Method for producing replicative influenza virus particles, cell composition (versions), cell culture composition and application thereof
SUBSTANCE: nucleic acid contains a gene segment of a influenza virus and a bacteriophage polymerase promoter or a complementary chain of said nucleic acid. What is described is a composition containing a cell or a material produced of a cell according to this invention, or a virus, or a material produced of a viral particle according to this invention. The invention can be used in medicine.
EFFECT: nucleic acid allows producing replicative viral particles without the use of an assistant virus.
26 cl, 6 dwg, 2 ex
This invention relates to the field of production of antiviral vaccines.
Influenza viruses (Orthomyxoviridae) are RNA viruses with negative-chain" with a segmented genome (Taubenberger and Layne, Molecular Diagnosis Vol.6 No.4 2001). They are divided into two groups: one that includes the influenza viruses a and b, and another consisting of influenza virus, on the basis of significant antigenic differences between their nucleoprotein and matrix proteins. These three types of viruses also differ in pathogenicity and organization of the genome. The type And detected in a wide range of warm-blooded animals, and types b and C are primarily human pathogens. Influenza a viruses are additionally classified by the antigenic characteristics of the hemagglutinin (ON) and glycoproteins surface NA that protrude from the surface of the virion. Currently, there are 15 subtypes and nine NA subtypes. The type of viruses And infect a large variety of animals, including birds, pigs, horses, humans and other mammals. Waterfowl serve as a natural reservoir for all known subtypes of influenza a viruses and are probably the source of genetic material for pandemic strains of the virus of human influenza.
In contrast to related paramyxoviruses, influenza viruses have a segmented RNA genome. Influenza viruses a and b have the same structure, while the flu virus is more divergent. If viruses type a and b, each contain eight discrete gene segments encoding at least one protein of each virus type contains seven discrete segments, with the merging of segments 4 and 6 types a and B. influenza a Viruses and covered ledges of three proteins: ON, NA and matrix protein 2 (M2). Influenza viruses have only one surface glycoprotein. Each segment RNA of influenza virus encapsidation the nucleoproteids (NP) education ribonucleoprotein (RNP) complexes. Three protein polymerase associated with one end of the RNP complex. RNP is surrounded by a membrane from the matrix protein (matrix 1) in the form of integral part. The phospholipid portion of this shell is formed from the cell membrane of the host. The viral particle is also found non-structural protein 2 (NS2).
Guidelines of the world Health Organization (who) are as follows. First indicate the type of virus (a, b and C), then the owner (if he is not the person), place selection, the number of allocations and year captures (separated by oblique strokes). For virus types And subtypes ON and NA is noted in parentheses). For example, the strains included in the newly developed trivalent vaccine for the season 2000-2001: a/Panama/2007/99 (H3N2), A/New Caledonia/20/99 (H1N1) and B/Yamanashi/16/98. 1977 was the detected two subtypes of influenza a virus, jointly circulating in humans: H1N1 and H3N2.
Influenza viruses accumulate point mutations during replication, as their RNA-polymerase complex has no corrective activity. Mutations that change amino acids in the antigenic parts of the surface glycoproteins, can confer a selective advantage to the viral strain, allowing him to evade pre-existing immunity. Molecule TO initiate infection by binding to receptors on certain cells of the host. Antibodies against the protein TO prevent binding of the receptor and are very effective in preventing re-infection by the same strain. ON may elude previously acquired immunity either through antigenic drift, in which mutations are circulating at the moment of the gene TO disrupt the binding of an antibody or by antigenic variation, when the virus gets TO the new subtype. Pressure antigenic drift are different for molecules and positively selected changes occur predominantly in the globular head of the protein. These changes accumulate to a greater extent than NA. Changes in other proteins of influenza virus occur more slowly. Similarly, the pressure antigenic drift is the highest adapted in the s-human strains of influenza, intermediate in adapted to singam and horses strains of influenza and best adapted to the birds strains.
Because influenza viruses have a segmented genome, co-infection with two different strains in the same host may lead to new rearranging influenza strains containing different combinations of the original gene segments. It is known that there are fifteen subtypes in wild birds, they provide a source ON and are new to people. The appearance in the bloodstream of a human influenza strain with a new subtype antigenic variability was the cause of the last two pandemics in 1957 and 1968 and, most likely, the cause of an influenza pandemic in 1918. To fit all that is known regarding the emergence of pandemic influenza viruses pandemic strain should have ON-antigenicity, which differs from the ON-antigenicity, prevailing at the moment; it may not circulate in the bloodstream of people within 60-70 years; and this virus must be transmitted from person to person. As in 1957, and 1968 pandemics occurred from variability in ON, and in both cases of the pandemic strains were closely related avian strains. Although one of the absolute requirements for a pandemic is something that should be changed, the extent to which it can or should, and in order to change this virus, is unknown. Only the pandemic viruses of 1957 and 1968 are available for immediate research, and pandemic virus of 1918 characterized using molecular archaeology. In 1957, three genes have been replaced by genes, such avian genes:, NA and subunit of the polymerase complex (RV). In 1968 were replaced only and RV.
Specific diagnosis of influenza can be accomplished through selection of the virus, the test of inhibition of haemagglutination (HI), detection of antigen by immunoassay, serological tests, demonstrations NA activity in the secretions or on the basis of molecular analyses. Samples can be collected in the form of saliva, nasopharyngeal (nasal) swab or nasopharyngeal lavage obtained by rinsing with buffered saline. Standard for the diagnosis of influenza was immunological characteristics after cultivation. Serological analysis provides accurate, but a retrospective method for the flu, as it requires the collection of serum in the acute period, and during recovery.
Influenza viruses can be grown in containing the germ and chicken eggs or in some systems tissue culture. Adding trypsin (for activation by cleavage) makes possible the reproduction of influenza virus in ledah kidney dogs Madin-Darby (MDCK) and other lines. The primary method of obtaining a vaccine is still the cultivation of influenza viruses in eggs. Cultivation of cell lines typically used for the initial allocation of human influenza virus (type a and type b). Many human influenza virus may be cultured directly in allantoine cavity containing the embryo eggs. Some influenza viruses a and b require initial cultivation in the amniotic cavity and subsequent adaptation to allantoine cavity. After highlighting the culture of the majority of isolates of influenza accurately identify using immunoassays or immunofluorescence assay. Molecules ON influenza viruses bind sialic acid residues on the surface of respiratory cells to achieve the occurrence of the virus.
Strains of influenza can be characterized antigenic using the ability of influenza viruses to agglutinate erythrocytes in vitro. Anti-antibodies can inhibit agglutination. Thus, the analysis of inhibition of haemagglutination (HI) is one of the standard methods used to characterize strains of influenza. HI-analyses are used to determine whether strains of samples immunologically related (i.e. cross-reactive with modern vaccine strains. Teruya serum, usually produced in ferrets, add the holes in a series of twofold dilutions, and technicians evaluate wells for analysis by comparative observations of suspended red blood cells with agglutinability erythrocytes. In most situations, using a panel of sera for mapping strains of samples with vaccine and reference strains, and in particular influenza season, and a huge number of strains of samples sequentially compare by using HI-analyses. Who provides guidance and collaborating centres provide guidance for the identification of antigenic characteristics of individual viral strains and can provide these strains to those who wish to receive them. Strains of samples distributed according to categories in accordance with immunological pedigrees, for example, A/Moscow/10/99 (H3N2)-like, A/New Caledonia/20/99 (H1N1)-like, and B/Beijing/184/93-like viruses. For example, strains of samples that are not amenable to description in HI-analyses, and laboratory workers should inoculate in ferrets to obtain strain-specific antisera. When a new anticavity ready again perform HI-analyses, as described. If this new serum reveals considerable gaps in cross-reactivity (usually defined as a fourfold difference between the sample and the vaccine strains), it is included in routine laboratory panel and is used to detect the new epidemic strains. Thus, HI-analyses are extremely important in achieving control of influenza virus for vaccine strain selection and are the most commonly used methods for evaluation of antigenic drift.
Strains of influenza can be characterized by genetic comparison of the sequences of individual gene segments, and again the guidelines of the who and collaborating centres provide guidance for the identification of individual identity RNA segment containing the gene of influenza virus; segments of the nucleic acid of influenza virus a and b, the coding of nucleoprotein (NP), basic polymerase 1 (RV), the basic polymerase 2 (RW), acidic polymerase (PA), hemagglutinin (ON), neuraminidase (NA), matrix proteins (M1 and M2) and nonstructural protein (NS1 and NS2), and segments of the nucleic acid of influenza virus With coding of nucleoprotein (NP), basic polymerase 1 (RV), the basic polymerase 2 (RW), hemagglutinin-neuraminidase-like glycoprotein (HN), matrix proteins (M1 and M2) and nonstructural protein (NS1 and NS2).
Requests for reference strains, for example, antigenic analysis, to compare sequences of nucleic acids and for the identification of vaccine viruses can be addressed to the who Collaborating Centre for Reference and Research on Influenza, 45 Poplar Road, Parkville, Victoria 3052, Australia (fax: +61 3 9389 1881, web site: http//www.influenza centre.org); the WHO Collaboratig Centre for Reference and Research on Influenza, National Institute of Infections Diseases, Gakuen 4-7-1, Musashi Murayama, Tokyo 208-0011, Japan (fax: -81 42 5610812 or +81 42 5652498); WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention 1600 Clifton Road, Mail stop G16, Atlanta, GA 30333, United States of America (fax: +1 404 639 23 34); or the WHO Collaborating Centre for Reference and Research on Influenza, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, England (fax: +44 208 906 4477). Updated epidemiological information available on the website of the who and in the geographical information system, FluNet, in . Awareness of the impact of influenza and the benefits to health and economic benefits of prevention are growing, and during the last decade has seen the use and benefits of vaccination, and the number of drugs against influenza increases significantly. Due to longer life expectancy in many countries, an increasing number of people are at risk of complications, the load on the health system during epidemics of influenza is increasingly recognized, and more frequent international travel has created opportunities for the spread of the virus, while the introduction of new products has increased the possibilities for prevention and treatment of this disease. About 50 countries have government-funded national immunization programmes, and the vaccine is available in many other countries. Specific recommendations in respect of the use of this vaccine varies, but usually include annual immunization for the elderly individuals and individuals older than 6 months who are at increased risk of severe disease because of pre-existing chronic medical condition. In some countries, the vaccine used to reduce the spread of influenza to individuals at increased medical risk. Member countries should consider the benefits of activities for preventing influenza in the context of their public health priorities in General. Inactivated vaccines are classified into several types, depending on whether they contain the whole viral particles, partially destroyed viral particles (split vaccine) or purified antigens shell (subunit vaccines). Some subunit vaccines have been combined with adjuvant or delivery system.
Few countries have licensed live attenuated influenza vaccines for specific groups of targets. Two different vaccine composition 1 used in healthy adults and children in the Russian Federation and the other live vaccine was intensively tested, but has not yet been licensed. Until more are available live attenuated vaccine, usually they are not recommended for the prevention of influenza.
Two Klas is and antiviral agents have been developed for the prevention and treatment of influenza. The M2 inhibitors, amantadine and rimantadine, are limited to the treatment of influenza a viruses, and it was reported that they are effective in preventing infection. Although both products cause some side effects to significant neurological side effects are more frequent with amantadine. Neuraminidase inhibitors such as zanamivir and oseltamivir, have been recently licensed for treatment of influenza types a and b in a number of countries, and it was reported that they are effective for prevention. In patients receiving both classes of antiviral agent, were detected resistant mutants. Although it is not considered at present an important public health problem, the situation may change if these drugs are used on a very large scale.
Who supports the global program of the international surveillance cooperation 110 national influenza centres located in 82 countries, and 4 collaborating centers for the study of influenza, located in Atlanta (United States), London (United Kingdom of great Britain and Northern Ireland), Melbourne (Australia) and Tokyo (Japan). These centers provide early system cautions against emerging strains with epidemic potential. This system is important because the effectiveness of VA the Qing influenza decreases, if they do not contain strains circulating at the present time. Who has published recommendations on the composition of the vaccines that can be found in the Weekly Epidemiological Record (for example, see publication 9, 2004, 79, page 88, or ), published by the world Health Organization, in February vaccine used in the Northern hemisphere and in September vaccine used in the southern hemisphere. Because the flu has a less pronounced seasonal distribution in the Equatorial regions, epidemiological consideration will affect which of these recommendations (February or September) are suitable for vaccines for use in Equatorial countries.
The centers of joint cooperation spend antigenic and genetic analyses of influenza isolates provided by the national centres. When there is evidence of variability of antigens is correlated with epidemiological data to assess the epidemiological value of options. Representative isolates compared with the existing vaccine strains using panels of human sera collected before and after vaccination to assess whether you can expect that existing vaccines will protect against these viruses. After the publication of the who annual recommendations regarding vaccine manufacturers provide p the bet fast-breeding strains to facilitate the generation of seed viruses for receiving the vaccine. Tests on the safety and effectiveness of influenza vaccines include the inactivation of the virus, microbial sterility, measurement of chemicals used for the destruction of the virus and confirm the recommended concentration of the antigen. It is recommended that the vaccine should comply with the who requirements, however, national regulatory authorities must approve the specific vaccine viruses used in each country. National office of public health are responsible for recommendations regarding the use of this vaccine. Who has published recommendations regarding the prevention of infections caused by influenza virus. (See WER No. 35, 2002, pp.281-288). Influenza vaccine was received in the containing germ and chicken eggs for more than 50 years, but recently significant efforts have been made to develop systems for cell culture to obtain vaccines. The generally accepted standard methodology contains the embryo of the chicken eggs is extremely time consuming and has several major drawbacks: requires millions of eggs; in the United States more than 100 million for the season, eggs should be inoculated and harvested separately; requires extensive cleaning with a number of stages of filtration and centrifugation in order to guarantee the release of protein from eggs to minimize the risk of allergies; requires many herds and production, which are difficult to automate and are time-consuming, not to mention the cost of time and exposure to pollution.
Thus, there is a long-standing need in the industry for the development of technologies for vaccine production, which demonstrates advantages over the existing technology of vaccine production, i.e. through the development of protocols cooking, which will use a special strains of cells capable of supporting growth of influenza virus and adapted to growth in automated bioreactors, biological media or in other systems, cell culture, to replace the existing methodology for the production of vaccines.
Often offered characterized by continuous cell lines such as Vero cells or other cells derived from primates, for use in the production of vaccines influenza viruses. However, the registration office in these days shy away from vaccines received in the cells of primates that are intended for use against humans. More and more such agencies recommend that all products derived from cells of primates (such as Vero)were free from the remaining intact cells, and expressed continued concern regarding the level of residual material, such as DNA cells of primates, products, manufactures of the x of these cells. Although the world Health Organization (who) considers acceptable limit of residual DNA from continuous cell lines 10 ng per dose for antiviral vaccines, parenteral, registering agencies continue to consider the level of risk generated on a random basis cellular material primates, such as DNA, for antiviral vaccines.
For a long time the fundamental study of influenza a viruses was hampered by the lack of availability of effective systems of reverse genetics. Although the earliest methods of reverse genetics for RNA viruses with negative-chain were actually designed for the flu, this virus entirely from recombinant DNA was achieved only recently.
Recombinant influenza virus was obtained after transfection of eukaryotic cells with a set of eight plasmids, of which each of the segments of genomic viral RNA (wrnc) transcribable RNA polymerase I, and a set of four additional plasmids expressing nucleoprotein (NP) and polymerase proteins PB1, PB2 and PA. Reported efficiency for virus using the 12-plasmid systems have been relatively low.
After five additional co-expression of plasmids encoding hemagglutinin (ON), neuraminidase (NA), matrix protein 1 and 2 (M1 and M2) and not traktornyy protein 2 (NS2), the titres in supernatant could be increased. Elegant modification of these 12 - and 17-plasmid systems is providing bidirectional vectors to reduce the amount of transfected plasmids to eight. With this system the viral RNA with negative circuit and mRNA with plus-circuit can be synthesized from the same plasmid.
The ability to produce recombinant influenza virus And facilitates future study of influenza virus, but still not found a practical solution to the use of recombinant influenza virus And obtained by means of reverse genetics, to a sufficiently high titers in the production of vaccines, due to the fact that most cellular systems, if not all, of the cellular system used in the production of vaccines, not allow, or allows only a small degree, to replicate the above recombinant viruses due to incompatibility between the polymerase involved in the reverse genetics systems, and the most frequently used types of cells.
The virus is a RNA virus with a negative circuit. This means that in one cycle of replication, are generated by three types of RNA: a negative semantic vrnc, positive semantic crnc and positive sense mRNA. Unlike viral RNA (wrnc) this mRNA is kopirovalnoy and has a poly(A)tail. The first is A-the remains of this poly(A)tail of mRNA correspond to a short stretch of U residues in the genome, which is considered a stop signal transcription/polyadenylation. It is believed that the polymerase when it reaches this stretch of U residues subjected to repeated cycles of reverse bias and thus creates a complete poly(A)tail of mRNA.
This invention provides a reverse genetics system for influenza virus, which can be applied in the types of cells of various types. Polymerase I is a nucleolar enzyme, which transcribers ribosomal RNA and in abundance is expressed in growing cells. rRNA, like vrnc, has no cap and poly(A)tail, and therefore polymerase I may be used to obtain wrnc from cDNA. Transcription of viral cDNA polymerase I allows you to generate virus-like RNA with the correct 5'- and 3'-ends. However, while the apparatus transcription polymerase II is often compatible with genes from different species, transcription polymerase I exhibits a strict, though not absolute possess narrow specificity. This possess narrow specificity reported by the interaction of transcription factors with the promoter and, to a lesser extent, protein-protein interactions between these factors. This possess narrow specificity based on polymerase I, reverse genetics systems is a major drawback for the development of vaccines, primarily because of a promotion the market polymerase I to cells of other species, than people, such as the promoter of a dog or bird polymerase I, have not yet been described, whereas in industry are often used well-defined dog (for example, kidney cells dogs Madin Darby (MDCK)or avian cells (fibroblasts of chicken embryo (CEF)to obtain a vaccine against influenza virus.
This invention provides a nucleic acid containing a gene segment of influenza virus and the promoter polymerase of the bacteriophage, or a complementary chain of the specified nucleic acid. In contrast to the opening Neuman & Kawaoka (Virology 287, 243-240, 2001), showing that, unlike non-segmented viruses, conspicuous exception, where, as believed, does not work polymerase T7, was the influenza virus, the generation of which includes the additional complexity of the synthesis of the eight viral RNA, together with the polymerase and nucleoprotein from cloned cDNA, the present invention provides significant flexibility (freedom) in respect of plasmid vectors for this is based on the polymerase of bacteriophage technology reverse genetics, and the elements they contain. For example, the authors of this invention used the RNA polymerase of bacteriophage T7 to obtain wrnc or crnc-like RNA molecules, but can be used in a variety of other RNA polymerases such as RNA polymerase is bacteriophage SP6. In a preferred embodiment, the invention provides a nucleic acid containing a gene segment of influenza virus and the T7 promoter, or a complementary chain of a specified nucleic acid, allowing the authors to establish a system of this invention for the expression of the gene segments of influenza virus under control of the T7 promoter. In one embodiment, the polymerase terminator is missing. Preferably, the specified nucleic acid was provided with one or two additional residues of guanine after promoter. To create a vaccine derived nucleic acid according to the invention, which contains a gene segment that is derived from influenza virus, which the who-recommended for vaccination. In a preferred embodiment, the invention relates to nucleic acid containing a gene segment of influenza a virus and the T7 promoter or the complementary chain of the specified nucleic acid.
In particular, in a bidirectional system, preferably a nucleic acid according to the invention does not contain the T7 terminator. Since this polymerase is preferably expressed from a plasmid, transtitional together with plasmids expressing the virus, the proposed system is not limited to certain types. Although the reverse genetics system based on Imereti T7 is used sometimes for release from the non-segmented viruses with negative-chain, the reverse genetics system for segmented influenza virus-based polymerase of the bacteriophage, have never been used successfully. One limiting factor in systems of reverse genetics using T7 polymerase for transcription cDNA, sometimes try to overcome by the introduction of the G residues at the site of transcription initiation to strengthen started by T7 polymerase transcription. This approach was used in the rescue, for example, RV, VSV and SV, however, Zobel et al (Karpova et al., 1994 Jul; 202(1):477-9; Nucleic Acids Res. 1993 Aug 11; 21(16):3607-14) indicate that both 5'-and 3'-ends of the gene segments of influenza virus And should not be specified for the proper functioning of the viral polymerase; thus, adding additional nucleotides at the sites of transcription and the introduction of the G residues at the site of transcription initiation are not necessary. However, unexpectedly, in a preferred embodiment of the invention, the authors obtained nucleic acid according to the invention, having at least one additional guanine residue after the T7 promoter, and even preferably, two additional residue guanine followed the T7 promoter. This invention is also related to the cells of the kidney of the dog Madin Darby (MDCK) or fibroblastoid cage of chicken embryo (CEF)containing the T7 polymerase. In particular, this image is the buy associated with the cell, provided with at least one nucleic acid according to the invention. This invention facilitates the use of multiplemodel system, such as 17-plasmid or 12-plasmid or 8-plasmid system, and because the invention is associated with the cell containing the nucleic acid according to the invention, optionally containing T7 polymerase, preferably expressed from a plasmid, transtitional together with one or more plasmid that can Express the gene segment of influenza virus according to the invention, this system is not limited to certain types. Also stated the use of cells according to the invention where the specified T7 polymerase contains a nuclear localization signal. In a preferred embodiment, the cell is not a cell primates, resulting in possible to avoid the introduction of DNA of primates in cellular material or vaccine derived from a nucleic acid or cells according to the invention. Preferably use the MDCK cell or cell CEF. An advantage of the invention is that this system reverse genetics don't need virus helper (helper virus), all viral particles obtained by transfection contain the desired nucleic acid and can be used without the development of procedures for cloning in the subsequent system received what I vaccine. This invention was first associated with can replicate viral particle containing nucleic acid according to the invention. In the US 5166057 such a viral particle capable of replication was not received, and other attempts using the T7 system for segmented influenza virus were also unsuccessful to create this invention. Composition of cell cultures with titres of ~104viral particles according to the invention can be easily obtained without replication of the virus in the culture of the transfected cells, which can be increased to >107when the virus is allowed to replicate. It is particularly important that the replication of the particles according to the invention is achieved without virus helper (helper virus). The composition of cell culture containing cells or material derived from cells according to the invention, or a virus or a material derived from a viral particle according to the invention, can be preferably used for the preparation of pharmaceutical compositions aimed at the generation of immunological protection against infection of a subject by the influenza virus. Definitely cell according to the invention were not obtained in the US 5166057. Thus, the invention relates also to a method of obtaining can replicate (replicative) particles of influenza virus, providing cultive the Finance cell at least one nucleic acid according to the invention. Preferably, at least one nucleic acid used in a specified way, contained at least one, and preferably seven or eight gene segments of influenza virus and the promoter polymerase of the bacteriophage or a complementary chain of the specified nucleic acid or nucleic acids. In addition, it is preferable that the specified segment does not contain terminator polymerase of the bacteriophage, preferably to such a segment has been provided with at least one additional residue guanine, followed by the promoter, or was provided with two additional guanine residues following the promoter. Preferably, these segments come from the influenza virus that are recommended by who for the purposes of creating a vaccine, for example, the gene segment of influenza virus A. Finally, this invention provides replicative particles of influenza virus produced by the method described here. Thus, the invention relates also to a method of generating immunological protection against infection of a subject by the influenza virus, providing an introduction to the needy in the subject compositions according to the invention. Such compositions preferably obtained in the form of vaccines, i.e. by mixing of viral particles or viral proteins derived from such particles (Subedi the ranks of the vaccine) with a suitable pharmaceutical carrier, such as saline or adjuvant (for example, aluminium salt or other commonly used excipient (see, for example, http://www.cdc.gov/nip/publications/pink/Appendices/A/Excipient.pdf.).
Captions to figures
Figure 1. The design used for reverse genetics system based on T7pol. Cm. the text is a detailed description of the cloning strategies.
Figure 2. FACS analysis of cells T, transfected with constructs encoding GFP-minigenome (0.6 ág), genes T7pol (0.6 ág) and polymerase of influenza a (each 1 μg). Left panel: % GFP-positive cells after 30 hours after transfection. Right panel: the level of GFP expression (mean fluorescence) in GFP-positive fraction. On the X-axis, shown transfetsirovannyh design GFP-minigenome or sense (S)or antisense (AS) orientation, with the specified number of additional nucleotides G. Black columns show cotransfection all 4 components of the polymerase complex of influenza virus a (PB2, PB1, PA and NP), white columns show the control transfection which may design pHMG-NP.
Figure 3. FACS analysis of cells T, transfected with 0.6 μg of antisense GFP-minigenome with two additional residues G, 4 µg structures polymerase of influenza virus a and 0.6 μg of each of the T7pol (C) wild-type, T7pol containing the nuclear localization signal (N), or both constructions in a 1:1 ratio (C/N). Le is th panel: % GFP-positive cells after 30 hours after transfection. Right panel: the level of GFP expression (mean fluorescence) in GFP-positive fraction.
Figure 4. FACS analysis of cells T or BSR-T7, transfected with 0.6 μg design, coding antisense GFP-minigenome with two additional G residues, and 4 µg structures polymerase of influenza virus A. Shows the level of GFP expression (mean fluorescence) in GFP-positive fraction of cells. The cells were transfusional a plasmid or transfusional a plasmid expressing T7pol containing the nuclear localization signal (T against 293 N or BSR-T7 against BSR-T7 N). Black columns show cotransfection all 4 components of the polymerase complex of influenza virus a (PB2, PB1, PA and NP), white columns show the control transfection, which was excluded design pHMG-NP.
Figure 5. FACS analysis of cells T, transfected with 0.6 μg design, coding antisense GFP-minigenome with two additional residues of G (AS-2G) or semantic GFP-minigenome (S-0G), and 0.6 μg of plasmids expressing T7pol with a nuclear localization signal, and 4 μg of plasmids expressing genes polymerase of influenza virus A. Left panel: % GFP-positive cells after 30 hours after transfection. Right panel: the level of GFP expression (mean fluorescence) in GFP-positive fraction. Black columns show cotransfection all 4 components of the polymerase complex, the virus is influenza a (PB2, PB1, PA and NP), white columns show the control transfection, which was excluded design pHMG-NP.
6. FACS analysis of cells T and MDCK, transfected with 0.6 μg constructs encoding GFP-antisense-minigenome with two additional residues of G (AS-2G), 0.6 ág plasmid expressing T7pol with a nuclear localization signal, and 4 μg of plasmids expressing genes polymerase of influenza virus A. Left panel: % GFP-positive cells after 30 hours after transfection. Right panel: the level of GFP expression (mean fluorescence) in GFP-positive fraction. Black columns show cotransfection all 4 components of the polymerase complex of influenza virus a (PB2, PB1, PA and NP), white columns show the control transfection, which was excluded design pHMG-NP.
Generation of recombinant influenza a using based on the RNA polymerase T7 reverse genetics system
For a long time the fundamental study of influenza a viruses was hampered by the lack of availability of effective systems of reverse genetics. Although the earliest methods of reverse genetics for RNA viruses with negative-chain were actually developed for influenza a (7, 18), this virus entirely from recombinant DNA had reached the UTO only recently (9, 20).
The virus is a RNA virus with a negative circuit. During the replication cycle of the virus are formed three types of RNA: a negative sense viral genomic RNA (brnc), positive sense RNA, complementary to that of the genomic RNA (crnc) and a positive sense of messenger-RNA (mRNA). While wrnc and crnc contain essentially unmodified ends of the mRNA is kopirovalnoy and has a poly(A)tail (16).
RNA polymerase I (PolI) is a nucleolar enzyme, which transcribers ribosomal RNA (rRNA) and in abundance is expressed in growing cells. Like vrnc, rRNA has no cap and poly(A)tail (23). Hobom et al. (19, 21, 29) successfully received such artificial wrnc influenza virus segments with precise 5'- and 3'-ends using PolI. Transcription of the cDNA, cloned in the context of a PolI promoter-terminator cassette was possible to generate wrnk-like molecules with the correct 5'- and 3'-ends (29). Subsequent studies, which included helper influenza virus, demonstrated that these genomic molecules wrnc could be recognized and replicated by the polymerase complex of influenza virus and be packaged in the virus, which is the offspring of influenza virus. This system has allowed us to generate influenza viruses containing mutations in one of the viral gene segments or additional gene segme is t, that allowed us to study viral genes and their products. The application of the helper virus, required the selection of virus-transfectant, which is quite time-consuming.
Neumann et al. created a system for PolI extraction of influenza a viruses entirely from cloned cDNA (20). cDNA encoding the full-size wrnc of the influenza a virus, cloned between the promoter and a PolI man and terminator PolI mouse. In principle, transfection of these eight plasmids in eukaryotic cells should lead to the synthesis of all eight wrnc flu. Embryonic stem cells of human kidney (T) was cotranslationally these eight expression plasmids and plasmids expressing viral nucleoprotein and polymerase proteins PB2, PB1 and PA from the promoter of RNA polymerase II (PolII). These wrnc synthesized cellular PolI, Packed in RNP and took more than 1×103plaque-forming units of infectious virus per ml (PFU/ml) of the supernatant. Cotransfected plasmids expressing other viral structural proteins, led to a substantial increase in the production of the virus, namely 3×104-5×107PFU/ml (20). Fodor et al. reported similar system for the extraction of influenza a (9). This system depended on the eight plasmids encoding all eight cDNA vrnc, flanked by a promoter and a PolI person, but it contained the serial is inost of the ribozyme of the hepatitis δ (HδVrib), not the sequence terminator PolI. These plasmids were cotranslationally in Vero cells with four plasmids expressing proteins PB1, PB2, PA and NP from the main late promoter type 2 adenovirus. Using equal amounts of each of the expression plasmids Fodor et al. said pistons 1-2 release of infectious viral particles from 106transfected cells (9). The authors of this invention have designed a similar system of reverse genetics for the production of recombinant influenza virus A/PR/8/34. The authors concluded that the titres of ~104can be obtained without replication of the virus in the culture of the transfected cells, and these credits can be increased to >107when the virus is allowed to replicate (4). Since these are run by polymerase PolI system required cotransfection 12-16 plasmids, it was necessary to use cell lines that could be transliterowany with high efficiency, for efficient production of recombinant virus.
Then Hoffmann et al. developed a bi-directional system PolI transcription-PolII to generate influenza virus from eight plasmids (12). In this bidirectional system cDNA wrnc was built between PolI promoter of human rights and the minimum sequence terminator PolI mouse. All this construction was built between PolI promoter and a polyadenylation site. This is made possible transcription wrnc and mRNA from PolI promoters and PolII, respectively, from a single design. Cotransfected eight plasmids PolI-PolII, each of which encodes one of the gene segments of influenza a virus in cells C, coculturing with kidney dogs Madin Darby, led to recovery of infectious influenza virus And with outputs of up to 2×107PFU/ml of the supernatant (12). Use one matrix for the synthesis of both mRNA and wrnc reduced the number of plasmids required for the generation of the virus. It was reported that the efficiency of generation of the virus in this system was similar to the efficiency of the unidirectional (12-16 plasmids) system PolI.
While the PolII promoters are often compatible with the transcription apparatus of various kinds, transcription from promoters PolI exhibits a strict, though not absolute possess narrow specificity. This possess narrow specificity reported by the interaction of transcription factors with the promoter and, to a lesser extent, protein-protein interactions between these factors (23).
This possess narrow specificity based on PolI (Pol I) reverse genetics systems forms a major drawback. The above-described reverse genetics system used a PolI promoter person, limiting the production of recombinant virus only cells derived from primates, such as CL the TCI T or Vero cells. Although the PolI promoters have been described for several species, including humans, mice, rats and pigs (8, 14, 17), they remain unknown for many other species. Cells of dogs and birds routinely used for studies of influenza virus a and receiving the vaccines, but the promoters PolI dogs and birds so far not been described. To improve the flexibility of the technology of reverse genetics for influenza virus authors of the present invention tried to develop a universal system of reverse genetics. The authors decided to create a system based on the expression of the gene segments of influenza a virus under the control of the promoter of the RNA polymerase of bacteriophage T7 (pT7). Because RNA polymerase of bacteriophage T7 (T7pol) can be delivered into cells by transfection or by using stably modified cell lines, this system is not limited to cells from species-specific.
The reverse genetics system based on T7pol used for non-segmented viruses with negative-chain. Schnell et al. were first in receipt of non-segmented viruses with negative-chain only from cloned cDNA (27). The cDNA clone was done encoding the full-size anti-genomic RNA of rabies virus (RV). This cDNA was flanked pT7 and sequence δVrib after sequence terminator T7pol (tT7). After transcription using T7pol exact 3'-who once genome receive the autolytic cleavage sequence δVrib on the 3'-end. This plasmid was cotranslationally with expression plasmids encoding the viral N protein and polymerase proteins L and R under the control of pT7 in cells expressing T7pol. This procedure resulted in the release of recombinant RV, but only about 1 out of 2×107transfected cells (27). Since then, similar systems have been described for the families Paramyxoviridae, Rhabdoviridae, and Filoviridae non-segmented NSV (10).
For successful retrieval of the non-segmented viruses with negative-chain cDNA, very often receive positive semantic antigenome RNA (crnc)and not negative semantic wrnc. Consider that the simultaneous presence of bare negative semantic wrnc and positive sense mRNA encoding viral proteins will lead to hybridization, preventing the Assembly of the genome in ribonucleoprotein complexes (RNP) (27). Viruses with negative-chain usually do not encounter such problem, as they always retain their genome in the form of RNP that prevents hybridization. Removing the Sendai virus (15), parainfluenza virus type 3 (6) and human metapneumovirus (11) was reported with cDNA coding antisense genomic RNA; however, the efficiency was significantly lower than the results with a positive sense RNA. This principle was also applied for the release of recombinant influenza virus. Hoffmann et al. (13) also predelli efficiency of production of recombinant influenza virus of antigenome positive sense RNA. In contrast to the non-segmented and segmented viruses with negative-chain that can replicate only in the cytoplasm, the virus could be obtained from both genomic and antigenomic vectors with similar efficiencies.
One of the limiting factors in systems release viruses using RT is the fact that the residues at positions +1 to +3 can affect transcription. Observed that transcription of the cDNA can be increased by the introduction of 2 or 3 residues G directly below RT (22). This observation was applied to obtain, for example, recombinant RV (27), vesicular stomatitis virus (28), respiratory Intellinova virus (3) and human metapneumovirus (11). Apparently, for these viruses additional G residues at one end of the genome did not affect viral replication, but had a positive effect on run T7pol transcription.
Systems based on T7pol used widely for studies of reverse genetics of influenza virus (18), but to date have not been described in the basis of plasmids recombinant influenza virus. Here the inventors for the first time describe the reverse genetics system based on T7pol to obtain a recombinant influenza virus.
Materials and methods
Cells and viruses
Cells of the kidney of the dog Madin Darby (MDCK) were cultured in media is EMEM (BioWhittaker), supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, 1.5 mg/ml sodium bicarbonate, 10 mm HEPES and nonessential amino acids. Cells T were cultured in DMEM (BioWhittaker), supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, 1 mm sodium pyruvate and nonessential amino acids. Cells BSR-T7, a line of cells, baby hamster, stably expressing the RNA polymerase T7 (2). Cells BSR-T7 were grown in DMEM, supplemented with 10% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, 1 mm sodium pyruvate and 0.5 mg/ml G418 (Life Technologies, Breda, The Netherlands). Influenza virus A/PR/8/34, adapted for replication in containing the embryo of the chicken eggs and unable to optimal replication in cultures of mammalian cells, was passively seven times at low multiplicity of infection in MDCK cells grown in medium Episerf (Gibco BRL), supplemented with 10 IU/ml penicillin and 10 μg/ml of streptomycin. After the seventh passage routinely received titres 108TCID50/ml.
Transfection of cells T
Transient mediated by calcium phosphate transfection of cells T performed essentially as described (24). Cells were sown on the day before transfection in gelatinisation cultural cups with a diameter of 100 mm to obtain 50% of confluent monolayers. After a night of transfection medium for transfection for anjali fresh environment supplemented with 2% FCS to obtain a virus or 10% FCS for all other transpency. Cells were incubated for 30-72 hours, after which supernatant collected and cells were analyzed on a fluorescence, if necessary. Plasmid pEGFP-N1 (Clontech, BD Biosciences, Amsterdam, The Netherlands) was transfusional in parallel in all experiments, and the percentage of fluorescent cells was measured in a FACSCalibur (Becton Dickinson) flow cytometer, confirming that the transfection efficiency was in the range of 95-100%. The virus-containing supernatant was osvetleni by centrifugation for 10 minutes at 300 × g. The titers of virus in the supernatant was determined either immediately or after storage at 4°C for less than one week, or at -80°C for no longer than one week.
Transfection of MDCK cells
Transient transfection of MDCK cells was performed essentially as described previously (1). Briefly, 240 μl of medium Optimem I (Gibco BRL) was added to 10 μl of Lipofectamine 2000 and incubated at room temperature for 5 minutes. To this mixture was added the intended amount of DNA, brought to a volume of 50 µl by using Optimem I. This mixture is incubated at room temperature for 20 minutes. After incubation were added 200 μl of culture medium MDCK (see above) without penicillin and streptomycin, and this mixture was added to 1×106of MDCK cells in suspension in a 6-hole tablet. The village is E. 5 hours incubation, the cells were washed twice in PBS and cultured in 2 ml of culture medium MDCK without penicillin and streptomycin. This medium was replaced by culture medium MDCK containing 2% FCS, after an overnight incubation.
Transfection of cells BSR-T7
For transient transfection of cells BSR-T7, 400,000 cells were sown in 6-hole culture Cup per day before transfection to obtain 50-70% confluent monolayers of. Serum-free DMEM (240 μl) was added to 10 μl of Lipofectamine 2000 and incubated at room temperature for 4 minutes. To this mixture was added DNA, brought to 50 μl of serum-free DMEM, and incubated at room temperature for 20 minutes. Before transfection the medium was replaced with 2 ml serum-free DMEM. After incubation, the transfection mixture for added dropwise to the cells and incubated for 5 hours at 37°C. After transfection cells were washed once with PBS and add 2 ml DMEM, supplemented with 2% FCS to obtain a virus or 10% FCS for FACS analysis.
Used eukaryotic expression vectors encoding T7pol (pAR3126 and pAR3132). Whereas plasmid pAR3126 encodes T7pol wild-type plasmid pAR3132 expresses T7pol containing the nuclear localization signal (NLS), which effectively directs T7pol in the nucleus of cells (5). Eukaryotic expression plasmids, which are expressed polymerase proteins of influenza a virus, used moter hydroxymethylglutaryl-coenzyme A-reductase mouse, pHMG-PB1, pHMG-PB2, pHMG-PA is pHMG-NP (25).
δVrib pPolI-Cat-RT (25) amplified using PCR and cloned into the sites XbaI-BamHI pSP72. The sequence tT7, split BamHI-EcoRV, and cloned into the BamHI sites-HpaI pSP72-δVrib obtaining pSP72-δVrib-tT7 (MS24). Oligonucleotide encoding RT, ligated in sites NdeI-XbaI pSP72-δVrib-tT7 in the appropriate context regarding introduced BbsI sites with obtaining vector pSP72-RT-δVrib-tT7 (MS25, figure 1). Open-reading frames green fluorescent protein (GFP), flanked NCR of segment 5 of influenza virus a/PR/8/34, cloned into the BbsI sites pSP72-RT-δVrib-tT7 using pSP-Hu-GFP-Mu (4) as a matrix. This GFP-minigenome cloned, as in sense and antisense orientations, and it contained 0/2/3 additional G residues directly below RT (figure 1).
For cloning the gene segments of influenza virus a/PR/8/34 in pSP72-RT-δVrib-tT7 bidirectional structure of the influenza virus A/PR/8/34, described by de Wit et al. (4)was used as template for PCR (fourth 3'-nucleotide corresponded to the sequences of influenza virus a/PR/8/34, reported in the database of the National Influenza sequence Database). Primers containing the restriction site AarI, used for cloning of segments 1, 2, 3, 4, 6, 7, 8, and ligation of blunt ends used for segment 5; these gene segments were cloned into the BbsI sites in antisense orientation, and they had 2 additional G residue after RT.
Dynapro the data vector pSP72-RT-δVrib-tT7-pCMV (MS65, 1) was obtained by cloning the promoter of CMV (pCMV) below tT7 to create the possibility of obtaining mRNA from the corresponding gene segments. pCMV amplified using PCR using primers containing restriction sites AseI. pSP72-RT-δVrib-tT7 was partially digested AseI and pCMV ligated below tT7 in the right direction for obtaining mRNA from a gene segment.
Segments of influenza virus a/PR/8/34 again cloned to obtain each of the bidirectional run T7pol structures of influenza virus a/PR/8/34.
The inventors have generated a set of bidirectional vectors of which was deleterows tT7. This was done by splitting pSP72-RT-δVrib-tT7-pCMV BamHI-BpeEI, processing enzyme maple and repeated legirovaniem obtaining pSP72-RT-δVrib-pCMV (MS90, figure 1).
Segments of influenza virus a/PR/8/34 again cloned to obtain each of the bidirectional run T7pol structures of influenza virus a/PR/8/34.
All plasmids sequenced using the kit for sequencing Big Dye Terminator v3.1 (Applied Biosystems) and a Genetic Analyzer 3100 (Applied Biosystems) according to the manufacturer's instructions.
The preparation of recombinant virus using the system on the basis of T7pol
Cells 293 T was transicional, as described above, 5 g of each of the unidirectional plasmids containing gene segment PR/8/34, 5 μg each of expressionnisme HMG-PB2, HMG-PB1, HMG-PA, HMG-NP and 15 µg pAR3132. Alternatively, the authors have transfusional 5 μg of each of the bidirectional plasmid containing the gene segment PR/8/34, and 15 µg pAR3132. Supernatant collected 72 hours after transfection and 1 ml was used to infect confluent monolayer of MDCK cells.
Infection and determine the titres
Before inoculation of MDCK cells were washed twice in PBS and 1 ml of the supernatant of cells T used for inoculation confluent monolayer of MDCK cells in 6-hole tablet; 40 μg of trypsin (2.5%, and Bio Whittaker) was added during infection. The tablets are kept at 37°C for 1 hour and washed two times with PBS, after which was added 2 ml of medium of EMEM (BioWhittaker), supplemented with 4% BSA, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mm glutamine, 1.5 mg/ml sodium bicarbonate, 10 mm HEPES, nonessential amino acids, and 20 μg/ml trypsin (environment for infection). 3 days after infection supernatant these cultures were collected and tested for activity as an indicator of infection of these cells. Determine the titers of virus were performed as described previously (26). Briefly, prepared tenfold serial dilution of supernatants transfected cells in the medium for infection. Before inoculation the cells were washed two times with PBS. 100 μl of the diluted culture supernatants used for inoculationofculture monolayer of MDCK cells in 96-well plates. After 1 hour at 37°C. these cells were washed again in PBS and each well was added 200 μl of fresh medium for infection. 3 days after infection supernatant these cultures were tested for activity as an indicator of infection of cells in separate wells. The titles of infection was calculated from 10 replicates in accordance with the method of Spearman-Karber (26).
Analyses of GFP-minigenome using unidirectional reverse genetics system based on T7pol
Designed unidirectional vector containing RT, δVrib and tT7. Open-reading frames GFP, flanked by non-coding regions (NCR) of segment 5 of influenza virus a/PR/8/34, cloned into pSP72-RT-δVrib-tT7 in semantic (S) and antisense (AS) orientation with 0, 2 or 3 additional G residues (figure 1 and Annex 2 and 3). These constructs were named S-0G, S-2G, S-3G, AS-0G, AS-2G and AS-3G, respectively. The inventors tested which of these possible structures led to the best performance.
The authors have transfusional cells T one of these GFP-minigenome (S-0G, S-2G, S-3G, AS-0G, AS-2G AS 3G), plasmid expression T7pol (pAR3132) and four plasmids expressing proteins RV, RV, PA and NP (pHMG-PB2, pHMG-PB1, pHMG-PA, pHMG-NP). As controls, the authors performed the same transfection of which were excluded pHMG-NP, which was supposed to lead to a lack of replication of this GFP-mi is igenom. 30 hours after transfection, these cells were analyzed for fluorescence in FACSCalibur. These results are depicted in figure 2. From the left panel you can see that the highest percentage of GFP-positive cells was observed after transfection with GFP-minigenome in antisense orientation, with two additional residues G.
Other constructs GFP-minigenome also gave the percentage of GFP-positive cells, but slightly lower. When comparing the average fluorescence of GFP-positive cells (figure 2, right panel), again GFP-minigenome in antisense orientation with two additional G residues showed the best performance. In this experiment, GFP-minigenome in sense orientation with two additional G residues was found very poor performance, and other structures were intermediate.
Although the inventors have observed some variation in the ratio of GFP-expressing cells and the levels of GFP expression between different plasmids GFP-minigenome from experiment to experiment (data not shown), GFP-minigenome in antisense orientation with two additional G residues was usually the most productive, and therefore, this design was selected for subsequent experiments.
Nuclear expression of T7pol against cytoplasmic expression T7pol
One problem is, which potentially had to be solved by the inventors, was the expression of T7pol. For reverse genetics paramyxovirus used T7pol, expressed primarily in the cytoplasm of cells, which is desirable because the replication paramyxovirus also takes place in the cytoplasm. Influenza viruses replicate in the cell nucleus, and therefore, expression T7pol in the cytoplasm is not the best choice. Thus, the inventors wanted to compare the level of expression of GFP ever used cytoplasmic version T7pol (plasmid AR3126), or used T7pol containing the nuclear localization signal (NLS plasmid pAR3132).
The results of this experiment are shown in figure 3. When using plasmid expression T7pol wild type, the average fluorescence of GFP positive cells was equal to 521. The level of expression of GFP could be increased significantly by using T7pol, which contains a nuclear localization signal (average fluorescence equal 1106). When combining designs T7pol with a nuclear localization signal and without nuclear localization signal (in the ratio of 1:1, while maintaining unchanged the total number transtitional plasmids) observed an intermediate level of GFP expression (mean fluorescence was equal to 775). In multiple independent experiments using a large variety is expressing GFP-minigenome plasmids these results were reproducible; observed 2-10-fold increase in the expression of GFP in the use of nuclear version T7pol (data not shown). Thus, in subsequent experiments, the authors used T7pol containing a nuclear localization signal.
Transient expression T7pol against stable expression T7pol
For some systems, reverse genetics of paramyxoviruses T7pol not served by plasmid transfection, and through the use of cell lines, which makes possible stable expression T7pol. For this purpose the available cells baby hamster (BSR-T7). The inventors tested whether cells BSR-T7 be used for transcription of minigenome influenza-GFP, which could then be replicated by the polymerase complex of influenza virus, leading to the expression of GFP (figure 4).
As can be seen in figure 4, the high fluorescence of GFP in cells T strongly depends on the expression T7pol. In cells BSR-T7 observed relatively high levels of expression of GFP after cotransfection GFP-minigenome with the polymerase complex of influenza virus unlike transpency, from which he was expelled plasmid pHMG-NP. It was found that after adding the plasmid expressing the nuclear version T7pol, expression of GFP was even higher. Relatively high levels of expression of GFP in cells BSR-T7 suggest that stable expression of T7pol the C is more effective than transient (temporary) expression due to transfection. However, the experiment in which nuclear T7pol was achieved by transfection suggests that for reverse genetics of influenza virus a stable cell line expressing nuclear T7pol, could be even more effective than T7pol wild-type.
The preparation of recombinant virus using the unidirectional reverse genetics system based on T7pol
Then gene segments of influenza virus a/PR/8/34 cloned in the vector pSP72-RT-δVrib-tT7 to generate recombinant influenza virus A/PR/8/34.
The authors have transfusional cells T eight constructs encoding gene segments of influenza virus a/PR/8/34, pT7pol (pAR3132), pHMG-PB1, pHMG-PB2, pHMG-PA and pHMG-NP. After transfection the medium was added trypsin to create a replication get viruses. 72 hours after transfection supernatant collected and used for inoculation of MDCK cells. 3 days after inoculation was performed TO test the supernatant of these cells MDCK as guidance on viral replication. ON test was positive. Then determined the titer of virus supernatants T and MDCK. It was shown that the titre of virus in the supernatant T was equal to 1.6×101TCID50/ml; it was shown that the titre of virus in the supernatant MDCK was equal to 2.0×107TCID50/ml. Slightly lower titers of virus in the cell is T and MDCK received, when trypsin was added to the cells D after transfection (data not shown). Thus, this shows for the first time the release of the influenza a virus using plasmid only with recombinant virus that does not use a PolI promoter.
Bidirectional system T7
Then, the inventors wanted to develop a bidirectional reverse genetics system under control RT. The plasmid vector was obtained by cloning pCMV in pSP72-RT-δVrib-tT7 with obtaining vector pSP72-RT-δVrib-tT7-pCMV (figure 1). Open-reading frames GFP, flanked by non-coding regions (NCR) of segment 5 of influenza virus a/PR/8/34, cloned into pSP72-RT-δVrib-tT7-pCMV in antisense (AS) orientation with 2 additional residues G. Since the inventors expected that this plasmid will lead to the expression of GFP without replication minigenome the polymerase complex of influenza virus (pCMV is in sense orientation relative to this minigenome), the authors have also made a similar structure, containing this minigenome (0 residues (G) in the sense orientation relative to RT (hence, in antisense orientation relative to the pCMV). These minigenome plasmids were transfusional cells T together with plasmids expressing nuclear T7pol and pHMG-PB1, pHMG-PB2, pHMG-PA and pHMG-NP. Cells were analyzed by FACS after 30 hours (figure 5).
Transfect the semantic GFP-minigenome (S-0G) part-time polymerase complex of influenza virus resulted in a very small number of GFP-positive cells (figure 5, the left panel) with a very low expression of GFP (figure 5, right panel). In the presence of a full complex of the polymerase of influenza virus ~7% of the cells were GFP-positive, with an average fluorescence of ~1200. Using this plasmid with antimuslim GFP-minigenome relatively large proportion of cells (~10%) expressed GFP in the absence of a full complex of the polymerase of influenza virus, but only at low levels (average fluorescence of GFP 182). After cotransfection full range polymerase of influenza virus, the proportion of cells expressing GFP was not increased, whereas the expression level of GFP per cell was significantly increased (mean fluorescence of GFP 1205). Thus, from this experiment, the authors were able to conclude that the bi-directional expression vector was functional; the authors observed low levels of expression of GFP without the need of the polymerase complex of influenza virus due to the formation of GFP mRNA from pCMV. It should be noted that this was confirmed by transfection of cells T only the plasmid AS-2G GFP-minigenome, leading to similar levels of expression of GFP (~19% of cells expressing at an average fluorescence 128, data not shown). In addition, the authors observed increased levels of expression of GFP in the presence of the polymerase complex of influenza virus as a result of replication minigenome transcribed from RT. Thus, vanaprasta expression plasmid RT-pCMV was functional.
Production of recombinant virus using bidirectional reverse genetics system based on T7pol
Then gene segments of influenza virus a/PR/8/34 cloned in the vector pSP72-RT-δVrib-tT7-pCMV to generate recombinant influenza virus A/PR/8/34.
The authors of this invention have transfusional cells T eight constructs encoding gene segments of influenza virus a/PR/8/34 and pT7pol (pAR3132). After transfection the medium was added trypsin to generate replication produced viruses. 72 hours after transfection supernatant collected and used for inoculation of MDCK cells. 3 days after inoculation was performed TO test the supernatant of these cells MDCK as guidance on viral replication. This test was negative, indicating that was not extracted recombinant virus.
From reporter assays of minigenome using bidirectional vectors for gene expression RV, RV, PA and NP, the authors obtained evidence that the protein expression of these plasmids was very low (data not shown). The authors suggested that the sequence tT7 let transcription from pCMV, leading to low production of the encoded genes. Thus, the authors generated a new bidirectional plasmid from which has been deleted sequence tT7 (pSP72-RT-δVrib-pCMV). GE the major segments of influenza virus a/PR/8/34 cloned in the vector pSP72-RT-δVrib-pCMV to generate recombinant influenza virus A/PR/8/34. In the initial attempts again did not producyrovtsa recombinant virus. However, after some optimization of the number of plasmids used for transfection, the authors successfully received recombinant virus. The number of plasmids used for this experiment were 10 μg of each of the designs, coding RW, RW, RA, and 5 μg of each of the constructs encoding NP, NA, MA and NS. Although titers of recombinant virus were neglectible in cells C, subsequent inoculation of MDCK cells resulted in a virus with an initial titer of 1.3×105TCID50/ml.
T7pol-system in MDCK cells
To provide additional evidence of the universal nature of the reverse genetics system based on T7pol the authors felt replication GFP-minigenome in MDCK cells, but not in cells C. Although experiments with cells BSR-T7 already provided proof that the reverse genetics system based on T7pol works in cells derived not from cells of primates (figure 4), MDCK cells more widely used for studies of influenza virus and getting the vaccine.
As can be seen in Fig.6, it was found that the reverse genetics system based on T7pol is functional in MDCK cells. Together with the results on the cells of BSR-T7 (figure 4), these experiments indicate that the reverse genetics system based on T7pol really is "UN the universal" system, applicable for a wide variety of cell types. From this experiment can also be concluded that it is now possible to obtain recombinant influenza virus from the cells, not the cells of primates.
Here the inventors for the first time showed the preparation of recombinant influenza virus A/PR/8/34 (MDCK-adapted strain NIBSC) using systems on the basis of T7pol in cells C. However, there are no assumptions that limit the application of these methods only in respect of the influenza a virus A/PR/8/34; they can be applied to all influenza viruses types a, b and C, as well as other segmented RNA viruses with negative-chain. There are also assumptions that limit the application of these methods only in the cells T, BSR-T7 and MDCK; T7pol can be supplied, for example, by transfection of a broad range of cell lines, which could then be produced recombinant virus.
There is also considerable flexibility (freedom) in respect of plasmid vectors for reverse genetics technology-based T7pol and the elements they contain. Here, the authors used the RNA polymerase of bacteriophage T7 to obtain wrnc or crnc-like RNA molecules, but could also be used in many other RNA polymerases such as RNA polymerase, bacteriophage SP6. Shown in the estuaries and the ü experiments RNA polymerase T7 was modified it contains the nuclear localization signal of the large T-antigen of SV40, but RNA polymerase may be modified using various other tones nuclear targeting (for example, the protein signals To hnRNP). Here the inventors used the sequence of the ribozyme of the hepatitis Delta, but were described by other sequences, ribozymes, which could be used alternatively. Finally, the system described here does not depend on the application expressing vectors polymerase proteins of influenza virus, based on the murine promoter hydroxymethylglutaryl-coenzyme A-reductase (pHMG-design); can be used polymerase proteins from a wide range of influenza viruses, and they can be expressed using a wide range expressing vectors.
Recombinant virus receive, as described above, based on the high molecular skeleton of the virus (e.g. derived from the vaccine strain A/PR/8/34) and NA genes ON the relevant epidemic virus (e.g., A/Moscow/10/99). After receiving recombinant virus by transfection, the virus is amplified in a suitable cell substrate (e.g., eggs, MDCK cells, Vero cells) to a sufficiently high quantities. After breeding in containing the germ and chicken eggs allantoin fluid is here clarify by centrifugation for 10 minutes at 1000 × g and filtered through a filter of 0.45 micrometers. After this, the virus is precipitated by centrifugation for 1.5 hours at 150000 × g at 4°C and resuspended in phosphate buffered saline (PBS). The virus then treated with 2% decanoyl-N-methylglucamide (MEGA), put on a layer of 25% sucrose in PBS and centrifuged for 1.5 hours at 250000 × g at 4°C. Then the upper layer, containing proteins and NA, dialist against PBS and the purity and quantity of this protein preparation is confirmed by using 12.5% of the LTO-polyacrylamide gels, colored Kumasi brilliant blue. Ferrets are subjected to immunization intramuscularly ~10 micrograms protein/NA. If desirable, vaccination can be performed using sequential multiple doses of or using adjuvants (MF59, ISCOM). Antibody titers against and NA in serum samples collected before and after vaccination, determined using assays of inhibition of haemagglutination, analysis of the neuraminidase inhibition, ELISA assays neutralization of virus etc. Vaccinated and control animals repeatedly subjected to immunization after 6 weeks after vaccination using dose infecting 50% of tissue culture cells 1×105(TCID50), influenza a/Moscow/10/99 or heterologous isolates of the virus. After immunization sample in the form of a smear from a nose and pharynx animals daily for 10 days and the number is irusa, secreted infected animals, define quantitative PCR analyses or determinations of amounts of the virus. Thus, the resulting vaccine-induced immunity may be confirmed by quantification of the increase of antibody titers and protection against infection introduced by the virus.
1. Method of production of replicative particles of influenza virus without the use of virus-assistant, providing for the cultivation of cells, transtitional 7 or 8 nucleic acids, wherein these nucleic acids contain, respectively, the gene segment of influenza virus and the promoter polymerase of the bacteriophage, or such nucleic acids contain, respectively, the complementary chain gene segment of influenza virus and the promoter polymerase of the bacteriophage, where the specified promoter polymerase of the bacteriophage is the promoter of the T7 polymerase, and where the cell is further provided with a polymerase of bacteriophage T7.
2. The method according to claim 1, whereby the nucleic acid used in a specified way, does not contain a terminator polymerase of the bacteriophage.
3. The method according to any one of claims 1 or 2, according to which the nucleic acid used in the criminal code of sannam the way provided with at least one additional residue guanine, followed by polymerase promoter of the bacteriophage.
4. The method according to claim 3, whereby the nucleic acid used in a specified way, with two additional guanine residues following the promoter polymerase of the bacteriophage.
5. The method according to any one of claims 1 to 4, according to which the transfection of cells carry out twelve unidirectional plasmids expressing eight nucleic acid wrnc of influenza virus, as well as detection of viral NP of influenza and polymerase protein PA, PB1 and RV.
6. The method according to any one of claims 1 to 5, according to which at least one nucleic acid used in a specified way, contains a gene segment of influenza virus obtained from influenza virus, which is recommended by the world Health Organization (who) for the purposes of vaccination.
7. The method according to any one of claims 1 to 6, according to which at least one nucleic acid used in a specified way, contains a gene segment of influenza virus A.
8. The method according to any one of claims 1 to 6, according to which the specified polymerase of bacteriophage contains a nuclear localization signal.
9. The method according to any one of claims 1 to 8, according to which the cell is used in a specified way, is not a cell of a Primate.
10. The method according to claim 9, whereby the cage is sportswea in a specified way is the MDCK cell or cell CEF.
11. Composition of cells, transfected with at least one nucleic acid specified in any one of claims 1 to 7, which is the producer of wrnc or crnc-like RNA molecules, without the use of virus-helper.
12. The composition of the cells transfected with all nucleic acids listed in any one of claims 1 to 7, which is the producer of replicative viral particles flu.
13. The composition of cell culture containing composition of cells or material obtained from the composition of cells according to claim 11 or 12, designed to produce pharmaceutical compositions aimed at the generation of immunological protection against infection of a subject by the influenza virus.
14. The use of a composition according to item 13 for obtaining pharmaceutical compositions aimed at the generation of immunological protection against infection of a subject by the influenza virus.
15. The way to generate immunological protection against infection of a subject by the influenza virus, providing in need thereof of a subject a composition according to item 13, where this composition contains material obtained from the composition of cells according to claim 11 or 12.
16. Nucleic acid containing the gene segment of influenza virus and the promoter polymerase of bacteriophage T7, for the expression of the gene segment of influenza virus under to the control of the specified T7 promoter, where this nucleic acid is provided with at least one additional guanine residue after the indicated promoter, or nucleic acid contains complementary chain gene segment of influenza virus and the promoter polymerase of bacteriophage T7 for the expression of the gene segment of influenza virus under control of the specified T7 promoter, where this nucleic acid is provided with at least one additional guanine residue after the indicated promoter, where this nucleic acid is used as a matrix for the production of viral material capable of generating immunological protection.
17. Nucleic acid according to item 16, is equipped with two additional guanine residues after the indicated promoter.
18. Nucleic acid according to item 16 or 17, which contains a gene segment derived from the influenza virus, which is recommended by the world Health Organization (who) for the purposes of vaccination.
19. Nucleic acid according to any one of p-18, which contains a gene segment of influenza virus A.
20. The cage is provided with at least one nucleic acid according to any one of PP-19, where the cell is a producer wrnc or crnc-like RNA molecules.
21. The cell according to claim 20, further provided with a polymerase of bacteriophage T7.
22. The cell according to item 21, where specified, the polymerase soderjitsya nuclear localization.
23. The cell according to any one of p-22, where the cell is not a cell of a Primate.
24. The cell according to item 23, which is the MDCK cell or cell CEF.
25. The cell according to any one of p-24 not containing virus helper (helper virus).
26. The use of cells according to any one of p-25 to obtain pharmaceutical compositions aimed at the generation of immunological protection against infection of a subject by the influenza virus.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biotechnology of veterinary medications. Vaccine against rabies virus represents allantoic fluid of chicken embryos. Fluid contains glycoprotein of rabies virus, as well as mixture if recombinant adenoviruses of birds, which carry gene of surface glycoprotein of rabies virus, one of which contains secreted form of surface glycoprotein of rabies virus, and second - membrane-bound one. Interaction of glycoproteins with animal organism is reached by peroral introduction of medication.
EFFECT: invention can be applied in veterinary.
3 dwg, 1 tbl, 6 ex
SUBSTANCE: strain is produced by cross breeding of sterile equine influenza virus A/horse/Prague/1/1956(H7N1) and cold-adapted vaccine strain A/17/California/09/38(N1) on the basis of an attenuation donor A/Leningrad/134/17/57(H2N2) and contains neuraminidase of pandemic influenza virus A//California/07/09(H1N1) and hemagglutinin of equine influenza virus A/horse/Prague/1/1956(H7N7). The strain RN1/09-swine A(H7N1) is deposited in the State collection of viruses of Institution of Russian Academy of Medical sciences of D.I. Ivanovsky Institute of virology of Russian Academy of Medical Science, No. GKV 2473 and can be applied for influenza virus neuraminidase N1 antibody assay.
EFFECT: higher assay accuracy.
2 dwg, 4 tbl, 1 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to molecular biology, immunology, virology and concerns a method of preparing a biologically active complex. A method is enabled by mixing an aqueous suspension of a modified rod-like virus particle with an aqueous solution of polypeptide. The modified virus particle is presented by a reaction product of 1 weight fraction of rod-like virus particle and 0.01-0.1 weight fractions of water-soluble polymer and cationic groups in an aqueous medium. 1 weight fraction of the modified rod-like virus particle is mixed with 0.1-25 weight fractions of polypeptide.
EFFECT: method can be used for preparing vaccine and therapeutic preparations for viral and other infectious diseases and other purposes.
3 tbl, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to molecular biology, immunology, virology. What is described is a biologically active complex which can be used for preparing vaccine and therapeutic preparations for viral and other infectious diseases and other purposes.
EFFECT: invention allows extending the range of biologically active complexes and improves biological activity of the complex in 1,6-1,7 times.
3 tbl, 5 ex
SUBSTANCE: bacteriophage Escherichia coli V32 strain is deposited in the Collections of Museum of microorganisms of Federal State Intuition of Science State Science Centre of Applied Microbiology and Biotechnology, No. Ph25 is specific and virulent for bacteriophage, and used as an option for identification of Escherichia coli bacteria serogroup O157 at the stage of bacterial colony analysis.
EFFECT: simple and available product not requiring heavy time and material consumption.
1 dwg, 4 ex
SUBSTANCE: invention can be used in laboratory diagnostics of Yersinia enterocolitica strains with using moderate bacteriophages FK-99, FK-100, FK-101 for intraspecific differentiation of a yersiniosis agent. The Yersinia enterocolitica 2012 strain is recovered from human in 1966 and deposited in the State collection of pathogenic bacteria of Russian Research Antiplague Institution "Microbe", No. KM-206. When used as a mentor strain, it enables the particle concentration of the phage preparations being equal to n·10-n·10 particles/ml.
EFFECT: use of the recovered phages in Yersinia enterocolitica on the indicator KM-206 offered under the invention provides the additional differentiation of lysogenic cultures from non-lysogenic and ensures the effectiveness of laboratory diagnostics of Yersinia enterocolitica.
1 tbl, 1 ex
SUBSTANCE: invention concerns PUUMALA and DOBRAVA viruses strains for preparing , vaccine preparations for specific prevention of hemorrhagic fever and renal syndrome (HFRS).
EFFECT: strains are replicable in a transferred renal cell culture of a VERO grivet with accumulating viral weight in the amount sufficient for preparing the vaccine preparations.
2 cl, 1 tbl, 2 ex
SUBSTANCE: donor strain producing an isotope-marked bacteriophage which is pre-recovered from lysogenic strain V.cholerae eltor in the 1 concentration n·108 PFU/ml is produced and added with 5 mcc/ml of isotope 3H-thymidine, and kept for one day in a thermostat at 37°C to produce marked cholera phage. The latter is introduced in the amount 1 ml in a test tube with an indicator recipient strain in the ratio 1:1 for lysogenisation, incubated at 37°C within one day; 0.1 ml of the derived mixture is seeded on an agar plate, grown at 37°C within 20-24 hours, and the donor strain is recovered. The residual portion of the mixture is used to record radioactivity of the bacteriophage and recipient strain. The positive result is shown by radioactivity of the marked bacteriophages within the range 475-1269 p/min, and of the recipient strains within the range 78-103 p/min.
EFFECT: invention allows higher efficiency of detection and recording of the cholera phage in a 3H-thymidine marked cholera germ cell.
3 tbl, 4 ex
SUBSTANCE: invention can be used in biotechnology, and can be used in practical health services for preventing seasonal influenza incidence in adults and children by a live influenza intranasal vaccine of the A/Solomon Islands/03/2006 (H1N1) influenza virus. Vaccine strain of B/60/Florida/04/181 (H1N1) is a reassortant prepared by cross breeding an epidemic B/Florida/07/04 virus and a cold-adapted thermally sensitive B/USSR/60/69 virus being a human-safe attenuation donor. The vaccine strain B/60/Florida/04/181 (H1N1) strain replicates actively in growing chicken embryos at optimum temperature 32°C, is characterised by thermal sensitivity and cold adaptation. The reassortant has inherited the genes coding surface virus antigens - hemagglutinin (HA) and neuraminidase (NA) from an epidemic parent virus, and six genes coding internal non-glycosylated proteins from the attenuation donor.
EFFECT: strain is areactogenic for adults and children in intranasal introduction.
SUBSTANCE: vaccine strain of A/17/Solomon Islands/06/9 (H1N1) is a reassortant prepared by cross breeding an epidemic A/Solomon Islands/03/2006 (H1N1) virus and a cold-adapted thermally sensitive A/Leningrad/134/17/57 (H2N2) virus being a human-safe attenuation donor. The vaccine strain A/17/Solomon Islands/06/9 (H1N1) strain replicates actively in growing chicken embryos at optimum temperature 32°C, is characterised by thermal sensitivity and cold adaptation, The reassortant has inherited the genes coding surface virus antigens - hemagglutinin (HA) and neuraminidase (NA) from an epidemic parent virus, and six genes coding internal non-glycosylated proteins from the attenuation donor. The A/17/Solomon Islands/06/9 strain is areactogenic for adults and children in intranasal introduction.
EFFECT: applicability in practical health services for preventing seasonal influenza incidence in adults and children by a live influenza intranasal vaccine of the strain of said influenza virus.
1 dwg, 3 tbl, 2 ex
SUBSTANCE: invention relates to isolated nucleic acid, encoding protein with activity of fluorescent biosensor for hydrogen peroxide detection, expression vector, containing said nucleic acid, cell of E.coli, producing protein, encoded by said nucleic acid, and isolated protein, with activity of fluorescent biosensor for hydrogen peroxide detection. Isolated protein, with activity of fluorescent biosensor for hydrogen peroxide detection, has amino acid sequence, presented in form SEQ ID NO: 2. Expression vector contains nucleic acid, encoding protein with activity of fluorescent biosensor for hydrogen peroxide detection, under control of regulatory elements, necessary for expression of nucleic acid in host cell. Cell of E.coli is modifies due to introduction into it of nucleic acid, encoding protein with activity of fluorescent biosensor for hydrogen peroxide detection, functionally connected with regulatory sequence.
EFFECT: claimed invention is applied for detection of hydrogen peroxide inside live cells.
4 cl, 3 dwg, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medicine and concerns agents and methods based on using fibronectin domain EDA. Substance of the inventions involves using a polypeptide comprising an amino acid sequence of the fibronectin domain EDA, or its TLR4-binding fragment, or a version of specified domain; for preparing a pharmaceutical composition stimulating an antigen- specific immune response.
EFFECT: improved stimulant property.
36 cl, 3 ex, 11 dwg
SUBSTANCE: substance of the invention involves a humanised human osteopontin antibody containing a variable region of a heavy chain consisting of the amino acid sequence SEQ ID NO:1 and a variable region of a light chain consisting of the amino acid sequence SEQ ID NO:3. Furthermore, the invention involves a polynucleotide containing a sequence coding the variable region of the respective light and heavy chains of the humanised antibody, an expression vector containing polynucleotide, a host cell, a medicine, a method of producing the humanised antibody, a medicine for treating an autoimmune disease, a method of treating, and application of the humanised antibody for producing a pharmaceutical agent.
EFFECT: advantage of the invention consists in creation of the humanised antibody exhibiting improved activity or stability, than activity and stability of standard human osteopontin antibodies.
13 cl, 14 ex, 1 tbl, 16 dwg
SUBSTANCE: substance of the invention involves a stem cell (SC) preparation with reprogrammed cell signalling containing a base SC preparation in a membrane and/or a nucleus and/or a cytoplasm of which there is implanted a protein or pharmaceutical preparation capable to regulate signalling pathways of the SC and nidus cells in a mammal's body, pre-encapsulated in a nanocontainer with dimensions of 100 nm, produced of a biodegradable material, intact for organelles and compartments of the SC of the base preparation. This material has the preset biodegradation time in a mammal's body to provide the programmed protein or pharmaceutical preparation recovery in an intra- or intercellular space and to implement thereby reprogramming of signalling transduction of the SC key genes in a required therapeutic pattern of physiological cell cycle events directly in a pathological body region or tissue.
EFFECT: provided target addition of the signalling substances strictly in the involved body region.
14 cl, 6 ex, 3 dwg
SUBSTANCE: antibody under the invention is produced from Nicotiana benthamiana leaves by transient transfection of expression vectors. It represents immunoglobulin G and a tetramer consisting of two light and two heavy peptide chains and them larger supramolecular assemblies. The antibody is recovered both in the glycated, and in non-glycated form. The antibody is capable to bind selectively HER2/neu protein, to inhibit cancer cell proliferation. Using the antibody under the invention enables diagnosing HER2-positive breast cancer.
EFFECT: invention can be used for preparing drugs and diagnostic agent for breast cancer.
8 dwg, 4 ex
SUBSTANCE: invention refers to DNA coding a modified antibody capable to identify and cross-link a TPO receptor, and containing two or more V-areas of the N-chain and two or more V-areas of the L-chain of an initial antibody connected directly or through a covalent or noncovalent linker of a smaller size in comparison with the initial antibody. DNA includes two or more DNA-sequences coding the V-areas of the L-chain and N-chains; DNA coding at least one of the V-areas of the L-chain and/or the N-chain includes specific nucleotide sequences presented in the description. The invention discloses DNA coding a compound showing an equal or more agonist action (ED50) compared with thrombopoietin (TPO); the compound can represent the modified antibody, the whole antibody or F(ab ')2 capable to identify specifically and cross-link the TPO receptor. Also, the invention relates to a vector containing specified DNA, an animal cell and a microorganism which contain DNA or the vector and produce the modified antibody or the compound with the TPO agonist activity. The invention describes methods of producing the modified antibody - TPO agonist and the compounds which involve cultivation of the transformed animal cell or microorganism containing DNA of the invention.
EFFECT: products of the invention can be applied as signal transduction TRO-agonists, and can be used as a prophylactic or therapeutic agent in blood disorders associated with connected with thrombocyte count reduction, thrombopenia accompanying chemotherapy in cancer or leukaemia.
33 cl, 61 dwg, 3 tbl, 8 ex
SUBSTANCE: disclosed is a conditionally defective particle of influenza virus with the absence of a nucleic acid segment of influenza virus selected from a group of segments, mainly coding acid polymerase (PA), basic polymerase 1 (PB1) and basic polymerase 2 (PB2). The conditionally defective particle of influenza virus is used for induction of influenza virus defence. Also, a method of producing such particles, a pharmaceutical composition containing such particles, its application and a method of induction of influenza virus defence are described.
EFFECT: higher efficacy.
27 cl, 4 dwg, 4 tbl, 2 ex
SUBSTANCE: in the method nutritional medium is used for cultivation of cells, transformed with the gene, encoding target product. One of peculiarities of nutritional medium is the following property: total concentration of amino acids is higher than approximately 70 mM; ratio of total molar quantity of glutamine to total quantity of asparagine is lower than approximately 2; ratio of total molar quantity of glutamine to total quantity of all amino acids is lower than approximately 0.2; ratio of total molar quantity of inorganic ions to total quantity of all amino acids is approximately from 0.4 to 1 or combined total quantity of glutamine and asparagine per unit of volume is in the range of approximately from 16 mM to 36 mM. Such system of cultivation ensures high levels of antibody production. Methods of cultivation can include change of temperature, usually reduction of temperature when culture reaches 20-80% of maximal cell density, pH value, osmolarity, level of chemical activator and their combinations. As alternative or supplement, described are methods-versions, in which levels of lactate and/or ammonium in the culture after reaching peak values decrease in the course of time.
EFFECT: invention ensures high output of antibodies with simultaneous reduction of undesirable products.
48 cl, 76 dwg, 27 tbl, 17 ex
SUBSTANCE: disclosed is a plasmid for producing a viral vector transporting multiple expression cassettes to a target. The plasmid contains genome nucleotide sequences packed into a polyvalent capsid and a number of cassettes to be transported. A method for producing a viral vector, a viral vector and an immunogenic composition are described besides.
EFFECT: group of inventions can be used for wide-ranging expression of target antigens.
23 cl, 3 dwg
SUBSTANCE: invention concerns a polypeptide exhibiting activity of telomerase reverse transcriptase (TERT), molecules of nucleic acid coding such polypeptide, a plasmid vector carrying said molecule of nucleic acid, an immortalised cell, transfected said molecule of nucleic acid, and applying such cell for producing an end substance.
EFFECT: invention allows creating the avian immortalised cell lines to be useable for making any end substance.
27 cl, 13 dwg, 9 ex
SUBSTANCE: it is described that immunogenicity of hemagglutinin (HA) molecule of influenza virus can be increased by substituting amino acids in the HA sequence. Substituting specific residues in HA such as asparagine introduction in position 223 in HA H5 allows ensuring more sensitive hemagglutination inhibition (HI) test provided by changing receptor specificity and/or ability to antibody-antigen linkage. The HA molecules having such substitutions can find application in creating diagnostic prototype viruses.
EFFECT: improved influenza virus vaccines.
9 cl, 3 dwg, 7 tbl, 7 ex