RU2366710C1 - Method for preparing genetically modified influenza virus and virus proteins and application thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method provides target modification of virulent properties of influenza virus by genetic modification of caspase and granzyme viral proteolysis sites. There is also described application of such genetically modified influenza virus as vaccines and therapeutic agents, translation and expression vectors of biologically active genetic elements, polypeptides and fragments thereof in an organism-recipient for metabolism targeting in bodies and tissues and as agents for selective elimination of damaged and tumour cells in an organism. The invention can be used in medicine.

EFFECT: well-directed attenuation or enhancement of viral virulence.

11 cl, 4 dwg, 2 tbl, 7 ex

Description

The invention relates to the field of biotechnology and medicine, in particular, a method for directionally changing the virulent properties of the virus and viral proteins.

There is a method of weakening the virulent properties of a virus by adapting it to multiply at a low temperature and obtaining vaccine strains based on it by recombination with a new actual virus strain [1, 2]. The resulting recombinant vaccine strain is applicable for intranasal administration, where the local temperature exceeds the viral optimum and weakens its reproduction. This method has three main disadvantages. First, the attenuating cold mutations of the adapted virus are random in nature and do not exclude the possibility of rapid genetic reversal to the original pathogenic phenotype of the virus. Secondly, the cold phenotype of the vaccine virus does not reduce the pathogenicity of the virus as such, but only reduces its ability to reproduce at elevated temperatures. Thirdly, to obtain a new attenuation donor, selection of new cold mutants with a random set of mutations that cannot be predicted and controlled by the signs of harmlessness, reproduction, compatibility with the genes of the actual virus, a recombinant donor, will be required.

Closest to the claimed technical solution relates to a method of weakening the virulence of viruses by the method of directed modification of hemagglutinin (HA) of the influenza virus, in which the site of proteolytic cleavage by cell trypsin-like proteases was subjected to a directed change [3]. Such directed modification of the HA protein made the virus insensitive or slightly sensitive to respiratory trypsin-like proteases, which led to the restriction of its reproduction in the infected organism and the weakening of its virulence. However, with such attenuation of the virus, difficulties arose with its reproduction and production, since the artificial use of activating proteases was required for the full reproduction and production of the virus.

The basis of the invention is the technical task to create a method for producing a genetically modified virus and viral proteins due to directed artificial modification of viral proteins in caspase sites, which would, while maintaining the ability of viruses to reproduce, use them as vaccine strains for the prevention of infectious diseases and increase immunity.

The problem is solved by the fact that, according to the invention, a method for producing a genetically modified influenza virus and viral proteins by introducing mutations, deletions, inserts into parts of the viral genome encoding caspase or granzyme sites or their motives, or creating additional caspase sites and their motifs for viral proteins by implementing site-directed mutagenesis and reverse genetics. During the modification process, the natural characteristics of sites / motifs are violated in such a way that the resulting modified influenza virus is an attenuated (i.e. attenuated) virus or the resulting modified influenza virus has increased virulent properties and the viral proteins are characterized by increased toxicity.

When introducing mutations, it is advisable to additionally incorporate foreign genes (chimeric viruses) that encode proteins, polypeptides and their fragments that enhance the expression, therapeutic, and prophylactic properties of the resulting viruses.

The problem is also solved by the fact that it is proposed, according to the invention, to use a genetically modified influenza virus and viral proteins to prevent infectious diseases and create antiviral immunity by introducing into the human or animal body inhalation, subcutaneous, cutaneous, intradermal, intramuscular, rectal, by mouth.

It is advisable to use them in aerosol form or in the form of a spray to optimize the therapeutic and prophylactic effect. It is also proposed to use them as producers for the production of viral and other proteins with useful properties, as substrates for the preparation of subunit and subvirion vaccines or their protectors and enhancers.

It is advisable to use the influenza virus as vectors for the directed transfer and expression in the recipient organism of new antigens, chimeric proteins, and therapeutic compounds of a polypeptide nature, as well as for targeted cell cytolysis and treatment of tumor and proliferative processes.

The technical result of the present invention is that the attenuation (attenuation) of the virus is achieved by directed artificial modification of the viral proteins at the sites (caspase or granzyme sites) of a specific proteolytic cleavage by proteolytic enzymes of the host cell, the so-called caspase or granzyme.

Due to artificial modification using site-directed mutagenesis of such sites in the NP or M2 protein in highly virulent, for example, avian influenza virus and using the reverse genetics method, mutant variants of a mature virus (the so-called recombinant virus) are obtained. As a result of the modification, mutant viral variants with reduced virulence (attenuated viruses) were synthesized, which, however, retained the ability to reproduce, or more virulent strains with enhanced virulence for chickens. At that time, in particular, as the initial wild type of chicken plague virus quickly killed chickens, the attenuated caspase mutants of this virus, reproducing well in the chickens, did not cause their death and, it is important to emphasize, induced full immunity and protected the chickens from death in the subsequent infection with a highly virulent wild virus.

The obtained mutant viruses can be useful for medicine and veterinary medicine as vaccine strains, as vectors for the directed transfer of foreign genes and their expression products in the recipient organism to target organs for the purpose of therapeutic effect on affected tissues and organs.

It is well known that many viral proteins in most human and animal viruses are susceptible to proteolytic cleavage by cellular caspase proteases or granzymes close to them. Caspases and granzymes are proteases that break down numerous cellular and viral proteins at specific sites (caspase sites) into aspartic acid residues (Asp) during programmed cell death (apoptosis) [4,5]. Some examples of amino acid sequences at the sites of caspase sites of viral proteins are shown in table 1.

Table 1 Viruses ^(**) Caspian sites ^(*) Herpes viruses DCVD Poxviruses LVAD Flu viruses DVDD, METD, MDID, SEAD, DSSD Flaviviruses TEVD, SGVD Coronaviruses Vvpd Baculoviruses DCMD (*) The table uses a single-letter code for amino acids (**) information is given in [6].

The primary structure of caspase sites in numerous proteins of various animals and microorganisms, including viruses, usually meets the rules EXD / Y, QXD / Y, LXD / Y or DXXD / Y, in which E is glutamic acid, D is aspartic acid, X is any amino acids, Y - any amino acids, but more preferably Gly, Ala, Thr, Ser and Asn [5]. It is known that the main nucleocapsid protein NP (mm 56 kD) of human influenza A viruses has a DETD ₁₆ / G motif in the N-terminal region, which is characteristic for cleavage by cell caspases. In the process of apoptosis of cells infected with human influenza viruses, NP protein is cleaved by caspases at this site with the formation of the apoptotic form of aNP (53 kD) and a short N-terminal 3 kD peptide. In avian influenza viruses, the NP protein contains G ₁₆ instead of D, which violates the caspase motif DETD ₁₆ / G and makes it resistant to proteolysis by cell caspases [7]. Another characteristic caspase (or possibly granzyme) motif VDVDD ₉₀ / G was found in the C-terminal region of the M2 viral protein (14 kD) [8], which acts as the viral proton channel. This caspase / granzyme site was identified in all studied influenza A viruses of both humans and birds and its cleavage leads to the formation of a shortened apoptotic product aM2 with m.m. 12 kDa in infected cells. The vast majority of human and animal viruses contain similar caspase sites or their motifs in the proteins encoded by them, but the function and role of these caspase sites in the reproduction of viruses and the pathogenesis of viral infection remains unclear [6].

As a prototype in the present invention, the natural avian plague virus A / FPV / Rostock / 34 (A / FPV / Ro) (H7N1) was used, which was highly pathogenic for chickens and chickens and caused their death when a small dose of the virus was introduced into the body of birds ( 1-5 PFU per individual). As noted above, the M2 and NP proteins of this avian virus have two caspase motifs. To carry out a directed modification of these motifs, the so-called reverse genetics technique was used, for which a complete set of DNA copies of all viral genes (PB1, PB2, PA, NP, HA, NA, M, NS) of the A / FPV / Ro virus inserted into plasmids was synthesized. pHH-21, and 4 pcDNA3 plasmids were additionally prepared with the built-in genes for the proteins PB1, PB2, PA and NP, used to enhance the formation of the recombinant virus in transfected cells. Using this set of plasmids, virus variants were obtained in the genes of which specific mutations were made using site-directed mutagenesis.

Using this technique of artificial mutation of viruses, 4 recombinant variants of the virus were obtained: wild unchanged virus (wtr) with mutation G16 → D in the caspase site of the NP gene (NPgd), with a deletion G16 in the caspase site of NP (NPdel) and mutation DD90 → NN in caspase site of the M2 gene (M2nn). Mutation G16 → D restored a full-fledged caspase site in the NP protein of avian virus, which is a characteristic feature of human influenza viruses, and made the NP protein sensitive to caspases. Deletion of the G16 residue at the caspase site of the NP protein damaged this site and enhanced its resistance to cleavage by cell caspases. The DD → NN mutation also violated the caspase (granzyme) motif in the M2 protein and forbade its cleavage by cell caspases. Thus, three artificial variants of the recombinant avian influenza virus were obtained.

Next, the resulting viruses were propagated in a dog cell culture (MDSK line) and the obtained mutant virus samples were tested for biological activity. To verify the biological properties of the obtained caspase mutants of the virus, cell cultures of various origin (the so-called in vitro system) and chickens (in vivo system), which are the natural host of the A / FPV / Ro / 34 influenza virus, were used.

During passages in cell cultures of human cells (intestinal cell line CACO-2), dogs (cell line of the kidney of a dog MDSK), chicken (primary culture of chicken embryo fibroblasts, CF), all viral variants propagated well and preserved the mutations for many passages. Thus, caspase virus mutants have proven to be stable species.

Next, the virulent activity of the obtained mutants was checked. To assess the virulence of viral mutants, their lethal effect on chickens was compared with respect to wild virus, which, as is known, has a high lethal effect on chickens. It has been found that various caspase virus mutants change their virulence differently. The NPgd and M2pp mutants had markedly reduced virulence, while the virulence of the NPdel mutant exceeded that of the wild type. Thus, by modifying caspase regions of the virus, it is possible to differentially influence virulence by enhancing or attenuating it, depending on the task and the beneficial use of such viral variants. Options with enhanced virulence can be used for targeted cell lysis in oncology, and attenuated viruses can be used as antiviral vaccines or vectors for the transfer and targeted delivery of genes of biologically active substances in the body.

In the final part of the invention, the possibility of using attenuated NPgd and M2pp mutants for the prevention and protection against death caused by a highly pathogenic wild virus is shown. To obtain such a technical result, the chickens were initially infected with mutant NPgd and M2pp viruses and then re-infected after some time with a lethal dose of the highly pathogenic wild chicken plague virus A / FPV / Ro / 34 (H7N1). If in the control group of chickens not infected with pre-mutant viruses, rapid death was observed, then in pre-infected caspase mutants there were no signs of the disease and 100% survival. These results show that caspase mutants of the virus cause the formation of full immunity and complete protection of the body from lethal doses of the virus and can be used as vaccines.

It is well known that foreign genes can be inserted into the genome of various viruses that colline biologically active polypeptides with useful medical properties, such as interferon, interleukin, cytokines, inducers or suppressors of cell division, oncolytics, hormones, etc. [9]. When infected with such chimeric viruses in the recipient organism, the built-in genes will be expressed at the virus propagation sites and biologically active polypeptides will be synthesized. As such carriers (delivery vectors), only attenuated viruses that do not cause serious pathology in the recipient organism can be used. Caspous virus mutants, possessing such qualities of non-pathogenic viruses, can be used as vector systems for targeted delivery and synthesis of various biologically active compounds of polypeptide nature to various organs and tissues of the body.

Next, with reference to the proposed drawings, a specific embodiment of the proposed method is described in detail, in which:

figure 1 depicts an analysis of the transfer, synthesis and proteolytic cleavage of caspases NP and M2 proteins in caspase mutants of the avian influenza virus;

figure 2 depicts the propagation of caspase mutants of the chicken plague virus and the expression of its proteins in cells of various origin;

figure 3 - the dynamics of the weight of chickens infected with variants of WTR, NPgd, NPdel and M2nn of bird plague virus;

figure 4 - dynamics of the propagation of human influenza viruses A / WSN / 33 (WT) and its caspase mutant NPG.

Examples of carrying out the invention

Example 1. Artificial production of mutant variants of avian influenza virus.

To obtain the recombinant wild-type virus (wtr), eight plasmids were used containing full copies of the chicken plague virus RNA segments A / FPV / Rostock / 34 (A / FPV / Ro / 34) (H7N1) integrated into the pHH21 plasmid at the BspMl (PB1 sites and NP), Bsal (PB2, PA, HA, and NA), or BsmBl (M and NS). In addition, 4 expression plasmids containing the genes of the viral proteins PB1 (pcDNA-PBl), PB2 (pcDNA-PB2), PA (pcDNA-PA) and NP (pcDNA-NP) were synthesized, which were used to enhance the synthesis of recombinant virus in transfected cells, as described in [10]. A suspension of 293T cells (~ 10 ⁶ cells) in Opti-M medium (Gibco BRL) was mixed with 0.5 ml of lipofectamine / 2000 complex and a set of 12 plasmids (1 μg of each plasmid) and incubated for 4 hours at 37 ° C. Next, 2 ml of a suspension of MBSA cells (1.5 × 10 ⁵ cells / ml) in DMEM medium containing 0.2% SCS, penicillin (50 U / m1), streptomycin (50 μg / ml) was added to transformed 293T cells, and continued co-incubation of cultures at 37 ° C. After 48-72 hours, the culture fluid containing the synthesized recombinant virus with a titer of 2 ⁵ -2 ⁷ GAU / ml (passage 1) was divided into portions and stored at -80 ° C. To obtain mutant viruses containing the NP gene with GLY ¹⁶ → ASP ¹⁶ mutations (NPgd), GLY ¹⁶ deletion (NPdel), or the M2 gene with mutation

AspAsp ⁹⁰ → AsnAsn (M ₂ nn), carried out site-directed mutagenesis of the plasmid pHH21-NP and pHH21-M using specific primers for these caspase mutations. Mutagenesis was performed using the Quick Change kit (Stratagene). A DNA-lipofectamine complex for the preparation of a mutant virus was prepared with the inclusion of mutant plasmids pHH21-NPgd, pHH21-NPdel and pHH21-M2nn.

Example 2. Molecular Characterization of Caspase Virus Mutants

After obtaining recombinant viruses, the sensitivity of the viral proteins NP and M2 to caspase proteolysis in infected cells was studied. To this end, MDSK cells were infected with viruses and incubated for 20-24 hours until the apoptotic phase, when, as shown previously [10], cell caspases were activated that specifically cleaved NP (56 kD) → aNP (53 kD) and M2 (14 kD) → aM2 (12 kD). Figure 1 presents the analysis of proteolytic cleavage by caspases of NP and M2 proteins in caspase mutants of avian influenza virus. The MDSC cell culture was infected with chicken plague influenza virus (MI 10) mutants WTR, NPdel, NPgd, M2nn and incubated for 19-23 hours when the phase of viral apoptosis developed in the cells. Cellular proteins were analyzed by polyacrylamide gel electrophoresis followed by registration of protein forms NP, aNP, M2 and aM2 by Western blot and enhanced chemiluminescence using monoclonal antibodies to these proteins and an anti-species peroxidase conjugate. In the NPgd mutant, the NP protein was cleaved by cell caspases. In the M2nn variant, the M2 protein acquired caspase resistance and was not cleaved by these enzymes in infected cells. As can be seen in Fig. 1, in the wild-type virus (wtr), the NP protein lacking a caspase site remained resistant to cell caspases and did not undergo proteolysis in infected cells, while the M2 protein having such a site was cleaved to form the apoptotic product aM2. A similar protein profile with resistant NP was detected in NPdel virus with deleted G16 in the NP protein. In the NPgd mutant, in which the full-fledged caspase site of the “human” type was restored by the G16 → D mutation, the typical proteolysis of NP → aNP was found together with the proteolysis of M2 → aM2. In the M2NN mutant, parallel to the resistant NP, M2 resistant to caspase proteolysis was revealed, which confirmed the damaging effect of the DD32 → NN mutation for the caspase / granzyme site. As in MDSK cells, the proteolysis of NP and M2 was detected in CASO-2 and CF cells. This fact indicated the presence of universal sensitivity to caspases of various origins in the viral proteins NP and M2, while the corresponding caspase sites remained in their composition. Thus, when caspase sites were damaged, viral proteins lost their sensitivity to caspases and, conversely, when caspase sites were recreated, proteolysis of caspases was resumed.

Example 3. Stability and reproductive properties of caspase mutants of the virus.

For practical use, it seems important to verify the stability of caspase mutations obtained in virus generations upon passage. To do this, we studied variants of the virus, which were either passaged three times in a culture of MDSK with a multiplicity of infection (MI) of 0.01, or twice in chicken embryos, and viruses isolated from cloacal washings of chickens on the 4-5th day after IM infection. RNA was isolated in the samples of the studied viruses, which was converted into viral DNA by reverse transcription with virus-specific primers, followed by sequencing of the regions of interest in the obtained DNA transcripts. It turned out that all the caspase mutants studied were stable and maintained site-directed mutations during replication both in cell culture, chicken embryos, and in chickens. The replication levels of the obtained recombinant viruses in mammalian cells (MDSK, CACO-2) and birds (CF) were compared. For this, an infectious focus technique was used in which the level of virus replication was estimated by the size of the infectious focus. In figure 2. shows the propagation of caspase mutants of the chicken plague virus in cells of various origins. Recombinant variants of the chicken plague virus were introduced into the culture of human intestinal cells (CACO-2 line), dog kidneys (MDCK line), chicken embryo fibroblasts (CF) in the amount of about 100 (CACO-2 and MDSK) or 10 (CF) of infectious viral particles per hole. After infection, the cell monolayer was covered with a layer of agarose prepared on DMEM and incubated for 24 (MDSK) or 50 (CACO-2, CF) hours. Then the cells were fixed and the viral foci were stained with virus-specific antibodies and a species-specific long-term peroxidase conjugate using a water-insoluble tetramethylbenzidine substrate. The size of the viral foci reflected the level of reproduction of the studied virus and the expression of its proteins in cell culture. It was found that in cultures of human cells (CACO-2) and birds (CF), all 4 variants of the avian virus formed foci of a similar size (Figure 2). Additionally investigated the levels of accumulation of the infectious virus in cell cultures in experiments with a multi-cycle infection (multiplicity of infection of 0.001 PFU per cell). It turned out that the yield of caspase mutants of the chicken plague virus in the cell culture of MDSK after 30-35 hours all four variants of the virus were similar, as shown in table 2.

Table 2. Virus variant Harvest virus (PFU / ml) 1. WTR 0.4 × 10 ⁸ 2. NPdel 0.8 × 10 ⁸ 3. NPgd 0.7 × 10 ⁸ 4. M2nn 0.4 × 10 ⁸

It was also found that the reproduction rate of the studied variants of the virus with a single-cycle infection (MI 1) was similar and only the NPdel virus was characterized by accelerated reproduction in cell cultures and reached its maximum level 1-3 hours earlier than other variants. It should be noted that the level of proteolytic cutting of NAO → HA1 / 2 in all the studied mutants practically did not differ and approached 100% cleavage in all the studied host systems, which is in good agreement with the data of other authors on the multi-arginine proteolytic site in the HA protein of this strain. It is also interesting that a similar reproduction of all 4 recombinant viruses was observed during reproduction in chicken embryos. These observations show that all the studied recombinant variants of the A / FPV / Ro / 34 virus retained the ability to reproduce similar to the wild type after amino acid substitutions at the sites of caspase sites of the NP and M2 proteins.

Example 4. Virulence of caspase variants of the virus

и М2nn (♦) вируса A/FPV/Ro/34 со множественностью инфекции 5 БОЕ на цыпленка. Для заражения использовали образцы вируса (пассаж 1), выращенного в культуре клеток МДСК. Инфекционность заражающего вируса определяли методом инфекционных фокусов в культуре МДСК. Момент инфицирования цыплят обозначен на чертеже вертикальной стрелкой. Далее ежедневно наблюдали за животными и контролировали прибавку веса и гибель. Для определения титров экскретируемого вируса через определенные дни после заражения брали клоакальное содержимое с помощью универсального зонда (Зонд 3Г Бу-«ЦМ+») и далее титровали методом инфекционных фокусов в культуре МДСК. Цыплят заражали внутримышечно одинаковой инфекционной дозой исследуемых вирусов (5 БОЕ на цыпленка) и наблюдали за динамикой прибавки веса и гибели цыплят.Результаты типичного эксперимента для вирусов wtr и NPgd показаны на Фиг.3. У цыплят, инфицированных вирусом дикого типа (wtr) и мутантом NPdel, отмечалось быстрое снижение веса птиц и их гибель через 4-5 дней после заражения (д.п.з.). Причем, более быстро, примерно на 1-2 дня раньше, умирали цыплята в группе, зараженной мутантом NPdel, что говорило о его более высокой вирулентности по сравнению с диким вирусом. Напротив, у цыплят инфицированных такой же дозой мутантных вирусов NPgd и М2nn, обнаруживалось лишь замедление прибавки веса, взъерошенность перьевого покрова, снижение активности в период 4-8 д.п.з., что являлось следствием развития гриппозного заболевания. В дальнейшем указанные симптомы быстро проходили, восстанавливался вес, и цыплята полностью выздоравливали. Смертность в этой группе животных не регистрировалась.In this section, we compared the virulence of the resulting recombinant viruses in experiments on 8-day-old chickens. Chickens (Lohmann Brown, line PK-13), 2 individuals in the group, were intramuscularly infected with the variants WTR (•), NPgd (▪), NPdel

and M2nn (♦) of A / FPV / Ro / 34 virus with a multiplicity of 5 PFU infection per chicken. For infection used samples of the virus (passage 1) grown in a cell culture of MDSK. The infectivity of the infecting virus was determined by the method of infectious foci in a culture of MDSK. The moment of infection of the chickens is indicated in the drawing by a vertical arrow. Next, animals were monitored daily and weight gain and death were monitored. To determine the titers of the excreted virus, certain days after infection, cloacal contents were taken using a universal probe (Probe 3G Bu- “CM +”) and then titrated by the method of infectious foci in the MDSC culture. The chickens were intramuscularly infected with the same infectious dose of the studied viruses (5 PFU per chicken) and the dynamics of weight gain and chickens death were observed. The results of a typical experiment for wtr and NPgd viruses are shown in Fig. 3. In chickens infected with wild-type virus (wtr) and NPdel mutant, a rapid decrease in the weight of birds and their death was observed 4-5 days after infection (d.p.z.). Moreover, more quickly, about 1-2 days earlier, chickens in the group infected with the NPdel mutant died, which indicated its higher virulence compared to the wild virus. On the contrary, in chickens infected with the same dose of mutant viruses NPgd and M2nn, only a slowdown in weight gain, disheveled feather cover, a decrease in activity during the period of 4-8 dpc was detected, which was a consequence of the development of influenza disease. Subsequently, these symptoms quickly disappeared, the weight recovered, and the chickens fully recovered. Mortality in this group of animals was not recorded.

Example 5. Protection against the lethal action of wild virus using caspase virus mutants.

Highly sensitive chickens for this infection were used to study the protective effect of caspase mutants. 8-day-old chickens of the line (Lohmann Brown, line PK-13) were infected with the virus in a volume of 0.15 ml intramuscularly in each leg and then the animals were monitored daily and weight gain and death were monitored. To determine the titers of the excreted virus, certain days after infection, cloacal contents were taken using a universal probe (Probe ZG Bu- “CM +”) and then titrated by the method of infectious foci in the MDSK culture. Fifteen days after the initial infection of the chickens with NPgd and M2nn viruses with a dose of 10 PFU per chicken, the second infection was repeated and infected with a high-fatal dose (100 PFU per chicken) of wild A / FPV / Ro / 34 virus. After reinfection with a lethal dose of wild virus, no signs of disease were detected and all chickens remained alive. In the study of cloacal swabs on the 3rd and 6th day after infection, the virus could not be detected in the preinfected NPgd and M2nn mutants, whereas in control chickens the virus content in the cloacal swabs on the 5th day after infection

amounted to 10 ³ -10 ⁵ PFU per ml of cloacal suspension. On the 9th day after the second infection, high titers of antiviral antibodies were determined in the blood serum of chickens, which worked in dilutions of 1 / 6400-1 / 21600 in the hemagglutination inhibition reaction. In the placebo control group (previously uninfected chickens), 100% death of animals was observed after 3-5 d.p.z. The data obtained show that mutant viruses NPgd and M2nn multiplied in the body of chickens and induced the development of protective immunity against wild chicken plague virus. Thus, the point mutations G16 → D and DD90 → NN in the caspase region of the NP and M2 proteins of avian influenza virus reduced the virulent properties of these strains, but retained their ability to induce a high level of immunity in the infected organism.

Example 6. Artificial preparation of a mutant human influenza virus.

The 16 plasmid method described by Neumann et al [11] was used. To obtain the recombinant wild-type virus (WT), eight plasmids were used containing complete copies of individual human influenza virus RNA A / WSN / 33 (H1N1) segments located under the human type 1 polymerase promoter (pPolI-WSN-PBl, pPolI-WSN-PB2 , pPolI-WSN-PA, pPolI-WSN-HA, pPolI-WSN-NP, pPolI-WSN-NA, pPolI-WSN-M, pPolI-WSN-NS) and eight expression plasmids containing the genes of the viral proteins PB1 (pcDNA774) , PB2 (pcDNA762), PA (pcDNA787), NP (pCASSG-WSN-NP0), NA (pCAGGS-WNA), Ml (pCAGGS-WSN-M!), M2 (pEP24c), NS2 (pCA-NS2) under the promoter β-actin cyplenic film (pCAGGS) or human cytomegalovirus (pcDNA). For cell transformation, a mixture was prepared containing 1 μg of all of the listed plasmids, except plasmids pPolI-WSN-PA, pEP24 c and pCA-NS2, which were taken in the amount of 0.25 μg, and the resulting DNA mixture was complexed with lipofectamine 2000 (Gibco BRL) , and the obtained DNA lipofectamine complex was used to transform 293T human cells. To obtain a caspase virus mutant (NPG variant) containing the NP gene with the ASP ¹⁶ → GLY ¹⁶ mutation, site-directed mutagenesis of the pPolI-NP-RT plasmid containing the complete NP gene of the A / WSN / 33 virus was performed using 5'-CGAACAGATGGAGAC primers C G G T GGAGAACGCCAG-3 'and 5'-CTGGCGTTCTCC A C C G GTCTCCATCTGTTCG-3' (modified bases are shown in bold, GLY triplets are underlined). When ASP was replaced by GLY, a unique AgeI restriction site arose in the NP gene, which made it possible to control the conservation of the mutation in the resulting recombinant virus during its subsequent passage. Mutagenesis was performed using DNA lipofektaminovy complex to produce a mutant virus having a mutation NPG ASP → GLY set «QuickChange" (Stratagene). Prepared as described above, but instead plasmids pPolI-WSN-NP plasmid taken pPolI-NP / GLY ¹⁶ -RT To transform 3 ml of a suspension of 293T cells (~ 10 ⁶ cells) in Opti-M medium (Gibco BRL) with 0.5 ml of DNA-lipofectamine / 2000 complex, they were added to culture plates (6 cm in diameter) and incubated for 4 hours at 37 ° C. Next to the transformed 293T cells was added 2 ml of a suspension of MDSK cells (1.5 × 10 ⁵ cells / ml) prepared on DMEM containing 0.2% FCS, penis illin (50 U / ml), streptomycin (50 μg / ml), and continued incubation at 37 ° C. After 24 hours, the medium was removed and the transformed 293T / MDSC mixed cell culture was incubated further for 48-72 hours in DMEM without FCS containing 0.3 μg / ml trypsin to activate the virus to be synthesized A culture fluid containing the synthesized recombinant virus and having a hemagglutinating titer of 2 ⁵ -2 ⁷ GAU / ml was fractionated in portions and stored at -80 ° C.

Example 7. Attenuation of the replicative properties of the human influenza virus by modifying the caspase site in the viral protein NP.

To assess the attenuation of caspase mutant influenza virus (NPG) of a person, the rate of its replication in cell culture was compared with the wild (initial; WT) variant of the virus. Virus replication rate was estimated by the level of virus accumulation in the culture fluid of infected cells. 2-day culture of MDCK cells were incubated for 1 hour with the virus under study multiplicity 1 virion per 10 ⁴ cells in cell culture. After 1-hour contact, the cells were thoroughly washed with medium and incubated at 37 ° C in DMEM containing 0.2 μg / ml acetylated trypsin (Sigma). Figure 4 presents the dynamics of reproduction of the human influenza virus. At the indicated time intervals (abscissa axis: the numbers 1, 2, 3, 4, 5, 6, 7, 8 show the time intervals - 12, 24, 48, 60, 72, 84, 96, 108 after infection, respectively) from the medium samples in which the amount of the virus was determined by the hemagglutinating activity of chicken erythrocytes and by the method of infectious foci in an MDSC cell culture. On the ordinate axis of Figure 4 shows the number of hemagglutinating units of the virus (GAE) per ml of culture fluid. Propagation and accumulation of viral progeny in wild virus occurred approximately 20 hours earlier and in larger quantities than in the NPG mutant during infection of the MDSC cell culture. Thus, these observations showed that the caspase mutant of the human influenza virus has reduced replication compared to the original wild strain, i.e. is attenuated.

Claims (11)

1. A method for producing a genetically modified influenza virus and viral proteins by introducing mutations, deletions, inserts into sections of the viral genome encoding caspase or granzyme sites or their motives, or creating additional caspase sites and their motifs in viral proteins by implementing site-directed mutagenesis and process reverse genetics. 2. The method according to claim 1, characterized in that the obtained modified influenza virus is an attenuated virus, and viral proteins are characterized by a weakening of toxicity. 3. The method according to claim 1, characterized in that the obtained modified influenza virus has increased virulent properties, and viral proteins are characterized by increased toxicity. 4. The method according to any one of claims 1 to 3, characterized in that when introducing mutations, foreign genes (chimeric viruses) are inserted into the region of the viral genome that encode proteins, polypeptides and their fragments, enhancing the expression, therapeutic and prophylactic properties of the resulting attenuated virus . 5. The use of genetically modified influenza virus and viral proteins obtained by the method according to any one of claims 1 to 4, for the prevention of infectious diseases and the creation of antiviral immunity. 6. The use of influenza virus obtained according to any one of claims 1 to 4, as a producer for the production of viral and other proteins with useful properties and as substrates for the preparation of subunit and subvirion vaccines or their protectors and enhancers. 7. The use of influenza virus obtained by the method according to any one of claims 1, 2, 4, as vectors for targeted transfer and expression in the recipient organism of new antigens, chimeric proteins and therapeutic compounds of a polypeptide nature. 8. The use of influenza virus and viral proteins obtained by the method according to any one of claims 1, 3, 4, for targeted cell cytolysis and treatment of tumor and proliferative processes. 9. The use of influenza virus and proteins according to claim 5 by introducing into the human or animal body inhalation, subcutaneous, cutaneous, intramuscular, rectal, parenteral, per os. 10. The use of influenza virus and proteins according to claim 7 by introducing into the human or animal body inhalation, subcutaneous, cutaneous, intramuscular, rectal, parenteral, per os. 11. The use of influenza virus and proteins of claim 8 by introducing into the human or animal body inhalation, subcutaneous, cutaneous, intramuscular, rectal, parenteral, per os.

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