt.% Protein component, 0-2 wt.% Amino acid component and 0-10 wt.% Hydroxyethylated starch. 5. The pharmaceutical composition according to claim 4, where the buffer solution contains 0.5-1.5 wt.% Monovalent salt, 0.01-5 wt.% L-carnosine, 1-5 wt.% Carbohydrate component, 0.1 -2 wt.% Protein component, 0.01-2 wt. % amino acid component and 1-10 wt.% hydroxyethylated starch. 6. The pharmaceutical composition according to claim 5, where the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose or lactose, the protein component is human recombinant albumin, casitone, lactalbumin hydrolyzate or gelatin, the amino acid component is arginine, glycine or monosodium glutamate. 7. The pharmaceutical composition according to any one of paragraphs. 3-6, where the subject is a mammal or bird. 8. The pharmaceutical composition according to claim 7, wherein the subject is a human. 9. A vaccine against influenza A and B, containing in an effective amount an attenuated influenza vector according to claim 1 or 2 and a pharmaceutically acceptable carrier. 10. The vaccine according to claim 9, containing 6.5-8.5 log EID 50 / ml attenuated influenza A virus according to any one of paragraphs. 1-5 or attenuated influenza vector according to any one of paragraphs. 6-14 and a buffer solution containing 0-1.5 wt.% Monovalent salt, 0-5 wt.% L-carnosine, 0-5 wt.% Carbohydrate component, 0-2 wt.% Protein component, 0-2 wt.% amino acid component and 0-10 wt.% hydroxyethylated starch. 11. The vaccine according to claim 10, where the buffer solution contains 0.5-1.5 wt.% Monovalent salt, 0.01-5 wt.% L-carnosine, 1-5 wt.% Carbohydrate component, 0.1- 2 wt.% Protein component, 0.01-2 wt. % amino acid component and 1-10 wt.% hydroxyethylated starch. 12. The vaccine of claim 11, wherein the monovalent salt is sodium chloride, the carbohydrate component is sucrose, trehalose or lactose, the protein component is human recombinant albumin, casitone, lactalbumin hydrolyzate or gelatin, the amino acid component is arginine, glycine or glutamate sodium. 13. The use of an attenuated influenza vector according to claim 1 or 2 for the prevention of influenza A and B in a subject. 14. The use of the pharmaceutical composition according to any one of paragraphs. 3-8 for the prevention of influenza A and B in a subject. 15. The use of claim 13 or 14, wherein the subject is a mammal or a bird. 16. The use of claim 13 or 14, wherein the subject is a human.

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