RU2701953C1 - Method of producing a polyvalent influenza vaccine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the biotechnology. Invention specifically relates to a method of producing a polyvalent influenza vaccine containing HPV, where haemagglutinin, neuraminidase and M1 protein genes are obtained from strain A/California/04/2009; haemagglutinin gene is obtained from strain A/Michigan/45/2015, and neuraminidase and M1 protein genes are obtained from strain A/California/04/2009; haemagglutinin gene is obtained from strain A/Novosibirsk/01/2014, and neuraminidase and M1 protein genes are obtained from strain A/California/04/2009; haemagglutinin gene is obtained from strain A/HongKong/4801/2014, and neuraminidase and M1 protein genes are obtained from strain A/California/04/2009; haemagglutinin gene is obtained from strain B/Phuket/3073/13, and neuraminidase and M1 protein genes are obtained from strain A/California/04/2009 or haemagglutinin gene is obtained from strain B/Brisbane/60/2008, and neuraminidase and M1 protein genes are obtained from strain A/California/04/2009.

EFFECT: invention allows to obtain HPV of influenza virus having high hemagglutinating activity, the method allows increasing the antigen yield, besides, during the method implementation the vitality and high productivity of the target proteins are preserved without use of controlled expression in HPV influenza cell-producers.

3 cl, 44 dwg, 15 tbl, 7 ex

Description

The invention relates to biotechnology. Specifically, the invention relates to a method for producing a multivalent influenza vaccine containing HPV, where the hemagglutinin, neuraminidase and M1 protein genes are derived from strain A / California / 04/2009; 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; 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; 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; the hemagglutinin gene is obtained from strain B / Phuket / 3073/13, and the neuraminidase and M1 protein genes are obtained from strain A / California / 04/2009 or the hemagglutinin gene is obtained from strain B / Brisbane / 60/2008, and the neuraminidase and M1 genes are obtained from strain A / California / 04/2009. EFFECT: invention makes it possible to obtain HPV of influenza virus having high hemagglutinating activity. Also, the method allows to increase the yield of antigen. In addition, during the implementation of the method, the viability and high productivity of the target proteins are preserved without the use of controlled expression in HPV-producing cells of influenza.

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 (VH) penetrates the cell by endocytosis of the virions, followed by the 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 at the end of 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 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, that is, the part of the population that did not have contact with the influenza serotype (H1N1) viruses 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 of 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 later named by WHO 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 was 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 HA 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 the influenza A virus. The HPV vaccine carrying the pandemic influenza A H1N 1 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 must 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 vaccination 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-BHpycoM, in whose genome the avian influenza A virus gene is 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 consequences of administering recombinant NDV are not well understood, and it is not clear whether NDV-based influenza vaccines 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-based 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 such vaccines has successfully passed the first stage of clinical trials in the United States, 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 their ability to infect non-dividing and differentiated cells - an extremely useful property for the purpose of gene therapy. The name lentiviruses (lat. Lenti = slow) is associated with a long period of time between infection and completion 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 envelope of the viral 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 cellular 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 with the help of 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, the 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 G of rabies virus, are an effective system for gene delivery to motoneurons during 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 human 293T cells [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 output of HPV production, but also complements the antigenic portrait of HPV antigens with conserved T and B cell epitopes, which allows inducing immunity not only against homologous, but also heterologous viruses [45, 46].

From UA 3975 dated 12/27/1994 a method for producing a multivalent influenza vaccine is known, including culturing the original strains of viruses, concentrating and purifying them, followed by mixing. Thus, this document does not refer to obtaining a vaccine containing HPV of genetically engineered origin.

From WO 2017210599 of 12/07/2017, a method for producing a flu vaccine comprising a mosaic virus is known. This document discloses the preparation of a vaccine based on a genetically engineered mosaic virus containing hemmagglutinin and neurominidase from different strains of the influenza virus.

The claimed invention differs from those known from the prior art in that the method for producing vaccines involves the production of HPV based on strains A / Califomia / 04/2009 (H1N1), A / Novosibirsk / 01/2014 (H3N2) and B / Phuket / 3073/2013. Thus, the claimed invention relates to a method for producing a multivalent influenza vaccine containing virus-like particles (HPV) of influenza virus constructed from surface proteins of the influenza virus: where the hemagglutinin, neuraminidase and M1 protein genes are derived from strain A / California / 04/2009; 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; 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; 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; the hemagglutinin gene is obtained from strain B / Phuket / 3073/13, and the neuraminidase and M1 protein genes are obtained from strain A / California / 04/2009 or the hemagglutinin gene is obtained from strain B / Brisbane / 60/2008, and the neuraminidase and M1 genes are obtained from strain A / California / 04/2009.

The technical result of the claimed invention is to obtain HPV of influenza virus having a large hemagglutinating activity. Also, the method allows to increase the yield of antigen.

In addition, the technical result is the preservation of the viability and high productivity of the target proteins without the use of regulated expression in cells producing HPV influenza. Which is unexpected. A specialist in the prior art, on the contrary, could assume that due to the overexpression of the proteins of the influenza virus, cells will not be able to pass the large number of passages necessary to obtain a working bank. In addition, since it is known from the prior art that when using adenoviral vectors carrying the HA A / PR / 8/34 (H1N1) gene, the infection of MDCK cells was generally detected only when using a vector carrying the gene from the A / PR / influenza strain 8/34 (H1N1); the viability and productivity of MDCK cells is lower than other types of mammalian cells (see RU 2565546), the preservation of viability for many passages is not obvious to a specialist when using MDCK cells.

The invention is illustrated by the following drawings.

In FIG. 1 shows a lentiviral vector plasmid carrying a cassette of the hemagglutinin genes of strain B, neuraminidase of strain A (H1N1) and M1 protein of strain A (H1N1).

In FIG. Figure 2 shows a lentiviral vector plasmid carrying a cassette of hemagglutinin genes of strain A (H3N2), neuraminidase of strain A (H1N1) and M1 protein of strain A (H1N1).

FIG. Figure 3 illustrates the 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).

FIG. 4 shows the result of electrophoretic separation in 1.5% agarose gel of amplified fragments of the HA and M1 genes.

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.

FIG. 5 illustrates the result of electrophoretic separation in a 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, 3, 4, 5, 6, 7, 8, 9, 10, 11 — independent clones of the plasmid pDrive_M1 (3830 bp + 771 bp);

13 - the base plasmid PDrive (3830 bp + 20 bp);

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

In FIG. Figure 6 shows 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, 2, 3, 4, 5, 6 — independent clones of the plasmid pDrive_HA (3830 bp + 1713 bp);

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

FIG. 7 illustrates the result of electrophoretic separation in a 1.5% agarose gel of independent clones of the recombinant plasmid pEntry carrying the NA gene.

1 - base plasmid pEntry;

2, 3, 4, 5, 6 — independent clones of the plasmid pEntry carrying the NA gene;

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

FIG. 8 shows the result of electrophoretic separation in 1.5% agarose gel of amplified DNA fragments of HA (H3N2) and HA (B).

1, 2, 3 - three PCR products for hemagglutinin H3N2 (607, 599 and 647 bp);

4, 5, 6 - three PCR products for hemagglutinin B (646, 629 and 638 bp);

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

FIG. 9 shows the result of electrophoretic separation in 1.5% agarose gel of amplified HA (H3N2) DNA fragments.

1 - PCR product for full-size hemagglutinin H3N2 (1719 bp);

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

In FIG. Figure 10 shows the result of electrophoretic separation in a 1.5% agarose gel of DNA fragments obtained after hydrolysis of the recombinant plasmid pEntry carrying the gene cassette HA (N3N2), M1 and NA, EcoRI restriction endonuclease (the lengths of the resulting fragments are indicated in parentheses):

1 - the base plasmid pEntry, carrying the cassette of the genes HA (N1N1), M1 and HA (9875 bp);

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 — independent clones of the plasmid pEntry carrying the cassette of the HA (N3N2), M1 and NA genes (22 bp + 1060 bp N. + 3033 bp + 5760 bp);

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

In FIG. 11 shows the result of electrophoretic separation in a 1.5% agarose gel of independent clones of the recombinant plasmid pLenti containing the HA, M1, and NA gene cassette:

1, 2, 3, 4, 6, 7, 8, 10, 11 - the base plasmid pLenti6.3 / TO / V5-DEST;

5, 9 - independent clones of the plasmid pLenti containing the gene cassette HA, M1 and NA;

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

In FIG. 12 you can see the result of electrophoretic separation in a 1.5% agarose gel of DNA fragments obtained after hydrolysis of the recombinant plasmid pLenti containing the HA (H1N1), M1 and NA gene cassette, Xhol, EcoRI, PstI and HindIII restriction enucleonucleases (the lengths of the resulting fragments are indicated in Table 2):

1, 2 - independent clones of the plasmid pLenti containing the gene cassette HA (H1N1), M1 and NA, hydrolyzed by XloI;

3, 4 - independent clones of the plasmid pLenti containing the gene cassette HA (H1N1), M1 and NA, hydrolyzed by PstI;

5, 6 - independent clones of the plasmid pLenti containing the gene cassette HA (H1N1), M1 and NA, hydrolyzed by EcoRI;

7, 8 - independent clones of the plasmid pLenti containing the gene cassette HA (H1N1), M1 and NA, hydrolyzed by HindIII;

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

In FIG. 13 you can see how electron-dense inclusions (arrow) are found in the MDCK cell nucleus (upper figure), giving it a “spotted” appearance, characteristic of cells infected with the influenza virus. HPV production in HEK 293FT cells is significantly less pronounced compared with MDCK cells (bottom figure).

In FIG. Figure 14 shows the main morphological forms of virus-like particles: spherical (white arrow) and filiform (black arrow) in MDCK cells.

FIG. 15 illustrates various forms of virus-like particles (arrows) in MDCK cells.

FIG. 16 illustrates apoptosis of MDCK cells. Numerous HPVs around and on the surface of a dying cell.

In FIG. 17A and 17B, hemagglutinin HA expression can be observed by candidate clones (immunoblotting). In FIG. 17A depicts clones of MDCK 1F1, 3C5, 5E4, and 5G7 cells. The negative control is MDCK cell lysate. FIG. 17B shows clones of MDCK cells 6D6, 6D12, 7F4, 9B3, 10C12, 10E7. The negative control is MDCK cell lysate.

FIG. 18A and 18B illustrate NA expression by candidate clones (immunoblotting). In FIG. 18B shows clones of MDCK 6D6, 6D12, 7F4, 9B3, 10C12, 10E7 cells. The negative control is MDCK cell lysate.

FIG. 19A and 19B show the expression of M1 by candidate clones (immunoblotting). In FIG. 19A depicts clones of MDCK 1F1, 3C5, 5E4, 5G7, and 6D6 cells. The negative control is MDCK cell lysate. FIG. 19B shows clones of MDCK cells 6D12, 7F4, 9B3, 10C12, 10E7. The negative control is MDCK cell lysate.

In FIG. 20, a schematic representation of pLenti6.3 / TO / V5-DEST plasmid DNA can be observed.

(https://tools.thermofisher.com/content/sfs/vectors/plenti6_3_to_v5_dest_map.pdf)

In FIG. Figure 21 shows the HPV elution profile in the Mustang Q column.

Filtrate samples were taken every 400 ml of filtration to determine flow rates. The purpose of determining flow rates was to control the Mustang Q overload, which leads to material loss during flushing or filtration.

In FIG. 22 shows an elution profile of material from Mustang Q using an EC.

In FIG. 23 is a Cellufine S profile of processing peak material obtained by EC.

In FIG. 24 shows the elution profile using Cellufine S material with a maximum value of Vo. A = loading 0.5 ml. B = loading 1 ml. C = loading 2 ml.

FIG. 25 illustrates the electrophoresis of the individual steps. The upper image refers to the HPV H1N1M1, showing the initial appearance, after ultrafiltration, concentration on Mustang Q, slip after Mustang Q, gel filtration, affinity chromatography, BSA control (70 kDa) + lysozyme (15 kDa).

The average image refers to the HPV H3N1M1, showing the initial appearance, the concentrate after ion exchange chromatography (Mustang Q), breakthrough after ion exchange chromatography, gel filtration, affinity chromatography, BSA control (70 kDa) + lysozyme (15 kDa).

The bottom image is for HPV HA (B) N1M1, showing the appearance after filtration, concentrate after ion exchange chromatography (Mustang Q), concentrate on Millipore concentrators, affinity chromatography, BSA control (70 kDa) + lysozyme (15 kDa).

In FIG. 26 shows the results of the purification of three types of HPV in one electrophoresis:

- HPV H1N1M1 after all stages of purification;

- HPV H3N1M1 after all stages of purification;

- HPV HA (B) N1M1 after all stages of purification;

- Human serum albumin;

- HSA + alpha 2-interferon.

FIG. 27 shows the results of the analysis of the purified HPV H1N1M1 HPV concentrate by electron microscopy.

FIG. 28 shows the results of an analysis of the purified HPV H3N1M1 HPV concentrate by electron microscopy.

FIG. 29 shows the results of an analysis of the concentrated HPV-HA (B) N1M1 HPV concentrate by electron microscopy.

FIG. 30 shows the result of electrophoretic separation in a 1.2% agarose gel of LR recombination products between pEntry_HA (H1N1-Mich) -M1-NA plasmid DNA and pLenti6.3 / TO / V5-DEST.

1, 2, 3, 5, 6, 7, 8, 11, 12, 13 - non-target products of recombination;

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)

In FIG. 31 shows a recombinant plasmid DNA map 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 is the M1 gene of strain A / California / 04/2009 (H1N1).

In FIG. 32 is a 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;

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

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

M1 is the M1 gene of strain A / California / 04/2009 (H1N1).

In FIG. 33 shows a recombinant map of 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 is the M1 gene of strain A / California / 04/2009 (H1N1).

In FIG. 34 along the ordinate axis the indicators are displayed (mg glucose / g disk) / h; along the abscissa axis are the dates of sampling.

Dates of measurement of parameters:

• March 30 = placing cells in a bioreactor

• April 01 = replacement for OptiMEM serum-free culture medium

• April 18 = temperature decreased from 37 ° C to 34 ° C

ο Changes have led to a decrease in glucose intake (as expected), but over time it has increased again

• April 18 = CO ₂ reduction from 5% to 2.5%

ο Glucose intake has not changed. The concentration of CO _{2 was} changed in order to better control the pH

• April 24 = glucose intake has changed slightly

• Under these conditions, NA activity was detected 3 weeks after sowing.

In FIG. 35 shows 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 lengths of the resulting fragments).

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).

FIG. 36 illustrates the result of electrophoretic separation in 1.2% agarose gel 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).

1, 2, 3, 4, 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)

In FIG. 37 shows 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 HA gene cassette (B / Brisbane / 60/2008), M1 (California / 04 / 2009) and NA (California / 04/2009), SalI and PstI restriction endonucleases (the lengths of the resulting fragments are indicated in parentheses).

1, 2, 3, 4, 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) (8164 bp + 1768 bp);

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

In FIG. 38 shows the result of electrophoretic separation in a 1.2% agarose gel of LR recombination products between pEntry_HA (B-Brisbane) -M1-NA plasmid DNA and pLenti6.3 / TO / V5-DEST.

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13 - non-target products of recombination;

11 - 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).

In FIG. Figure 39 shows 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 (shown in brackets the length of the resulting fragments).

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

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

3 - plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-NA, hydrolyzed by XhoI (13496 bp);

4 - plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-NA, hydrolyzed by PstI (11686 bp + 1810 bp);

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

In FIG. 40 shows 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 gene cassette HA (HongKong / 4801), M1 (California / 04/2009) and NA (California / 04/2009), SalI and PstI restriction endonucleases (the lengths of the resulting fragments are indicated in parentheses).

1, 2, 3, 4, 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 bp);

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

FIG. 41 shows the result of electrophoretic separation on a 1.2% agarose gel of LR recombination products between pEntry_HA (HongKong / 4801) -M1-NA plasmid DNA and pLenti6.3 / TO / V5-DEST.

1, 2, 3 - non-target products of recombination;

4, 5, 6, 7 — target product of recombination pEntry_HA (HongKong / 4801) -M1-NA;

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

Fig. 42 illustrates 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 (A / HongKong) -M1-NA, restriction endonucleases XhoI, EcoRI, PstI and HindIII (in parentheses the length of the resulting fragments).

1 - plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed by PstI (11682 bp + 1761 bp);

2 - plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed with HindIII (5576 bp + 3344 bp + 2590 bp + 584 bp + 556 bp N. + 481 bp + 312 bp);

3 - plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed by EcoRI (9087 bp + 4356 bp);

4 - plasmid pLenti6.3 / TO / HA (A / HongKong) -M1-NA, hydrolyzed by XhoI (8063 bp + 5380 bp);

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

In FIG. 43 shows the result of electrophoretic separation in 1.2% agarose gel of independent clones of the recombinant plasmid pEntry_HA (H1N1-Mich) -M1-NA containing the HA gene cassette (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009 ) and NA (California / 04/2009).

1, 2, 3, 4, 5, 6 — independent clones of the plasmid pEntry_HA (H1N1-Mich) -M1-NA carrying the HA gene cassette (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04 / 2009) and NA (California / 04/2009);

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

In FIG. 44 shows the result of electrophoretic separation in 1.2% gel of DNA fragments obtained after hydrolysis of independent clones of the recombinant plasmid pEntry_HA (H1N1-Mich) -M1-NA containing the HA gene cassette (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, 2, 3, 4, 5 - independent clones of the plasmid pEntry_HA (H1N1-Mich) -M1-NA carrying the HA gene cassette (A / Michigan / 45/2015 (H1N1) pdm09), M1 (California / 04/2009) and NA (California / 04/2009) (8164 bp + 1711 bp);

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

The invention is also illustrated by the following 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 / 3073/2013

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.

EXAMPLES

For the development of producer cells, a vector with tetracycline-regulated expression of the 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 Vis of the collection of FBUN 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. PCR and RT-PCR

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 KCl, 1.5 mM MgCb, UMM 2-mercaptoethanol, 0.1% Triton X-100), 0.2 mm each dNTP, oligonucleotide primers in an amount of 20 pmol each, 5 u Taq polymerase, 1 μl of DNA solution. The reaction was carried out in a programmable thermostat GeneAmp PCR-system 6700 using the following program:

Number of cycles Temperature (° C) Time one 95 5 minutes 94 30 seconds twenty 55 30 seconds 72 1-2 minutes one 72 10 minutes

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:

Number of cycles Temperature (° C) Time one fifty 30 minutes one 95 15 minutes 94 45 seconds 40 55 45 seconds 72 1-2 minutes one 72 10 minutes

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 ° С to an optical density of Ds65 = 0.8 - 0.85 under 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 DNA 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. The selection of plasmid DNA 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:

Number of cycles Temperature (° C) Time 96 10 Seconds thirty fifty 15 seconds 60 4 minutes

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, USA).

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 attRl 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 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 at ZAO Evrogen Ru using pEntry plasmid suitable for LR recombination with pLenti6.3 / TO / V5-DEST plasmid DNA.

2.2. Obtaining and analysis of recombinant pEntry plasmids containing 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 by means of the Oligo program (version 6.31) of 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 this work are shown in Table 1.

Table 1.

Oligonucleotide primers used in this work for PCR, RT-PCR and sequencing

No. Primer Name Primer Sequences (5'-3 ') one HA_SalI_upper GTCGACACCATGAAGGCAATACTAGTAGTTCTGC 2 HA_EcoR_lower GAATTCTCATTAAATACATATTCTACACTGTAGAGACC 3 HA_upper ACCATGAAGGCAATACTAGTAGTTCTGC

four HA_lower TCATTAAATACATATTCTACACTGTAGAGACC 5 HAH1N1 _fr1 _lower AGAGACTTTGTTGGTCAGCACT 6 H A_H1N1 _fr2_upper GATAAAGGGAAAGAAGTCCTCG 7 HA_H1N1 _fr2_lower TCTGTGTGCTCTTCAGGTCG 8 H A_H1N1 _fr3_upper ATGGATGGTACGGTTATCACC 9 NA_SalI_upper GTCGACACCATGAATCCAAACCAAAAGATAATAA one NA_EcoRI_lower GAATTCTCATTACTTGTCAATGGTAAATGGCA one NA_upper ACCATGAATCCAAACCAAAAGATAATAA one NA_lower TCATTACTTGTCAATGGTAAATGGCA one NA_frl_lower ATGGAGAGGGAACTTCACCA one NA_fr2_upper GACAGGAGCCCATATCGAAC one NA fr2 lower TTGTCTCCGAAAATCCCACT one NA_fr3_upper GACCGTGGGTGTCTTTCAAC one M1_SalI_upper GTCGACACCATGAGTCTTCTAACCGAGGTCG one M1_EcoRI_lower GAATTCTTATCACTTGAATCGCTGCATCT one M1_upper ACCATGAGTCTTCTAACCGAGGTCG 2 M1_lower TTATCACTTGAATCGCTGCATCT 2 HA_H3N2_SalI_upper GTCGACACCATGAAGACTATCATTGCTTTTGAGC 2 HA_H3N2_PstI_lower CTGCAGTTATCAAATGCAAATGTTGCACC 2 HA_H3N2_upper ACCATGAAGACTATCATTGCTTTGAGC 2 HA_H3N2_ lower TTATCAAATGCAAATGTTGCACC 2 HA_H3N2_fr1_1оwer GGTGAACCCCCCAAATGTA HA_H3N2_fr2_upper ACCCAGCATTGAACGTGACT HA_H3N2_fr2_lower TCCCTCAGAATTTTGATGCC 2 HA_H3N2_fr3_upper TAGAAAATGGTTGGGAGGGA 2 HA_B_SalI_upper GTCGACACCATGAAGGCAATAATTGTACTACTCAT HA_B_Zsp2I_lower ATGCATTCATTATAGACAGATGGAGCATGAAAC 3 HA_B_upper ACCATGAAGGCAATAATTGTACTACTCAT 3 HA_B_lower TCATTATAGACAGATGGAGCATGAAAC HA_B_fr 1 _lo wer TTTTGTTATCTGAATGGAACCC

HA_B_fr2_upper CCATACATTTGTGCAGAAGGAG 3 HA_B_fr2_lower CTCTTAAGGTCTGCAGCAACTG 3 HA_B_fr3_upper AAGGAATGATTGCAGGTTGG 3 CMV_upper CGCAAATGGGCGGTAGGCGTG 3 V5 (C-term) _lower ACCGAGGAGAGGGTTAGGGAT

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 by PCR or RT-PCR (Fig. 4). To obtain HA, we used a pair of HA_SalI_upper and HA_EcoRI_lower primers and a plasmid containing influenza virus 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 and 6). 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, clones of recombinant plasmid DNA pDrive with a confirmed nucleotide sequence of the inserted HA, M1, and NA genes were used.

Further, HA, M1, and NA fragments isolated from recombinant plasmids at the SalI and EcoRI sites were cloned into the pEntry plasmid previously hydrolyzed with these restriction enzymes (Fig. 7). 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).

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). 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 compared to the H1N1 hemagglutinin sequence, Fig. 10) and sequencing.

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

The influenza B virus hemagglutinin gene was synthesized at ZAO Evrogen Ru. The synthesized 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 based on the available nucleotide sequences in the GISAID EpiFlu database (Fig. 17).

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 was synthesized by CJSC Evrogen Ru (Russian Federation). The nucleotide sequence of the gene is represented by SEQ ID NO: 6.

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).

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 was synthesized by CJSC Evrogen Ru (Russian Federation). The nucleotide sequence of the gene is represented by SEQ ID NO: 7.

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 based on the available nucleotide sequences in the GISAID EpiFlu database (Fig. 19).

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 was synthesized by CJSC Evrogen Ru (Russian Federation). The nucleotide sequence of the gene 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 at ZAO Evrogen Ru as part of a vector plasmid.

Next, the obtained branded 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), carrying 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 branded plasmid DNA pLenti6.3 / TO / V5-DEST (Fig. 10) with plasmid DNA pEntry_ HA (HongKong / 4801) -M1- NA. 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 (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 by restriction analysis and sequencing.

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).

The artificial gene encoding the hemagglutinin of strain B / Brisbane / 60/2008 (H3N2) was calculated and synthesized in ZAO Evrogen Ru 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 with data of hydrolysis 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 (Fig. 10) with plasmid DNA pEntry_ HA (B- Brisbane) -M1-NA. 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 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 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 at ZAO Evrogen Ru 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 SalI and PstI restriction endonucleases 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 (Fig. 10) with pEntry_HA plasmid DNA (H1N1-Mich ) -M1-NA. 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 Sali HA PstI IRES M1 WPRE CMV NA attL2

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

• IRES - site of the internal landing of ribosomes;

• CMV - cytomegalovirus promoter;

• NA - the gene encoding the hemagglutinin of the influenza virus (California / 04/2009);

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

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

• NA is the gene encoding the influenza virus neuraminidase (California / 04/2009).

The gene encoding the hemmagglutinin of strain B / Brisbane / 60/2008 (H3N2) was synthesized at EvrogenRu CJSC as part of a vector plasmid, and the gene is 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-NA (California / 04/2009) was digested with SalI and PstI restriction endonucleases. Using the indicated restriction endonucleases, the plasmid DNA obtained from ZAO EvrogenRu 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 (Fig. 3). 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, clones of recombinant plasmid DNA pEntry_HA (B-Brisbane) -M1-NA with the confirmed nucleotide sequence of the inserted gene encoding hemagglutinin were used.

3.3.2. Obtaining and analysis of the 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 HA gene cassette (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_HA (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 clone 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 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. 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-NA, hydrolyzed by HindIII;

2: plasmid pLenti6.3 / TO / HA (B-Brisbane) -M1-NA, 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).

Design Length, bp - HindIII EcoRI Xhoi PstI pLenti6.3 / TO / HA (B-Brisbane) -M1-NA 13496 5580; 3344; 2639; 584; 556; 481; 312 9091; 4405 13496 11,686; 1810

3.3.3. Obtaining and analysis of recombinant plasmids 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 that for 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 by the gene encoding the hemagglutinin of strain A / HongKong / 4801/2014 (H3N2) .

The gene encoding the hemagglutinin of strain A / HongKong / 4801/2014 (H3N2) was synthesized at ZAO EvrogenRu, 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 plasmid synthesized by EvrogenRu CJSC, containing the hemagglutinin gene of the required strain, were hydrolyzed 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 (Fig. 8) and sequencing. For further work, a clone without nucleotide substitutions was selected.

3.3.4. Obtaining and analysis of the 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 . Competent E. coli strain Stbl3 cells were transformed with the products of a recombination reaction. 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 clonal variant pLenti6.3 / TO / HA (A / HongKong) -M1-NA does not contain nucleotide substitutions with respect 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 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 (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 with 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).

Design Length, bp - PstI HindIII EcoRI Xhoi pLenti6.3 / TO / HA (A / HongKong) -M1-NA 13443 11,682; 1761 5576; 3344; 2590; 584; 556; 481; 312 9087; 4356 8063; 5380

3.3.5. Obtaining and analysis of recombinant plasmids 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. Information on the nucleotide sequence of the hemagglutinin gene was obtained from the GISAID EpiFlu database (Fig. 11). 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 a part of plasmid DNA at CJSC EvrogenRu. Then, the obtained plasmid DNA was hydrolyzed at the SalI and PstI restriction endonucleases sites. In addition, plasmid DNA pEntry_HA-M1-NA (California / 04/2009) was hydrolyzed by 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 by SalI and PstI restriction endonucleases, and sequencing). A clone without nucleotide substitutions was used for further work.

3.3.6. Obtaining and analysis of the 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. Clonal plasmid DNA variants 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 target plasmids pLenti6.3 / TO / HA (H1N1-Mich) -M1-NA (Fig. 14). One of the plasmid DNA clones was hydrolyzed with restriction endonucleases XhoI, EcoRI, PstI and HindIII (Fig. 15). 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 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 (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).

Design Length, bp - HindIII EcoRI Xhoi PstI pLenti6.3 / TO / HA (B-Brisbane) -M1-NA 13492 5580; 3344; 2639; 584; 556; 481; 312 9091; 4405 13496 11,686; 1810

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, LR recombination of the proprietary plasmid DNA pLenti6.3 / TO / V5-DEST and previously synthesized plasmid DNA pEntry containing the gene cassette (Fig. 11). 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 of 40 analyzed were identified (Figure 12). The correctness of the nucleotide sequence is confirmed by sequencing.

Table 2.

Restriction analysis of recombinant plasmid DNA pLenti6.3 containing the target gene cassette HA (H1N1), M1 and NA

Design Length, bp - Xhoi EcoRI PstI HindIII pLenti6.3 / TO / ON (Н1N1) _М1_NA 1349 4220.9219 4348, 9091 1753, 11686 312, 481, 556, 584, 2582, 3344, 5580 pLenti6.3 / TO / V5- DEST 9351 9351 463.8888 1580, 7771 312, 481, 556, 584, 3344, 4074 pLenti6.3 / TO / Gus 9538 9538 9538 9538 312, 481, 556, 584, 3344, 4261

Thus, a lentiviral plasmid vector was created, carrying a cassette of the hemagglutinin, neuraminidase and M1 protein genes for influenza A virus (H1N1pdm09) and expressing a 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 = H1 protein: ACS45035 gene bank No. (H1N1)

• NA = N1 protein: No. of gene bank 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 the influenza virus.

Restriction sites of cassette expression vectors

Design The lengths of the resulting fragments, bp - A fIII Xhoi EcoRI PstI HindIII pLenti6.3 / TO / HA (H1N1) -M1-NA 13439 3656, 9783 4220, 9219 4348, 9091 1753, 11686 312, 481, 556, 584, 2582, 3344, 5580 Restriction site coordinates 13439 294, 10077 4130, 8350 2516, 6864 2525, 4278 308, 864, 1448, 1929, 4511, 10091, 13435

Restriction sites of cassette expression vectors

Design The lengths of the resulting fragments, bp - A fIII Xhoi EcoRI PstI HindIII pLenti6.3 / TO / HA (H3N2) -M1-NA 13436 3656, 9780 13436 22, 230, 1060, 3033, 9091 1750, 11686 312,481,556, 584, 1054, 1525, 3344, 5580 Restriction site coordinates 13436 294, 10074 8347 2516, 2746, 3806, 3828, 6861 2525, 4275 308, 864, 1448, 1929, 2983, 4508, 10088, 13432

Restriction sites of cassette expression vectors

Design The lengths of the resulting fragments, bp - A fIII Xhoi EcoRI PstI Hind iii 13505 3482, 4520, 48, 43 66, 1247, 312, 481, 556, 656, 8985 9091 12258 584, 746, 936, 6367 966, 3344, 5580 Restriction site coordinates pLenti6.3 / TO / HA (B) -M1-NA - A fIII Xhoi EcoRI PstI HindIII 13505 294, 3896, 2516, 2564, 2525, 308, 864, 3776, 8416 6930 3772 1448, 1929, 0143 2865, 3611, 4577, 10157, 13501

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

SEQ ID NO: 1 — 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. FOE method for determining the number of transducing units by immunochemical staining of transduced cells

A monolayer of the culture of target cells intended for transduction is grown in 96-well plates in a supporting medium (DMEMD00 units / ml penicillin, 100 μg / ml streptomycin, 300 μg / ml L-glutamine, 10 μg / ml DEAE-dextran (Sigma ), 5 μg / ml trypsin TRSK). 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 CO ₂ 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 monoclonal antibody preparation is being prepared for the HA1 protein of influenza A virus 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 = FOESp * 10N + 1 FOE / ml, where N is the number of 10-fold dilution of the pseudovirus in which the FFU was recorded.

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, the pseudovirus tube is removed from the nicotemperature 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% CO ₂ 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 assessing 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 HPV flu 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, it is necessary to dilute the washed red blood cells 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. FOE method for determining the number of cells expressing a reporter (target) gene by 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 CO ₂ 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 dilution of 1: 5000 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 of HPV-producing 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 solutions of 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 using a JEM 1400 electron microscope (Jeol, Japan); photography and image analysis were carried out 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 into the genome of producer cells

To isolate cell nuclei, cells are scraped off from a monolayer, approximately 106-107 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 MgCl2). 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 reagents FRT-50 F. Option. 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 conduct N reactions, 10 * (N + 1) μl of PCR mixture-FL, 5 * (N + 1) μl of PCR buffer-B, and 0.5 * (N + 1) mkh 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. Analysis of HPV flu production by H1N1M1 clones by electron microscopy

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).

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 filamentous form of particles prevails. In addition, in some areas groups of particles having a pleomorphic shape are found (Fig. 15).

The size of the spherical particles is typical for the influenza virus and is 80-120 nm, filiform HPV with a diameter of 90-100 nm and a length (on sections) up 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. Cell death by apoptosis was major (Fig. 16).

The results of the study by electron microscopy show that the morphology of virus-like particles in the cell culture of MDCK and HEK-293FT 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 the 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.

Table 3.

Detection of the influenza A virus hemagglutinin gene (H1N1pdm09) in PCR of the genomic DNA of influenza HPV producing cells

No. Clone Sample Type Ct one H9 MDCK DNA 26.47 2 H1 MDCK DNA 25.09 3 AT 12 MDCK DNA 26.08 four G10 MDCK DNA 29.41 5 G12 MDCK DNA 27.12 5 DNA- Negative control > 35 6 RT- Negative control > 35 7 PCR- Negative control > 35 8 k + N1 Positive control 19.42

Table 4.

Detection of the influenza A (H1N1) virus neuraminidase gene in PCR of the genomic DNA of influenza HPV producing cells

No. Clone Sample Type Ct one H9 MDCK DNA 29.64 2 H1 MDCK DNA 28.92 3 AT 12 MDCK DNA 29.47 four G10 MDCK DNA 32.84 5 G12 MDCK DNA 9.12 5 DNA- Negative control > 35 6 RT- Negative control > 35 7 PCR- Negative control > 35 8 k + Nl Positive control 18.82

Thus, the results of PCR diagnostics of cellular 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. Only pLenti6.3 plasmid was used as a lentiviral vector; 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 * 105 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 is taken from each well to evaluate the HPV production in the DGA. 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 into 75cm ² bottles, and the cell suspension is transferred to form a cell bank of the producers of HPV influenza virus.

Table 5.

The number of unpainted MDCK cells per 1000 cells of the monolayer

clone A (H1N1) clone A (H3N2) clone AT H1 N1 H1 N1 H1 N1 F4 450 465 A3 300 290 AT 5 690 700 C1 560 590 H12 670 610 E11 630 590 E9 350 400 E4 580 620 E4 680 610 D7 640 700 G7 730 750 H11 510 470 H8 750 670 D12 340 310 G3 680 650

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 HPV productivity assessment of the fifteen MDCK clones.

Table 6.

RGA titer of the supernatant of MDCK clones of HPV producing cells of influenza A (H1N1), A (H3N2) and B

A (H1N1) A (H3N2) AT clone RGA title clone RGA title clone RGA title F4 1:64 A3 1:64 AT 5 1:32 C1 1:32 H12 1: 128 E11 1:32 E9 1:32 E4 1:32 E4 1:16 D7 1:16 G7 1:64 H11 1:16 H8 1:64 D12 1: 128 G3 1:16

Five clones transfected with the 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 close 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 products of expression 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 agglutination of the latter.

Studies have also shown that MDCK cells are the best producers of HPV for the influenza virus, ensuring an HPV titer of RHA in the range of 1:64 to 1: 256, while clones obtained 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.

5.4. Methods of quality control of the culture of producer cells and HPV flu

To control the quality of producer cell cultures, twelve clones were selected for HA production intensity and growth characteristics. These clones were designated as: 5E4, 7F4, 10C12, 10E7, 3C5, 6D6, 9B3, 5G75G7, 6D12, 1F1. Part of the population of each clone was cryopreserved, and the other part was cultured for further analysis of the HPV flu.

The HPV flu proteins were separated on a 4-12% Bis-Tris gel with morpholine propanesulfonic acid buffer (hereinafter IX MOPS) (Invitrogen reagents). The gel was transferred to PVDF membranes and blocked. Table 7 lists the reagents and concentrations that were used to detect influenza virus proteins by Western blotting.

Table 7.

Reagents and Concentrations Used in Western Blotting

Antibody / protein goal Provider Test concentration Protein ON Positive control Protein Science 100 ng NA protein Positive control “A and Dee System” 100 ng Rabbit Antibodies to M1 Determination of protein M1 "Enzyme" 1: 500 Antibodies to rams to NA NA protein determination “A and Dee System” 1: 1000 Rabbit Antibodies to HA Definition of protein HA Protein Science 1: 500 IgGHRP goat antibodies Antibody Detection Santa Cruz 1: 15000 rabbits rabbits Rabbit Antibodies KlgGHRP Rams Determination of sheep antibodies Santa Cruz 1: 15000

In FIG. 17A and 17B show the results of analysis of HA expression by candidate clones by Western blotting method. A negative control (lysate of non-transduced MDCK cells) was used to detect non-specific antibody binding. The expected molecular weight of HA protein after lysis is approximately 80 kDa (arrow). The expected mass of non-lysed polyprotein is approximately 140 kDa. Negative control - MDCK cell lysate shows non-specific binding. A negative control was used to detect non-specific antibody binding. Negative control - MDCK cell lysate shows non-specific binding, and the NA positive control band indicates active antibody binding. The proteins synthesized in MDCK cells are glycosylated, so they will rise higher than non-glycosylated proteins. In FIG. 18A shows the active expression of HA by candidate clones 1F1, ZC5, 5G7, and FIG. 18B shows the active expression of NA by candidate clone 6D12. In FIG. 19A and 19B show the results of the analysis of M1 expression by candidate clones by Western blotting. A negative control (lysate of non-transduced MDCK cells) was used to detect non-specific antibody binding. The expected molecular weight of the processed M1 protein is approximately 25 kDa (arrow). In FIG. 19A shows active expression of M1 by candidate clones ZC5, 6D6 and weak expression of M1 by candidate clones 1F1 and 5G7. The negative control clone 7 MDCK shows non-specific binding. At the same time, massive bands of non-lysed protein are visible. This may be due to the fact that the processing of proteins of the transfected baseline cell line may be ineffective. In FIG. 19B shows the active expression of M1 by candidate clone 6D12. Negative control of MDCK cell lysate shows a lack of specific binding.

Table 8 summarizes the expression of hemagglutinin, neuraminidase, and Ml protein by candidate clones. Plus signs indicate the level of expression. Additional advantages indicate more active expression.

Table 8.

Summary of Western Blotting for Protein Expression

Clone number Western Blotting: ON Western Blotting: NA Western blotting: M1 5E4 (- / +) (-) (-) 7F4 (++) (+) (-) 10C12 (++) (++) (+) 10E7 (++) (-) (+) ZC5 (+++) (+++) (+++) 6D6 (++) (+) (++) 9VZ (++) (++) (+) 5G7 (+++) (+++) (++) 6D12 (+++) (++) (++) 1F1 (++) (++) (+)

5.5. Protocol for the study of the biological properties of producer cells of HPV influenza

5.5.1. Assessment of cell growth parameters.

The purpose of this analysis is to determine cell growth parameters. In particular, the cell population doubling time was determined (doubling time). Each candidate clone was cultured in 6 wells of a 12-well plate. 2 x 105 cells were placed in each well. Every 24 hours, cells were collected and counted in two wells. After plating, cells from each well were harvested after 24 hours, 48 hours and 72 hours. At each time point, the average values of two wells were determined, and the obtained value reflected the total number of cells formed within 24 hours. Harvesting cells after 24 hours was considered time point 0 to standardize the day of planting cells on the previous day. Doubling times were calculated on the doubling-time, com website using all time points.

Doubling ranges in a wide range - from about 18 hours to 41 hours. Since these cells will be grown in a bioreactor, production will be more efficient in the case of rapid cell multiplication and vice versa. The best performance was in clones 5G7, 1F1, 6D12. 4.3.2.

Rating of individual cell clones.

Data on protein expression and growth characteristics are summarized in Table 9. The table shows the expression levels of HA / NA / M1 proteins and the growth rate of clones in the form of doubling time.

The rating of candidate clones is determined by two factors. The first factor is the presence of all three HSPT proteins (HA, NA, M1) according to Western blotting. HPV will not form without these three proteins. The second factor is the doubling time. This is due to the cultivation of the cell line in the bioreactor. Slow cell line growth will limit the use of the reactor. For these two indicators, the 5G7 clone candidate was the best, as it showed active expression of all three proteins and the best doubling time. Clones 6D12 and 1F1 were rated 2 and 3. Clone candidate 3C5 was interesting due to the high level of protein expression. However, the doubling time for clone 3C5 was the worst.

Table 9.

Candidate Clone Rating Summary

Clone number Western Blotting: ON Western Blotting: NA Western blotting: M1 Growth indicators (doubling time), hour Clone candidate rating 5E4 (- / +) (-) (-) 27.73 7F4 C ++) (+) (-) 30.86 10C12 (++) (++) (+) 28.82 10E7 (++) (-) (+) 27.53 ZC5 (+++) (+++) (+++) 41.18 6D6 (++) (+) (++) 35.1 9VZ (++) (++) (+) 25.04 5G7 (+++) (+++) (++) 17.84 one 6D12 (+++) (++) C ++) 28.73 2 1F1 (++) (++) (+) 22.64 3

5.6. Evaluation of clones producing HPV H1N1M1.

Each of the H1N1M1 clones with the best rating was evaluated for sterility, viability after thawing and mycoplasma contamination. It was found that each of the clones was not contaminated by bacteria, fungi and mycoplasma, and their viability after thawing was more than 85%.

5.6.1. Inoculation of HPV-producing cells in the bioreactor.

We used the FibraStage bioreactor, which is an inexpensive, non-laborious, single-use cell culture system designed to produce proteins, viruses, or cell mass from substrate-dependent and suspension cell cultures. The system uses 500 ml disposable bottles with pre-placed FibraCel discs, which are a unique solid matrix for cell growth. Cells remain fixed in the disc layer throughout the process, which makes it easy to replace the medium and extract the final product. The system excludes autoclaving, purification, lengthy training, costly startup, cross-contamination between batches and requires only the use of a standard CO ₂ incubator.

A stable cell line from clone 6D12 expressing HPV flu was used for plating in two Fibra-Stage systems. Cells were cultured in high glucose DMEM (Hyclone) supplemented with 10% PBS / 4 mM glutamine / 1 mM sodium pyruvate. 21108 cells were placed in each Fibra-Stage system. The total volume of the medium was 450 ml. One capacity of the Fibra-Stage system contains 10 g of Fibra-disks. The density of cell seeding on Fibra-disks is 2 * 107. The following parameters were set on the control panel: 1.5 mm / s with a position at the top of 10 seconds and a position at the bottom of 2 minutes. The CO ₂ incubator was set to a temperature of 37 ° C and a CO ₂ concentration _of 5%. After plating the cells, the following medium was added to the Fibra-Stage: DMC high glucose (Hyclone) supplemented with 5% PBS, 4 mM glutamine / 1 mM sodium pyruvate / 1 mM HEPES.

5.6.2. Growth and maintenance of cells producing HPV flu.

To control the conditions and viability of the cells, daily medium samples were taken and evaluated using a NOVA Bio Profile analyzer. The glucose / glutamine / lactate / pH levels were carefully monitored.

These parameters were supported in the following limits:

Glucose start: 5 g / l replace medium if less than 1.5 g / l Glutamine start: 4 mmol / L replace medium if less than 1 mmol / l Lactate start: less than 1 mmol / l replace medium if more than 4 g / l in pH start: 7.2 replace medium if less than 6.8

As the cells grow, the glucose concentration will fall below 1.5 g / l until other parameters shift beyond the specified limits of the norm. When the glucose level drops below 1.5 g / l, the medium is completely replaced. Subsequently, as the cells grow, all glucose will be consumed within 24 hours. This occurred approximately 3 weeks after placement of the cells in the Fibra-Stage. At this point, the temperature drops from 37 ° C to 34 ° C. The incubator setting changes from 5% CO ₂ to 2.5%. The aim of the changes is to increase the production of HPV and inhibit cell growth. These changes in conditions will slow down cell growth for several days. However, glucose intake will start to rise again. At this point, the concentration of PBS in the medium also changes from 5% to 2.5%. Within 24 hours, the pH from 7.2 will drop to 6.8. Complete replacement of the medium will restore pH 7.2.

Each Fibra-stage reactor contains 10 grams of fiber discs. The initial glucose level is the amount of glucose in the fresh medium. Cells grow for approximately 24 hours without changing the medium. During this time, glucose concentration is measured and recorded. In FIG. Figure 34 shows the number of milligrams of glucose consumed per gram of disks per hour in FibraStage container No. 1.

5.6.3. Cell collection and quantification of Fibra-Stage samples for HA.

Samples were taken after two weeks of cell culture in the Fibra-Stage. After two weeks, the HA content in the samples was low, but after three weeks the RGA titer was 1: 128, which is an acceptable concentration of HA for purification. By the end of the Fibra-Stage cycle, the RGA titer was approximately 1: 256.

For purification, two separate cell pools prepared from each device — No. 1 and No. 2 — were prepared.

Table 10.

The diagram of the indicators of the maximum dilution of HA in the material of the cell pools of the RGA titer Volume ml ON ng / ml

Cell Pool No. 1 64 3500 301 1053 Cell Pool No. 2 128 6865 294 2018

EXAMPLE 6. Refinement of the technology for purification of HPV influenza to obtain monovalent beams from the supernatant of cells producing HPV influenza

6.1. Protocols for the selection and justification of the parameters of the HPV flu purification process to obtain monovalent beams from the supernatant of MDCK influenza HPV producing cells

6.1.1. Protocol 1

Schematic diagram of the isolation of HPV from the cell pool after cultivation:

1. Cell pool material 3.5 L

2. Lightening Filter Sartopore 2

3. Treatment with benzonase 10 U / ml overnight at + 4 ° C

4. Direct loading into a capsule 5 ml Mustang Q ion exchange chromatograph

5. EC, S-500

6. Cellufine S Affinity Chromatography

Lightening The cell pool material was clarified using a Sartopore 2 filter 0.8 / 0.45 μm and treated with benzonase (10 U / ml) overnight at 4 ° C. After treatment with benzonase, the concentration of NaCl in the clarified material was adjusted to 250 mM.

Mustang Q. 3580 ml of conditioned and clarified material was loaded into a 5 ml capsule of Mustang Q XT 5MSTGQPM6 (Pall Corp.). The loading rate was 20 ml / min and the elution rate was 10 ml / min. Trapped HPV was eluted with a step gradient of 40% (B = 3 M NaCl in 50 mM Tris-HCl, pH 7.4).

The HPV elution profile in the Mustang Q column is shown in FIG. 21.

Size exclusion chromatography (SEC). The eluted material from the Mustang Q column was subjected to EC for buffer exchange and further purification. 15 ml of material from a Mustang Q column with a maximum concentration of the material was loaded onto a K16 column with 120 ml Sephacryl S-500. The loading speed and resolution were 2 ml / min. Tris-HCI 50 mM + 100 mM NaCI buffer was used as the mobile phase. In FIG. 22 shows an EC profile of the peak material of a Mustang Q XT capsule. A sample with a maximum Vo score was obtained.

Table 11.

Breakthrough performance in the form of RGA titer according to the results of the analysis of the filtrate / washing samples from the Mustang Q column

Mustang Q Volume ON ON (ng / ml) filtration in ml (title RGA) 400 16 75,2 800 16 75,2 1200 16 75,2 1600 16 75,2 2000 16 75,2 2400 16 75.2 2800 16 75,2 3200 16 75,2 3600 16 75,2 Washing 16 75,2

Affinity chromatography. A 1 ml SEC column with a maximum Vo value was loaded onto a Cellufine S column with a volume of 1 ml. After the unbound material was obtained, a 40% step gradient was applied to displace the bound material. The peak sample after treatment with a step gradient of 40% was dialyzed using 1 L of buffer A (50 mM Tris HCl + 100 mM NaCl, pH 7.4). In FIG. 23 shows a Cellufine S profile for processing peak material obtained by EC. Peak material was obtained after treatment with a step gradient of 40%. The peak sample after treatment with a step gradient of 40% was dialyzed using 1 L of buffer A (50 mM Tris HCl + 100 mM NaCl, pH 7.4).

In FIG. 24 shows a profile of 2 ml of EC material with a peak value of Vo loaded onto a 1 ml Cellufine column. In order to identify the overload of the column, several runs were carried out through the same column (with leaks between runs) of different volumes of EC material with a peak value of Vo.

In a run with a load of 0.5 ml of the second (HPV), the maximum optical density (OD) was 10 milli units in an elution of 2 ml. In a run with a load of 1 ml, second (HPV), the maximum OD was 20 milli-units under elution conditions of 2.3 ml, and in a run with a load of 2 ml of second (HPV), the maximum OD was 40 mil-units under elution 3, 5 ml There is a direct correlation between the loaded volume and the value, which means there is no overload of the HPV saturation column. At each stage, samples were taken and analyzed for NA activity. The results obtained are shown in Table 12.

The amount of HA can be calculated using the positive control of HA that was used on each plate. The yield (%) was calculated by the total amount of antigen after each stage relative to the previous one. It was also noted that in the last step, 2 ml was loaded into the Cellufine S column from 37 ml of material after the SEC column. Normalization shows that during the purification, the activity of HA decreased after the Mustang Q column to 78%, after EC - to 42%, there were practically no losses after the Cellufine S column (yield - 97%). Thus, the total loss of antigen during purification and concentration according to Protocol 1 was 67%.

Table 12.

Indices of NA according to the results of isolation of HPV material of cell pool No. 1

Samples RGA title Volume ON (ng / ml) Total ON (mcg) The output of the antigen relative to the previous stage,% Material collected 16 3500 301 1053 one hundred Clarified Material (CH) 16 3580 301 1077 one hundred Benzonase Treated CH 16 3580 301 1077 one hundred Maximum Mustang Q 1024 17.5 48128 842 78 Sterile material EC 2048 37 9626 356 42

Sterile material Cellufine S 512 8.5 2406 twenty 97 The output of the antigen,% 33

6.1.2. Protocol 2.

In order to increase the yield of antigen during chromatographic purification and concentration, some modifications were introduced into the process.

Ultrafiltration of the supernatant of producer cells.

Ultrafiltration of the supernatant of producer cells containing HPV H1N1 Ml influenza is carried out in two stages by pumping the material through a pressure vessel with a volume of 1 JI pump under a pressure of 2 bar. At the first stage, at the outlet of the pressure vessel, the Sartorius stedim biotech filter capsule Sartopure GF + 1.2 pm is installed, through which the material is pumped. In a second step, material is pumped through a Sartorius stedim biotech Sartopure GF + 0.65 pm filter capsule. The ultrafiltration procedure takes place at room temperature. A 1 ml sample was taken for subsequent analysis.

Concentration of HPV flu.

Concentration of HPV influenza H1N1M1 is carried out by ion exchange chromatography. Ion-exchange chromatography is carried out using the GE Akta Purifier chromatographic system, consisting of P-900 gradient pumps, a UV-900 detector, a pH / C-900 pH conductivity meter and a conductivity test system with Voh-900 inlet and outlet valves. The system is managed by the Unicorn software. A 5 ml Mustang Q XT5 ion exchange capsule from Pall Corporation is used as a sorption chromatographic column. Detection is carried out at two wavelengths - 210 nm and 280 nm.

Conditions for ion exchange chromatography (IOX).

The total pressure in the chromatographic system does not exceed 0.8 MPa. The maximum flow rate of the eluent is 10 ml / min. Al solution: 1 M NaOH, Solution A2: 50 hM Tris-HCl, pH 7.5, Solution B1: ultrafiltered cell supernatant containing HPV influenza, Solution B2: 50 mM Tris-HCl, pH 7.5 + 1M NaCl.

Before chromatography begins, the capsule is equilibrated with A1 solution (at least 6 column volumes) to constant values of optical density, pH and conductivity, after which the flow of the eluate is stopped for 30 minutes. After keeping the capsule in solution A1 after 30 minutes, the capsule is washed with solution A2 (at least 10 column volumes) until constant values of optical density, pH and conductivity occur. After washing through a capsule, a solution of B1 containing HPV influenza is pushed through in a volume not exceeding 100 volumes of the capsule. The biological material adsorbed onto the capsule is eluted with solution B2. The formed chromatographic peak is collected in a separate container in a volume not exceeding 15 ml (not more than 3 column volumes). A sample of 500 μl was taken from the collected concentrate for subsequent analysis.

Intermediate analysis of HPV concentrate and breakthrough after IOC.

The concentrate and the breakthrough from the IOC are analyzed in a hemagglutination reaction (RGA) with rooster red blood cells. In the conical wells of the plate, 50 μl of the analyzed sample and 50 μl of a erythrocyte solution in 0.01 phosphate-saline buffer with the addition of 0.135 M NaCl, pH 6.5-7 are mixed. As a result of serial two-fold dilutions of the analyzed sample in red blood cells, the HPA RGA titer is determined. The filled tablet is aged for 30 minutes at a temperature of +4 ° C. The RGA titer is determined in three samples: ultrafiltrofan supernatant, concentrate after IOX, slip after IOX.

Primary chromatographic purification of HPV flu.

The initial purification of influenza HPV from the concentrate is carried out by gel permeation chromatography (Gel filtration) using a GE Akta Purifier chromatographic system consisting of P-900 gradient pumps, a UV-900 detector, a pH meter and conductivity meter pH / C-900, and Sample inlet systems with Voh-900 inlet and outlet valves. The system is managed by Unicom software. As a preparative column, Pharmacia Fine Chemicals 250 × 25 mm is used, which is filled with a Sepharose CL-4B sorbent (Pharmacia Fine Chemicals). Detection is carried out at two wavelengths - 210 and 280 nm.

Chromatography conditions.

The flow rate of the eluent is 5 ml / min. Solution A1: 50 mM Tris-HCl, pH 7.5. Solution A2: HPV flu concentrate. Before cleaning, the chromatographic column is balanced by passing solution A1 to constant values of optical density, pH and conductivity.

Collect the target fraction with the HPV flu.

No more than 5 ml (0.04 sorbent volume) of solution A2 is introduced into the column and the chromatographic process is started using solution A1 as eluent and observing the optical density. Fractions are collected in separate containers - only three fractions, the first of which contains HPV flu. 500 μl of each fraction is selected for analysis in the RGA.

Secondary chromatographic purification of HPV flu.

Secondary purification of the HPV flu from the initially isolated HPV fraction is carried out by the Pseudo-affinity chromatography method using a GE Akta Purifier chromatographic system consisting of P-900 gradient pumps, a UV-900 detector, a pH / C-900 pH meter and conductometry, and an input system samples with inlet and outlet valves Вох-900. The system is managed by the Unicorn software. A 10 ml Thermo Scientific column is used as a preparative column, which is filled with a Cellufine Sulfate sorbent (JNC Corporation). Detection is carried out at two wavelengths - 210 and 280 nm.

Chromatography conditions.

Solution A1: 2 M NaCl, Solution A2: 50 mM Tris-HCl, pH 7.5, Solution B1: Sample with HPV influenza fractionated at the stage of gel filtration. Solution B2: 50 cM Tris-HCl, pH 7.5 + 0.6 M NaCl. Before chromatography begins, the column is washed with A1 solution (at least 2 column volumes) to constant values of optical density and conductivity. Next, the column is washed with A2 solution (at least 2 column volumes) until constant values of optical density, pH and conductivity occur. The flow rate is changed from 5 ml / min to 1.5 ml / min. After washing, a solution of B1 containing HPV flu is passed through the column in a volume not exceeding 2.5 column volumes. The biological material adsorbed onto the column is eluted with solution B2. The formed chromatographic peak is collected in a separate container in a volume not exceeding 10 ml (not more than 1 column volume). A sample of 500 μl was taken from the collected concentrate for subsequent analysis. Analysis of the purified preparation.

The finished sample is analyzed for the degree of concentration, hemagglutinating and neuraminidase activity, as well as for the degree of purification in analytical gel filtration and step electrophoresis.

6.2. Final HPV flu treatment results

Table 13 presents the results of evaluating the concentration and activity of HPV of three types. In all three cases, concentration was 200 times, while the maximum activity in the RGA was 1: 2048 for HPV-H1N1M1 and HPV-NA (B) N1M1, with hemagglutinin concentrations of 450 and 540 μg / ml, respectively. HPV-H3N1M1 at a hemagglutinin concentration of 450 μg / ml showed less agglutinating activity - 1: 1024.

Table 13.

The results of the concentration and purification of HPV-H1N1M1

Stage number The name of the technological stage of cleaning HPV-H1N1M1 Volume ml Caption, GAE Hemagglutinin concentration, mcg / ml 0 Harvesting HPV supernatant of MDCK producer cells (H1-N1-M1) 1000 64 - one Filter clarification at GF + 1.2 μm, Sartopure GF + 0.65 μm 950 16 - 2 HPV Concentration by Ion Chromatography on Mustang Q XT5 thirty 128 - 3 HPV gel filtration on a Sepharose CL-4B 60 128 - four HPV affinity chromatography on Cellufine Sulfate 5 2048 450

Table 14.

The results of the concentration and purification of HPV-HA (B) N1M1

Stage number The name of the technological stage of cleaning HPV-NA (V) N1M1 Volume ml Caption, GAE Hemagglutinin concentration, mcg / ml 0 Harvesting HPV supernatant of MDCK producer cells (HA (B) -Nl-Ml) 1000 32 - one Filter clarification Sartopure at GF + 1.2 μm, Sartopure GF + 0.65 μm 950 8 - 2 HPV Concentration by Ion Chromatography on Mustang Q XT5 thirty 256 - 3 HPV gel filtration on a Sepharose CL-4B 60 128 - four HPV affinity chromatography on Cellufine Sulfate 5 2048 540

Lemmley electrophoresis results.

The process of purification and concentration of HPV was controlled in electrophoresis for both individual stages of the process and only purified samples (Fig. 25). Samples were denatured in SDS and beta-mercaptoethanol with boiling for 10 minutes. Upper gel - 5%, Lower gel - 15%. The electrophoregram figures show that the initial product is quite clean, after concentration, the manifestation of impurity proteins is visible, which disappear after gel filtration. In FIG. 26 presents the results of the purification of three types of HPV in one electrophoresis. Traces of impurity proteins are not recorded, and the maximum optical density of hemagglutinin bands corresponds to HPV-NA (V) N1M1, then the lower optical density corresponds to HPV-H1N1M1, and the smallest of the three HPVs is recorded for HPV-H3N1M1.

Determination of the amount of residual cellular DNA in a purified concentrate of three types of HPV flu. DNA concentration was measured by two methods.

Method 1 (fluorimetric) using Qubit © dsDNA HS (High Sensitivity) Assay Kit, ThermoFisher Scientific, USA and Qubit fluorimeter (1.0). DNA from an HPV suspension was isolated using the PureLink Genomic DNA Mini kit (Invitrogen).

Method 2 (RT-PCR) using the resDNASEQ Quantitative MDCK DNA Kit PCR kit, AB-4464335 to quantify dog DNA, 100 DNA reactions from the HPV suspension were isolated using the PureLink Genomic DNA Mini kit (Invitrogen). Table 15 presents the results of measuring residual cell DNA in HPV after all stages of concentration and purification. Measurements according to method 1 give rather high values of impurity DNA for HPV-H1N1M1 and HPV-H3N1M1 equal to 87 and 98 ng / ml, respectively; in the HPV concentrate HA (B) N1M1, the level of impurity DNA is below the detection limit for this method (<10). The PCR method is more sensitive and specific for the detection of cellular DNA, in our measurements we used a special kit for the detection of DNA of MDCK cells. The results indicate a good agreement between the measurements obtained by method 1 and method 2 for Bn4-H3N1M1 and HPV-HA (B) N1M1, and significantly lower values for impurity cell DNA for the HPV-H1N1M1 concentrate. This difference is possibly due to the presence of double-stranded cellular RNA, which also fluoresces in method 1. Recalculation per dose of the vaccine shows that the total DNA mass of the three types of HPV in one dose is 3.39 ng, which does not exceed the threshold value of 10 ng. Attention should be paid to the fact that in the finished LF, cellular DNA is not detected at all, which is probably due to the fact that DNA is adsorbed on aluminum hydroxide.

Electron microscopic characterization of the purified concentrate of three types of HPV flu.

In FIG. 27-29 presents the results of the analysis of the purified HPV concentrate by electron microscopy. On HPV, H1N1M1 (Fig. 27) is present in abundance of spines of hemagglutinin. HPVs have an irregular shape of dumbbells, with spikes on the heads of dumbbells, the diameter of the heads is 100 nm, the length of the dumbbells is 200-300 nm. HPV concentration H1N1M1-1010 pieces / ml. On HPV H3N1M1 (Fig. 28) spikes of hemagglutinin are present in abundance. HPVs have an irregular shape of dumbbells, with spikes on the heads of the dumbbells, the diameter of the heads is 100 nm, the length of the dumbbells is 200-300 nm. The concentration of HPV is 109 units / ml.

Table 15.

The results of measuring impurity cell DNA in the HPV of three types

HPV name The measured concentration of HA and DNA in purified HPV concentrate Estimated DNA and GA mass for a single dose of vaccine (0.5 ml) hemagglutinin, m kg / ml DNA according to method 1, ng / ml DNA according to method 2, ng / ml DNA according to method 2, ng GA, mcg H1N1M1 450 87 13.67 0.45 15.0 H3N1M1 450 98 82.96 2.76 15.0 HA (B) N1M1 540 <10 6.51 0.18 15.0 The measured concentration of unbound DNA in the finished LF 0,0 Estimated total weight per vaccine dose 3.39 45.0

On HPV, H3N1M1 (Fig. 29) has an abundant spike of hemagglutinin. HPVs have an irregular shape of dumbbells, with spikes on the heads of dumbbells, the diameter of the heads is 100 nm, the length of the dumbbells is 200-300 nm. The concentration of HPV is 109 units / ml.

After the development of the technology for producing and culturing HPV producing cells, the most important step in creating the finished dosage form is the technology for purifying HPV influenza to obtain monovalent beams from the supernatant of the HPV influenza producing cells. We investigated two protocols for the concentration and purification of HPV flu from the supernatant of producer cells in order to ensure maximum yield of HPV and a minimum content of impurity cellular proteins and DNA. Both protocols contain the main stages of purification - clarification by filtration, concentration in ion exchange chromatography, purification of the concentrate by gel filtration and pseudo-affinity chromatography. The resulting HPV semi-finished product is concentrated by volume in 200-400 times. The HPV preparation purified according to protocol No. 1 is active in RGA with a titer of 1: 512, while concentration and purification according to protocol No. 2 provides a larger titer in RGA from 1: 1024 to 1: 2048, and the hemagglutinin concentration is about 500 μg / ml, while impurity cell proteins are not detected in the electrophoresis of monovalent HPV beams. The level of impurity cellular DNA in the preparations purified according to protocol No. 2 in terms of a single dose of GLF is 3.4 ng. An electron microscopic examination of HPV showed that after concentration and purification according to protocol No. 2, an HPV concentration of 109-1010 pieces / ml is achieved, the HPV itself has an irregular shape mainly in the form of clubs and dumbbells, and there are rarely spherical or icosahedral HPVs.

Thus, the developed and applied methods for the concentration and purification of HPV from the supernatant of producer cells make it possible to create a pharmacopoeial product.

EXAMPLE 7. Preparation of a multivalent influenza vaccine

The obtained monovalent HPV concentrates (purified HPV containing hemaglutinin H1N1; purified HPV containing hemagglutinin H3N2; purified HPV containing hemagglutinin B) as described above in the examples are diluted in PBS (pH 7.4) to the required concentration of HPV. Mixing diluted monovalent concentrates purified HPV. Carrying out a sterilizing filtration of the resulting mixture of three types of HPV. Preparation of a suspension of aluminum hydroxide in PBS. Conducting information on a suspension of HPV and aluminum hydroxide, sorption.

Release of the finished product.

Technological scheme for the production of Trivalent recombinant influenza vaccines (vaccine for the prevention of influenza [recombinant]), suspension for subcutaneous administration of 0.5 ml

Stage name Check Point Direct transfection with a cassette of three genes 1. Decision of the WHO meeting on the choice of influenza vaccine composition for the upcoming epidemic season 2. Request to the GISAID database - 3. Design and synthesis of the target gene (gene cassette) Chromatography, sequencing (CJSC "Evrogen Ru") 4. Cloning of the target gene into the vector pLenti6.3 / TO / V 5-DEST 5. Plasmid expressing the gene cassette of HA, NA, Ml pLenti6.3 / TO / HA-Ml-NA Restriction analysis, sequencing 6. Transfection of MDCK cell culture in 24-well plates with Lipofectamine 2000 + pLenti6.3 / TO / HA-Ml-NA. Transduction Efficiency 1-10% Measurement of hemagglutinating units in the supernatant in each well; Immunochemical staining of a monolayer of transduced cells in each well to control the expression of target proteins using monoclonal antibodies
• Anti-Influenza A Neuraminidase ab # 91646
• Anti-Influenza A ML protein ab # 20910
• Anti-Influenza A HI-Hemagglutinin ab # 169855
• Anti-Influenza A H3-Hemagglutinin ab # 66187
• Anti-Influenza In Hemagglutinin ab # 769815 7. Selection of clones of HPV producing cells by limiting dilutions in 96-well culture plates • by RGA titer in the supernatant of MDCK cell clones
• according to the proliferation rate of MDCK cell clones
• for immunochemical staining using MKAT clones of MDCK cells
• in electron microscopy of MDCK cell clones 8. Selection of candidates for clones transferred to the bank • lack of mycoplasma contamination of the selected clone-producer of HPV Transfer of cell cultures to the Bank for cryopreservation, characterization and deposition Creation and certification of banks of producers of virus-like particles Creation and certification of banks of producers of virus-like particles 9. Authenticity Sequencing of HA, NA, M1 fragments of genes in the cell genome 10. Genetic stability Karyological analysis 11. Genetic uniformity of the main bank Karyological analysis 12. A complete description of the biological characteristics of producer cells Morphology, karyological characteristics, growth properties, productivity in the RSA

13. A complete description of the biological characteristics of expression vectors Plasmid restriction map 14. Physiological measures to ensure and control the expression of the cloned gene in the producer cell Description of the conditions of cultivation of producer cells, RGA, immunochemical staining using MKAT to HA, NA, M1 15. Description of the genetic markers of producer cells Description of the HA-M1-NA gene cassette and primers 16. Design, genetic parameters and structure of the expression vector Map of expressed genes and regulatory regions in plasmids 17. The origin and identification of the cloned gene Gene identification number in the database 18. Description of the nucleotide sequence of the inserted gene and adjacent parts of the vector Description of the sequence of regions of plasmids with integrated genes and their flanking sites 19. The results of the mapping using restriction enzymes of PCR fragments of the gene cassette PCR of fragments of the HA-M1-NA gene cassette from producers and their restriction by specific endonucleases 20. Data on the stability of the expression system during storage of the main bank of cells obtained with or without exceeding the number of passages used for production Real-time PCR of the HA-M1-NA gene cassette - the average number of copies of the gene cassette in the genome of the producer cell 21. Lack of extraneous contaminants Mycoplasma control, sterility control 22. Creation of the main bank of cell culture MDCK (H1N1M1) At least 150 ampoules in a jar (of which 50 ampoules are used for controls). In each ampoule from 5 to 8 million cells 23. Report on the establishment of banks 24. The study of growth properties The study of morphology (light and electron microscopy), the determination of the method of cultivation, the timing of the formation of the monolayer, the proliferation index and viability 25. Lack of extraneous contaminants Monitoring viral contamination in cell cultures; Control of viral contamination in laboratory animals; control of viral contamination in chicken embryos; determination of viral contamination by electron microscopy 26. Species identity Determination of the stability of the karyotype (karyological analysis); Determination of the spectrum of isoenzymes of glucose-6-phosphate dehydrogenase, lactate dehydrogenase and nucleoside phosphorylase; Real-Time Polymerase Chain Reaction (PCR-RV) 27. Tumorigenicity control The method of invasiveness in organ cultures; Control on immunodeficient animals 28. Genetic stability Real-time PCR of target genes (HA, NA, Ml) 29. Genetic homogeneity of the bank Real-time PCR of target genes (HA, NA, Ml) 30. Physiological measures to ensure and control the expression of the cloned gene in the host cell Description of the conditions for the production of HPV, immunochemical staining of producer cells using MKAT and RGA supernatant

31. Data on the stability of the expression system during storage of the cell bank The productivity of producer cells by antigen in ELISA, RGA, RTGA HPV with reference serums 32. The study of oncogenicity of cell cultures Assessment of oncogenic properties in accordance with the recommendations “Certification of transplantable cell cultures” (Methodical recommendations. - M.: “FSBI GISK named after L. Tarasevich” of the Ministry of Health and Social Development of Russia, 2011) Bank transfer to production GLF production 33. Recovery of producer cells:
HPV containing hemagglutinin H1N1 HPV containing hemagglutinin H3N2 HPV containing hemagglutinin B after cryopreservation Producer Cell Viability Assessment 34. Production time (sowing production banks, cultivation, collection of supernatants):
HPV containing hemagglutinin H1N1 HPV containing hemagglutinin H3N2 HPV containing hemagglutinin B Determination of RGA titer in the supernatant 35. Concentration by ion exchange chromatography of a pool of supernatants: HPV containing hemagglutinin H1N1 HPV containing hemagglutinin H3N2 HPV containing hemagglutinin B 36. Gel filtration of concentrates: HPV containing hemagglutinin H1N1 HPV containing hemagglutinin H3N2 HPV containing hemagglutinin B 37. Affinity chromatography of gel filtrate fractions:
HPV containing hemagglutinin H1N1 HPV containing hemagglutinin H3N2 HPV containing hemagglutinin B 38. Obtaining monovalent banks of concentrates (liquid semi-finished product): purified HPV containing hemagglutinin H1N1 purified HPV containing hemagglutinin H3N2 purified HPV containing hemagglutinin B Determination of hemagglutinin concentration in ORID, total protein concentration, virus-like particle size, mycoplasma control, cell DNA and cell protein impurity content, ratio of HI, NA and Ml proteins; presence of NA, purity of HPV 39. Dilution of monovalent concentrate banks:
purified HPV containing hemagglutinin H1N1 purified HPV containing hemagglutinin H3N2 purified HPV containing hemagglutinin B in PBS (pH 7.4)

40. Mixing diluted monovalent beams of concentrates: purified HPV containing hemagglutinin H1N1 purified HPV containing hemagglutinin H3N2 purified HPV containing hemagglutinin B 41. Sterilizing filtration of the resulting mixture of three types of HPV: sterility, total protein HPV containing hemagglutinin H1N1
HPV containing hemagglutinin H3N2
HPV containing hemagglutinin B through a 0.22 μm membrane filter 42. Prefabricated Trivalent Recombinant Influenza Vaccine 43. Preparation of a suspension of aluminum hydroxide in the FSBR 44. Reduction of a suspension of HPV and aluminum hydroxide, sorption 45. Sorbed prefabricated Recombinant influenza vaccine 46. Release of the finished product Description, authenticity, mechanical inclusion, pH, sealing, recoverable volume, sedimentation stability time, sterility, presence of mycoplasma, pyrogenicity, abnormal toxicity, immunogenicity, antigen sorption completeness, aluminum hydroxide content, packaging, labeling, storage, shelf life

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Claims (3)

1. A method of producing a multivalent influenza vaccine, comprising obtaining MDCK cells producing surface proteins of the influenza virus, the first MDCK producer cell containing the lentiviral plasmid vector pLenti6.3 / TO / HA (H1N1) -M1-NA containing the hemagglutinin, neuraminidase genes and M1 influenza virus protein (H1N1) shown in FIG. 3 or FIG. 33; a second MDCK producer cell containing the lentiviral plasmid vector pLenti6.3 / TO / HA (B) -M1-NA containing the influenza B virus hemagglutinin gene as well as the influenza virus (H1N1) protein neuraminidase and M1 genes shown in FIG. 1 or FIG. 32; a third MDCK producer cell containing the pLenti6.3 / TO / HA (H3N2) -M1-NA lentiviral plasmid vector containing the cloned hemagglutinin gene from the H3N2 subtype strain, as well as the neuraminidase and M1 genes of the subtype strain (H1N1) shown in FIG. . 2 or FIG. 31; production and purification of HPV, where HPV is obtained by separate cultivation of these three cell cultures MDCK; wherein the purification includes clarification of the cell pool, benzonase treatment, HPV elution with a stepwise gradient, size exclusion chromatography, affinity chromatography, concentration by ion exchange chromatography and additional HPV purification in two stages by gel permeation chromatography and pseudo-affinity chromatography after purification; dilution of the obtained monovalent concentrates of purified HPV in PBS (pH 7.4) to the desired concentration; mixing diluted monovalent concentrates of purified HPV; sterilizing filtration of the resulting mixture of three types of HPV; preparation of a suspension of aluminum hydroxide in FSBR; conducting information on a suspension of HPV and aluminum hydroxide, sorption; release of the finished product. 2. The method according to p. 1, where the incubation of the culture of MDCK cells producing HPV influenza virus is carried out with an inoculum concentration of 3 × 103 cells / ml in bioreactors at a temperature of 37 ° C for 2-3 days in DMEM growth medium containing 10% embryonic calf serum, before the appearance of a monolayer, then the obtained producer cells are washed with phosphate buffer solution, OptiMEM supplementation medium is added with the addition of 5 μg / ml trypsin TRSC, the HPV time is performed at a temperature of (37 ± 1) ° С for 72 hours in the regime continuous stirring at scab rotation 10-11 rev / min, until the activity RGA 1:64 and above, the procedure of HPV flu developments can be repeated as long as the HPV harvest in the supernatant of the results of the RGA does not become less than 1:64. 3. The method according to p. 1, where the HPV contains genes for hemagglutinin, neuraminidase and M1 protein obtained from strain A / California / 04/2009; 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; 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; 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; the hemagglutinin gene is obtained from strain B / Phuket / 3073/13, and the neuraminidase and M1 protein genes are obtained from strain A / California / 04/2009 or the hemagglutinin gene is obtained from strain B / Brisbane / 60/2008, and the neuraminidase and M1 genes are obtained from strain A / California / 04/2009.

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