Recombinant pseudo adenoviral particle based on human being adenovirus genome of 5-th serotype for induction of specific immunity to influenza virus a of h3n2 subtype and method of its use

FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use. Provided particle contains expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus, haemagglutinin gene of A/Perth/16/2009(H3N2) strain with pre-optimised for expression in human being cells nucleotide sequence presented in SEQ ID NO:2. The specified haemagglutinin gene of influenza virus of A/Perth/16/2009(H3N2) strain is cloned in expressing cassette containing polyadenylation signal SV40 under control of cytomegalovirus promoter. Presented invention may be used for induction of specific immunity to influenza virus A of H3N2 subtype during injection in efficient quantity.

EFFECT: providing intense expression of recombinant haemagglutinin of the specified influenza virus.

6 cl, 9 dwg, 1 tbl, 4 ex

 

The technical field

The invention relates to the field of biotechnology and relates to recombinant vectors, which can be used in the pharmaceutical industry for the production of vaccines, in particular for influenza vaccines.

Prior development

The vaccine against human influenza is the most important link in the implementation of anti-epidemic measures to reduce morbidity among people during seasonal outbreaks. Especially important vaccines based on the so-called "pandemic" influenza virus strains. Pandemic subtypes of influenza virus is able to spread globally, often with a high mortality of affected people. Epidemiological monitoring of the circulation of influenza viruses is carried out by the who (world Health Organization), which announces the strains recommended for inclusion in the seasonal composition of influenza vaccines in certain areas (the Resource is available: www.who.int).

For the manufacture of a vaccine requires highly immunogenic antigens in its composition. Typically, the attenuation or inactivation of strain in the production of classical vaccines reduces its immunogenicity. Therefore, at the present stage of development of biotechnology is much more technologically and immunobiological effective alleycatallies genetic vaccines.

To date, all antigens included in the vaccine, according to the method of obtaining can be divided into two main groups - native and recombinant, the latter in connection with the development of genetic engineering and biotechnology are beginning to displace the antigens obtained by classical methods. To obtain antigens classical methods should initially be isolated from patients with influenza-the flu virus.

The basis of any vaccine preparation are various antigens of pathogens, necessarily has immunogenic properties. The main specific immunogenicity of influenza virus is its hemagglutinin, this is taken into account in the development of immunobiological drugs against influenza.

Thus, the known vaccine strain influenza a/60/Brisbane/08/83 for production of live intranasal influenza vaccine for adults and for children (RF Patent No. 2 422 517). This strain is a live and attenuated by the method of reasontly with donor strain, harmless to people that deprived him of pathogenicity, but retained its immunogenicity of hemagglutinin. Strain propagated in chicken embryos. As can be seen, the strain is a live attenuated, that is, it is possible to reversion to the original pathogenic form, for cultivation requires the use of chick embryos, which is uneconomical, inefficient, in smosna reassurace in other influenza viruses in the case of contaminated embryos in addition, the residual amount of chicken protein preparations can cause allergic reactions.

From classical variants of known vaccine strain influenza A/8/Perth/16/2009(H3N2) for the production of inactivated influenza vaccine (Patent RF №2458124). This strain of pathogenic and requires compliance with special care during handling in the laboratory and on the production and manufacturing process required step inactivation to transmit this virus in the inanimate condition, which, however, dramatically reduces immunogenicity.

To solve the problems associated with native strains of different microorganisms intended for the production of vaccines, promising direction for the use of safe vectors expressing the genes for antigens of the pathogen (Abdulhaqq S.A.,D.B. Weiner DNA Vaccines: developing new strategies to enhance immune responses, 2008, No. 42, s.219-232 - Vaccines: the development of new strategies to enhance the immune response); (J.A. Zaia, The status of gene vectors for the treatment of diabetes, Cell Biochem. Biophys, 2007, No. 48 (2-3), .183-90. Status of genetic vectors for the treatment of diabetes). One of the most safe for the body recognized by the vectors constructed based on the human adenovirus (Van Kampen K.R. et al., Safety and immunogenicity of adenovirus - vectored nasal and epicutaneous influenza vaccines in humans, Vaccine, 2005, No. 23(8), s-36 - safety and immunogenicity adenomectomy nasal and acogny influenza vaccines in humans).

Recombinant adenoviral vectors have the following useful characteristics: not pathogenic because of their genome deleted region responsible for pathogenicity, can transducible as dividing and postmitotic cells, DNA of adenovirus remains extrachromosomal form capable of reproduction only in special cell lines in vitro, eliminated from the body within 4-5 weeks, induce both cellular and humoral immune response, the process of obtaining a new recombinant adenovirus is just a few weeks, they provide a high level of expression of a target gene in the target cell.

Today known adenoviral vector expressing recombinant hemagglutinin of influenza a (patent Application WO No. 2008/157419). This design is chosen by the author for the prototype, as the closest to the invention (prototype).

Due to the fact that at the priority date of the application, nothing was known about the strain of influenza virus A/Perth/16/2009(H3N2), because for the first time this pathogen was isolated and characterized during the flu epidemic in 2009, the nucleotide sequence of its hemagglutinin gene could not be used to construct recombinant adenovirus particles. Accordingly, the applicants have not had the opportunity to create an adenoviral construct with Oh is richersounds cultural-morphological properties, the level of expression, immunogenic and protective properties. This is a significant drawback of the claimed design, as it cannot be used for the induction of specific immunity to influenza virus subtype H3N2, in particular to the strain A/Perth/16/2009(H3N2), which prevents the creation of influenza vaccine products based on it.

Disclosure of inventions

The technical problem of the invention is aimed at creating recombinant pseudoadenoviral particles on the basis of the genome of a human adenovirus 5-th serotype producing recombinant hemagglutinin of influenza virus that can provide an enhanced expression of recombinant hemagglutinin of influenza virus A/Perth/16/2009(H3N2), allowing its use for induction of specific immunity to influenza a subtype H3N2.

The technical problem is solved by constructing recombinant pseudoadenoviral particle on the basis of the genome of adenovirus human serotype 5 containing expressing cassette inserting the gene for hemagglutinin of influenza virus, the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) use gene hemagglutinin with pre-optimized for expression in human cells nucleotide sequence, providing increased expression of the gene gemigo inina influenza virus strain A/Perth/16/2009(H3N2). Moreover, an optimized nucleotide sequence of the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) is SEQ ID NO: 2, and the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) - optimized nucleotide sequence cloned in expressing cassette under the control of the promoter and contains a polyadenylation signal, wherein the promoter is a cytomegalovirus promoter and a polyadenylation signal SV40 is. Expressing cassette in this technical solution is in the deleted E1 genome of a human adenovirus serotype 5. The method of using recombinant pseudoadenoviral particles on the basis of the genome of adenovirus human serotype 5, producing the hemagglutinin of influenza virus A/Perth/16/2009(H3N2,) is the introduction created recombinant pseudoadenoviral particles in an effective amount for the induction of specific immunity to influenza a subtype H3N2.

List of figures

Figure 1 - submitted:

SEQ ID NO: 1 - non-optimized nucleotide sequence of the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2);

SEQ ID NO: 2 - optimized for expression in human cells nucleotide sequence of the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2);

Protein - amino acid sequence encoded by nucleotidyltransferase SEQ ID NO: 1 and SEQ ID NO:2.

Figure 2 - the scheme of expressing the recombinant cassette pseudoadenoviral particles with the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2).

Figure 3 - shows electrophoregrams PCR analyses of DNA recombinant pseudoadenoviral particles, based on the human adenovirus serotype 5 with insert optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) (SEQ ID NO: 2).

Figure 4 - shows the results of immunoblot analysis. Tracks indicate the result of the analysis with the lysate of cells NECK line.

Figure 5 is represented the diagram, the columns show the level antihemagglutinin antibodies identified in RTCA to the influenza virus A/Perth/16/2009(H3N2).

Figure 6 - presents survival curves immunized mice after infection with a lethal dose of influenza virus strain A/Perth/16/2009(H3N2).

7 - presents non-optimized nucleotide sequence of the hemagglutinin gene of A/Perth/16/2009(H3N2).

On Fig - presents-optimized nucleotide sequence of the hemagglutinin gene of A/Perth/16/2009(H3N2).

Figure 9 - presents the amino acid sequence of the hemagglutinin gene of A/Perth/16/2009(H3N2).

It is well known that the genetic code has degeneracy, that is, one amino acid may be coded from one to six different triplets (kadono is) of different nucleotides, which, as a rule, have a difference only in the last nucleotide (Dubinin, N.P. General genetics, M., 1970 s.204-207). Each such codon corresponds to a single tRNA, which is in the process of protein synthesis delivers the appropriate amino acids to the ribosome. However, in different organisms the set of tRNA is different, and there is preferential use of one of the several codons that encode the same amino acid. Data on the frequency of use of codons in different public organisms (for example, a resource www.kazusa.or.jp).

Based on the above biological characteristics of transcription protein in organisms, the author was solved in the nucleotide sequence of the hemagglutinin gene to replace the individual nucleotides in the codons that do not entail replacement of the amino acids, taking into account the preference of the use of tRNA codons in humans, that is, to use gene hemagglutinin with pre-optimized for expression in human cells a nucleotide sequence that, ultimately, led to increased expression of the target gene by introduction of the vector with the insert received hemagglutinin gene in the human body.

To solve the technical challenge:

1) to optimize the nucleotide sequence of the hemagglutinin gene of the virus is influenza A/Perth/16/2009(H3N2) to increase the expression of this gene in human cells;

2) to construct a recombinant pseudoadenoviral particle with the target transgene by embedding the obtained optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2) human adenovirus serotype 5;

3) show cultural - morphological features obtained recombinant pseudoadenoviral particle;

4) show an improvement in the expression of optimized hemagglutinin gene in vitro;

5) show enhanced immunogenicity and protective properties obtained by the claimed invention recombinant pseudoadenoviral particles on the basis of human adenovirus 5 serotype to the influenza virus A/Perth/16/2009(H3N2), and accordingly, to justify their use for the induction of specific immunity to influenza a subtype H3N2.

The following examples illustrate the solution of a technical problem.

Examples of carrying out the invention

Example 1.

This example shows the process of constructing recombinant pseudoadenoviral particles on the basis of the genome of adenovirus human serotype 5 with insert optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009 (H3N2).

At the first stage to improve the expression of the recombinant transgene pseudoadenoviral particle sequence of the hemagglutinin gene of influenza virus strain A/Perth/I 6/2009(H3N2), using the data on the basis of who recommendations (taken from official public source NCBI, GenBank, USA, No. GQ293081.1; which data are available: www.ncbi.nlm.nih.gov on the basis of known information about the degeneracy of the genetic code and the frequency of use of certain types of codons in humans has been pre-optimized for expression in human cells.

To do this, in the nucleotide sequence of the hemagglutinin gene of influenza virus strain A/Perth/I 6/2009(H3N2) was replaced by "inconvenient" for human tRNA codons so that did not happen amino acid replacement sequence. When selecting used the well-known data on the frequency of occurrence of codons in humans (http://www.kazusa.or.jp/codon/). For example, the amino acid leucine (L) is encoded by six codons - UUA, UUG, CUA, CUC, CUG, CUU, but the man is most often used CUC (20%) and CUG (40%), so they were replaced by all the remaining four codon, in the appropriate proportions (CUC - 24.7% and CUG - 56.4 per cent).

In the same way I had done with all other codons that are not in the singular encoding one amino acid.

The figure 1 presents:

SEQ ID NO: 1 - non-optimized nucleotide sequence of the hemagglutinin gene of influenza virus strain strain A/Perth/16/2009(H3N2);

SEQ ID NO: 2. - optimized for expression in human cells nucleotide sequence of the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2);

Protein - amino acid PEFC is the gene sequence of the hemagglutinin of influenza virus A/Perth/16/2009(H3N2), encoded by the nucleotide sequences SEQ ID NO:

1 and SEQ ID NO: 2.

Numbers above the sequences indicate the number of nucleotides in the sequence.

Conducted for the purpose of optimization, replacement of nucleotides in the nucleotide sequence of SEQ ID NO: 1 non-optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2) nucleotides in the nucleotide sequence of SEQ ID NO: 2 optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2) darkened gray rectangles.

The letter amino acid is located under three nucleotides compared two nucleotide sequences (SEQ ID NO: 1 and SEQ ID NO: 2), which it is encoded.

All letter designations nucleotide and amino acid standard and well-known.

Thus, presented on figure 1 unoptimized and optimized nucleotide sequence of the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) consist of 1701 pairs of nucleotides, located underneath one another and compared (top sequence is not optimized hemagglutinin gene, underneath optimized sequence). Only two nucleotide sequences encode 566 amino acids (amino acid sequence shown under the title "protein"), both of which presents the nucleotide sequence of SEQ ID NO: 1 and SE ID NO: 2 encode the same amino acid sequence of the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2), that is containing substances that occur with optimized nucleotide sequence of the hemagglutinin gene of the above strain of influenza virus does not differ from the native, which is required for optimization of the nucleotide sequence.

Thus, the bottom (SEQ ID NO: 2) nucleotide sequence represents a unique optimized for expression in human cells nucleotide sequence of the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2).

Received a unique optimized cDNA sequence of hemagglutinin gene of influenza virus human synthesized well-known chemical method and ligated in the well-known helper plasmid for further perchlorovinyl. Thus, the synthesized nucleotide sequence contains the optimized codons, which do not affect the amino acid composition of the ha, but has greatly improved the level of its expression in human cells.

In the next step, received recombinant pseudoadenoviral particle-based genome of a human adenovirus 5-th serotype containing an insert of the optimized gene sequence of the hemagglutinin of influenza virus strain A/Perth/16/2009(H3N2) - SEQ ID NO: 2.

All of the following cloning was performed using well-known techniques, opican the x in Sambrook J. et al., Molecular cloning: a laboratory manual, 3rd ed., Russell, 2001, vol. 1, 2, 3 - Molecular cloning-a laboratory manual.

Obtaining construction of recombinant pseudoadenoviral particles on the basis of the genome of a human adenovirus fifth serotype (size 70-80 nm) was performed by the method of homologous recombination between the well-known plasmid pJM17 (McGrory W.J. et al., A simple technique for the rescue of early regioni mutations into infectious human adenovirus type 5, Virology, V. 163, No. 2, 1988 - a Simple technique for removal of early region 1 into infectious human adenovirus type 5), containing the genomic part of the human adenovirus serotype 5 with broken E1 region, and a helper plasmid pACCMVpLpA (Roth M. G., Methods in cell biology, No. 43, s - Methods in cell biology.), (Go' mez-Foix, A. et al., Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism, J. Biol. Chem., 1992, №267 (35), 15, s-25134 - Provided by adenovirus gene transfer of the muscle glycogen phosphorylase in hepatocytes alters the regulation of glycogen metabolism). Previously in the Shuttle plasmid pACCMVpLpA was periglomerular optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2) from the helper plasmid, resulted in pACCMVpLpA with expressing cassette with the target transgene (gene hemagglutinin), flanked by parts of the adenovirus genome that are involved in further recombination. Homologous recombination of conduction and in the cell line NECK (for example, No. 300192, CLS, Germany) after cotransfection its plasmid pJM17 and pACCMVpLpA. Because the plasmid pJM17 contains the bacterial site of initiation of replication (Ori) and the gene for resistance to ampicillin inside the area E1 of the genomic part of the adenovirus, this plasmid could not be packaged into an adenoviral virions, and thus prevented the replication of adenoviruses "wild type". After recombination of the Ori and the gene for resistance to ampicillin disappeared, being replaced by a cassette with the target transgene. Recombinant DNA was packaged in the virion capsid, resulting recombinant pseudoadenoviral particles are not able to multiply in nopermission cell cultures, including, in normal human cells, due to the lack of E1 in the genome of the recombinant pseudoadenoviral particles.

Figure 2. Diagram expressing the recombinant cassette pseudoadenoviral particles gene

14

hemagglutinin of influenza virus strain A/Perth/16/2009(H3N2). Pictured:

1 - adenoviral nucleotide sequence (is);

2 - the promoter of cytomegalovirus (→);

3 - transgene (---);

4 - the polyadenylation signal SV40 (Simian vacuolating virus 40) (.........).

To verify compliance with the stated properties of the created designs recombinant pseudoadenoviral of particles based on human adenovirus serotype 5 with the insertion of optimizion the frame hemagglutinin gene of influenza virus type a strain A/Perth/16/2009(H3N2) (SEQ ID NO: 2) was performed as described below PCR (polymerase chain reaction) by known standard methods.

Checked the following properties created design:

1. The authenticity of the recombinant pseudoadenoviral particles, i.e., the availability of the genome of adenovirus human serotype 5. This was performed PCR for adenovirus (DNA sequence encoding hexon adenovirus human serotype 5).

Been using a pair of primers to confirm the presence of the genome (gene hexane) recombinant adenovirus.

ADH12-Reverse

5'-CTCAAAAGTCATGTCTAGCGC-3'

ADHH-Forward

S'-AACTTCCAGCCCATGAGCCG-3'

2. The lack of pathogenicity, i.e. the inability to cause disease adenovirus in humans, E1 region demeterova the resulting recombinant pseudoadenoviral particles, in contrast to adenovirus "wild type"that can cause disease. To confirm the absence of the field E1 PCR was performed.

Been using a pair of primers to the region E1, which should not have created recombinant pseudoadenoviral particles.

KD25-Forward

5'-CGCGGGAAAACTGAATAAGAGGA-3'

wtE1 R-Reverse

5'-ATGTCGGGCGTCTCAGG-3'

3. The presence of insertion optimized hemagglutinin gene (SEQ ID NO: 2). This used the PCR to the hemagglutinin of influenza virus with primers for the target gene hemagglutinin of influenza virus A/Perth/16/2009(H3N2) - optimized for expression in human cells.

H3opt - forward

5' - CGT GCA CCA TCC CGG AAC AG - 3';

H3opt-reverse

5' - TAG AGT TTG TCG AAC TGC TC - 3'

p> The results of PCR with the above primers are represented on a single electrophoregram (Figure 3).

In the figure 3. depicted electrophoregram PCR analyses of DNA recombinant pseudoadenoviral of particles based on human adenovirus serotype 5 with insert optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) (SEQ ID NO: 2).

Bright horizontal stripes on the track - a positive result of the reaction, the absence of bands on the track - negative result.

Tracks: 1, 2, 3 - negative control, DNA of adenovirus and positive control, respectively, with primers for the transgene. Tracks 4, 5, 6 - negative control, DNA of adenovirus and positive control, respectively, with primers for exon of human adenovirus fifth serotype.

Tracks 7, 8, 9 - negative control, DNA of adenovirus and positive control, respectively, with primers on the E1 region of adenovirus person fifth serotype.

M - molecular weight marker Gene Ruler™ DNA Ladder Mix, Fermentas. The analysis was performed in 0.8% of agarose gel.

Thus, according to the results presented on figure 3, the obtained recombinant pseudoadenoviral particle was analyzed by PCR using pairs of primers complementary to the corresponding target transgene (SEQ ID NO: 2), the gene of hexane of human adenovirus five the CSOs serotype, as well as the E1 region of adenovirus to control the possible presence of replication-competent viral particles (revertants wild-type adenovirus).

Shown in the figure the results of PCR analysis show that the E1 region, which should be demeterova the recombinant pseudoadenoviral particles is not detected (negative result on track 8), the DNA sequence encoding hexon adenovirus human serotype 5 is detected (positive result on track 5), gene optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2)(SEQ ID NO: 2) is detected (positive for track 2).

According to the results of the PCR concluded that the obtained recombinant pseudoadenoviral (no E1 region of adenovirus) particle-based adenovirus human serotype 5 (hexon adenovirus human serotype 5) insert optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) (is optimized hemagglutinin gene - SEQ ID NO: 2), indicating that under this construction the claimed invention requirements.

Example 2.

This example describes the culture of the morphological features of the constructed recombinant pseudoadenoviral particles on the basis of the genome of adenovirus human serotype 5 with insert optimized gene to gemigo inina influenza virus strain A/Perth/16/2009(H3N2),SEQ ID NO: 2.

All the used culture methods are well known (Fresno R. and other Culture of animal cells. Methods, M., Mir, 1989, s).

Recombinant pseudoadenoviral particles are collected in the cell line NEC (human embryonic kidney kidney of a human embryo) (for example, No. 300192, CLS, Germany). The genome of this cell type contains the E1, which allows recombinant pseudoadenoviral particles similar to remote areas to gather and multiply in the cells that line. The Assembly of particles is accompanied by lysis of the cells, from the moment transduction prior to lysis of the cells and obtain a new generation of recombinant pseudoadenoviral particles with the insertion of a gene hemagglutinin of influenza virus passes 48 hours.

For growth of the cell line NECK in the adhesion culture, you must use the DMEM (e.g., Invitrogen, No. 52100-047, USA)containing 25 mm glucose, 4 mm L-glutamine and 10% fetal bovine serum, special incubator with a temperature of+37°C and 5% CO2.

The number of recombinant pseudoadenoviral particles capable of transducible (activity) cell line NECK, was determined using the standard method of titration by plaque, the result has got the title of the particles, expressed as plaque-forming units per ml (PFU/ml).

To evaluate the activity of monolayer cell line NECK with confluently 50-70%were infected with a suspension of infected cells, at 10 PFU of virus in culture cascadilla 15 see in two days, infected cells were removed, concentrated by low-speed centrifugation, suspended in phosphate buffer (pH 7,2-7,4) and destroyed with 3-times freezing-thawing. The resulting suspension was osvetleni by centrifugation at 2000 rpm for 10 min at 4°C and was titrated by plaque.

Thus, established the activity of recombinant pseudoadenoviral particles from 5×107up to 3×108PFU/ml (plaque-forming units per ml of culture NEC).

Example 3.

The ability for enhanced expression of recombinant pseudoadenoviral particles optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) was determined in vitro standard method of Western blot turns (Western blot) using monoclonal antibodies to the hemagglutinin.

For this cell line NECK was transducible recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene, and recombinant pseudoadenoviral particles with not optimized for broadcast codons and recombinant pseudoadenoviral particles having the target gene is not related to the hemagglutinin. One day after transduction, the cells of all samples were washed 2 times with phosphate-saline buffer, pH=7.4 and Lisi is ovale in the buffer for drawing, containing dodecyl sodium sulphate and dithiothreitol. Samples rise 7 min at +95°C and cooled to rooms temperature. Next, the resulting lysates of control and transduced cells were subjected to electrophoresis in polyacrylamide gel under denaturing conditions.

As a positive control was used purified hemagglutinin of influenza A (Sion Biological Inc., cat. No. 40043-V08H). After conducting gel, the proteins from the gel were transferred to a special membrane for holding the immunoblot analysis. The immunoblot analysis was performed according to the standard scheme using antibodies to the hemagglutinin of influenza virus A/Perth/16/2009(H3N2) (Sion Biological Inc., cat. No. SEK11056-1). The results of the immunoblot analysis shown in figure 4.

The figure 4 shows the results of the immunoblot analysis. Tracks indicate the analysis with the lysate of cells NECK line:

1 - positive control (purified hemagglutinin of influenza A (Sion Biological Inc., cat. No. 40043-V08H);

2 - negative control (recombinant pseudoadenoviral particles, producing hemagglutinin);

3 - recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 2;

4 - recombinant pseudoadenoviral particles with the insertion of a non-optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 1.

Thus, is igure 4 noticeable staining band detection on track 1 (positive control) and lane 3 (in the lysate of the cell line 293, transduced recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene, SEQ ID NO:

2. In the lysate of cells transduced with recombinant pseudoadenoviral particles with hemagglutinin with non-optimized codons, SEQ ID NO: 1 to track 4, we observed a very weak band, indicating a very low level of expression, in the case of the negative control on track 2 staining was not.

The results of the immunoblot analysis allow to conclude about the significantly enhanced level of expression of recombinant pseudoadenoviral particles optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2) compared to non-optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), which corresponds to the solution according to the invention the task of creating recombinant pseudoadenoviral particles on the basis of the genome of adenovirus human serotype 5, strenuously produce the hemagglutinin of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 2.

Example 4.

In this example discusses how the use of recombinant pseudoadenoviral particles on the basis of the genome of adenovirus human serotype 5, producing the hemagglutinin of influenza virus A/Perth/16/2009(H3N2) by entering them in an effective amount for the induction of specific immunity to the influenza virus is the subtype H3N2.

To confirm usage were immunized mice. The best indicators of immunogenicity and protective properties of the recombinant pseudoadenoviral particles were obtained with the dose of 2×107PFU/mouse, with no side effects at the time of introduction and in the remote period were noted.

To immunize mice with recombinant pseudoadenoviral particles animals were divided into groups of 10 animals under light ether anesthesia intranasally immunized once recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2), SEQ ID NO: 2, at a dose of 2×107PFU/mouse. As control was used groups of mice treated with recombinant pseudoadenoviral particles do not carry the transgene, and physiological NaCl solution. Drug comparisons were recombinant pseudoadenoviral particles with the insertion of a non-optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2), SEQ ID NO: 1.

Blood samples are taken for analysis of serum was carried out three weeks after immunization.

Immunogenicity of recombinant pseudoadenoviral particles with insert hemagglutinin gene was determined by the level antihemagglutinin antibodies in the serum in standard rcga of response inhibition of haemagglutination) (table 1 and figure 5).

Table 1 shows the titers of specific antibodies to the influenza virus A/Perth/16/2009(H3N2) in serum at 21 days after immunization of mice.

Table 1
The name of an administered drugAntihemagglutinin antibodies in rtga (mean geometric titer)
Recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 23429,77±863,5
Recombinant pseudoadenoviral particles with the insertion of a gene hemagglutinin NA3 (not optimized) of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO:1203,44±61,8
Recombinant pseudoadenoviral particles without insertion of the transgene<8
The control substance (physiological NaCl solution)<8

According to table 1 it is evident that the immunogenicity of recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 2 is much superior immunogenicity of recombinant pseudoactivity the x particles with the insertion of a non-optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 1. Control substances were neimenovani.

The figure 5 presents the diagram, the columns show the level antihemagglutinin antibodies identified in RTCA to the influenza virus A/Perth/16/2009(H3N2), while immunization was performed with the following substances:

1 - recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 2;

2 - recombinant pseudoadenoviral particles with an insert is not optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 1;

3 - recombinant pseudoadenoviral particles without insertion of the transgene;

4 is a physiological NaCl solution.

Results the charts in figure 5 show a significant improvement in antihemagglutinin antibodies in the case of immunization with recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 2, compared with recombinant pseudoadenoviral particles with the insertion of a non-optimized hemagglutinin gene of the same strain, SEQ ID NO: 1, and the control substances (titles antihemagglutinin antibodies were absent).

Also three weeks after immunization for the study of protective properties of the recombinant human pseudoactivity the x particles simulated lethal infection by infecting laboratory mice a high lethal dose of influenza virus strain A/Perth/16/2009(H3N2) intranasally under light ether anesthesia. Observation of the animals was carried out for 16 days after infection.

The figure 6 presents survival curves immunized mice after infection with a lethal dose of influenza virus A/Perth/16/2009(H3N2). Immunization was performed:

1 (◆) - recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 2;

2 (■) - recombinant pseudoadenoviral particles with an insert is not optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO:1;

3 (▲) recombinant pseudoadenoviral particles without insertion of the transgene;

4- physiological NaCl solution.

Presented in figure 6, the data indicate the presence of the protective properties of the recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 2, as none of the immunized mice when infected with a lethal dose of the virus A/Perth/16/2009(H3N2) were not lost (100% survival). In the case of immunization with recombinant pseudoadenoviral particles with the insertion of a non-optimized hemagglutinin gene of influenza virus A/Perth/16/2009(H3N2), SEQ ID NO: 1, and without insertion of the transgene protective properties were much weaker survival were, respectively, 32% and 9%. Thus, in the control group, floor who were saline, was observed in 100% death of the mice for 12 days.

Thus, the results of the experiments concluded that created recombinant pseudoadenoviral particles with insert optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2), SEQ ID NO: 2, producing the hemagglutinin of influenza virus strain A/Perth/16/2009(H3N2), have reinforced immunogenic and protective properties against specific strains of the pathogen influenza virus, which allows their use for the induction of specific immunity to influenza a subtype H3N2 in the body.

Conclusion. Based on the examples and tables the results of the research concluded that the goal of the project according to the invention is performed. Created recombinant pseudoadenoviral particle with insert optimized hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2), with activity 5×107up to 3×108PFU/ml and increased level of gene expression of the hemagglutinin of influenza virus strain A/Perth/16/2009(H3N2), reinforced immunogenic and protective properties, which allows its use for induction of specific immunity to influenza a subtype H3N2 in the body, which also confirms the industrial applicability of the claimed invention.

1. Recombinant pseudoadenoviral particle-based Geno is and adenovirus human serotype 5 for the induction of specific immunity to influenza a subtype H3N2, contains expressing cassette inserting the gene for hemagglutinin of influenza virus,
characterized in that
as hemagglutinin gene of influenza virus used in gene hemagglutinin of influenza virus strain A/Perth/16/2009(H3N2) SEQ ID NO:2 with pre-optimized for expression in human cells nucleotide sequence, with increased gene expression of hemagglutinin of influenza virus A/Perth/16/2009(H3N2).

2. Recombinant pseudoadenoviral particle according to claim 1, characterized in that the hemagglutinin gene of influenza virus strain A/Perth/16/2009(H3N2) - optimized nucleotide sequence cloned in expressing cassette under the control of the promoter and containing the polyadenylation signal.

3. Recombinant pseudoadenoviral particle according to claim 2, wherein the promoter is a cytomegalovirus promoter.

4. Recombinant pseudoadenoviral particle according to claim 2, wherein the polyadenylation signal is SV40.

5. Recombinant pseudoadenoviral particle according to claim 1, wherein expressing the cassette is located in the region of the deletion of E1 genome of a human adenovirus serotype 5.

6. The method of using recombinant pseudoadenoviral particles according to claim 1 on the basis of the genome of adenovirus human serotype 5, producing the hemagglutinin of influenza virus A/Prth/16/2009(H3N2) by introducing the individual in an effective amount for the induction of specific immunity to influenza a subtype H3N2.



 

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FIELD: biotechnologies.

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FIELD: medicine.

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FIELD: medicine.

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32 cl, 8 dwg, 7 tbl

FIELD: chemistry.

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FIELD: chemistry.

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7 cl, 7 dwg, 4 tbl, 7 ex

FIELD: chemistry.

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FIELD: medicine.

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FIELD: medicine.

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14 cl, 3 ex, 5 tbl

FIELD: medicine, pharmaceutics.

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FIELD: biotechnologies.

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6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

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FIELD: medicine, pharmaceutics.

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21 cl, 41 dwg, 31 ex

FIELD: biotechnologies.

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FIELD: biotechnologies.

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14 cl, 1 tbl, 4 ex

FIELD: chemistry.

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SUBSTANCE: what is described is a differential diagnostic technique for respiratory viral infections by multiplex reverse transcription and real-time PCR. The technique provides analysis of each test sample for the presence of nucleic acids of 11 respiratory viruses in 3 reaction mixtures. The technique enables differential detection of nucleic acids of primary ARVI agents - influenza A and B viruses, coronaviruses, type 1, 2, 3, 4 parainfluenza viruses, adenoviruses, respiratory syncytial virus, rhinoviruses and enteroviruses in biological samples. The presence of nucleic acid of any respiratory virus in the test sample is determined by an increasing fluorescent signal of specific dye in one of the reaction mixture.

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38 cl, 14 dwg, 25 tbl, 16 ex

FIELD: medicine.

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.

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FIELD: biotechnologies.

SUBSTANCE: characterised is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype and method of its use as a component for production of vaccine for influenza virus A of H1N1 subtype. Presented recombinant particle contains expressing cassette including SV40 polyadenylation signal and cytomegalovirus promoter with influenza virus haemagglutinin gene being included. As influenza virus haemagglutinin gene, haemagglutinin gene of strain A/California/07/2009(H1N1) is used with pre-optimised for expression in human being cells nucleotide sequence provided in SEQ NO:2. These inventions allow raising specific immunity to influenza virus A of H1N1 subtype by provision of overexpression of haemagglutinin gene of influenza virus A/California/07/2009(H1N1).

EFFECT: improvement of the method.

6 cl, 9 dwg, 1 tbl, 4 ex

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