RU2680537C1 - Lentivirus plasmida (options), method for its obtaining (options), set of primers for obtaining lentivirus plasmid vector (options) - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: lentiviral vectors designed to carry the A (H1N1) and A (H3N2) influenza virus protein gene cassette and type B coding influenza virus protein sequences for transduction of mammalian cells. Method describes how to obtain these vectors and kits for their production.EFFECT: obtained lentiviral vectors are used further to create cell-producers of virus-like particles (HPV) of the influenza virus.15 cl, 6 tbl, 6 ex, 34 dwg

Description

The invention relates to biotechnology, and can be used to obtain lentiviral vectors in the form of plasmids carrying cassettes of influenza virus protein genes of subtypes A (H1N1), A (H3N2) and type B, coding sequences of influenza virus proteins, for transduction of mammalian cells and to create producer cells of virus-like particles (HPV) of influenza virus.

The inventive lentiviral plasmids are used for transduction into the genome of the target cell, namely, the MDCK cell culture, as having the highest productivity in comparison with other cell cultures. The criteria are two characteristics of the clones of the producers of HPV influenza virus - the level of antigen production and the growth rate of producer cells.

Human influenza is a highly contagious disease. The transmission of the virus occurs by airborne droplets, as well as by contact with objects contaminated with the pathogen (toys, door handles, etc.), followed by contact with the upper respiratory tract or conjunctival mucosa. Cell receptors, to which human influenza viruses are preferred, are expressed on epithelial cells throughout the respiratory tract: in the nasal mucosa, paranasal sinuses, pharynx, trachea, bronchi, bronchioles and alveoli, but their number is different in different areas [1].

The influenza virus has a negative genome consisting of 8 fragments of single-stranded virion RNA (vRNA) encoding at least 11 proteins. If all the fragments are arranged in decreasing molecular weight, the first three fragments encode proteins of the polymerase complex (PB2, PB1 and RA), the fourth - hemagglutinin (HA), the fifth - nucleoprotein (NP), the sixth - neuraminidase (NA), the seventh - matrix protein (Ml) and non-structural protein (M2), the eighth encodes two proteins (NS1 and NS2). The second fragment of PB1 encodes another protein PB1-F2, resulting from alternative splicing of mRNA, this protein causes apoptosis through a violation of the structure of mitochondria [2]. In the virion, all vRNA fragments are in the form of a ribonucleoprotein (vRNP) - vRNA associated with the NP protein and proteins of the polymerase complex [3]. The composition of the polymerase complex exhibiting the activity of an RNA-dependent RNA polymerase includes proteins PB1, PB2 and RA. Influenza virus (HBV) penetrates the cell by endocytosis of the virions, followed by transfer of all 8 fragments to the ribonucleoprotein (RNP), first into the cytoplasm and then into the nucleus of the infected cell [4, 5].

The interaction of vRNP, M1, HA, and NA plays an important role in the packaging process of the virus. Viral RNA segments contain unique packaging signals that partially overlap the reading frames in specific RNA segments, which allows coordination of the packaging of the viral genome. HA and NA are important structures for determining the shape of the virion, however, the mechanism of selective selection of vRNP fragments upon their packing into virions is still not known [6, 7]. The most important types of HBV antigens of types A and B, whose variability contributes to the emergence of new epidemics, are HA and NA. The first is responsible for the interaction of the virus with the surface of the cell and during the process of reproduction in the body causes the induction of neutralizing antibodies. Enzyme NA is antigenically different from HA. Antibodies to NA do not neutralize the infectivity of the virus (except for very high concentrations), but they strongly slow down the release of the virus from infected cells, and these antibodies can play an important role in reducing the replication of the virus in vivo and in preventing the spread of infection [8].

A distinctive feature of GV is the high variability of the genome, which allows it to evade specific immunity and, as a result, cause annual epidemics, less often pandemics when a virus with a new genotype appears in the population, to which the majority of the population does not have specific immunity [9]. One of the reasons for the high variability of the virus is associated with RNA-dependent RNA polymerase. This enzyme makes one mistake for every 10,000 nucleotides and, unlike DNA-dependent DNA polymerase, cannot correct them. If a single cell infected with hepatitis B produces about 10,000 new viral particles, then, in theory, 10,000 new viral mutants will be produced by this error rate. The consequence of this is the antigenic drift of the hepatitis B virus, due to which the surface proteins of the virus change so strongly in 3-4 years that specific immunity to the previous variants cannot protect a person from infection with new mutant strains [10]. In this case, they speak of antigenic drift of the VG. The presence of a segmented genome is the cause of another type of antigenic variability of type A HB - antigenic shift - the appearance of viruses with a new set of genes. This becomes possible as a result of the exchange of RNA segments between genetically different hepatitis B viruses upon infection of a single cell by them [11]. Currently, most researchers share the view that pandemic HBs result from the exchange of genes between human and animal type A HBs. This hypothesis was convincingly confirmed in 2009, when molecular genetic analysis showed that the new pandemic strain of HB type A (H1N1) pdm 09 is a triple reassortant carrying the HB gene of birds, humans and pigs [12]. However, it remains unclear why, out of the 16 subtypes of HA and 9 subtypes of NA that are common in avian VH, human hepatitis V contain only three HA subtypes and two NA subtypes. So, the Spanish Spaniard pandemic of 1918 was caused by AH type H (H1N1), the Asian flu pandemic of 1957 by the A (H2N2) virus, the 1968 Hong Kong flu pandemic by the A (H3N2) virus. The cause of the following two pandemics - the “Russian” flu in 1978 and the “swine” flu in 2009 - was again virus A (H1N1), although the viruses and the Spanish flu virus of 1918 differ significantly in molecular biological and antigenic characteristics. from friend.

HA is the main surface GH glycoprotein. It initiates the infection by binding to the cellular receptor, causing the fusion of the viral and endosomal membranes, necessary for the release of RNP into the cytoplasm of the infected cell. The receptor specificity of HA is an important factor in determining the range of hosts. The role of the GH receptor on the host cells is performed by sialic acids. Sialic acid is an N-acetyl derivative of neuraminic acid, or amino sugar, attached to many different proteins on the surface of a cell. Sialic acid is always the last residue in the sugar chain that attaches to a protein; penultimate is galactose. Human HBV is predominantly associated with sialic acid, which is associated with galactose by an a2-6 bond [13]. These receptors are abundant on epithelial cells in the human trachea.

In contrast, avian HBs primarily recognize sialic acids associated with galactose A2-3 bonds, which are common on epithelial cells in the intestines of waterfowl (the main replication site for avian birds). Apparently, the lack of receptors for hepatitis B viruses in the human upper respiratory tract may be one of the factors preventing the productive transmission of hepatitis B viruses from person to person.

Proteolytic activation of HA is a determining factor in the pathogenicity of HBV A [14]. On low pathogenic viruses have a cleavage site, usually being agrigine, but sometimes lysine [15]. The molecule is cut using trypsin-like proteases, which are present only in the epithelial tissues of the respiratory tract and intestinal tract. Highly pathogenic hepatitis B viruses have a cleavage site consisting of 4 amino acid residues; the HA molecule is cut using furin, a member of the serine-endoprotease family [16]. The ubiquity of this enzyme in the cells explains the ability of the hepatitis B virus to replicate in various tissues.

From 3 to 5 million people get the flu every year in the world. Influenza epidemics occur with a frequency of 1-3 years, but epidemic outbreaks occur annually and cause great damage to the health of the population, leading to huge financial costs for the treatment and rehabilitation of patients, especially among high-risk groups.

Influenza A (H1N1) pandemic in the 1918-1920s ("Spaniard"). The pandemic, called the “Spanish disease”, began at the end of the First World War - in 1918, it was not possible to determine exactly where it appeared, but, in any case, Spain was not the primary focus of the epidemic. The name "Spaniard" appeared by chance. Since military censorship of both struggling parties did not allow reports of an epidemic that had begun in the army and among the population, the first news of it appeared in print in May-June 1918 in neutral Spain.

The first wave of the pandemic was relatively mild, the severity increased in the next two, and the mortality rate reached 3% (during normal outbreaks it is less than 0.1%). The main cause of death was pneumonia, often complicated by a bacterial infection [17].

A pandemic circled the globe three times. The first wave began in late April 1918 in Paris and broke out almost immediately across Europe. In May, the Spaniard appeared in northern Africa, and in June - in India. This temporarily ended the further development of the epidemic. The number of cases of the disease, which reached quite large numbers with very low mortality, began to decrease. In August, the number of cases fell sharply, which was taken as the end of the pandemic.

After several months of calm, the second wave of the pandemic came, during which the incidence was lower, but the mortality rate was higher. First, the disease appeared on the west coast of Africa in Sierra Leone in late August 1918, from where the disease spread to the entire west coast of Africa. In North America, the second wave began in October 1918 in Boston, where, as it was believed then, it was brought by soldiers returning from Europe. By the end of the second wave of populated areas, only Madagascar, Australia and New Caledonia remained untouched, apparently due to rational and strictly implemented preventive measures. The end of the second wave is attributed to the end of December 1918.

The third wave of the pandemic began in February - March 1919, capturing this time the island territories that had survived until then. This outbreak was also characterized by high mortality. It did not end in different places at the same time, and in some areas it was recorded until June and even until August 1919. The pandemic completely stopped only in 1920. It was impossible to determine the exact number of people who had the flu. Apparently, no less than 550 million people were sick, and, according to various estimates, from 20 to 50 million people died - more than 2.5% of the total population of the planet at that time, which amounted to 1850 million people [17].

Hong Kong influenza pandemic A (H3N2) (1968-1970). This pandemic developed in three waves (1968, 1969 and 1970) and was caused by the new serotype A virus (H3N2). The disease appeared in Singapore in early August 1968 and by September had already spread to other neighboring countries, in particular to India and northern Australia, but in most of these areas it was clinically mild. In the fall of 1968, a new virus reached Europe, but even before the start of winter, not a single large outbreak was recorded there. The most dangerous was the second wave of the pandemic. The third wave of the pandemic made itself felt at the end of 1970. Then the epidemic waves plummeted. In terms of mortality, the Hong Kong flu pandemic, like the previous one, was relatively mild [17].

Pandemic of Russian influenza A (H1N1) (1977-1978). In May 1977, the A (H1N1) virus again caused an outbreak in the region of the Russian-Chinese border, which reached North America in 1978. The virus was close to the isolates of the 1950s in terms of antigenic and molecular biological characteristics. [18, 19]. This pandemic was unusual. In early 1978, serotype (H3N2) viruses caused epidemics in Western Europe, Africa and Asia, and the serotype A (H1N1) influenza virus settled on the American continent (USA and Canada), but by March in many countries, including in the USSR, strains of influenza A (H1N1), A (H3N2) and influenza B viruses were isolated simultaneously.

The vast majority of cases in the epidemic of 1977-1978. accounted for people under the age of 20 years, i.e. to that part of the population that did not have contact with serotype influenza viruses (H1N1) that disappeared from circulation more than 20 years ago. In contrast, people over 30 made up only 20% of patients, although their share in the total population exceeded 50%, i.e., given the low incidence in this epidemic in general, people of mature and old age who had previous contact with H1N1 flu viruses practically did not hurt.

The first pandemic in the XXI century. It began, as before, unexpectedly, although the whole decade has been preparing for it. Two samples, independently collected in Southern California in mid-April, at the Centers for Disease Control (CDC), Atlanta, USA, identified swine-derived influenza virus, originally called S-OIV (Swine-Origin Influenza Virus, which was subsequently proposed by WHO named A (H1N1) pdm09). The virus has spread around the planet with such speed that on June 11, 2009, WHO raised the level of preparedness for an influenza pandemic to phase 6, officially announcing the first influenza pandemic in the 21st century. [twenty]. By that time, there were 30,000 confirmed cases in 74 countries. In less than a month, WHO has confirmed more than 77,000 cases.

The antigenic pandemic virus A (H1N1) pdm09 is similar to the classic swine virus and the triple reassorted swine virus A (H1N1), which has the genes of bird and human viruses and has been circulating in the United States for more than 10 years. At the same time, there was practically no serological intersection of the pandemic virus with seasonal influenza A (H1N1) virus. A characteristic feature of the 2009 pandemic was the disproportionate defeat of children and young people compared with older age groups [78; 79]. Probably, this age distribution is associated with the presence of immunity to this virus in the elderly population [82]. Thus, it has been shown that in the USA 33% of people over 60 have antibodies that cross-react with A (H1N1) pdm09 in RTGA and neutralization reactions [21].

The most important measure of protection against influenza infection and limiting its spread is vaccination. Modern influenza vaccines induce, as a rule, the formation of antibodies to the surface antigens of the influenza virus - hemagglutinin and neuraminidase.

Existing influenza vaccines can be divided into two types: attenuated (live) and inactivated, including subunit ones. All of them are widely used in vaccination of the population and have proven themselves well. Attenuated vaccines are influenza viruses with reduced virulence. In the preparation of inactivated subunit vaccines, epidemiologically relevant strains of the virus are also used, although the use of highly pathogenic strains is limited by high biological safety requirements. Traditional methods for producing vaccine strains of influenza A virus have several disadvantages.

The use of both attenuation based on the adaptation of viruses to the organism of a heterologous host and reassortment during coinfection with epidemic strains and attenuation donors does not always allow maintaining a balance between the level of virulence of the original virus and its immunogenicity. Excessive attenuation can lead to strains that have lost the ability to reproduce in human respiratory tract cells.

An alternative way to obtain vaccine strains is to use reverse genetics methods. Reverse genetics allows you to recreate a biologically active viral particle by co-infection of permissive cell lines with plasmids containing genes encoding viral proteins. By making changes to these genes, you can change the virulence and antigenic properties of the influenza virus. Using reverse genetics methods, reassortants of influenza viruses can be obtained. For example, permucid eukaryotic cells are transfected with plasmids encoding segments of the genome of a pandemic or circulating seasonal strain and an attenuated vaccine strain of influenza A virus. As a result, complete virus virions are assembled that carry a set of proteins of both the vaccine and pathogenic strains. Using this method, it was possible to obtain and study the influenza A virus (H1N1), which caused a pandemic in 1918 (the “Spanish flu”).

Reverse genetics methods can reduce the virulence of a virus by introducing mutations into various viral genes. For example, mutations in two genes encoding the polymerase proteins PB1 and PB2 of the avian influenza virus A / guinea fowl / Hong Kong / WF 10199 (H9N2) led to the loss of the pathogenicity of the virus for chickens. The deletion of the non-structural protein NS1 ensured the attenuation of influenza A virus. The vaccine obtained in this way successfully passed the first phase of clinical trials. The introduction of mutations into the M2 protein, necessary for the formation of the ion channel, also leads to attenuation of the virus. A change in the amino acid sequence at the site of cutting the highly pathogenic H5 influenza A virus using targeted mutagenesis led to its acquisition of characteristics of low pathogenic viruses.

Reverse genetics methods have proven themselves in obtaining attenuated strains of influenza viruses. However, the use of reassortment in the case of vaccine strains raises the issue of biosafety in connection with the possibility of mutations that restore or increase the virulence of the virus. In addition, the widespread use of live attenuated influenza vaccines is of concern because of the possible reassortment of live vaccines with circulating strains of human influenza viruses. In preparative quantities, vaccine strains of the influenza virus are most often obtained in chicken embryos, which makes it impossible to vaccinate persons allergic to chicken protein. The dependence of the technological process on the productivity of the chicken herd also refers to the disadvantages of vaccines obtained using chicken embryos.

Solving the problems associated with the use of chicken embryos and the need to attenuate pathogenic strains of the influenza virus can be done using recombinant subunit vaccines. One of the new approaches to obtaining subunit vaccines against influenza viruses is the use of various expression systems for the rapid production of individual viral proteins in preparative quantities [22].

In one popular expression system, influenza antigens are produced in insect cells using baculovirus vectors carrying the genes of the target antigens. The most widely used virus is multiple nuclear polyhedrosis of the California scoop (AcMNPV). To work with AcMNPV, Sf9 cell lines obtained from the ovary of Spodoptera frugiperda caterpillars are usually used. Various influenza A antigens can be produced in this system. Immunization of mice with recombinant H5N1 influenza virus AN obtained in a baculovirus expression system resulted in the induction of a high level of neutralizing antibodies. However, to achieve significant antibody levels, either an adjuvant or prime boost immunization with an inactivated H5N1 influenza virus or recombinant adenovirus carrying the influenza HA gene was required.

The disadvantage of recombinant subunit vaccines, as well as traditional subunit vaccines, is their low immunogenicity and, as a consequence, the need for multiple vaccinations and the use of adjuvants.

Virus-like particles (HPVs) are antigenic determinants of virions without fragments of genomic RNA. Due to the lack of genetic material, HPVs are not able to infect human and animal cells, which ensures their safety. The surface proteins of influenza HPV can present conformational epitopes to the cells of the immune system as native virions. A number of studies have shown that the participation of the internal protein of the influenza virus, M1, plays a key role in the formation of influenza HPV. This protein binds to the lipid portion of the apical domain of the plasma membrane, interacts with the surface glycoproteins of the influenza virus and initiates the assembly and budding of the HPV containing the lipid membrane of the host cell with the three transmembrane proteins of the influenza virus incorporated into it.

Influenza HPV obtained in various expression systems. For the efficient release of influenza HPV containing HA from mammalian cells, either simultaneous expression of NA or the addition of exogenous NA is necessary. This is due to the ability of active NA to break down sialic acids on the surface of the cell membrane. In insect cells, influenza HPV containing HA can be obtained even in the absence of NA expression, since in these cells sialic acids do not bind to N-glycans during post-translational modification.

One approach to producing influenza HPV in insect cells involves the use of recombinant baculoviruses. In animal models, it was shown that surface influenza antigens in the HPV composition obtained using recombinant baculoviruses induced the production of both anti-hemagglutinating and virus-neutralizing antibodies and effectors of the cellular immune response. In addition, the influenza HPV vaccine induced protective immunity against homologous and heterologous strains of influenza A virus. The HPV vaccine carrying the pandemic influenza A H1N1 antigens (2009) passed the second phase of clinical trials in 4,563 healthy adult volunteers and showed safety and immunogenicity [23].

The use of recombinant baculoviruses for the expression of influenza virus proteins in insect cells leads to the accumulation of not only HPV, but also baculoviruses in the culture fluid. Since these structures are close in size, there are problems with the purification of HPV from baculovirus particles. Influenza HPV can be obtained in mammalian cells using other DNA and RNA virus vectors. For example, a system was developed for the production of influenza HPV in Vero cells using DNA vectors carrying the HA, NA, M1 and M2 proteins of the influenza virus. The use of the modified Ankara vaccinia virus for the production of HPV containing H5N1 influenza virus proteins (HA, NA, Ml) in mammalian cells is described. Such HPVs are able to form a protective immune response in mice.

Thus, obtaining HPV is a promising area in the development of a new type of influenza vaccine.

Viral vectors are recombinant viruses in the genome of which a target gene with a set of regulatory elements is embedded. Among the existing systems for the delivery of antigens, viral vectors occupy a special place because they have the following properties: they have a natural mechanism of interaction with the cell and penetration into it; transfer foreign genetic material to the cell nucleus; able to provide long-term antigen expression; the viral envelope protects the genetic material encoding the antigen.

Viral vector vaccines effectively activate cytotoxic T-lymphocytes, which is especially important when vaccinating against intracellular pathogens. Such vaccines can realize a wide spectrum of action due to the induction of a T-cell response to conservative epitopes that are potentially able to provide protection against various strains of pathogens (including influenza virus).

Viral vectors have the ability to activate innate immunity by binding genetic material or their envelope proteins to pattern recognition receptors (TLR, RIG-1, etc.). Viral vectors are recognized by TLRs such as TLR2, TLR3, TLR4, TLR7, TLR8, TLR9.

During the interaction of these receptors with ligands, various transcription factors are activated, which leads to the formation of a focus of inflammation and the rapid activation of the body's defense reactions.

When choosing a viral vector for genetic immunization, the following criteria should be followed: such a vaccine should not cause symptoms of the disease, it should be safe for people with weakened immune systems, the elderly and children; own recombinant virus proteins should not cause a strong immune response; the viral vector should be simple for genetic manipulation and allow large fragments of foreign DNA to be included; the resulting preparations should have a high titer and provide a high level of expression of the target antigens; upon immunization, the DNA of the viral vector must not integrate into the genome of the host cell, and the vector itself must be completely excreted after the induction of the immune response. In addition, the presence of a pre-existing immune response to the proteins of the viral vector in immunized individuals is undesirable, since it can significantly reduce the level of the immune response to the target antigen.

Not all viruses possess the properties necessary to create effective vectors. To create influenza vaccines based on viral vectors, poxviruses, Newcastle disease virus and adenoviruses are currently the most widely used.

Poxviridae are DNA viruses with a large genome. Among the poxviruses used as a viral vector, the vaccinia virus is most popular, the advantages of which include the simplicity and low cost of production, as well as its high packing capacity (up to 25 kb). For vaccines, attenuated vaccinia viruses are used, such as the modified Ankara vaccinia virus (MVA) and the attenuated NYVAC strain derived from the Copenhagen strain. Attenuation of MVA was achieved by repeated passage in chicken embryo fibroblasts, which led to the loss of a number of genes that were not essential for replication in bird cells and to a decrease in reproduction in human cells. The attenuation of the NYVAC strain was achieved by deletion of 18 genes, as a result of which the virus became replicatively defective for human cells.

A serious drawback of vaccinia virus-based vectors is the preexisting immunity to this virus that has formed in the human population as a result of immunization against smallpox. Therefore, it is advisable to use vectors based on poxviruses such as canary pox virus (Sapaguroch) and poultry pox virus (Flowpox), to which there are no pre-existing antibodies in the human population. Immunization of chickens and ducks with the recombinant Flowpox-BHrusoM, in the genome of which the HA gene of the bird influenza A virus was introduced, protected the birds from infection with lethal doses of homologous influenza viruses. The high packing capacity of poxviruses allows several transgenes to be introduced into the genome, for example, the HA and NP genes of influenza A. However, Sapaguroch and Flowpox induce a weaker immune response to target antigens than the vaccinia virus and require repeated administration or use of adjuvants.

Newcastle disease virus (NDV) belongs to the Paramyxoviridae family. This is a virus with a non-segmented single-stranded RNA gene that contains six genes encoding seven proteins: NP, P and V proteins, M protein, fusion protein, or F protein, HA-NA and large polymerase protein L. Since the expression level of each viral protein decreases in the direction from the 3'- to the 5th end of the genome; when using NDV as a vector, the expression level of a foreign gene can be controlled by its position in the viral genome. The level of virulence and tropism of NDV depend on the site of cutting the protease F-protein, necessary for the fusion of the viral membrane and cell membrane.

NDV in nature only infects birds, therefore, there are no antibodies to this virus in humans. Therefore, the problem of a preexisting immune response for this viral vector is not worth it. However, a significant drawback of the NDV vaccine vector is that the effects of the introduction of recombinant NDV are not well understood, and it is not clear whether influenza vaccines based on NDV are safe for humans. In addition, NDV is characterized by low packing capacity and the difficulty of obtaining vectors carrying several target antigens. Preparative amounts of NDV are obtained in chicken embryos, which, as noted above, has several disadvantages.

Recombinant adenoviruses (Adenoviridae) are the best-studied and most commonly used recombinant viral vectors. Adenovirus virions are composed of a double-stranded DNA molecule surrounded by a protein capsid. Many types of adenoviruses are characterized in detail, including at the genetic level. The nucleotide sequence of the genomes of most of them is completely deciphered. Detailed data on the structure, physicochemical and biological properties of adenoviruses make it possible to use them to create recombinant vaccines and gene therapeutic drugs. Of the genetic vaccines undergoing clinical trials, recombinant adenovirus vaccines account for about 24%.

Adenoviruses possess such important properties for vaccine vectors as the ability to provide a high level of expression of the target transgene in the target cell and to transduce both dividing and postmitotic cells. In this case, the adenovirus DNA remains in an extrachromosomal form. Adenoviruses are able to accumulate in cell culture in high titers. The process of obtaining a new recombinant adenovirus takes several weeks, which may allow you to respond to changing epidemiological conditions in the shortest possible time.

Currently, vaccines based on recombinant adenoviruses against various serotypes of influenza A virus are being developed in various countries. One of these vaccines has successfully passed the first stage of clinical trials in the USA, proved to be safe for humans and highly immunogenic with respect to influenza A H5N2 virus.

The problem of creating influenza vaccines that form a heterosubtypic immunity that can protect against various variants of the influenza virus is relevant, and in recent years new approaches have appeared to solve it. Conformational conformational epitopes of HA were found to be conservative for various subtypes of the influenza A virus, on which antibodies of a wide spectrum of action can be produced both after the infection and during immunization with live vaccines. Immunization with recombinant adenoviral vectors imitates infection of the cells of the mucous membrane of the upper respiratory tract, providing expression of antigens with a native tertiary structure, which allows the formation of such cross-antibodies. Recombinant adenovirus vaccines are also able to form a strong T-cell immune response, which is characterized by a wider than humoral spectrum of action.

Lentiviruses induce a wide variety of pathologies in various animal species. A common feature of these viruses is the ability to infect non-dividing and differentiated cells - an extremely useful property for gene therapy. The name lentiviruses (lat. Lenti = slow) is associated with a long period of time between the infected and the end of the disease, which can last several months or even years. Viruses belonging to the genus Lentivirus are found in primates, ungulates and cats.

In general, various types of cells are targets of lentiviruses, which suggests the potential widespread use of these viruses: from anti-cancer strategies to vaccination and antiviral immunity. Since lentiviruses act on non-dividing cells, they must have a mechanism for overcoming the intact nuclear membrane to access the cellular genome. Among retroviruses, lentiviruses have acquired the most effective mechanism for achieving this goal, and this property is used in gene therapy [24].

The life cycle of the virus is divided into two main phases: the first is the transfer of the viral genome into the host cell, and the second is its expression and reproduction of the virus. Of primary interest for gene therapy purposes is the first phase, together with the early stages of the viral life cycle. Such a process is defined in terms of gene transduction (in gene therapy) or single-round infection (in virology). In the case of retroviruses, infection begins after the binding of a specific cell receptor to viral envelope proteins, and ends with the integration of the synthesized DNA into the genome of the host cell. The binding of the cell receptor and the viral Env protein triggers the fusion of the cell membrane and the shell of the virus particle, as a result, the viral nucleoprotein complex (the viral genome in combination with viral and cellular proteins) enters the cytoplasm of the target cell. The problem of infection of non-dividing cells is to cross the nuclear membrane for access of the viral genome to the host DNA. This phase, key for the purpose of gene therapy, is called nuclear imports. In viral infections, the nuclear membrane is an additional obstacle that viruses overcome either by moving during cell division or by overcoming nuclear pores. The latter option is the only one in the case of non-dividing cells, however, the mechanism is still not clear [24].

Despite the fact that most retroviruses are able to infect non-dividing cells, it is lentiviruses that are most effective for nuclear import [24].

The development of lentiviral vectors is based on the tendency to optimize the efficiency of gene transfer and, at the same time, to reduce the potential danger associated with the use of retroviral vectors. During optimization, the third and fourth generations of vectors were developed, which are characterized by a minimal risk of formation of replicable recombinants. And the introduction of SIN mutations not only reduces the likelihood of the formation of such recombinants, but also reduces the non-specific activity of enhancers of the viral promoter [24].

Further efforts to optimize the design of lentiviral vectors are aimed at improving the integration of the target gene into the genome of the target cell. Retroviruses have an unusual ability to place heterologous envelope proteins on their envelope, called pseudotyping, as a result of which viral particles can acquire new cell tropism and intracellular behavior. Pseudotyping is characteristic of all retroviruses, but the most interesting is the pseudotyping of lentiviruses capable of transducing non-dividing and differentiated cells [25]. Lentiviral pseudotyped vectors are produced on a mammalian cell culture by co-transfection with packaging and vector plasmids proper.

Another property of pseudotyped lentiviral vectors is the ability to influence the intracellular behavior of the virus. To achieve effective effects on cells in the absence of major activation signals, a number of strategies have been developed based on artificially constructed membranes other than VSVg. A common feature of these strategies is the use of special molecules located on the surface of a viral particle and triggering an activation signal upon binding to a cellular receptor. The signal temporarily activates the cell and allows it to be infected. This strategy has been successfully applied for transduction of resting B and T cells using enveloped measles virus proteins and lentiviruses with cytokines on the surface [26, 27].

An example of the successful use of lentiviral vectors for retrograde VEGF transport in model mice with amyotrophic lateral sclerosis (ALS, Charcot's disease) is described [28]. Charcot's disease is a disease characterized by progressive degeneration of motor neurons in the brain and spinal cord. In the treatment of this disease, one of the most important difficulties is the delivery of the therapeutic agent to motor neurons, for which a lentiviral system for delivering the gene directly to neutrons due to the tropism of rabies virus envelope proteins has been developed. In the study in question, a vector based on equine infectious anemia virus (EIAV) was created, pseudotyped by rabies virus G protein. Model mice with neurodegeneration were injected with a lentiviral vector preparation that provides VEGF expression in various muscle tissues before and during the progressive degeneration of neurons. According to the test results, expression of the reporter gene was recorded in the spinal cord and brain, and a noticeable extension of the development of the disease was established for both experimental groups. Thus, it was shown that lentiviral vectors based on EIAV, pseudotyped by envelope glycoproteins of G rabies virus, are an effective delivery system genes for motoneurons with intramuscular injection. The study also showed the therapeutic effect of VEGF on model mice: even at the stage of death of half of the motor neurons, corresponding to the usual diagnosis, the use of the viral vector for VEGF transfer provided not only an extension of the life of experimental animals, but a partial restoration of function. Microscopic and histological studies did not reveal any pathologies of the spinal cord and brain after the injection of the drug, including abnormal tissue vascularization. In addition, the immune response to the delivery system was negligible even five weeks after the injection.

Thus, discoveries and research in the field of virology allow us to expand the use of technology of lentiviral pseudotyped vectors to obtain a wide variety of producer cells of therapeutic drugs and, in particular, for the purposes of gene therapy.

Recently, the use of non-infectious virus-like particles (HPVs), which spontaneously assemble themselves due to the interaction of viral structural proteins, has created a good prospect for improving vaccines against a wide range of viruses that cause diseases in humans [29]. Influenza HPV expressed by recombinant baculoviruses, which are multicomponent antigens including hemagglutinin and matrix protein M1, with or without neuraminidase, can induce an immune response against homologous or heterologous strains of the influenza virus, which is described in detail [30, 31, 32, 33, 34, 35, 36]. In addition, the reverse genetics method licensed in Europe for the development of seasonal influenza vaccines uses a mammalian cell culture system rather than RCE [37]. The use of mammalian cell cultures, such as Vero or MDCK, adaptive hosts for vaccine viruses, has several advantages, not only due to the increased flexibility and consistency of the production process, but also due to the possibility of glycosylation of viral antigens in their natural form, which cannot be achieved in RCE or baculovirus systems. In eukaryotic cells, protein glycosylation is involved in the proper assembly and orientation of newly translated proteins, which plays an important role in protein function. The generation of HPV influenza in mammalian cells was previously obtained as a result of transient co-expression of hemagglutinin and neuraminidase in 293 T cells of humans [38]. In order to improve production characteristics, not only hemagglutinin and neuraminidase proteins, but also matrix proteins M1 and M2 were included in the composition of the HPV flu, since they play an important role in the assembly of viral particles and their budding [39, 40, 41, 42, 43, 44] . The inclusion of M1 and M2 proteins in influenza HPV produced in mammalian cells not only increases the yield of HPV production, but also complements the antigenic portrait of HPV with antigens, with conserved T and B cell epitopes, which allows inducing immunity not only against homologous, but also heterologous viruses [45, 46].

The closest analogue to the claimed group of inventions is the development of the American company Lentigen Corporation, which constructed a non-integrating non-replicating lentiviral vector (patent application of Russia No. 2012140691, publ. 03/27/2014). In this vector, structural proteins themselves assemble into a virus-like particle (VLP) when polynucleotide sequences are expressed in a vector-transduced cell. The vector has the heterologous env gene of the influenza A, B virus, Ebola virus and Marburg. HPV contains the proteins of the above viruses (see also US 20120135034).

The objects of the present invention are lentiviral plasmid vectors carrying cassettes of the hemagglutinin, neuraminidase and influenza virus M1 protein genes, and expressing HA proteins of the influenza virus subtypes A (H1N1), A (H3N2) and type B, NA (H1N1) and M1 of the influenza virus subtype A (H1N1). Moreover, the hemagglutinin gene of the influenza virus subtype AH1N1, AH3N2 or B is the gene of the strains recommended by WHO for the corresponding epidemic season.

Preferably, the present invention relates to a lentiviral plasmid vector pLenti6.3 / TO / HA-M1-NA containing the attL1 SalI gene cassette of PstI AgeI IRES Ml XmaI WPRE CMV / TO EcoRI NA MluI XmaI attL2, where

attL1 and attL2 are the necessary sites in the vector plasmid pEntry for transfer of genetic material to the vector plasmid pLenti6.3 / TO / V5-DEST at the attR1 and attR2 sites using LR recombination;

IRES - site of the internal landing of the ribosome;

WPRE - Woodchuck Posttranscriptional Regulatory Element, a regulatory element for increasing the export of mRNA from the nucleus and enhancing transgenic expression;

CMV / TO - a hybrid promoter consisting of a cytomegalovirus promoter and two tandem tetracycline operators, for regulated highly efficient expression of target genes;

SalI / PstI - recognition sites of the corresponding restriction endonucleases to replace HA in the gene cassette, if necessary;

EcoRI / MluI - recognition sites of the corresponding restriction endonucleases to replace the NA in the gene cassette, if necessary;

XmaI / XmaI - recognition sites of the corresponding restriction endonucleases to remove NA from the gene cassette, if necessary;

AgeI / XmaI - recognition sites of the corresponding restriction endonucleases to remove M1 and NA from the gene cassette, if necessary;

HA - hemagglutinin of influenza virus with Kozak sequence and stop codons;

M1 - matrix protein of influenza virus with a Kozak sequence and stop codons;

NA - neuraminidase of influenza virus with the Kozak sequence and stop codons, intended for delivery of the indicated cassette to the producer cell.

Most preferably, the present invention relates to a lentiviral plasmid vector pLenti6.3 / TO / HA (H1N1) -M1-NA containing a cassette of influenza virus hemagglutinin genes of strain A / California / 04/2009 (H1N1) or A / Michigan / 45/2015 ( H1N1), neuraminidase of strain A / California / 04/2009 (H1N1) and M1 protein of strain A / California / 04/2009 (H1N1), intended for integration with the producer cell gene shown in FIG. 3 - Lentiviral expression vector of the gene cassette pLenti6.3 / TO / HA (H1N1) -M1-NA carrying the cassette of the hemagglutinin genes of strain A (H1N1), neuraminidase of strain A (H1N1) and M1 of protein of strain A (H1N1) or FIG. 34 - Map of the recombinant plasmid DNA pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA.

The main genetic elements contained in the structure of plasmid DNA:

IRES - site of the internal landing of the ribosome;

WPRE - Woodchuck Posttranscriptional Regulatory Element, a regulatory element for increasing the export of mRNA from the nucleus and enhancing transgenic expression;

CMV / TO - a hybrid promoter consisting of a cytomegalovirus promoter and two tandem tetracycline operators, for regulated highly efficient expression of target genes;

HA (H1N1-Mich) - hemagglutinin gene of strain A / Michigan / 45/2015;

NA — neuraminidase gene of strain A / California / 04/2009 (H1N1);

M1 - gene M1 of strain A / California / 04/2009 (H1N1) or to the lentiviral plasmid vector pLenti6.3 / TO / HA (H3N2) -M1-NA containing the cloned hemagglutinin gene from strain A / Novosibirsk / 01/2014 or A / HongKong / 4801/2014, as well as the genes for neuraminidase and M1 protein of strain A / California / 04/2009 (H1N1), intended for integration with the producer cell gene shown in FIG. 2 - Lentiviral vector plasmid carrying the cassette of hemagglutinin genes of strain A (H3N2), neuraminidase of strain A (H1N1) and M1 protein of strain A (H1N1)

or FIG. 32 Map recombinant plasmid DNA

pLenti6.3 / TO / HA (A / HongKong) -M1-NA.

The main genetic elements contained in the structure of plasmid DNA:

IRES - site of the internal landing of the ribosome;

WPRE - Woodchuck Posttranscriptional Regulatory Element, a regulatory element for increasing the export of mRNA from the nucleus and enhancing transgenic expression;

CMV / TO - a hybrid promoter consisting of a cytomegalovirus promoter and two tandem tetracycline operators, for regulated highly efficient expression of target genes;

HA (A-HongKong) - hemagglutinin gene of strain A / Hong Kong / 4801/2014 (H3N2);

NA — neuraminidase gene of strain A / California / 04/2009 (H1N1);

M1 - M1 gene of strain A / California / 04/2009 (H1N1)

to the lentiviral plasmid vector pLenti6.3 / TO / HA (B) -M1-NA containing the hemagglutinin gene of influenza B strain B / Phuket / 3073/13 or B / Brisbane / 60/2008, as well as the genes for neuraminidase and Ml protein of the strain A / California / 04/2009 (H1N1), intended for integration with the producer cell gene of FIG. 1 - Lentiviral vector plasmid carrying a cassette of hemagglutinin genes of strain B, neuraminidase of strain A (H1N1) and M1 protein of strain A (H1N1) or FIG. 33 - The result of electrophoretic separation in 1.5% agarose gel of DNA fragments obtained after recombinant hydrolysis

plasmids pLenti6.3 / TO / HA (H1H1-Mich) -M1-NA, restriction endonucleases XhoI, EcoRI, PstI and HindIII (the lengths of the resulting fragments are indicated in parentheses).

1: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, hydrolyzed by XhoI (13496 bp);

2: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, hydrolyzed by EcoRI (9091 bp + 4405 bp);

3: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, hydrolyzed with HindIII (5580 bp + 3344 bp + 2639 bp + 584 bp + 556 bp N. + 481 bp + 312 bp);

4: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, hydrolyzed by PstI (11686 bp + 1810 bp). (Recombinant map of plasmid DNA pLenti6.3 / TO / HA (B-Brisbane) -M1-NA.

The main genetic elements contained in the structure of plasmid DNA:

IRES - site of the internal landing of the ribosome;

WPRE - Woodchuck Posttranscriptional Regulatory Element, a regulatory element for increasing the export of mRNA from the nucleus and enhancing transgenic expression;

CMV / TO - a hybrid promoter consisting of a cytomegalovirus promoter and two tandem tetracycline operators, for regulated highly efficient expression of target genes;

HA (B - Brisbane) - hemagglutinin gene of strain B / Brisbane / 60/2008;

NA — neuraminidase gene of strain A / California / 04/2009 (H1N1);

M1 - M1 gene of strain A / California / 04/2009 (H1N1))

Also objects of the present invention are methods for producing such lentiviral plasmid vectors.

Preferably, the present invention relates to a method for producing a lentiviral plasmid vector pLenti6.3 / TO / HA-M1-NA.

Most preferably, the present invention relates to a method for producing a lentiviral plasmid vector pLenti6.3 / TO / HA (H1N1) -M1-NA, including the preparation of HA, M1 and NA genes, using HA_SalI_upper-GTCGACACCATGAAGGATATACATATATATATATATATATATATATATATATATATATATATAGATACATATATATAGATTCATACATATATATAGATTCATACATATATATAGTAGTACTATCATATAGATACATATAGTAGTACTATCATATATAGTAGCATATATAGATACATACTAGTAGTTCTGCATATATAGATACATACTAGTAGTTCTGCATATATAGATACATACTAGTAGTTCTGCATATATAGATACATACTAGTAGTTCTACC containing the cDNA of the influenza virus subtype (H1N1) as a template; to obtain M1, a pair of primers M1_SalI_upper - GTCGACACCATGAGTCTTCTAACCGAGGTCG and M1_EcoRI_lower - GAATTCTTATCACTTGAATCGCTGCATCT and viral RNA (H1N1) as a matrix are used; to obtain NA, we use a pair of primers NA_SalI_upper - GTCGACACCATGAATCCAAACCAAAAGATAATAA and NA_EcoRI_lower - GAATTCTCATTACTTGTCAATGGTAAATGGCA and the gene cassette as a matrix, then the obtained viral genome fragments are inserted into the palliative cellulose restriction digestion assay, I use restriction enzyme restriction analysis (QRI the works use clones of recombinant plasmid DNA pDrive with a confirmed nucleotide sequence of the inserted HA, M1 and NA genes, further fragments of HA, M1 and NA isolated from recombinant plasmids at SalI and EcoRI sites of cells they are poured into the pEntry plasmid previously hydrolyzed with these restriction enzymes, then the pLenti6.3 / TO / V5-DEST plasmid DNA is recombined with the recombinant pEntry plasmid DNA, and then clones are selected by PCR analysis with CMV_upper primers - CGCAAATGATGTGTAGTGGGCGTAGTGGGCGTAGTGTGCGTGCGTGCGTGCGTGTGCGTGCGTGCGTGCGTAG PCR product 1869 bp long; primers NA_upper - ACCATGAATCCAAACCAAAAGAGATAATAA and V5 (C-term) _lower - ACCGAGGAGAGGGTTAGGGAT - PCR product 1538 bp long and extended restriction analysis using restriction endonucleases XhoI, EcoRI, PstI and HindIII.

Also most preferably, the present invention relates to a method for producing a lentiviral plasmid vector pLenti6.3 / TO / HA (H3N2) -M1-NA, comprising obtaining the HA (H3N2), M1 and NA genes, individual HA (H3N2) genes are obtained using OT- PCR in two rounds, the first round is performed independent three reactions RT-PCR for three short overlapping DNA fragments for each type of aT using the following primer pairs: HA_H3N2_SalI_upper - GTCGACACCATGAAGACTATCATTGCTTTGAGC and HA_H3N2_fr1_lower - GGTGAACCCCCCAAATGTA, HA_H3N2_fr2_upper - ACCCAGCATTGAACGTGACT and HA_H3N2_fr2_lower - TCCCTCAGAATTTTGATGCC, HA_H3N2_fr3_upper - TAGAAAATGGTTGGGAG GGA and HA_H3N2_PstI_lower - CTGCAGTTATCAAATGCAAATGTTGCACC, in the second round is performed common PCR to three overlapping DNA fragments with the addition of external primers: HA_H3N2_SalI_upper - GTCGACACCATGAAGACTATCATTGCTTTGAGC and HA_H3N2_PstI_lower - CTGCAGTTATCAAATGCAAATGTTGCACC, then the resulting amplification product ON inserted into plasmid pDrive followed by selection of clones with no amino acid substitutions relative to the reference strain , at the next stage, the HA (H3N2) fragment is excised at SalI and PstI sites from the selected clone and inserted into the recombinant plasmid pEntry, previously hydrolyzed by these restriction enzymes (instead НА NA (H1N1)), then recombine plasmid DNA pLenti6.3 / TO / V5-DEST with plasmid DNA recombinant pEntry, then select clones using PCR analysis with primers CMV_upper - CGCAAATGGGCGGTAGGCGTG TCATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATATAT 1869 bp; primers NA_upper - ACCATGAATCCAAACCAAAAGAGATAATAA and V5 (C-term) _lower - ACCGAGGAGAGGGTTAGGGAT - PCR product 1538 bp long and extended restriction analysis using restriction endonucleases XhoI, EcoRI, PstI and HindIII.

Also most preferably, the present invention relates to a method for producing a lentiviral plasmid vector pLenti6.3 / TO / HA (B) -M1-NA, comprising obtaining the genes HA (B), M1 and NA, individual HA (B) genes are obtained using OT- Two-round PCR, in the first round, three independent RT-PCR reactions are performed to obtain three short overlapping DNA fragments for each type of HA using the following primer pairs: HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_frl_lower - TTTTGTATT; HA_B_fr2_upper - CCATACATTTGTGCAGAAGGAG and HA_B_fr2_lower - CTCTTAAGGTCTGCAGCAACTG; HA_B_fr3_upper - AAGGAATGATTGCAGGTTGG and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC, in the second round is performed common PCR to three overlapping DNA fragments with the addition of external primers: HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC, then the resulting amplification product ON inserted into plasmid pDrive followed by selection of clones with no amino acid substitutions relative to the reference strain, in the next step, the HA fragment is excised at the SalI and PstI sites from the selected clone and inserted into the recombinant pEntry plasmid, previously hydrolyzed by res trittases (instead of HA (H1N1)), then recombine plasmid DNA pLenti6.3 / TO / V5-DEST with plasmid DNA recombinant pEntry, then select clones using PCR analysis with primers CMV_upper - CGCAAATGGGCGGTAGTACGTACGTAGTACGTACGTAGTACGTAGTGTACGTAGTGTAGTGTACGTAGTGTACGTAGTGTACGTAGTACGTAGTACGTAGTGCTACCTACCTACCTAGCTACCTAGCTACCTAG 1869 bp product; NA_upper - ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower - ACCGAGGAGAGGGTTAGGGAT - 1538 bp PCR product and extended restriction analysis using restriction endonucleases XhoI, EcoRI, PstI and HindIII.

In addition, the objects of the present invention are sets of primers that were used to obtain lentiviral plasmid vectors of the present invention.

The present invention relates to sets of primers. To the set of primers for obtaining the lentiviral plasmid vector pLenti6.3 / TO / HA (H1N1) -M1-NA, consisting of the following pairs of primers: HA_SalI_upper - GTCGACACCATGAAGGCAATACTAGTAGTTCTGC and HA_EcoRI_lower - GAATTCATAGTAGAACATTCAGTA; M1_SalI_upper - GTCGACACCATGAGTCTTCTAACCGAGGTCG and M1_EcoRI_lower - GAATTCTTATCACTTGAATCGCTGCATCT; NA_SalI_upper - GTCGACACCATGAATCCAAACCAAAAGATAATAA and NA_EcoRI_lower - GAATTCTCATTACTTGTCAATGGTAAATGGCA; CMV_upper - CGCAAATGGGCGGTAGGCGTG and HA_lower - TCATTAAATACATATTCTACACTGTAGAGACC; NA_upper is ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower is ACCGAGGAGAGGGTTAGGGAT.

Also, a kit for primer lentivirus vector plasmid pLenti6.3 / TO / HA (H3N2) -Ml-NA, consisting of the following pairs of primers: HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_frl_lower - TTTTGTTATCTGAATGGAACCC; HA_B_fr2_upper - CCATACATTTGTGCAGAAGGAG and HA_B_fr2_lower - CTCTTAAGGTCTGCAGCAACTG; HA_B_fr3_upper - AAGGAATGATTGCAGGTTGG and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC; HA_H3N2_SalI_upper - GTCGACACCATGAAGACTATCATTGCTTTTGAGC and HA_H3N2_PstI_lower - CTGCAGTTATCAAATGCAAATGTTGCACC; CMV_upper - CGCAAATGGGCGGTAGGCGTG and HA_lower - TCATTAAATACATATTCTACACTGTAGAGACC; NA_upper is ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower is ACCGAGGAGAGGGTTAGGGAT.

Also, to the set of primers for obtaining the lentiviral plasmid vector pLenti6.3 / TO / HA (B -) - M1-NA, consisting of the following pairs of primers: HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_frl_lower - TTTTGTGATGTCCTGTGTCCTGTGTCCTGTGTCCGTGGTCCTGTGTGTCCGTGTGCA HA_B_fr2_upper - CCATACATTTGTGCAGAAGGAG and HA_B_fr2_lower - CTCTTAAGGTCTGCAGCAACTG; HA_B_fr3_upper - AAGGAATGATTGCAGGTTGG and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC; HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC; CMV_upper - CGCAAATGGGCGGTAGGCGTG and HA_lower - TCATTAAATACATATTCTACACTGTAGAGACC; NA_upper is ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower is ACCGAGGAGAGGGTTAGGGAT.

The technical result of the claimed invention is the preservation of the viability and high productivity of the target proteins without the use of controlled expression in producer cells of HPV influenza. Which is unexpected. A specialist in the prior art, on the contrary, could have assumed that due to the overexpression of the proteins of the influenza virus, the cells will not be able to pass the large number of passages necessary to obtain a working bank.

For the development of producer cells, a vector with controlled tetracycline regulated expression of target genes was selected. The technology of tetracycline-regulated expression of the target gene in mammalian cells is based on the binding of tetracycline to the Tet repressor, which leads to the repression of the CMV / TO promoter that controls the expression of the target gene. The CMV / TO hybrid promoter contains a CMV promoter and two tetracycline operators. If a tetracycline repressor is expressed in the cell, the latter blocks the tetracycline operator. After the introduction of tetracycline, the tetracycline repressor itself is blocked, and the tetracycline operator is released, providing expression of the target gene.

However, when studying the properties of HPV influenza producing cells, it unexpectedly turned out that they retained the viability and high productivity of the target proteins without the use of controlled expression. Therefore, the tetracycline repressor was not introduced and the tetracycline operator in the cells of the influenza virus protein developed by us, although it is present in the vector construct, it is released and is not involved in the regulation of expression. The presence of such an element in the construction of the vector will allow, if necessary, to modify the producer cell and provide tetracycline regulated expression of the target genes, if necessary.

Example 1. The method of obtaining lentiviral vectors

1.1. Viruses, bacterial strains

The bacterial strains of Escherichia coli XL2-Blue {recAl endAl gyrA96 thi-1 hsdR17 supE44 relAl lac [F 'proAB lacqZΔM15 Tn10 (Tet') Amy Cam '] c, d} and Stb13 {F mcrB mrrhsdS20 (rB'- mB ') recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20 (StrR) xyl-5 Kleumtl-1}; Influenza virus of strains A and B from the collection of FBSI SSC VB "Vector".

1.2. Isolation of viral RNA

Viral RNA was isolated using the QIAamp MinElute Virus Spin Kit (QLAGEN, USA) in accordance with the manufacturer's recommendations. To 200 μl of virus-containing liquid, 25 μl of Protease and 200 μl of AL solution were added, mixed and placed in a thermostat at 56 ° C for 15 minutes. Then, 250 μl of ethanol (96%) was added and mixed. The solution was applied to the sorbent layer of the column and centrifuged at 6000 g for 1 minute in an Eppendorf centrifuge. The eluate was removed and the sorbent layer of the column was washed with 500 μl of AW1 solution by centrifugation at 6000 g for 1 minute in an Eppendorf centrifuge. The eluate was removed and the sorbent layer of the column was washed with 500 μl of AW2 solution by centrifugation at 6000 g for 1 minute in an Eppendorf centrifuge. The eluate was removed and the sorbent layer of the column was washed with 500 μl of ethanol (96%) by centrifugation at 6000 g for 1 minute in an Eppendorf centrifuge, followed by further centrifugation at 13000 g for 3 minutes. RNA material was eluted from the column with 100 μl of AE solution by centrifugation at 13000 g for 1 minute in an Eppendorf centrifuge.

1.3. PCRiOT-PCR

, 1.5 мМ

,

2-меркаптоэтанол, 0.1% Тритон Х-100), 0.2 мМ каждого dNTP, олигонуклеотидные праймеры в количестве 20 пмоль каждого, 5 ед.а. Taq-полимеразы, 1 мкл раствора ДНК. Реакцию проводили в программируемом термостате GeneAmp PCR-system 6700 с использованием следующей программы:For PCR, the reaction mixture, with a volume of 50 μl, contained a buffer for PCR (SibEnzyme, Russia) (60 mm Tris-HCl pH 8.5, 25 mm

, 1.5 mm

,

2-mercaptoethanol, 0.1% Triton X-100), 0.2 mM each dNTP, oligonucleotide primers in an amount of 20 pmol each, 5 units a. Taq polymerase, 1 μl of DNA solution. The reaction was carried out in a programmable thermostat GeneAmp PCR-system 6700 using the following program:

RT-PCR was performed using the Qiagen OneStep RT-PCR Kit (Qiagen, USA) in accordance with the manufacturer's recommendations. The reaction mixture, with a volume of 50 μl, contained lxQIAGEN OneStep RT-PCR Buffer, 0.4 mM each dNTP, oligonucleotide primers in an amount of 30 pmol each, 2.0 μl QIAGEN OneStep RT-PCR Enzyme Mix, 5 μl viral RNA. The reaction was carried out in a programmable thermostat GeneAmp PCR-system 6700 using the following program

1.4. Agarose gel electrophoretic analysis of nucleic acids

Electrophoretic analysis of DNA fragments was carried out in horizontal plates at a voltage of 6-10 V / cm for 1 hour using 1.0-2.0% agarose gel in TAE buffer. The gel was stained with ethidium bromide (0.2 μg / ml) and photographed using an Image Station 440CF imaging system (Kodak, United States).

1.5. Elution of DNA fragments from agarose gel

Elution of DNA fragments from agarose gel was performed using the Gel Extraction Kit (Qiagen, USA) in accordance with the manufacturer's recommendations. A triple volume of QG solution was added to the excised agarose gel strip containing the desired DNA fragment to melt the agarose, mixed and placed in a thermostat at 50 ° C for 10 minutes until the gel was completely dissolved. Then, one volume of isopropanol was added and mixed. The solution was applied to the sorbent layer of the column and centrifuged at 13000 g for 1 minute in an Eppendorf type centrifuge. The eluate was removed and the sorbent layer of the column was washed with 750 μl of PE solution by centrifugation at 13000 g for 1 minute in an Eppendorf centrifuge. DNA material was eluted from the column with 10-50 μl of TE solution by centrifugation at 13000 g for 1 minute in an Eppendorf centrifuge.

1.6. Ligation

The reaction mixture, with a volume of 20 μl, contained 0.1 μg of vector plasmid DNA and 0.5 μg of a DNA fragment hydrolyzed with the corresponding restriction enzymes (the vector and fragment, after restriction, were purified using the Gel Extraction Kit (section 1.6.)), 50 units aa. T4 phage DNA ligases (SibEnzyme, Russia), 2 μl of 10-fold concentration ligase buffer (SibEnzyme, Russia). The reaction mixture was incubated for 6 hours at 16 ° C.

1.7. LR recombination

The reaction mixture, with a volume of 10 μl, contained 0.15 μg of pLenti6.3 / TO / V5-DEST vector plasmid DNA and 0.15 μg of pEntry vector plasmid DNA containing the target DNA fragment, 2 μl of Gateway LR Clonase II Plus Enzyme Mix. The reaction mixture was incubated for 2 hours at 25 ° C. Next, 1 μl of Proteinase K was added to the reaction mixture, followed by incubation for 10 minutes at 37 ° C.

1.8. Preparation of competent E. coli cells

An individual colony grown on a petri dish on an agar medium was seeded overnight in 5 ml of JLB broth. Then, 500 μl of the obtained overnight culture was added to 50 ml of fresh LB broth and incubated at 37 ° C to an optical density of Ds65 = 0.8–0.85 with vigorous aeration. The resulting cell suspension was cooled in ice for 30 minutes, then precipitated by centrifugation at 3000 rpm at 4 ° C for 10 minutes in a JA-20 rotor in a J2-21 centrifuge (Beckman, USA). The supernatant was carefully removed, the pellet was resuspended in 15 ml of RF-1 buffer and incubated in ice for 15 minutes. Cells were besieged by centrifugation at 3000 rpm at 4 ° C for 10 minutes in a JA-20 rotor in a J2-21 centrifuge, after which the supernatant was carefully removed. Cells were resuspended in 3 ml of RF-2 buffer and incubated in ice for 10 minutes. Then the cells were packaged in 1.5 ml tubes of 200 μl and stored in a kelvinator at minus 70 ° C.

1.9. Transformation of competent E. coli cells

Competent cells with a volume of 200 μl were thawed in ice, 20 μl of ligase mixture or 10 μl of LR recombination product, or 5 μl of a solution containing plasmid DIC at a concentration of 0.1 μg / ml were added, gently mixed and incubated for 30 minutes in ice. Next, the cells were subjected to heat shock. For this, microtubes were placed in a microthermostat with a temperature of 42 ° C for 2 minutes, then cooled to 0 ° C on ice for 5 minutes. Then the cells were scattered on Petri dishes with the appropriate antibiotic.

1. 10. Isolation of plasmid DIC in the analytical version

Plasmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen, USA) in accordance with the manufacturer's recommendations. 3 ml of overnight culture was besieged by centrifugation at 6800 g for 3 minutes. The supernatant was removed, the pellet was resuspended in 250 μl of solution P1. Then, 250 μl of a solution of P2 was added to the lysate, gently mixed and incubated for 5 minutes at room temperature. After that, 350 μl of N3 solution was added to the suspension, gently mixed and precipitated at 13000 g for 10 minutes. The supernatant was applied to the sorbent layer of the column and centrifuged at 13000 g for 1 minute in an Eppendorf centrifuge. The eluate was removed and the sorbent layer of the column was washed with 500 μl of PB solution by centrifugation at 13000 g for 1 minute in an Eppendorf centrifuge. The eluate was removed and the sorbent layer of the column was washed with 750 μl of PE solution by centrifugation at 13000 g for 1 minute in an Eppendorf centrifuge. DNA material was eluted from the column with 50 μl of EB solution by centrifugation at 13000 g for 1 minute in an Eppendorf centrifuge.

1.11. DNA hydrolysis by restriction endonucleases

The reaction mixture contained 0.1-5.0 μg of DNA in a volume of at least 10 μl per 1 μg of DNA, restriction endonuclease at a rate of 1-5 e.a. per 1 μg of DNA, 1/10 of the total reaction mixture of the corresponding buffer at a 10-fold concentration (SibEnzyme, Russia). Hydrolysis was carried out at 37 ° C for 2-4 hours.

1.12. DNA sequencing

The sequencing reaction was carried out using the BigDye 3.1 kit (Applied Biosystems, USA) in accordance with the manufacturer's instructions. Each 5 μl reaction mixture contained the following components: 2 μl of the solution from the sequencing reaction kit, 5 pmol of the oligonucleotide primer, and 0.5 μg of DNA. The reaction was carried out in a programmable thermostat GeneAmp PCR-system 6700 using the following program:

After amplification, the reaction mixture was purified from unincorporated fluorescently labeled nucleotides by purification on Sephadex G-50 superfine. Sequencing was performed on both DNA strands. The decoding of the primary sequencing data (chromatograms) was performed using the Sequencher 4.0.5 program. (Gene Codes, USA).

1.13. Purification of plasmid DNA from endotoxins

Plasmid DNA was purified using the EndoFree Plasmid Maxi Kit (Qiagen, USA) in accordance with the manufacturer's recommendations. 100 ml of overnight culture was besieged by centrifugation at 6000 g at 4 ° C for 15 minutes. The supernatant was removed, the pellet was resuspended in 10 ml of P1 solution. Then, 10 ml of a solution of P2 was added to the lysate, gently mixed and incubated for 5 minutes at room temperature. After that, 10 ml of a solution of PZ was added to the suspension to isolate plasmid DNA, gently mixed, the lysate was transferred to a QIAfilter Cartidge and incubated for 10 minutes at room temperature, followed by filtration of the lysate. 2.5 ml of ER solution was added to the clarified lysate to remove endotoxins, mixed and incubated in ice for 30 minutes. The lysate was then transferred onto a pre-equilibrated 10 ml QLT QLAGEN-tip 500 column. After releasing the column by gravity, it was washed with 60 ml of QC solution. The purified DNA was eluted from the column with 15 ml of QN solution into a clean tube, 10.5 ml of isopropanol was added, mixed and precipitated at 15000 g at 4 ° C for 30 minutes. The precipitate was washed with 70% ethanol and precipitated at 15000 g at 4 ° C for 10 minutes. The precipitate was then dried at room temperature and dissolved in a suitable volume of TE solution. Plasmid DNA concentration was measured spectrophotometrically with an Ultrospec 3000 pro instrument (GE Healthcare Life Sciences, CIIIA).

Example 2. Protocols for obtaining vectors

2.1. Design and synthesis of HA, Ml, and NA gene cassettes

Design of the gene cassette for subsequent production of the corresponding lentiviral vector. Schematically, the gene cassette consists of the following elements:

attL1 and attL2 are the necessary sites in the vector plasmid pEntry for transfer of genetic material to the vector plasmid pLenti6.3 / TO / V5-DEST at the attR1 and attR2 sites using LR recombination;

IRES - site of the internal landing of the ribosome;

WPRE - Woodchuck Posttranscriptional Regulatory Element, a regulatory element for increasing the export of mRNA from the nucleus and enhancing transgenic expression;

CMV / TO - a hybrid promoter consisting of a cytomegalovirus promoter and two tandem tetracycline operators, for regulated highly efficient expression of target genes;

SalI / PstI - recognition sites of the corresponding restriction endonucleases to replace HA in the gene cassette, if necessary;

EcoRI / MluI - recognition sites of the corresponding restriction endonucleases to replace the NA in the gene cassette, if necessary;

XmaI / XmaI - recognition sites of the corresponding restriction endonucleases to remove NA from the gene cassette, if necessary;

AgeI / XmaI - recognition sites of the corresponding restriction endonucleases to remove M1 and NA from the gene cassette, if necessary;

HA - hemagglutinin of influenza virus with the Kozak sequence and stop codons;

M1 - matrix protein of influenza virus with a Kozak sequence and stop codons;

NA - neuraminidase of influenza virus with a Kozak sequence and stop codons.

Thus, in the developed constructions of vectors after recombination of the cassette of influenza virus genes into pLenti6.3 / TO / V5-DEST, the genes for hemagglutinin and matrix protein of the influenza virus are expressed from the hybrid promoter CMV / TO, which is part of the vector plasmid, and the virus neuraminidase gene influenza is expressed from the hybrid promoter CMV / TO, which is part of the gene cassette. In this case, mRNA with the HA and M1 genes is capped by the WPRE regulatory element, which is part of the gene cassette, and the mRNA with the NA gene is capped by the WPRE regulatory element, which is part of the vector plasmid. The translation of the matrix protein is carried out from the site of internal landing of the ribosome. Tetracycline-dependent regulation of the expression of target genes is absent.

It is known that optimization of the codon composition of genes can increase the rate of protein synthesis on the corresponding mRNA and, as a result, increase the level of synthesis of the target protein in mammalian cells. Therefore, the HA, M1, and NA genes that make up the gene cassette were calculated with a modification of the codon composition optimized for expression in mammalian cells.

The gene cassette was synthesized as part of the pEntry plasmid, suitable for LR recombination with plasmid DNA pLenti6.3 / TO / V5-DEST.

2.2. Obtaining and analysis of recombinant pEntry plasmids containing the individual genes HA (H1N1), M1 and NA

To create recombinant DNA molecules containing the HA, M1, and NA genes of the influenza virus, the oligonucleotide primers were calculated at the first stage of work. The design of the primers was carried out using the program "Oligo" (version 6.31) of the company "Molecular Biology Insights", USA. It has now been established that the sequence near the initiator codon affects the level of translation of eukaryotic mRNA. The adenine residue at the -3 position has a dominant effect to achieve a high level of translation; therefore, the Kozak sequence optimized for gene expression in eukaryotic cells was inserted into the nucleotide sequence of the calculated primers.

Also, restriction endonuclease recognition sites were introduced into the oligonucleotide primer structure, allowing embedding of DNA fragments into vector plasmids at the “sticky” ends using SalI and EcoRI restriction endonucleases. The choice of these enzymes is due to the lack of recognition sites in the nucleotide sequence of cloned genes (analysis of the nucleotide sequence of genes for the presence of restriction endonuclease hydrolysis sites was carried out using the Vector NTI computer program (version 9.0.0) from InforMax, USA).

The primary sequences of the oligonucleotide primers used in the work are shown in table 1.

GTCGAC - SalI restriction endonuclease site

GAATTC - restriction endonuclease site EcoRI CTGCAG - restriction endonuclease site

PstI ATCCAT - Zsp2I restriction endonuclease site

The individual HA, M1, and NA genes were obtained as a result of PCR or RT-PCR (Fig. 4 - Electrophoretic separation of amplified fragments of the HA and M1 genes in 1.5% agarose gel):

1. PCR product for a full-sized matrix protein (777 bp);

2. PCR product for full-size hemagglutinin (1719 bp);

M.DNA marker, length in bp shown on the left side.

To obtain HA, we used a pair of HA_SalI_upper and HA_EcoRI_lower primers and a plasmid containing influenza HA cDNA (A / California / 04/2009 (H1N1) as a template; for M1, we used a pair of primers Ml_SalI_upper and Ml_EcoRI_lower and viral RNA in H1 ( ; for NA, a pair of NA_SalI_upper and NA_EcoRI_lower primers and a gene cassette were used as template.

Next, the obtained viral genome fragments were inserted into the pDrive plasmid (Qiagen, USA) using a proprietary kit according to the protocol of the manufacturer. Clones were selected by restriction analysis using restriction endonucleases SalI and EcoRI (Fig. 5 - The result of electrophoretic separation in 1.5% agarose gel of amplified DNA fragments obtained after hydrolysis of the recombinant plasmid pDrive_M1 SalI and EcoRI

1, 12. PCR - product M1 (777 bp);

2-11. Independent clones of the plasmid pDrive_M1 (3830 bp + 771 bp);

13. Base plasmid pDrive (3830 + 20 bp);

M.DNA marker, length in bp shown on the left side.

and FIG. 6 - The result of electrophoretic separation in 1.5% agarose gel of amplified DNA fragments obtained after hydrolysis of the recombinant plasmid pDrive_HA SalI and EcoRI:

1-6. Independent clones of the plasmid pDrive_HA (3830 bp + 1713 bp);

M.DNA marker, length in bp shown on the left side).

The correct nucleotide sequences of all recombinant plasmids were confirmed by sequencing. The determination of the nucleotide sequence was carried out on an automatic sequencer 310 Genetic analyzer (Applied Biosystems, USA). For further work, we used clones of recombinant plasmid DNA pDrive with a confirmed nucleotide sequence of the inserted HA, M1, and NA genes.

Further, the HA, M1, and NA fragments isolated from recombinant plasmids at SalI and EcoRI sites were cloned into the pEntry plasmid previously hydrolyzed with these restriction enzymes (Fig. 7 - Electrophoretic separation of independent clones of the recombinant plasmid pEntry carrying the NA gene in 1.5% agarose gel:

1. The base plasmid pEntry;

2-6. Independent clones of the plasmid pEntry, carrying the NA gene;

M.DNA marker, length in bp shown on the left side).

The structure of the obtained plasmids was confirmed by restriction analysis (restriction endonucleases SalI and EcoRI) and sequencing.

Obtaining and analysis of recombinant plasmids pEntry containing the gene cassette HA (H3N2), M1 and NA.

Individual HA (H3N2) genes were obtained by RT-PCR in two rounds. In the first round, three independent RT-PCR reactions were performed to obtain three short overlapping DNA fragments for each type of HA using the following pairs of primers for H3N2: HA_H3N2_SalI_upper and HA_H3N2_frl_lower, HA_H3N2_fr2_upper and HA_H3N2_fr2_erpper_ HArH3_2_pper for B: HA_B_SalI_upper and HA_B_frl_lower, HA_B_fr2_upper and HA_B_fr2_lower, HA_B_fr3_upper and HA_B_Zsp2I_lower (Fig. 8 - The result of electrophoretic separation in 1.5% agarose gel of amplified DNA fragments N2) ON and (3)

1-3. Three PCR products for hemagglutinin H3N2 (607, 599 and 647 bp);

4-6. Three PCR products for hemagglutinin B (646, 629 and 638 bp);

M.DNA marker, length in bp shown on the left side).

In the second round, total PCR was performed for three overlapping DNA fragments for H3N2 with the addition of external primers (HA_H3N2_SalI_upper and HA_H3N2_PstI_lower) (Fig. 9 - Electrophoretic separation of amplified DNA fragments of HA (H3N2) in 1.5% agarose gel:

1. PCR - the product of full-sized hemeglutinin H3N2 (1719 bp);

M.DNA marker, length in bp shown on the left side).

Next, the obtained amplification product of HA was inserted into the pDrive plasmid, followed by selection of the clone without amino acid substitutions relative to the reference strain by sequencing.

In the next step, the HA (H3N2) fragment was excised from SalI and PstI sites from the selected clone and inserted into the recombinant pEntry plasmid carrying the gene cassette previously hydrolyzed with these restriction enzymes (instead of HA (H1N1)). The structure of the obtained recombinant plasmid was confirmed by restriction analysis using EcoRI (the H3N2 hemagglutinin sequence has three additional EcoRl recognition sites in comparison with the H1N1 hemagglutinin sequence, Fig. 10_ - Electrophoretic separation of DNA fragments obtained after hydrolysis of recombinant plasmid p in 1.5% agarose gel carrying a cassette of genes HA (H3N2), M1 and NA, endonuclease restriction EcoRI (the lengths of the resulting fragments are indicated in parentheses):

1. The base plasmid pEntry, carrying the cassette of the genes HA (H1N1), M1 and NA (9875 bp);

2-13. Independent clones of the plasmid pEntry carrying the gene cassette HA (H3N2), M1 and NA (22 bp + 1060 bp + 3033 bp + 5,760 bp);

M.DNA marker, length in bp shown on the left side.

Obtaining and analysis of recombinant plasmids pEntry containing the gene cassette HA (B), M1 and NA

The synthesized influenza B hemagglutinin gene was excised from the Sad and Pstl sites from the selected clone and inserted into the recombinant pEntry plasmid carrying the gene cassette previously hydrolyzed with these restriction enzymes (instead of HA (H1N1)). The structure of the obtained recombinant plasmid was confirmed by restriction analysis and sequencing.

Example 3

3.1.1 Design and synthesis of the hemagglutinin gene strain A / HongKong / 4801/2014 (H3N2)

The design of the nucleotide sequence of the hemagglutinin gene strain A / HongKong / 4801/2014 (H3N2) was carried out on the basis of the available nucleotide sequences in the GISAID EpiFlu database (Fig. 17 - Print screen of the hemagglutinin strain A / HongKong / 4801/2014 (H3N2) from the site http://platform.gisaid.org/epi3/frontend#3ab6d3).

Further, the primary nucleotide sequence of the gene encoding hemagglutinin was optimized, taking into account the frequency of occurrence of various codons to increase the level of gene expression in the eukaryotic expression system. The nucleotide sequence of the gene, which is represented by SEQ ID NO: 6, was synthesized.

3.1.2 Design and synthesis of the hemagglutinin gene of strain B / Brisbane / 60/2008

The design of the nucleotide sequence of the hemagglutinin gene strain B / Brisbane / 60/2008 was carried out on the basis of the available nucleotide sequences in the GISAID EpiFlu database (Fig. 18 Print-screen of the hemagglutinin strain of strain B / Brisbane / 60/2008 from http: //platform.gisaid .org / epi3 / frontend # 3ab6d3).

Further, the primary nucleotide sequence of the gene encoding hemagglutinin was optimized, taking into account the frequency of occurrence of various codons to increase the level of gene expression in the eukaryotic expression system. The nucleotide sequence of the gene, which is represented by SEQ ID NO: 7, was synthesized.

3.1.3 Design and synthesis of the hemagglutinin gene of strain A / Michigan / 45/2015 (H1N1) pdm09

The design of the nucleotide sequence of the hemagglutinin gene strain A / Michigan / 45/2015 (H1N1) pdm09 was carried out on the basis of the available nucleotide sequences in the GISAID EpiFlu database (Fig. 19 - Print-screen of the hemagglutinin sequence of strain A / Michigan / 45/2015 (H1N1) pdm09 from the site http://platform.gisaid.org/epi3/frontend#3ab6d3).

Further, the primary nucleotide sequence of the gene encoding hemagglutinin was optimized, taking into account the frequency of occurrence of various codons to increase the level of gene expression in the eukaryotic expression system. The nucleotide sequence of the gene was synthesized, which is represented by SEQ ID NO: 8.

3.2.1 Cloning of the hemagglutinin gene of strain A / HongKong / 4801/2014 (H3N2) into the vector pLenti6.3 / TO / HA-M1-NA (California / 04/2009).

The artificial gene encoding the hemagglutinin of strain A / HongKong / 4801/2014 (H3N2) was calculated and synthesized as part of a vector plasmid.

Next, the obtained proprietary vector plasmid containing the hemagglutinin gene of strain A / HongKong / 4801/2014 (H3N2) was hydrolyzed with restriction endonucleases SalI and PstI and inserted into the recombinant plasmid pEntry_HA-M1-NA (California / 04/2009), which previously carried the gene cassette hydrolyzed by these restriction endonucleases (instead of HA (California / 04/2009)). The structure of the obtained recombinant plasmid was confirmed by restriction analysis and sequencing.

To obtain recombinant plasmid DNA. pLenti6.3 / TO / HA (HongKong / 4801) -M1-NA, LR recombination of the proprietary plasmid DNA pLenti6.3 / TO / V5-DEST with plasmid DNA pEntry_HA (HongKong / 4801) -M1-NA was performed. The products of the recombination of two plasmids were analyzed by electrophoretic analysis, restriction analysis, and sequencing. As a result, one maple variant of the plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA was selected (a schematic representation of the plasmid is shown in the passport), the plasmid was prepared and the endotoxins were purified using the “EndoFree Plasmid Maxi Kit” (“ Qiagen ", Germany). Next, the authenticity of the isolated plasmid DNA was verified using restriction analysis and sequencing (Fig. 20 - Schematic representation of plasmid DNA pLenti6.3 / TO / V5-DEST (https://tools.thennofisher.com/content/sfs/vectors/plenti6_3_to_v5_dest_map. pdf)

3.2.2 Cloning of the hemagglutinin gene of strain B / Brisbane / 60/2008 into the vector pLenti6.3 / TO / HA-M1-NA (California / 04/2009).

An artificial gene encoding the hemagglutinin of strain B / Brisbane / 60/2008 (H3N2) was calculated and synthesized as part of a vector plasmid.

Next, the obtained branded vector plasmid containing the gene, hemagglutinin strain B / Brisbane / 60/2008 was hydrolyzed with restriction endonucleases SalI and PstI and inserted into the recombinant plasmid pEntry_HA-M1-NA (California / 04/2009) carrying the gene cassette preliminarily hydrolyzed with data restriction (instead of HA (California / 04/2009)). The structure of the obtained recombinant plasmid was confirmed by restriction analysis and sequencing.

To obtain recombinant plasmid DNA pLenti6.3 / TO / HA (B-Brisbane) -M1-NA, LR recombination of branded plasmid DNA pLenti6.3 / TO / V5-DEST with plasmid DNA pEntry_HA (B-Brisbane) -M1-NA was performed . The products of the recombination of two plasmids were analyzed by electrophoretic analysis, restriction analysis, and sequencing. As a result, one clonal variant of the plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-NA (a schematic representation of the plasmid is shown in the passport) was selected, the plasmid was prepared and the endotoxins were purified using the “EndoFree Plasmid Maxi Kit” (“ Qiagen ", Germany). Next, the authenticity of the isolated plasmid DNA was verified by restriction analysis and sequencing.

3.2.3 Cloning of the hemagglutinin gene of strain A / Michigan / 45/2015 (H1N1) pdm09 into the vector pLenti6.3 / TO / HA-M1-NA (California / 04/2009).

The artificial gene encoding the hemagglutinin of strain A / Michigan / 45/2015 (H1N1) pdm09 was calculated and synthesized as part of a vector plasmid.

Next, the obtained branded vector plasmid containing the hemagglutinin gene of strain A / Michigan / 45/2015 (H1N1) pdm09 was digested with restriction endonucleases SalI and PstI and inserted into the recombinant plasmid pEntry_HA-M1-NA (California / 04/2009) carrying the cassette previously hydrolyzed by these restriction endonucleases (instead of HA (California / 04/2009)). The structure of the obtained recombinant plasmid was confirmed by restriction analysis and sequencing.

To obtain pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA recombinant plasmid DNA, LR recombination of pLenti6.3 / TO / V5-DEST branded plasmid DNA with pEntry_HA (H1N1-Mich) -M1-NA plasmid DNA was performed . The products of the recombination of two plasmids were analyzed by electrophoretic analysis, restriction analysis, and sequencing. As a result, one clonal variant of the plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA (a schematic image of the plasmid is shown in the passport) was selected, the plasmid was prepared and the endotoxins were purified using the “EndoFree Plasmid Maxi Kit” (“ Qiagen ", Germany). Next, the authenticity of the isolated plasmid DNA was verified by restriction analysis and sequencing.

3.3.1 Obtaining and analysis of recombinant plasmids pEntry containing the gene cassette HA (B / Brisbane / 60/2008), M1 (California / 04/2009) and NA (California / 04/2009)

Recombinant plasmid DNA pEntry_HA-M1-NA (California / 04/2009) was used as a vector plasmid. This plasmid contains a gene cassette consisting of the following structural elements:

- attL1 and attL2 are the nucleotide sequences necessary for LR recombination between pEntry and pLenti6.3 / TO / V5-DEST plasmids;

- IRES - site of the internal landing of ribosomes;

- CMV - cytomegalovirus promoter;

- HA - a gene encoding the hemagglutinin of the influenza virus (California / 04/2009);

- SalI / PstI - recognition sites of the corresponding restriction endonucleases for replacing HA (California / 04/2009) in the gene cassette, if necessary;

- M1 - gene encoding the matrix protein of the influenza virus (California / 04/2009);

- NA is a gene encoding an influenza virus neuraminidase (California / 04/2009).

The gene encoding the hemmagglutinin of strain B / Brisbane / 60/2008 (H3N2) was synthesized as a part of a vector plasmid, with the gene flanked by SalI / PstI restriction endonuclease recognition sites for further cloning into the desired vector. In addition, the nucleotide sequence of the gene was optimized to increase its expression in eukaryotic cells. The nucleotide sequence of the hemagglutinin gene of strain B / Brisbane / 60/2008 (H3N2) from the GISAID EpiFlu database was used for optimization; optimization of the nucleotide composition of the gene does not lead to amino acid substitutions in the corresponding protein.

To obtain recombinant plasmid DNA pEntry_HA (B-Brisbane) -M1-NA, the vector plasmid pEntry_HA-M1-MA (California / 04/2009) was digested with SalI and PstI restriction endonucleases. Using the indicated restriction endonucleases, the plasmid DNA containing the gene encoding the hemagglutinin of strain B / Brisbane / 60/2008 was hydrolyzed. Next, the hydrolyzed vector plasmid DNA and the hydrolyzed DNA fragment encoding hemagglutinin were purified by isolating the corresponding fragments from the agarose gel using the GelExtractionKit kit (QIAGEN, Germany) in accordance with the manufacturer's recommendations. The obtained DNA fragments were ligated, competent E. coli XL-10Gold cells were transformed. Then, plasmid DNA was isolated from individual E. coli colonies. Clones were selected by restriction analysis using restriction endonucleases SalI and PstI. The correct nucleotide sequences of all recombinant plasmids were confirmed by sequencing. The determination of the nucleotide sequence was performed on a 310 Geneticanalyzer automatic sequencer (AppliedBiosystems, USA). For further work, we used clones of recombinant plasmid DNA pEntry_HA (B-Brisbane) -M1-NA with a confirmed nucleotide sequence of the inserted gene encoding hemagglutinin (Fig. 21 - Electrophoretic separation of independent clones of the recombinant plasmid pEntry_NA (B-Bris in 1.2% agarose gel) -M1-MA, containing the gene cassette HA (B / Brisbane / 60/2008), M1 (California / 04/2009) and NA (California / 04/2009):

1-5. Independent clones of the plasmid pEntry_HA (B-Brisbane) -M1-NA carrying the gene cassette HA (B / Brisbane / 60/2008), M1 (California / 04/2009) and NA (California / 04/2009);

M.1 KbDNA marker, length in bp shown on the left side (SibEnzyme, Russia) and (Fig. 22 - The result of electrophoretic separation in 1.2% gel of DNA fragments obtained after hydrolysis of independent clones of the recombinant plasmid pEntry_HA (B-Brisbane) -M1-NA containing the gene cassette HA ( B / Brisbane / 60/2008), M1 (California / 04/2009) and NA (California / 04/2009), restriction endonucleases SalI and PstI (the lengths of the resulting fragments are indicated in parentheses):

1-5. Independent clones of the plasmid pEntry_NA (B-Brisbane) -M1-NA carrying the gene cassette HA (B / Brisbane / 60/2008), M1 (California / 04/2009) and NA (California / 04/2009) (8164 bp . + 1768 bp);

M.1 KbDNA marker, length in bp shown on the left side (SibEnzyme, Russia).

3.3.2 Preparation and analysis of recombinant plasmid pLenti6.3 containing the gene cassette HA (B / Brisbane / 60/2008), M1 (California / 04/2009) and NA (California / 04/2009)

To obtain recombinant plasmid DNA pLenti6.3 containing the gene cassette HA (B / Brisbane / 60/2008), M1 (California / 04/2009) and NA (California / 04/2009), LR recombination was performed between the branded plasmid DNA pLenti6. 3 / TO / V5-DEST and plasmid DNA pEntry_NA (B-Brisbane) -M1-NA containing the gene cassette. Then, competent E. coli strain Stbl3 cells were transformed with a mixture of plasmid DNA obtained after recombination. Then, plasmid DNA was isolated from individual E. coli colonies. Potentially one maple variant was the target. To confirm the structure of the obtained plasmid, an extended restriction analysis was performed using restriction endonucleases XhoI, EcoRI, PstI, and HindIII (the lengths of the obtained DNA fragments after hydrolysis correspond to theoretical lengths. In addition, the correct nucleotide sequence was confirmed by sequencing. For further work, this plasmid was developed and purified from endotoxins using the EndoFreePlasmidMaxiKit kit (QIAGEN, Germany).

The result of electrophoretic separation in a 1.5% agarose gel of DNA fragments obtained after hydrolysis of the recombinant plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-NA, restriction endonucleases XhoI, EcoRI, PstI and HindIII (Fig. 23 - Electrophoretic separation result in a 1.2% agarose gel of LR products of recombination between plasmid DNA pEntry_HA (B-Brisbane) -M1-NA and pLenti6.3 / TO / V5-DEST:

1-10, 12, 13. non-targeted recombination products;

11. The target product of recombination pLenti6.3 / TO / HA (B-Brisbane) -M1-NA;

M.1 KbDNA marker, length in bp shown on the left side (SibEnzyme, Russia)

and (Fig. 24). The table shows the theoretical lengths of DNA fragments formed after hydrolysis by the corresponding restriction endonucleases.

1: plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-MA, hydrolyzed with HindIII;

2: plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-MA, hydrolyzed by EcoRI;

3: plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-NA, hydrolyzed by XhoI;

4: plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-NA, hydrolyzed by PstI;

M. DNA marker 1 Kb (SibEnzyme, Russia).

3.3.3 Preparation and analysis of recombinant plasmid pEntry containing the gene cassette HA (A / HongKong / 4801/2014 (H3N2)), M1 (California / 04/2009) and NA (California / 04/2009)

The scheme for producing recombinant plasmid DNA

pEntry_HA (HongKong / 4801) -M1-NA is similar to the scheme for obtaining pEntry_HA (B-Brisbane) -M1-NA.

PEntry_HA-M1-NA plasmid DNA (California / 04/2009) was also used as a vector plasmid, in which the gene encoding the hemagglutinin of strain California / 04/2009 was replaced with the gene encoding the hemagglutinin of strain A / HongKong / 4801/2014 (H3H2).

The gene encoding the hemagglutinin of strain A / HongKong / 4801/2014 (H3N2) was synthesized with optimization of the codon composition to increase the level of gene expression in eukaryotic cells. Information on the natural nucleotide sequence of the hemagglutinin encoding gene is obtained from the EpiFlu GISAID database.

The plasmid DNA pEntry_HA-M1-NA (California / 04/2009) and the synthesized plasmid containing the hemagglutinin gene of the desired strain were digested with SalI and PstI restriction endonucleases. After that, the vector plasmid and the gene encoding hemagglutinin were purified from agarose gel. Two obtained DNA fragments were ligated, then competent E. coli cells of strain XL-10Gold were transformed with a ligase mixture. Further, plasmid DNA was isolated from individual E. coli colonies. The authenticity of the obtained plasmid DNA was confirmed by restriction analysis and sequencing. For further work, a clone without nucleotide substitutions was selected (Fig. 25 - The result of electrophoretic separation in 1.2% gel of DNA fragments obtained after hydrolysis of independent clones of the recombinant plasmid pEntry_HA (HongKong / 4801) -M1-NA containing the HA gene cassette (HongKong / 4801 ), M1 (California / 04/2009) and NA (California / 04/2009), restriction endonucleases SalI and PstI (the lengths of the resulting fragments are indicated in parentheses):

1-5. Independent clones of the plasmid pEntry_HA (HongKong / 4801) -M1-NA carrying the gene cassette HA (HongKong / 4801), M1 (California / 04/2009) and NA (California / 04/2009) (8164 bp + 1711 p .n.);

M.1 KbDNA marker, length in bp shown on the left side (SibEnzyme, Russia) and (Fig. 26 - The result of electrophoretic separation in 1.2% agarose gel of LR products of recombination between plasmid DNA pEntry_HA (HongKong / 4801) -M1-NA and pLenti6.3 / TO / V5- DEST:

1-3. Inappropriate recombination products;

4-7. The recombination product of interest is pEntry_HA (HongKong / 4801) -M1-NA;

M.1 KbDNA marker, length in bp shown on the left side (SibEnzyme, Russia).

3.3.4 Preparation and analysis of recombinant plasmid pLenti6.3 containing the gene cassette HA (A / HongKong / 4801/2014 (H3N2)), M1 (California / 04/2009) and NA (California / 04/2009)

To obtain plasmid DNA pLenti6.3 / TO / HA (A / HongKong) -M1-NA, LR recombination was performed between plasmid DNA pLenti6.3 / TO / V5-DEST and plasmid DNA pEntry_HA (HongKong / 4801) -M1-NA . Transformed competent E. coli strain Stbl3 cells with recombination reaction products. Next, plasmid DNA was isolated from individual E. coli clones. As a result of electrophoretic analysis of the isolated plasmid DNA, it was found that potentially 4 clones are the target products of recombination. Next, one of the clones was sequenced and restriction analysis was performed using restriction endonucleases XhoI, EcoRI, PstI and HindIII. The selected maple variant pLenti6.3 / TO / HA (A / HongKong) -M1-NA does not contain nucleotide substitutions relative to the theoretical sequence, in addition, the lengths of DNA fragments obtained after hydrolysis by the indicated restriction endonucleases correspond to theoretical lengths. For further work, this plasmid was developed and purified from endotoxins using the EndoFreePlasmid MaxiKit kit (QIAGEN, Germany).

The result of electrophoretic separation in a 1.5% agarose gel of DNA fragments (Fig. 27) obtained after hydrolysis of the recombinant plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, restriction endonucleases XhoI, EcoRI, PstI and HindIII. The table shows the theoretical lengths of DNA fragments formed after hydrolysis by the corresponding restriction endonucleases:

1. Plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed by PstI,

2. Plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed by HindIII;

3. Plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed by EcoRI;

4. Plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed by XhoI;

M. DNA marker 1 Kb (SibEnzyme, Russia)

3.3.5. Preparation and analysis of recombinant plasmid pEntry containing the gene cassette HA (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009) and NA (California / 04/2009)

To obtain recombinant plasmid DNA pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, at the first stage of the work, a gene encoding the hemagglutinin of strain A / Michigan / 45/2015 (H1N1) pdm09 was synthesized. Hemagglutinin gene nucleotide sequence information was obtained from the EpiFlu GISAID database. Further, work was done to optimize the codon composition of the indicated gene in order to increase its expression level in eukaryotic cells. The gene was calculated and synthesized as part of plasmid DNA. Then, the obtained plasmid DNA was hydrolyzed at the SalI and PstI restriction endonucleases sites. In addition, the plasmid DNA pEntry_HA-M1-NA (California / 04/2009) was hydrolyzed with the indicated restriction endonucleases. After hydrolysis, vector DNA and a fragment encoding the hemagglutinin of strain A / Michigan / 45/2015 (H1N1) pdm09 were isolated from an agarose gel using the GelExtractionKit kit (QIAGEN, Germany), the isolated fragments were ligated, and competent E. coli cells were transformed with a ligase mixture strain XL-10Gold. Clonal plasmid DNA variants were isolated from individual E. coli colonies. The authenticity of the isolated plasmid DNA was confirmed by restriction analysis (hydrolysis with SalI and PstI restriction endonucleases and sequenced. A clone without nucleotide substitutions was used for further work (Fig. 28 - Electrophoretic separation of independent clones of the recombinant plasmid pEntry_NA (H - Н1М1 in 1.2% agarose gel) M1-NA containing the gene cassette HA (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009) and NA (California / 04/2009):

1-6. Independent clones of the plasmid pEntry_HA (H1N1-Mich) -M1-NA carrying the gene cassette HA (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009) and NA (California / 04/2009);

M.1 Kb DNA marker, length in bp shown on the left side (SibEnzyme, Russia) and (Fig. 29 - The result of electrophoretic separation in 1.2% gel of DNA fragments obtained after hydrolysis of independent clones of the recombinant plasmid pEntry_NA (H1N1-Mich) -M1-NA containing the gene cassette HA ( A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009) and NA (California / 04/2009), restriction endonucleases SalI and PstI (the lengths of the resulting fragments are indicated in parentheses):

1-5. Independent clones of the plasmid pEntry_HA (H1N1-Mich) -M1-NA carrying the gene cassette HA (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009) and NA (California / 04/2009) ( 8164 bp + 1711 bp);

M.1 Kb DNA marker, length in bp are shown on the left side (SibEnzyme, Russia) and (Fig. 30 - Result of electrophoretic separation in 1.2% agarose gel of LR products of recombination between plasmid DNA pEntry_HA (H1N1-Mich) -M1-NA and pLenti6.3 / TO / V5- DEST:

1-3, 5-8, 11-13. Inappropriate recombination products;

4, 9. Target product of recombination pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA;

M.1 KbDNA marker, length in bp shown on the left side (SibEnzyme, Russia)

3.3.6. Preparation and analysis of recombinant plasmid pLenti6.3 containing the gene cassette HA (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009) and NA (California / 04/2009).

Recombinant plasmid DNA pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA was obtained according to the scheme described above. At the first stage of the work, LR recombination was performed between the obtained plasmid DNA pEntry_HA-M1-NA (California / 04/2009) and plasmid DNA pLenti6.3 / TO / V5-DEST, then the competent E. cells were transformed with a mixture of plasmids obtained after the recombination reaction. coli strain Stbl3. Maple variants of plasmid DNA were isolated from individual E. coli colonies. As a result of electrophoretic analysis, it was found that potentially two clonal variants of plasmid DNA are the target plasmids pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA. One of the plasmid DNA clones was hydrolyzed with restriction endonucleases XhoI, EcoRI, PstI and HihdIII. As a result, it was shown that the length of DNA fragments obtained after hydrolysis corresponds to the theoretical lengths of hydrolysis products. The correct nucleotide sequence of the recombinant plasmid was confirmed by sequencing. For further work, this plasmid was developed and purified from endotoxins using the EndoFreePlasmid MaxiKit kit (QIAGEN, Germany).

The result of electrophoretic separation in a 1.5% agarose gel of DNA fragments obtained after hydrolysis of the recombinant plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, restriction endonucleases XhoI, EcoRI, PstI and HindIII. The table shows the theoretical lengths of DNA fragments formed after hydrolysis by the corresponding restriction endonucleases.

1: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, hydrolyzed by PstI;

2: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, hydrolyzed with HindIII;

3: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA, hydrolyzed by EcoRI;

4: plasmid pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA hydrolyzed by XhoI;

M. DNA marker 1 Kb (SibEnzyme, Russia) - (Fig. 30).

Example 4. Protocols for the study of physico-chemical and molecular biological properties of lentiviral vectors

To obtain recombinant plasmid DNA

pLenti6.3 / TO / HA (H1N1) -M1-NA containing the HA, M1, and NA gene cassette performed LR recombination of the branded plasmid DNA pLenti 6.3 / TO / V5-DEST and the previously synthesized pEntry plasmid DNA containing the gene cassette (FIG. 11 - The result of electrophoretic separation in 1.5% agarose gel of independent clones of the recombinant plasmid pLenti containing the gene cassette HA, M1 and NA:

1-4, 6-8, 10, 11. Base plasmid pLenti 6.3 / TO / V5-DEST;

5, 9. Independent clones of the plasmid pLenti containing the gene cassette HA, M1 and MA;

M.DNA marker, lengths are shown on the left side).

When selecting clones using PCR analysis (primers CMV_upper and HA_lower - PCR product with a length of 1869 bp; primers NA_upper and V5 (C-term) _lower - PCR product with a length of 1538 bp) and extended restriction analysis with Using restriction endonucleases XhoI, EcoRI, PstI, and HindIII (Table 2), 2 target clones from 40 analyzed clones were revealed (Fig. 12 - Electrophoretic separation of DNA fragments obtained after hydrolysis of recombinant plasmid pLenti containing the HA gene cassette in 1.5% agarose gel). (H1N1), M1 and NA, restriction endonucleases XhoI, EcoRI, PstI and HindIII (the lengths of the resulting fragments cops are shown in table.2):

1, 2. Independent clones of the plasmid pLenti containing the gene cassette HA, M1 and NA, hydrolyzed by XhoI;

3, 4. Independent clones of the plasmid pLenti containing the gene cassette HA, M1 and NA, hydrolyzed by PstI;

5, 6. Independent clones of the pLenti plasmid containing the HA, M1 and NA gene cassette, hydrolyzed by EcoRI;

7, 8. Independent clones of the plasmid pLenti containing the gene cassette HA, M1 and NA, hydrolyzed by HindIII;

M.DNA marker, length in bp shown on the left side.

The correctness of the nucleotide sequence is confirmed by sequencing.

Thus, a lentiviral plasmid vector was created, carrying a cassette of the hemagglutinin, neuraminidase and Ml protein of the influenza A virus (H1N1pdm09), and expressing the polyprotein consisting of HA (H1), NA (N1) and M1 proteins.

The H1N1M1 plasmid construct includes three sections of influenza virus proteins linked together using two 2A linkers. As a result, expression of one long protein is achieved, which is then divided into three functional proteins during processing. Reference sequences for influenza virus proteins are:

- HA protein = H1: gene bank No. ACS45035 (H1N1)

Protein NA = N1: Gene Bank No. AGU92835 (H1N1)

- Protein Ml = Ml: gene bank No. AAT70589 (H5N1)

The resulting three lentiviral vectors were subsequently used to create producer cells of virus-like particles (HPV) of influenza virus

Restriction sites of cassette expression vectors

Restriction sites of cassette expression vectors

Restriction sites of cassette expression vectors

The following nucleic / amino acid sequences are used in plasmids:

SEQ ID NO: I — Sequence of hemagglutinin H1 DNA;

SEQ ID NO: 2 — Sequence of Neuraminidase N1 DNA;

SEQ ID NO: 3 - The sequence of protein M DNA;

SEQ ID NO: 4 - The nucleic acid sequence of hemagglutinin HA (H3N2) from strain (H3N2);

SEQ ID NO: 5 - The sequence of the nucleic hemagglutinin of influenza virus B from strain.

Example 5. Selection of cell lines producing HPV influenza based on cell lines of higher mammals (HEK293FT, Vero, MDCK). Creation of an industrial producer strain for the synthesis of HPV.

Three packaging plasmids containing HIV protein genes were used. The lentiviral vector, plasmid pLenti6.3, is delivered to the target cell by transfection of complexes with lipofectamine, it contains only non-structural elements of the HIV genome. Refusal to use packaging plasmids completely eliminates the possibility of the formation of lentiviral particles.

The mammalian HEK 293FT, Vero, and MDCK mammalian cells were used as target cells for transduction of influenza virus proteins by genes.

The MDCK cell culture was isolated from the kidney cells of an adult healthy female Cocker Spaniel in 1958.

HEK293 cell culture was obtained in the early 70s in the Netherlands as a result of the transformation of normal human embryonic kidney cells with genetic material of type 5 adenovirus, the size of the insertion of the viral genome into the cell genome is approximately 4,500 bp The adenovirus E1A gene is expressed in these cells and is involved in the transactivation of certain viral promoters, and therefore these cells produce a large amount of protein. The widespread use of HEK293 cells for scientific purposes is associated with their highest transfection ability, which reaches an efficiency of 100%. The HEK293FT cell variant contains a large T-antigen of the SV40 virus, which provides episomal replication of plasmids containing the SV40ori replication site. This property provides a high level of amplification of transfected plasmids inside 293FT cells and subsequent transient expression of the target gene product. HEK293 cell culture is not widely used in the production of viral vaccines.

5.1. Methods of transfection, transduction and protocols describing the procedure

5.1.1 method of determination of the number of transducing units by ilshunochemical staining of transduced cells

A monolayer of the culture of target cells intended for transduction is grown in 96-well plates in a support medium (DMEM D00 u / ml penicillin, 100 μg / ml streptomycin, 300 μg / ml L-glutamine, 10 μg / ml DEAE-dextran ( Sigma), 5 μg / ml trypsin TRSC). Separately, 10-fold serial dilutions of the pseudovirus suspension are prepared in sterile microtubes by adding 50 μl of the pseudovirus suspension to 450 μl of dilution support medium. 10-fold dilutions of the pseudovirus suspension are added to the wells of a plate with a monolayer of MDCK cells. Each dilution of the pseudovirus is introduced into 4 wells of 100 μl. After 18 hours of incubation in a CO2 atmosphere at a temperature of 37 ° C, the medium is removed from the wells and the plate is washed 2 times with the PBS. 100 μl of chilled acetone (80%) is added for 10-15 minutes, after which acetone is removed, the plate is rinsed twice with the FBI. A 1: 5000 dilution of monoclonal antibodies to the HA1 protein of influenza A virus is prepared in the FBI containing 1% bovine serum albumin and 0.1% Tween 20, and 100 μl is added for 60 min in each well, after which the wells are washed 4 times and covered with affinity purified horseradish peroxidase conjugated goat anti-mouse IgG antibodies. After 60 minutes of incubation, the wells are washed 4 times, a substrate solution of 3-amino-9-ethylcarbazole (AEC) is introduced. After 30 minutes of incubation, the substrate solution is removed, the plate is washed 1 time with the FBI, and infected pink-brown colored cells are counted in an inverted microscope. The number of transducing units is calculated after averaging the number of focal-forming units (stained infectious centers) over 4 wells according to the formula T = FFU _With p * 10 ^{N + 1} FFU / ml, where N is the number of 10-fold dilution of the pseudovirus in which the FFU was counted .

5.1.2 Protocol describing the procedure for producing HPV influenza producing cells by transduction of mammalian cells (HEK293FT, Vero, MDCK)

Mammalian cells (HEK293FT, Vero, MDCK) to be transduced are grown in complete culture medium. On the day of transduction, a pseudovirus tube is removed from the low temperature refrigerator, thawed and, if necessary, diluted to the required multiplicity of infection. Do not shake up the suspension of the pseudovirus. The culture medium is removed from the cells, a suspension of pseudovirus is carefully added to the monolayer of cells. To increase transduction activity, Polybrene® can be added to a final concentration of up to 10 μg / ml. Gently shake the tablets and incubate at 37 ° C in a humidified 5% C02 atmosphere overnight. The next day, the virus-containing suspension is removed and replaced with fresh complete nutrient medium, incubation is continued at 37 ° C overnight. The next day, the culture medium is removed and replaced with a medium containing blasticidin. The medium is replaced every 3-4 days until antibiotic-resistant colonies can be identified (usually after 10-12 days). At least 5 antibiotic-resistant colonies are selected and each is transferred to a separate culture vial. After the formation of the monolayer, HPV production in the supernatant is evaluated for each clone in the hemagglutination reaction (RGA) and in an electron microscope.

5.2 Methods for evaluating cell cultures and their expression constructs (analysis of reporter gene expression, integration of the genetic construct into the genome of target cells) in the production of immunobiological preparations.

5.2.1 Method for determining the concentration of influenza HPV in the hemagglutination reaction

For the production of RGA, a 1% suspension of washed rooster erythrocytes is used. To prepare a suspension of washed red blood cells, 1 ml of fresh blood of a rooster is used, to which 9 ml of FBI is added, the suspension is centrifuged at 1200 rpm, the supernatant and the sediment of white blood cells are removed, the procedure is repeated 2 more times. To obtain a 1% suspension of red blood cells from 1 ml of blood, the washed red blood cells must be diluted in 60 ml of the FBI, which corresponds to a concentration of 60,000 cells / ml.

In 96-well round-bottom plates, 50 μl of phosphate buffered saline (PBS) is added to each well. Serial twofold dilutions of the studied samples of the culture supernatant of the HPV influenza producing cells are prepared by transferring 50 μl of the sample, 50 μl is removed from the wells of the last row. 50 μl of a 1% suspension of rooster erythrocytes is added to all wells of the plate, the plate is slightly swayed to mix the samples in the wells, closed and incubated for 60 minutes at room temperature, after which the results of agglutination are recorded. The RGA titer is considered the largest dilution of the test serum, in which erythrocyte agglutination ("umbrella") is observed,

5.2.2 Method of FOE to determine the number of cells expressing the reporter (target) gene, immunochemical staining of transduced cells

A monolayer culture of influenza HPV producing cells is grown in 96-well plates in a support medium (DMEMD00 units / ml penicillin, 100 μg / ml streptomycin, 300 μg / ml L-glutamine, 10 μg / ml DEAE-dextran (Sigma), 5 μg / ml trypsin TRSC) by incubation in a CO2 atmosphere at a temperature of 37 ° C. The medium is removed from the wells and the plate is washed 2 times with the FBI. 100 μl of chilled acetone (80%) is added for 10-15 minutes, after which acetone is removed, the plate is rinsed twice with the FBI. A 1: 5000 dilution of monoclonal antibodies to the influenza virus protein is prepared in the FBI containing 1% bovine serum albumin and 0.1% Tween 20, and 100 μl is added for 60 min to each well, after which the wells are washed 4 times and covered with affinity purified conjugated peroxidase horseradish goat antibodies against mouse IgG. After 60 minutes of incubation, the wells are washed 4 times, a substrate solution of 3-amino-9-ethylcarbazole (ABS) is introduced. After 30 minutes of incubation, the substrate solution is removed, the plate is washed once with PBS, and the transduced pink-brown colored cells are counted in an inverted microscope.

5.2.3 Electron microscopy method for producing HPV influenza cells

Samples of HPV influenza producing cells were fixed in a 4% solution of paraformaldehyde at + 4 ° C for 48 hours. Then, they were additionally fixed with a 1% solution of osmium acid, dehydrated according to standard methods in increasing concentration of ethyl alcohol and acetone, and poured into the mixture of epon-araldite. Ultrathin sections were prepared on a Reichert-Young microtome (Austria). The obtained sections were contrasted with uranyl acetate and lead citrate. Ultrathin sections were examined with a JEM 1400 electron microscope (Jeol, Japan), photographing and image analysis were performed using the built-in digital camera Veleta (SIS, Germany) and the iTEM software package (SIS, Germany).

5.2.4 PCR Method for Determining Target HA, NA Genes Integrated in the Genome of Producer Cells

To isolate cell nuclei, cells are scraped off from a monolayer, approximately 10 ⁶ -10 ⁷ cells, washed twice in a cold FBI, using centrifugation at 1500 rpm. The cell pellet was resuspended in 500 μl of hypotonic buffer solution, pipetting several times, and incubated on ice for 15 minutes (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl ₂ ). 25 μl of detergent (10% NP4O) is added and vortexed for 10 seconds. After that, the homogenate is centrifuged for 10 minutes at 10,000 rpm at 4 ° C, the supernatant is removed, and the precipitate containing the nuclear fraction is used to extract the genomic DNA of producer cells.

Genomic DNA is extracted from the nuclear fraction of HPV influenza producing cells using the AmpliSens RIBO-sorb reagent kit. 450 μl of lyse solution are added to the tubes. Pipette 100 μl of test samples of producer cells into test tubes with a lysing solution using a separate filter tip for each sample. Mix by pipetting. Incubate at room temperature for 3 minutes. Tightly closing the lids, mix thoroughly on a vortex. Precipitated drops on a vortex. Place the tubes in a thermostat with a temperature of 60 ° C for 10 minutes. Mix, then precipitate drops on a vortex. Resuspend the sorbent, vigorously mixing on a vortex. 25 μl of a resuspended sorbent is added to each tube with a separate tip, and the caps are tightly closed.

Mix on a vortex, put in a tripod for 1 min, mix again and leave in a tripod for 5 minutes. Centrifuge the tubes in a microcentrifuge for 1 min at 7000 g. Without capturing the sorbent, remove the supernatant from each tube with a separate tip without a 200 μl filter, using a vacuum aspirator. Add 400 μl of washing solution to the tubes, close the lids tightly, mix on a vortex until the sorbent is fully resuspended. Centrifuge the tubes in a microcentrifuge for 30 s at 7000 g. Without capturing the sorbent, remove the supernatant from each tube with a separate tip without a 200 μl filter, using a vacuum aspirator. Add 400 μl of washing solution to the tubes, close the lids tightly, mix on a vortex until the sorbent is fully resuspended. Centrifuge the tubes in a microcentrifuge for 30 s at 7000 g. Without capturing the sorbent, remove the supernatant from each tube with a separate tip without a 200 μl filter, using a vacuum aspirator. Repeat the washing procedure. Place the tubes with open caps in a thermostat with a temperature of 60 ° C for 15 minutes to dry the sorbent. 50 μl of elution buffer A are added to the tubes. Vortex is mixed until the sorbent is fully resuspended. They are placed in a thermostat with a temperature of 60 ° C for 5 minutes. Allowed to increase the volume of elution to 120 μl. Centrifuge the tubes in a microcentrifuge for 2 min at 12 thousand g. The supernatant contains purified DNA. Samples are ready for the PCR reaction.

Real-time PCR amplification with detection is carried out on reagent kits manufactured by the Central Research Institute of Epidemiology of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare. The total volume of the reaction mixture is 30 μl, including the sample volume of cell DNA is 10 μl. The required number of tubes with the PCR mixture-FL is selected to amplify the DNA of the studied and control samples. On the surface of the wax, 10 μl of PCR buffer A is added, and it should not fall through the wax and mix with the PCR mixture-FL. 10 μl of DNA samples are added to the prepared tubes. They put control reactions. Preparation of tubes for amplification using PCR-kit reagent kits option FRT-50 F. The total volume of the reaction mixture is 25 μl, including the volume of DNA sample - 10 μl. Defrost the required number of tubes with PCR mixture-FL. The contents of the tubes are mixed with PCR mixture-FL, PCR buffer-B and polymerase (TaqF) and the droplets are precipitated by short-time centrifugation (1-2 s) using a centrifuge / vortex. Select the required number of tubes or strips for amplification of cDNA of the studied and control samples. To carry out N reactions, 10 * (N + 1) μl of PCR mixture-FL, 5 * (N + 1) μl of PCR buffer-B, and 0.5 * (N + 1) micron polymerase (TaqF) are mixed in a separate tube ) Mix the prepared mixture on a vortex and precipitate drops by short-term centrifugation using a centrifuge / vortex. Pipette 15 μl of the prepared mixture into each tube. 10 μl of cDNA obtained in the RNA reverse transcription reaction is added to the prepared tubes. They put control reactions. Amplification with detection is carried out in the "real time" mode. You should program the device (an amplifier with a real-time detection system) to perform amplification and detection of the fluorescent signal in accordance with the instructions for the reagent kit, taking into account the recommended channels for fluorophores. Set the tubes in the cells of the reaction module of the device. Before installation in a plate type amplifier, it is necessary to precipitate droplets from the walls of the tubes by short-term (1-3 s) centrifugation on a centrifuge / vortex. Run the amplification program with the detection of the fluorescent signal. At the end of the program, they begin to analyze and interpret the results.

Results. The research protocols of the obtained cell cultures-producers

5.3.2.1 Electron microscopy analysis of HPV production of influenza H1N1M1 clones

Further confirmation of the maximum HPV productivity in MDCK cells was obtained by examining the presence of virus-like particles (HPV) in MDCK and HEK-293FT cell cultures by electron microscopy. On ultrathin sections, cells have large nuclei and a complete set of cytoplasmic organoids, a distinctive feature of the morphology of MDCK cells is the presence of a large number of endosomal structures, the formation of numerous outgrowths of the cytoplasm of various shapes and microvilli. Cells retain the epithelioid type of structure. In the nuclei of some cells, aggregates of electron-dense particles are identified, giving the nucleus a “spotted” appearance (Fig. 13 — electron-dense granular inclusions (arrow) are found in the nucleus of MDCK cells (upper figure), giving it a spotted appearance characteristic of those affected by the influenza virus cells, HPV production in HEK293FT cells is significantly less pronounced compared to MDCK cells (bottom figure).

On the surface and in the surrounding area of approximately 40% of MDCK cells and 5% of HEK293FT cells, particles morphologically resemble the influenza virus are detected (Fig. 13). Two types of virus-like particles are distinguished: spherical and filiform (Fig. 14 - The main morphological forms of virus-like particles: spherical (white arrow) and filiform (black arrow) in MDCK cells). The filamentous form of particles prevails. In addition, in some areas groups of particles are found having a pleomorphic shape (Fig. 15 - Various forms of virus-like particles (arrows) in MDCK cells).

The size of the spherical particles is typical for the influenza virus and is 80-120 them, filiform HPV with a diameter of 90-100 nm and a length (on sections) to a micrometer. Virus-like particles have a well-defined membrane membrane, on which spikes are visible with a length of approximately 15 nm and a diameter of about 5 nm. A characteristic feature was a significant decrease in the electron density of the inner part of the “virion” corresponding to the location of the viral genome, compared with the typical influenza virus.

About 10% of the cells in the culture are damaged, numerous autophagosomes accumulated in them, chromatin compaction, fragmentation of nuclei, and the formation of apoptotic bodies were traced. Apoptotic cell death was major (Fig. 16 — MDCK cell apoptosis around and on the surface of a dying cell).

The results of the study by electron microscopy show that the morphology of virus-like particles in the MDCK and HEK-293FT cell culture is not fundamentally different from the morphology of the influenza virus cultured on the same cell cultures. A feature of the structure of HPV is a decrease in the electron density of their inner part.

Example 6. Protocols for determining in PCR target genes HI, N1, integrated into the genome of the producer cell

Tables 3, 4 show the results of PCR detection of the presence of target genes HI, N1, respectively, in the nuclear fraction of HPV producing cells. The measured threshold value Ct for the five studied clones is significantly less than the value detected for a positive control sample, which indicates the presence of built-in genes of the influenza virus in the genome of the producer cell. Based on data on the maximum productivity of HPV influenza on an MDCK culture, the determination of target genes in PCR was performed only for this culture.

Thus, the results of PCR diagnostics of cell DNA indicate the integration of influenza virus genes into the genome of the studied clones of HPV producing cells.

Further, only MDCK cells were used as target cells for the transduction of influenza virus genes, which showed maximum productivity among the three studied mammalian cell lines — HEK 293FT, Vero, and MDCK. As the lentiviral vector, only the plasmid pLenti6.3 was used; three packaging plasmids were not used. At this stage, three vector plasmids pLenti6.3 / TO / HA (HlNl) -Ml-NA, pLenti6.3 / TO / HA were used to obtain HPV producer cells of the influenza virus subtypes A (H1N1) and A (H3N2) and type B (H3N2) -M1-NA, pLenti6.3 / TO / HA (B-Phuket) -M1-NA, the structure of which is described above.

Protocol describing the procedure for producing producer cells.

The day before transfection, 1-3 * 10 ⁵ MDCK cells are plated on a T25 culture flask. According to the protocol for the transfecting reagent Lipofectamine 2000, a mixture of plasmid and transfectant is prepared in 200 μl of maintenance medium and incubated at room temperature for 10-15 minutes. After this, the cell monolayer is washed once with a phosphate buffer solution and the prepared complex of lipofectamine and plasmid is applied to the cell monolayer. Incubation is carried out in a thermostat (37 ° C) for 3 hours, after which 5 ml of support medium is added to the culture vial. On the 3rd day after transfection, the monolayer of cells is reseeded in several 24-well plates. After 3, 5, 7, and 9 days of incubation of the plates in a CO ₂ atmosphere at 37 ° C, an aliquot of the nutrient medium was taken from each well to evaluate the production of HPV in the RGA. The hole in which the highest level of production is observed is seeded by limiting dilutions into several 96-well plates. After 1-2 weeks, 10-15 clones with maximum production by RGA are selected. Each of the selected clones is seeded into several wells of a 96-well plate, and the presence of expression products of all three genes is confirmed by immunochemical staining of the monolayer. Selected several clones with maximum HPV production are scattered into vials, and HPV production is evaluated by electron microscopy. The most productive clone is selected according to the RGA, immunochemical staining and electron microscopy, which is scattered on 75 cm ² vials, and the cell suspension is transmitted to form a cell bank of influenza virus HPV producers. Clones isolated by limiting dilutions retained their ability to produce HPV influenza at the same level as in the case of transduction with lentiviral pseudoviruses. Table 6 shows the results of the HPV productivity assessment of the fifteen MDCK clones.

Five clones transfected with plasmid pLenti6.3 / TO / HA (HlNl) -Ml-NA produce HPV with a titer in the range from 1:16 to 1:64. HPV of the influenza virus subtype A (H3N2) is produced with greater hemagglutinating activity, the titer in the RGA varies from 1:32 to 1: 128. Five clones transfected with plasmid pLenti6.3 / TO / HA (B-Phuket) -M1-NA showed the least productivity. Thus, clones of HPV producing cells obtained from MDCK cells by transfection with a single vector plasmid retain HPV productivity similar to that of clones obtained as a result of infection with lentiviral pseudoviruses.

The study showed the possibility of obtaining highly productive HPV flu influenza MDCK cell clones without the use of lentiviral pseudovirus particles as a system for delivering the target gene to the genome of the target cell.

At the first stage of the study, according to the results of immunochemical staining, it was found that 99.5-99.9% of cells subjected to triple co-infection with pseudoviruses contained the expression products of the hemagglutinin, neuraminidase and M1 protein genes. The studied clones of MDCK, Vero, and HEK 293FT cell cultures transduced with lentiviral pseudoviruses with the genes hemagglutinin, neuraminidase, and influenza A (H1N1) M1 protein showed the presence of influenza HPV production determined in the RGA of the supernatant of these cells. Only in the composition of HPV hemagglutinin molecules are able to ensure the polyvalency of the construct with respect to sialosides on the erythrocyte membrane and cause the agglutination of the latter.

Studies have also shown that MDCK cells are the best producers of HPV for the influenza virus, providing an HPV titer of RGA in the range of 1:64 to 1: 256, while clones derived from HEK293FT and Vero cells are characterized by low productivity. The results of the study by electron microscopy show that the morphology of the virus-like particles produced in the culture of MDCK and HEK-293FT cells is not fundamentally different from the morphology of the influenza virus. On the surface and in the surrounding area of approximately 40% of MDCK cells and 5% of HEK293FT cells, particles are morphologically reminiscent of the influenza virus. Two types of virus-like particles are distinguished: spherical and filiform. A feature of the structure of HPV is a decrease in the electron density of their inner part. The results of the study of cellular DNA of MDCK cells by PCR indicate the integration of influenza virus genes into the genome of the studied clones of HPV producing cells.

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List of sequences:

SEQ ID NO: 1 - Sequence of hemagglutinin H1 DNA

SEQ ID NO: 2 - The sequence of neuraminidase N1 DNA

SEQ ID NO: 3 - M1 DNA Protein Sequence

SEQ ID NO: 4 - The nucleic acid sequence of hemagglutinin HA (H3N2) from strain A / Novosibirsk / 01/2014 (H3N2)

SEQ ID NO: 5 - The sequence of the nucleic hemagglutinin of influenza B virus from strain B / Phuket / 3 073/2013

SEQ ID NO: 6 - The nucleotide sequence of the artificial hemagglutinin gene strain A / HongKong / 4801/2014 (H3N2)

SEQ ID NO: 7 - The nucleotide sequence of the artificial hemagglutinin gene strain B / Brisbane / 60/2008

SEQ ID NO: 8 - The nucleotide sequence of the artificial hemagglutinin gene of strain A / Michigan / 45/2015 (H1N1) pdm09

Claims (15)

1. Lentiviral plasmid vector pLenti6.3 / TO / HA (H1N1) -M1-NA, containing a cassette of the hemagglutinin genes for influenza A (H1N1), neuraminidase A (H1N1) and M1 protein A (H1N1), designed for integration with the cell gene producer shown in FIG. 3 or FIG. 34. 2. Lentiviral plasmid vector according to claim 1, where the genes for hemagglutinin, neuraminidase and M1 protein are obtained from strain A / California / 04/2009. 3. The lentiviral plasmid vector according to claim 1, wherein the hemagglutinin gene is obtained from strain A / Michigan / 45/2015, and the genes for neuraminidase and M1 protein are obtained from strain A / California / 04/2009. 4. Lentiviral plasmid vector pLenti6.3 / TO / HA (H3N2) -M1-NA, containing the cassette of the hemagglutinin genes of influenza A (H3N2), neuraminidase A (H1N1) and M1 protein A (H1N1), designed for integration with the cell gene producer shown in FIG. 2 or FIG. 32. 5. Lentiviral plasmid vector according to claim 4, where the hemagglutinin gene is obtained from strain A / Novosibirsk / 01/2014, and the genes for neuraminidase and M1 protein are obtained from strain A / California / 04/2009. 6. Lentiviral plasmid vector according to claim 4, where the hemagglutinin gene is obtained from strain A / HongKong / 4801/2014, and the genes for neuraminidase and M1 protein are obtained from strain A / California / 04/2009. 7. Lentiviral plasmid vector pLenti6.3 / TO / HA (B) -M1-NA, containing a cassette of the hemagglutinin genes of influenza virus B, neuraminidase A (H1N1) and M1 protein A (H1N1), designed for integration with the producer cell gene, shown in FIG. 1 or FIG. 33. 8. Lentiviral plasmid vector according to claim 7, where the hemagglutinin gene is obtained from strain B / Phuket / 3073/13, and the genes for neuraminidase and M1 protein are obtained from strain A / California / 04/2009. 9. Lentiviral plasmid vector according to claim 7, where the hemagglutinin gene is obtained from strain B / Brisbane / 60/2008, and the genes for neuraminidase and M1 protein are obtained from strain A / California / 04/2009. 10. A method of obtaining a lentiviral plasmid vector according to claims. 1, 2, 3, including the generation of HA, M1, and NA genes, HA HA is used using a pair of primers HA_SalI_upper - GTCGACACCATGAAGGCAATACTAGTAGTTCTGC and HA_EcoRI_lower - GAATTCTCATTAAATACATATTCTACACTGTAGAGACC and a plasmid containing HNA / 2009 cdNA / ; for M1 using a primer pair M1_SalI_upper - GTCGACACCATGAGTCTTCTAACCGAGGTCG and M1_EcoRI_lower - GAATTCTTATCACTTGAATCGCTGCATCT and viral RNA (H1N1) as a template; for NA using primer pair NA_SalI_upper - GTCGACACCATGAATCCAAACCAAAAGATAATAA and NA_EcoRI_lower - GAATTCTCATTACTTGTCAATGGTAAATGGCA and cassette of genes as a template, then the resulting fragments of the viral genome vstra pDrive plasmid (Qiagen, USA); clones are selected by restriction analysis using restriction endonucleases SalI and EcoRI; recombinant plasmid DNA clones pDrive with the confirmed nucleotide sequence of the inserted HA, M1 and NA genes are used for further work, fragments are then isolated from recombinant plasmids HA, M1, and NA at SalI and EcoRI sites are cloned into the pEntry plasmid, previously hydrolyzed with these restriction enzymes, then pLenti6.3 / TO / V5-DEST plasmid DNA is recombined with recombinant pEntry plasmid DNA, and then clones are selected by PCR analysis with CMV_upper primers — CGCAAATGGGCGGTAGGCGTG and HA_lower — TCATTAAATACATATTCTACACTGTAGAGACC — 1869 bp PCR product; primers NA_upper - ACCATGAATCCAAACCAAAAGAGATAATAA and V5 (C-term) _lower - ACCGAGGAGAGGGTTAGGGAT - PCR product 1538 bp long and extended restriction analysis using restriction endonucleases XhoI, EcoRI, PstI and HindIII. 11. The method of obtaining lentiviral plasmid vector according to claims. 4, 5, 6, including obtaining the HA (H3N2), M1, and NA genes, the individual HA (H3N2) genes are obtained by RT-PCR in two rounds; in the first round, three independent RT-PCR reactions are carried out to obtain three short overlapping fragments DNA for each type of aT using the following primer pairs: HA_H3N2_SalI_upper - GTCGACACCATGAAGACTATCATTGCTTTGAGC and HA_H3N2_fr1_lower - GGTGAACCCCCCAAATGTA, HA_H3N2_fr2_upper - ACCCAGCATTGAACGTGACT and HA_H3N2_fr2_lower - TCCCTCAGAATTTTGATGCC, HA_H3N2_fr3_upper - TAGAAAATGGTTGGGAGGGA and HA_H3N2_PstI_lower - CTGCAGTTATCAAATGCAAATGTTGCACC, in the second round is performed common PCR to three overlapping DNA fragments with the addition of external pr Yamers: HA_H3N2_SalI_upper - GTCGACACCATGAAGACTATCATTGCTTTTGAGC and HA_H3N2_PstI_lower - CTGCAGTTATCAAATGCAAATGTTGCACC, then the obtained amplification product is inserted into the plasmid p site with the following amino acid and the following sequence is replaced with the PDrive H1 I site pEntry recombinant plasmid, previously hydrolyzed with these restriction enzymes (instead of HA (H1N1)), then recombine pLenti6.3 / TO / V5-DEST plasmid DNA with recombinant pEntry plasmid DNA, then select clone in using PCR analysis with primers CMV_upper - CGCAAATGGGCGGTAGGCGTG and HA_lower - TCATTAAATACATATTCTACACTGTAGAGACC - PCR product with a length of 1869 bp; primers NA_upper - ACCATGAATCCAAACCAAAAGAGATAATAA and V5 (C-term) _lower - ACCGAGGAGAGGGTTAGGGAT - PCR product 1538 bp long and extended restriction analysis using restriction endonucleases XhoI, EcoRI, PstI and HindIII. 12. The method of obtaining lentiviral plasmid vector according to claims. 7, 8, 9, including obtaining the HA (B), M1, and NA genes, the individual HA (B) genes are obtained by RT-PCR in two rounds; in the first round, independent three RT-PCR reactions are performed to obtain three short overlapping fragments DNA for each type of HA using the following pairs of primers: HA_B_SalI_upper - GTCGACACCATGAAGGGCAATAATTGTACTACTCAT and HA_B_fr1_lower - TTTTGTTATCTGAATGGAACCC; HA_B_fr2_upper - CCATACATTTGTGCAGAAGGAG and HA_B_fr2_lower - CTCTTAAGGTCTGCAGCAACTG; HA_B_fr3_upper - AAGGAATGATTGCAGGTTGG and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC, in the second round is performed common PCR to three overlapping DNA fragments with the addition of external primers: HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC, then the resulting amplification product ON inserted into plasmid pDrive followed by selection of clones with no amino acid substitutions relative to the reference strain, in the next step, the HA fragment is excised at the SalI and PstI sites from the selected clone and inserted into the recombinant pEntry plasmid, previously hydrolyzed by res trittases (instead of HA (H1N1)), then recombine plasmid DNA pLenti6.3 / TO / V5-DEST with plasmid DNA recombinant pEntry, then select clones using PCR analysis with primers CMV_upper - CGCAAATGGGCGGTAGGACTGTACGTACGTACGTAGTACGTAGTGTAGTGTAGTGTACGTAGTGTACGTAGTGTACGTAGTGGTACGTAGTGTACGTAGTGCTACCTGCTACCTACCTAGCTACCTAGCTACCTAGCTACCTAG 1869 bp product; NA_upper - ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower - ACCGAGGAGAGGGTTAGGGAT - 1538 bp PCR product and extended restriction analysis using restriction endonucleases XhoI, EcoRI, PstI and HindIII. 13. A set of primers for obtaining lentiviral plasmid vector according to claims. 1, 2, 3, consisting of the following primer pairs: HA_SalI_upper - GTCGACACCATGAAGGCAATACTAGTAGTTCTGC and HA_EcoRI_lower - GAATTCTCATTAAATACATATTCTACACTGTAGAGACC; M1_SalI_upper - GTCGACACCATGAGTCTTCTAACCGAGGTCG and M1_EcoRI_lower - GAATTCTTATCACTTGAATCGCTGCATCT; NA_SalI_upper - GTCGACACCATGAATCCAAACCAAAAGATAATAA and NA_EcoRI_lower - GAATTCTCATTACTTGTCAATGGTAAATGGCA; CMV_upper - CGCAAATGGGCGGTAGGCGTG and HA_lower - TCATTAAATACATATTCTACACTGTAGAGACC; NA_upper is ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower is ACCGAGGAGAGGGTTAGGGAT. 14. A set of primers for obtaining lentiviral plasmid vector according to claims. 4, 5, 6, consisting of the following primer pairs: HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_fr1_lower - TTTTGTTATCTGAATGGAACCC; HA_B_fr2_upper - CCATACATTTGTGCAGAAGGAG and HA_B_fr2_lower - CTCTTAAGGTCTGCAGCAACTG; HA_B_fr3_upper - AAGGAATGATTGCAGGTTGG and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC; HA_H3N2_SalI_upper - GTCGACACCATGAAGACTATCATTGCTTTTGAGC and HA_H3N2_PstI_lower - CTGCAGTTATCAAATGCAAATGTTGCACC; CMV_upper - CGCAAATGGGCGGTAGGCGTG and HA_lower - TCATTAAATACATATTCTACACTGTAGAGACC; NA_upper is ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower is ACCGAGGAGAGGGTTAGGGAT. 15. A set of primers for obtaining lentiviral plasmid vector according to claims. 7, 8, 9, consisting of the following primer pairs: HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_fr1_lower - TTTTGTTATCTGAATGGAACCC; HA_B_fr2_upper - CCATACATTTGTGCAGAAGGAG and HA_B_fr2_lower - CTCTTAAGGTCTGCAGCAACTG; HA_B_fr3_upper - AAGGAATGATTGCAGGTTGG and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC; HA_B_SalI_upper - GTCGACACCATGAAGGCAATAATTGTACTACTCAT and HA_B_Zsp2I_lower - ATGCATTCATTATAGACAGATGGAGCATGAAAC; CMV_upper - CGCAAATGGGCGGTAGGCGTG and HA_lower - TCATTAAATACATATTCTACACTGTAGAGACC; NA_upper is ACCATGAATCCAAACCAAAAGATAATAA and V5 (C-term) _lower is ACCGAGGAGAGGGTTAGGGAT.

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