Attenuated cold-adapted influenza virus strain a/pr/8/59/m2 (h1n1) applicable for vaccinal influenza virus strains as attenuation donor, vaccinal influenza virus strains a/59/m2/california/66/2211 (h2n2) and a/59/m2/tokyo/67/22111 (h2n2)

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions concern the influenza virus strains A/PR/8/59/M2 (H1N1), A/California/1/66 (H2N2) and A/Tokyo/3/67 (H2N2). The vaccinal strains A/59/M2/California/66/2211 (H2N2) and A/59/M2/Tokyo/67/22111 (H2N2) are reassortants prepared by crossing epidemic viruses A/California/1/66 (H2N2) and A/Tokyo/3/67 (H2N2) with cold-adapted heat-sensitive virus A/PR/8/59/M2 (H1N1). The strains A/59/M2/California/66/2211 (H2N2) and A/59/M2/Tokyo/67/22111 (H2N2) are characterised by heat sensitivity, cold adaption, safety and immunogenicity for laboratory animals. The reassortants has inherited two genes coding surface virus antigens (hemagglutinin and neuraminidase) from the epidemic virus and the rest six genes coding non-glycated proteins, from the attenuation donor A/PR/8/59/M2 (H1N1).

EFFECT: presented inventions can be used in preparing a live influenza intranasal vaccine for adults and children.

3 cl, 2 dwg, 4 tbl, 1 ex

 

The invention relates to medical Virology and can be used in health care. Created donor of production to obtain the vaccine strains of live influenza vaccine for the prevention of influenza (Ortomyxoviridae, genus Influenza) subtype A(H2N2) among adults and children in the event of his return to circulation. Also received two vaccine strain of influenza virus subtype A(H2N2) A/59/M2/California/66/2211 (H2N2) and A/59/M2/Tokyo/67/22111 (H2N2) on the basis of the proposed donor of production.

Known donor of production And/Leningrad/134/17/57 (H2N2) to obtain harmless live intranasal vaccine for adults [Alexandrova, G. I., Klimov A. I. Live vaccine against influenza. - SPb.: Science.- 1994.-151]. The specified strain A/Leningrad/134/17/57 (H2N2) on the antigenic properties similar to the virus A(H2N2) circulating in 1957-1968 years, and in the preparation of vaccine virus A(H2N2) method of classical reasontly encounter methodological difficulties, since hyperimmune serum to donor A/Leningrad/134/17/57 (H2N2) cross-reacts with epidemic A(H2N2) viruses, not allowing to form reassortants.

The problem to be solved by the claimed invention is to obtain a new donor of production with an antigenic structure that is different from A(H2N2).

This strain was created as a result of careful study of many variants of objects of study, this donor �attenuatio A/PR/8/59/M2 (H1N1). The prototype of the obtained strain A/PR/8/59/M2 (H1N1) was a well-known donor of A production/PR/8/59/1 (H1N1) [Egorov AYu, Medvedeva THOSE Polezhaev Fl, et al. Peculiarities of obtaining and characterization of a cold-adapted A/PR/8/34 influenza virus variant // Acta Virol.- 1984.- Vol.28.- No.1.- R. 19-25]. The reason for creating a new donor was the following (the disadvantages of the known strain): a prototype A/PR/8/59/1 (H1N1) is resistant to chemotherapy Adamantine row (amantadine, rimantadine).

From the literature it is known that the stability of influenza viruses of type a to the adamantanes develops as a result of a single mutation in any of the following positions of the amino acids in the M2 protein: 26 (leucine), 27 (valine), 30 (alanine), 31 (series) or 34 (glycine) [Belshe RB, Smith MH, Hall CB, et al. Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection // J Virol.- 1988.-Vol.62.- P. 1508-1512; D. M. Weinstock and G. Zuccotti. Adamantane Resistance in Influenza A // JAMA.- 2006.- Vol.295, No. 8, R. 934-936]. According to genome-wide sequencing, prototype A/PR/8/59/1 (H1N1) virus is resistant to adamantanes due to the presence of amino acids threonine and arginine at positions 27 and 31 of the M2 protein, respectively.

Resistance to these drugs limits the use of the vaccine strain of the virus on the basis for mass immunization of the population, because of rimantadine is the most readily available tool for the treatment of influenza infection in case of unforeseen reaction to vaccination.

To eliminate this drawback is not�bhodemon was to modify the donor of A production/PR/8/59/1 (H1N1) by introducing two mutations in the M2 protein of the virus: replacement of threonine (Thr) valine (Val) and arginine (AGD) on the series (Ser) at positions 27 and 31, respectively.

New donor of A production/PR/8/59/M2 (H1N1) was obtained by genetic engineering after modifying a set of plasmids carrying the full-size DNA copy of the eight segments of the "wild" virus A/Puerto Rico/8/34 (H1N1) [Hoffmann, E., Neumann, G., Hobom, G., et al. "Ambisense" approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template // Virology.- 2000. -Vol.267.- No.2.- P. 310-317; Subbarao, K., Chen, H., Swayne, D. et al. Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics // Virology.- 2003.- Vol.305.- No.1.- R. 192-200]. The resulting strain A/PR/8/59/M2 (H1N1) in amino acid sequences of the internal genes of the identical virus A/PR/8/59/1 (H1N1), with the exception of M2 protein, which contained two mutations (Thr-27-Val and Arg-31 to Ser), which is responsible for its adamantane-sensitivity.

It is known that the strain A/PR/8/59/1 (H1N1) virus is attenuated for humans, as it has the properties of temperaturesalinity and holodnodeformirovannoj [Alexandrova, G. I., Klimov A. I. Live vaccine against influenza. - SPb.: Science.- 1994.-151]. We received a donor/PR/8/59/M2 (H1N1) also has signs of temperaturesalinity and holodnodeformirovannoj. Therefore, a new donor is also attenuated for humans.

A sample process

Strain A/PR/8/59/M2 (H1N1) was obtained using the methods of reverse genetics, which included the following. Was determined the nucleotide sequence of the six internal genes known donor A/PR/8/59/1 (HN1) using an automatic sequencer. Further, the obtained sequence was compared with the nucleotide sequence of the genes of the virus A/PR/8/34 (H1N1), cloned in vectors for reverse genetics [Hoffmann, E., Neumann, G., Hobom, G., et al. "Ambisense" approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template // Virology.- 2000. -Vol.267.- No.2.- R. 310-317].

Found amino acid differences are shown in Table 1. Through targeted point mutagenesis have been modified six genes of the virus A/PR/8/34 (H1N1) so that their amino acid sequence was identical to the virus A/PR/8/59/1 (H1N1). Next, in the M gene were introduced additional mutations AC-792,793-GT and A-805-G, is responsible for the adamantane-sensitivity of the virus. Mutagenesis of DNA copies of the genes of the virus A/PR/8/34 (H1N1) was performed using the mutagenesis kit "QuikChange XL Multi site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to manufacturer's instructions.

New donor of A production/PR/8/59/M2 (H1N1) virus was obtained by transfection of the cell line T a set of eight plasmids: six modified plasmids carrying the internal genes of influenza A/PR/8/59/1 (H1N1), and two plasmids that carry genes for hemagglutinin and neuraminidase of influenza A/PR/8/34 (H1N1). Next, the resulting virus accumulated in 10-day-old developing chicken embryos under optimal conditions: 33°C, 48 hours.

Using the full-genome sequencing of influenza virus was determined complete nucleate�tial sequence of strain A/PR/8/59/M2 (H1N1), confirming the identity of the amino acid sequence of its internal genes of the original virus A/PR/8/59/1 (H1N1), with the exception of two positions in the M2-protein (Val-27 and Ser-31).

The nucleotide sequence of the gene of hemagglutinin and neuraminidase was identical to the virus A/PR/8/34 (H1N1).

New donor of A production/PR/8/59/M2 (H1N1) is characterized by signs of temperaturesalinity and holodnodeformirovannoj characteristic of the source of the virus A/PR/8/59/1 (H1N1). He has a reduced level of reproduction in chicken embryos at 39°C (RCT39the difference in the activities of the reproduction of the clone at 33°C and 39°C is 6.4 lg EID50/0.2 ml), and actively propagated at a temperature of 26°C (RCT26the difference in reproductions at 33°C and 26°C is 2.3 lg EID50/0.2 ml). Thus, these two signs modified donor does not differ from the original donor of A production/PR/8/59/1 (H1N1) (table 2).

Attenuatio new donor was assessed by the intensity of its reproduction in the respiratory tract (nasal passages and lungs) mice of the CBA in comparison with the original virus A/PR/8/59/1 (H1N1) virus and virus wild type A/PR/8/34 (H1N1). Reproduction in the nasal passages of mice all studied viruses ranged from 3.9-5.3 lg EID50/ml.

"Wild type" virus was actively published in the lungs. Noted reduced levels of reproduction in the lungs of mice donor attenua�AI A/PR/8/59/M2 (H1N1) and its prototype in comparison with wild virus (1,5 Ig EID 50/ml vs. 6,8 Ig EID50/ml, respectively) (table 2). The decrease in the level of reproduction of influenza virus in the lungs of mice by more than 10,000-fold compared with wild virus is essential to established a donor.

The sensitivity of the new donor of A production/PR/8/59/M2 (H1N1) to the drugs Adamantine row was evaluated by titration of the virus in the culture of MDCK cells in the presence or absence of growth in the environment of the adamantane derivative of rimantadine (with 0.5 or 5.0 μg/ml). Virus titer was expressed in Ig TCID50/ml, the sensitivity of the virus to rimantadine was evaluated by the degree of reduction in viral titer in the presence of rimantadine compared to the titer of the virus without medication. As control of adamantane-resistant virus took "wild" strain of influenza virus A/PR/8/34 (H1N1).

From table 3 it can be seen that the addition of rimantadine to the new donor of A production/PR/8/59/M2 (H1N1) led to a significant reduction in virus titer in cell culture MDCK. Thus, the addition of rimantadine at a concentration of 0.5 μg/ml reduced the virus titer of 1.8 Ig TCID50and the increase of drug concentration to 5.0 mg/ml resulted in a reduction in virus titer of 2.3 Ig TCID50i.e. 200 times.

The control virus A/PR/8/34 (H1N1) did not show sensitivity to rimantadine, maintaining a high titer even in the presence of 5.0 μg/ml of the drug.

Thus, the claimed strain A/PR/859/M2 (H1N1) according to its characteristics meet the requirements requirements for donors of production for live influenza vaccine.

Strain A/PR/8/59/M2 (H1N1) deposited 10.03.2011 years in the Public collections of the CPS pathogens of viral infections, rickettsial FBSI SRC VB "Vector" under number V-654.

The prototype obtained on the basis of a new donor of A production/PR/8/59/M2 (H1N1) vaccine strains is known holodnodeformirovannye strain of influenza virus A/Moscow/21/17/65 (H2N2), which was part of a live influenza vaccine for children in 1967 [Alexandrova, G. I., Mikocka B. A., E. A. Sirotenko, etc. the Results of the study of a specialized option of live influenza vaccine for immunization of preschool and primary school age // Bulletin of Academy of Sciences USSR. - 1968. - No. 9. - P. 41-45]. This vaccine strain was prepared by serial passages at reduced temperature of epidemic virus isolated in 1965.

However, in recent years the circulation (1966-1967) H2N2 viruses have undergone significant antigenic changes, and vaccine strain A/Moscow/21/17/65 (H2N2) in the case of return to the human population of viruses circulating in the late H2N2 wave will not be able to induce a protective response in vaccinated people [Lindstrom SE, Cox NJ, Klimov A. Genetic analysis of human H2N2 and early H3N2 influenza viruses, 1957-1972: evidence for genetic divergence and multiple reassortment events // Virology.- 2004.- Vol.328.- p.101-119].

It is known that in the end of the epidemic cycle of circulating H2N2 viruses divided�ü into two branches, significantly different antigenic properties.

The problem to be solved by the claimed invention is to provide a potentially pandemic vaccine strains for adults and children with antigenic structure of the virus H2N2, in force at the end of the epidemic cycle - viruses A/California/1/66 (H2N2) and A/Tokyo/3/67 (H2N2) belonging to two different branches of H2N2 viruses of the end of the epidemic wave 1966-1967

Vaccine strain of influenza virus A/59/M2/California/66/2211 (H2N2) obtained by genetic reasontly epidemic virus A/California/1/66 (H2N2) with holodnodeformirovannym temperaturecontrolled donor of A production/PR/8/59/M2 (H1N1), with subsequent selection when lowered to 26°C temperature incubation in the presence of antiserum to the donor of production.

Vaccine strain A/59/M2/California/66/2211 (H2N2) is temperaturecontrolled (the difference in rates of infectious activity at incubation temperature of 33°C and 39°C is 7.5 lg EID50/0.2 ml) and holodnodeformirovannym (the difference in rates of infectious activity at incubation temperature of 33°C and 26°C is 3.1 lg EID50/0.2 ml).

Responsible for the antigenic specificity of the surface protein of the vaccine strain is a hemagglutinin (ha) - hi the identical virus A/California/1/66 (H2N2), a antiserum which is fully neutralized.

<> Method genome-wide sequencing established that the vaccine strain A/59/M2/California/66/2211 (H2N2) inherited six genes encoding internal proteins (RV1, RV2, PA, NP, M, NS), from a donor of A production/PR/8/59/M2 (H1N1), and surface proteins hemagglutinin and neuraminidase (NA) from epidemic virus A/California/1 /66 (H2N2).

Accordingly, the vaccine strain A/59/M2/California/66/2211 (H2N2) is characterized by a combination of useful features necessary to vaccine strain: antigenic specificity of epidemic virus A/California/1/66 (H2N2), genome structure, optimal for reassortant vaccine strains, temperaturesalinity and holidaytravelwatch, which correlates with attenuata for man, characteristic of the donor of production.

The morphology of strain - polymorphic typical of influenza virus.

CHARACTERIZATION of STRAIN

Infectious activity during reproduction in developing chicken embryos at 33-34°C for 48 hours - 9,2 lg EID50/ML. Hemagglutinins activity -1:1024.

Strain shows genetic stability of biological signs after 5 passages in chicken embryos (when using large infecting doses).

An example of obtaining the vaccine strain A/59/M2/California/66/2211 (H2N2) are presented in the attached passport.

The strain is deposited in the collections of the Institute virologist�and them. D. I. Ivanovsky No. 2652.

PASSPORT STRAIN

1. The name of the strain A/59/M2/California/66/2211 (H2N2).

2. Series: series 1 (first).

3. Setter reassortment; feature parental viruses:

a) epidemic virus A/California/1/66 (H2N2);

b) the donor of production: A/PR/8/59/M2 (H1N1).

4. The number of passages: 5 in the process of growth re-assortment.

5. Characterization of the strain prior to lyophilization:

(a) the optimal conditions of reproduction: 33°C, 48 hours).

b) hemagglutinins activity: 1:1024;

b) infectious activity: 8.5 lg EID50/0.2 ml;

d) sensitivity to inhibitors: inhibitorscoating in hi with normal serum of horses and Guinea pigs;

d) the difference in rates of infectious activity at 33°C / 39°C: 7,5 lg EID50/0.2 ml;

(e) the difference in rates of infectious activity at 33°C / 26°C: 3,1 lg EID50/0.2 ml;

g) structure of the genome of reassortant according to partial sequencing of genes:

genes from epidemic virus: NA;

genes from the donor of production: RV2, RV1, PA, NP, M, NS.

6. Antigenic specificity of hemagglutinin:

hemagglutinin in hi the identical virus A/California/1 /66 (H2N2), rat antiserum which is fully neutralized. Antigenic specificity of neuraminidase:

neuraminidase is identical to A/California/1/66 (H2N2) according to the complete sequencing of genes.

<> 7. Characteristics of the strain after lyophilization:

a) the date of lyophilization - April 25, 2011;

b) the volume of material in the ampoule - 1.0 ml;

b) infectious activity of 7.7 lg EID50/0.2 ml;

8. Bacteriological control: posting date: June 1, 2011, the result is sterile.

9. Harmless to mice and Guinea pigs by subcutaneous and intraperitoneal administration: harmless.

Vaccine strain A/59/M2/Tokyo/67/22111 (H2N2) obtained by genetic reasontly epidemic virus A/Tokyo/3/67 (H2N2) with holodnodeformirovannym temperaturecontrolled donor of A production/PR/8/59/M2 (H1N1), followed by selection at lowered to 26°C temperature incubation in the presence of antiserum to the donor of production.

Vaccine strain A/59/M2/Tokyo/67/22111 (H2N2) is temperaturecontrolled (the difference in rates of infectious activity at incubation temperature of 33°C and 39°C is 6.9 lg EID50/ml) and holodnodeformirovannym (the difference in rates of infectious activity at incubation temperature of 33°C and 26°C is equal to 3.2 lg EID50/ml).

Responsible for the antigenic specificity of the surface protein of the vaccine strain is a hemagglutinin (ha) - hi the identical virus A/Tokyo/3/67 (H2N2), a antiserum which he completely neutralized.

Method genome-wide sequencing established that the vaccine strain A/59/M2/Tokyo/67/22111 (2N2) inherited six genes encoding internal proteins (RV1, RV2, PA, NP, M, NS), from a donor of A production/PR/8/59/M2 (H1N1), and surface proteins hemagglutinin and neuraminidase (NA) from epidemic virus A/Tokyo/3/67 (H2N2).

Accordingly, the vaccine strain A/59/M2/Tokyo/67/22111 (H2N2) is characterized by a combination of useful features necessary to vaccine strain: antigenic specificity of epidemic virus A/Tokyo/3/67 (H2N2), genome structure, optimal for reassortant vaccine strains, temperaturesalinity and holidaytravelwatch, which correlates with attenuata for man, characteristic of the donor of production.

The morphology of strain - polymorphic typical of influenza virus.

CHARACTERIZATION of STRAIN

Infectious activity during reproduction in developing chicken embryos at 33-34°C for 48 hours - 9,0 lg EID50/ml. Hemagglutinins activity -1:1024.

Strain shows genetic stability of biological signs after 5 passages in chicken embryos (when using large infecting doses).

An example of obtaining the vaccine strain A/59/M2/Tokyo/67/22111 (H2N2) are presented in the attached passport.

The strain is deposited in the collections of the Institute of Virology. D. I. Ivanovsky No. 2653.

PASSPORT STRAIN

1. The name of the strain A/59/M2/Tokyo/67/22111 (H2N2).

2. Series: series 1 (first).

3. Setter �assortative; characteristics of the parental viruses:

a) epidemic virus A/Tokyo/3/67 (H2N2);

b) the donor of production: A/PR/8/59/M2 (H1N1).

4. The number of passages: 5 in the process of growth re-assortment.

5. Characterization of the strain prior to lyophilization:

(a) the optimal conditions of reproduction: 33°C, 48 hours).

b) hemagglutinins activity: 1:1024;

b) infectious activity: 8.8 lg EID50/0.2 ml;

d) sensitivity to inhibitors: inhibitorsfatty in hi with normal serum of horses and Guinea pigs;

d) the difference in rates of infectious activity at 33°C / 39°C: 6,9 lg EID50/0.2 ml;

(e) the difference in rates of infectious activity at 33°C / 26°C: 3,2 lg EID50/0.2 ml;

g) structure of the genome of reassortant according to partial sequencing of genes:

genes from epidemic virus: NA;

genes from the donor of production: RV2, RV1, PA, NP, M, NS.

6. Antigenic specificity of hemagglutinin:

hemagglutinin in hi the identical virus A/Tokyo/3/67 (H2N2), rat antiserum which is fully neutralized. Antigenic specificity of neuraminidase:

neuraminidase is identical to the virus A/Tokyo/3/67 (H2N2) according to the complete sequencing of genes.

7. Characteristics of the strain after lyophilization:

a) the date of lyophilization - April 25, 2011;

b) the volume of material in the ampoule of -1.0 ml;

b) infectious activity of 7.5 lg e�D 50/0.2 ml;

8. Bacteriological control: posting date: June 1, 2011, the result is sterile.

9. Harmless to mice and Guinea pigs by subcutaneous and intraperitoneal administration: harmless.

Table 1
Nucleotide and amino acid differences of the internal genes of the donor of A production/PR/8/59/1 (H1N1) and genes of the virus A/PR/8/34 (H1N1), cloned in vectors for reverse genetics.
GenenucleotideProteinamino acid
position59/11PR-wt2position59/11PR-wt2
RV2342AndGRV2105NoMet
779GAnd 251ArgLys
923GAnd299ArgLys
1106And360TightSer
1537AndG504llVal
1660TG545LeuVal
1916AndG630LysArg
2132AndG702LysArg
RV1196GAndRV158AspAsn
549And175AsnLys
639GAnd205MetHe
647AndG208LysArg
670AndG216 SerGly
1335G437CysTrp
294GAndPB1-F259ArgLys
297GAnd60ArgGin
RA65TRA14AlaVal
497AndG158LysArg
1101And T359LysAsn
1672And550lleLeu
NP98GAndNP18GlyGlu
328T95SerPro
1102G353LeuVal
1318AndG425lleVal
1334And430AsnThr
M434AndGM1137ThrAla
476AndT151SerCys
NS94TNS123AlaVal
329And71AspGlu
575GAndNS2 25GlyGlu
763AndG88lleVal
1donor of A production/PR/8/59/1 (H1N1);
2the genes of the virus A/PR/8/34 (H1N1), cloned in vectors for reverse genetics.

td align="center"> 6,8
Table 2
Phenotypic characterization of a new donor of A production/PR/8/59/M2 (H1N1) and the original donor A/PR/8/59/1 (H1N1) in comparison with the virus wild type A/PR/8/34 (H1N1)
VirusesIndicators of reproduction in RCA ((Ig EID50/ml)Virus titer (Ig EID50/ml) in the lungs and nasal passages of micePhenotype
RCT26RCT39nasal passageslight
A/PR/8/346,51,05,3non-ca, non-ts, non-att
A/PR/8/59/12,87,33,91,5Ca, ts, att
A/PR/8/59/M22,36,44,31,5Ca, ts, att

Table 3
Study the sensitivity of the new donor of A production/PR/8/59/M2 (H1N1) virus to rimantadine in comparison with the virus wild type A/PR/8/34 (H1N1)
VirusThe titer of the virus, Ig TCID50/mlThe reduction in virus titer in the presence of rimantadine, Ig TCID50
without rimantadinerimantadine 0.5 μg/mlrimantadine 5,0 mg/ml0.5 μg/mlA 5.0 mg/ml
A/PR/8/348,88,68,60,20,2
A/PR/8/59/M28,66,86,31,82,3

Example 1. The results of the STUDY of IMMUNOGENICITY AND PROTECTIVE EFFICACY of the VACCINE STRAINS H2N2 IN EXPERIMENTS ON MICE

Immunogenicity of vaccine strains H2N2

Immunogenicity of the vaccine strains A/59/M2/CA/66/2211 (H2N2) (59/Cal/2211) and A/59/M2/Tokyo/67/22111 (H2N2) (59/Current/22111) was evaluated by two-fold immunization of mice of the CBA (18 animals per group) vaccine strains, taken in doses of 100 Mead50and 1000 Meade50(50% bear mouse infective doses). Control animals received the drug or placebo (PBS). Taking samples of blood serum were performed at day 28 after the first vaccination and at 28 days after the second immunization. Titles antihemagglutination antibodies were detected in the reaction of hemagglutination-inhibition (HAI). As antigens in hi used epidemic virus A/California/1/66 (H2N2) and A/Tokyo/3/67 (H2N2). Table 4 shows that immunization with vaccine strains resulted in the formation of humoral antibodies not only against homologous virus, but also against heterologous strains. Immunization of mice with a higher dose of 1000 Meade50caused the formation of significantly higher titers of both homologous and hetero�ulichnyh antibodies. Control animals did not develop any antibodies to the virus H2N2.

Table 4
Immunogenicity of vaccine strains H2N2, introduced in doses of 100 Mead50and 1000 Meade50in experiments on mice
Vaccine groupDose, MFA50The geometric mean return of credits antihemagglutination of hi antibodies in the antigen H2N2
A/California/1/66A/Tokyo/3/67
Day 28Day 56Day 28Day 56
59/Cal/22111006,8156,65,012,3
100022,3255,85,041,2
59/Current/221111005,00,5 10,260,3
10006,942,520,6122,8
Placebo-5,05,05,05,0

Protective efficacy of the vaccine strains H2N2

Protective efficacy of the vaccine strains A/59/M2/CA/66/2211 (H2N2) and A/59/M2/Tokyo/67/22111 (H2N2) was evaluated by experimental infection of immunized mice epidemic virus A/California/1/66 (H2N2) and A/Tokyo/3/67 (H2N2), taken in a dose of 105EID50. 9 mice from each vaccine group were infected with influenza A/California/1/66, and the remaining 9 virus A/Tokyo/3/67 (H2N2). At 3 days after challenge in 4 mice from each group were taken organs: lungs, nasal passages, brain, spleen. The titer of virus in organs of mice was determined by titration of tissue homogenates in developing chicken embryos by the method of limiting dilutions. For the remaining 5 mice in the group were watching, weighing every 2 days for 14 days after infection. As can be seen from figure 1, immunized with a higher dose of the vaccine strains (1000 Meade50) mice were �totally protected from reproduction as homologous, and heterologous wild type virus. Mice immunized with a lower dose of 100 Mead50were fully protected against homologous reproduction of the virus, whereas heterologous virus was detected in the lungs of mice in low titers. These titers were significantly lower than those in mice of the control groups (p<0,001 according to the Mann-Whitney test).

The study of the dynamics of weight changes of mice immunized with strains A/59/M2/CA/66/2211 (H2N2) and A/59/M2/Tokyo/67/22111 (H2N2), also indicates the protective effect of vaccination. As can be seen from figure 2, the decrease in weight in vaccinated mice did not exceed 7.5%, while in mice of the control group weight loss reached 15-18% at 8-10 days after the challenge.

Thus, the study of vaccine strains A/59/M2/CA/66/2211 (H2N2) and A/59/M2/Tokyo/67/22111 (H2N2), obtained using a new donor of A production/PR/8/59/M2 (H1N1), have proven their immunogenicity and prophylactic efficacy against a disease caused by the homologous virus and heterologous H2N2 strain.

1. Attenuated, holodnodeformirovannye strain of influenza virus, Ortomyxoviridae, genus Influenza, A/PR/8/59/M2 (H1N1), deposited in the Public collections of the CPS pathogens of viral infections, rickettsial FBSI SRC VB "Vector" under number V-654, which is the donor of production to obtain the vaccine strains of live influenza vaccine, PaTiPa(H2N2).

2. Reassortant vaccine strain of influenza virus, Ortomyxoviridae, genus Influenza, A/59/M2/CA/66/2211 (H2N2), deposited in the Institute of Virology.D.And.Ivanovo No. 2652 obtained by the donor of production according to claim 1, and used to receive live influenza intranasal vaccine for adults and for children.

3. Reassortant vaccine strain of influenza virus, Ortomyxoviridae, genus Influenza, A/59/M2/Tokyo/67/22111 (H2N2), deposited in the Institute of Virology.D.And.Ivanovo No. 2653 obtained by the donor of production according to claim 1 and used to obtain live influenza intranasal vaccine for adults and for children.



 

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FIELD: biotechnology.

SUBSTANCE: proposed primers comprise endonuclease cleavage sites, flanking genomic regions encoding the glycoproteins Gn and Gc, and the nucleoprotein N, for obtaining libraries of genes encoding glycoproteins Gn and Gc and N nucleoprotein of Rift Valley fever virus.

EFFECT: invention can be used in creating a bank of nucleotide sequences encoding immunodominant proteins of Rift Valley fever virus Gn, Gc and N, which can be used for creation of diagnostic and vaccine preparations based on recombinant technologies.

3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology and can be used for the identification of heteromultimeric ubiquitins possessing an ability to bind with a ligand-antigen. The method includes the contact of a totality of heterodimeric modified ubiquitins, including two ubiquitin monomers, bound to each other by a head-to-tail scheme, with the potential ligand in a display way. Each of the said monomers is modified in a different way and contains 5-8 substitutions in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 SEQ ID NO:1. After that, a heterodimeric modified protein, which has bound with the ligand with the binding affinity Kd in the range of 10-7-10-12 M and the monovalent binding activity. Claimed are DNA-libraries, responsible for obtaining a population of the said heteromultimeric ubiquitins, as well as libraries of proteins, obtained by the expression of the said DNA-libraries.

EFFECT: invention makes it possible to obtain the novel bonding proteins based on heteromultimeric ubiquitin, capable of specific high affinity binding with the selected ligands.

8 cl, 17 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented a composition and a method for producing it. The characterised composition contains: an effective amount of a viral antigen, which represents a live attenuated rotavirus pre-processed in 0.1% human serum albumin, and a pharmaceutically acceptable buffer. A method for producing a composition involves growing Vero cell culture pre-cultured in the presence of 5% foetal calf serum and 0.1% human serum albumin, infecting the above Vero cell culture with the live attenuated rotavirus, propagating the virus in the cell culture and adding a pharmaceutically acceptable buffer to the above virus.

EFFECT: presented inventions can be used to prevent rotavirus infection and/or rotavirus gastroenteritis.

11 cl, 17 dwg, 4 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.

EFFECT: characterised inventions can be used to prevent infection, caused by porcine Torque teNo virus.

23 cl, 53 dwg, 5 tbl, 24 ex

FIELD: biotechnology.

SUBSTANCE: characterised strain was isolated from diseased pigs and produced by serial passages on sensitive hetero- and homologous cell cultures and deposited in the collection of the FSBI "Federal Animal healthcare centre" under the registration number of FMD virus strain A No.2187/Kuti/2013 (production). The presented strain is reproduced in monolayer cell culture of porcine kidney (PK), passaged cell cultures of kidney of Siberian mountain ibex (SMIK-30), VPK-21 and IB-RS-2. During 18÷24 hours of incubation the virus yield in the said cell cultures reaches the values of 6.0÷7.0 lg TCD50/cm3. At high multiplicity of infection (1÷10 TCD/cell) causes TCID50 after 5 hours, while maintaining the original characteristics when passaging in cell cultures for 5 passages.

EFFECT: invention can be used to monitor the antigenic and immunogenic activity and for producing biological products for diagnostics and specific prophylaxis of FMD of type A.

6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology. The subject of the invention is a new strain of the Sendai virus Sen293nsk1 adapted to effective replication in the human cell culture HEK293. The produced strain of the Sendai virus possesses lower virulence for laboratory mice and higher ability to destroy human tumour cells. The strain is supposed to be used experimentally as a therapeutic oncolytic preparation for treating malignant diseases. The invention can be used in treating oncologic diseases.

EFFECT: invention enables providing higher clinical effectiveness in oncologic diseases by using the murine Sendai virus non-pathogenic for humans, possessing an increased oncolytic activity and intensifying anti-tumour immunity.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.

EFFECT: obtaining pharmaceutical compositions, which include poxviruses.

11 cl, 3 dwg

FIELD: biotechnology.

SUBSTANCE: method of detection and differentiation of a genome of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates comprises: extraction of the DNA from the biological material, posing a multiplex polymerase chain reaction using synthesised primers complementary to regions of genes M130R and M151R of the myxoma virus of rabbit, and having the following nucleotide composition: MF 5'TGG-AGC-TTT-TCA-AGC-ATT 3', MR 5'ATA-TCT-CGG-CTC-TAG-GGC-GAG 3', MZ 5' [FAM]-AG-CGT-CGG-ACG-TCT-TCG-TT-[RTQ1] 3', VF 5'AGC-CCT-ATA-AAC-CCG-TAG-ACG-AAC 3', VR 5'CAA-GCT-TTT-TTT-TAT-CCT-CGT-CCG 3', VZ 5' [R6G]-TCG-ACG-GTT-TCG-TCC-GCC-TTC-TTG-[BHQ2] 3', DNA amplification of the virus and evaluation of the reaction. The present inventions may be used in veterinary virology for the detection and differentiation of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates.

EFFECT: increase in accuracy.

2 cl, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.

EFFECT: preparing the new avian astrovirus.

11 cl, 5 dwg, 4 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing influenza A or B viruses in a cell culture, and a composition of the cell culture for preparing influenza A or B viruses.

EFFECT: invention provides the serum-free culture medium, avoids the need for the stage of cell culture medium replacement, prepares the influenza viruses with the high live virus recovery and can be used for the active immunisation of individuals and for producing the antibodies for various applications, including passive immunisation and diagnostic immunoassays.

22 cl, 24 dwg, 47 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to veterinary science and biotechnology. Immunogenic compositions containing a canine influenza virus and a canine respiratory coronavirus, can contain Bordetella bronchiseptica, pertactin, canine para-influenza virus and serotype 2 canine adenovirus; they are effective for treating or preventing the complex of infectious respiratory diseases in dogs.

EFFECT: using the immunogenic compositions is accompanied by no immunologically adverse action between various viral and bacterial antigens.

20 cl, 3 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing an immunogenic composition. The method provides preparing a recombinant haemagglutinin (HA) molecule of influenza A virus subtype H5 (HA H5) and mixing the recombinant molecule with a pharmaceutically acceptable carrier and/or excipient. The recombinant molecule contains an asparagine substitute in position 223 of amino acid sequence as compared to the respective wild-type molecule. Substitution of specific residues in HA, such as asparagine introduction in position 223 in HA H5 enables increasing haemagglutination inhibition reaction sensitivity as a result of receptor specificity and/or ability to binding of antibody-antigen.

EFFECT: invention can be used in medicine.

14 cl, 3 dwg, 7 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for producing viral-like influenza virus particles (IVP) in a plant or a part thereof. The method involves the expression of a new influenza virus protein HA in plants and purification thereof. The invention also aims at IVPs containing the influenza virus protein HA and herbal lipids. The invention also refers to nucleic acids coding an improved influenza virus HA, as well as to vectors. The IVPs can be used in developing the influenza vaccines or for the treatment of existing vaccines.

EFFECT: presented group of inventions can be used in medicine.

18 cl, 44 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to immunology, and can be used for preventing influenza. That is ensured by combined single administration of cytokine that is ensured by interferon gamma, and an inactivated anti-influenza vaccine. Interferon gamma is administered intermuscularly or subcutaneously in a dose of 0.5 ml 100,000 International Units±10% into the upper one-third of one shoulder; then the inactivated anti-influenza vaccine 0.5ml based on vaccine influenza viral strains (H5N1) Orniflue containing heamagglutinin in a minimum dose of 15 mcg/dose for this vaccine is administered intramuscularly into the upper one-third of the other shoulder.

EFFECT: method enables increasing immunogenicity and protective activity of the vaccine ensured by single vaccination and reducing side effects on the antigen by the immune response modulation.

9 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with an influenza virus strain. The vaccine strain B/60/Massachusetts/2012/10 is a reassortant, obtained by crossing the "wild" virus B/Massachusetts/2/2012 with the cold-adapted temperature-sensitive virus B/USSR/60/69 -attenuation donor. The strain B/60/Massachusetts/2012/10 is deposited in the State Collection of Viruses FSBI D.I.Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health under No 2740, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (HA) and neuraminidase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza morbidity among adults and children.

4 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with a strain of influenza virus. The vaccine strain A/17/Indiana/2011/72 (H3N2v) - reassortant, obtained by crossing of the "wild" virus A/Indiana/10/2011 (H3N2v) with the cold-adapted temperature-sensitive virus A/Leningrad/13 4/17/57 (H2N2) - attenuation donor. The strain A/17/Indiana/2011/72 (H3N2v) is deposited in the State Collection of Viruses FSBI D. I. Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health, under No 2739, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature-sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (NA) and neuramindase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza disease morbidity among adults and children.

4 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medical virology and concerns a vaccinating influenza virus strain. The characterised strain B/60/Wisconsin/2010/125 is a reaccortant produced by crossing the epidemic virus B/Wisconsin/1/2010 with the cold-adapted heat-sensitive virus B/USSR/60/69 that is an attenuation donor. The strain B/60/Wisconsin/2010/125 is characterised by heat-sensitivity and cold-adaptation. The reassortant has inherited genes coding the surface antigens, haemagglutinine (HA) and neuraminidase (NA), from the epidemic virus and rest six genes coding internal non-glycosylated proteins, from the attenuation donor. The strain is deposited in the State Collection of Viruses of Federal State Budgetary Institution D. I. Ivanovskiy Research Institute of Virology, under No. 2723.

EFFECT: invention can be used in practical health care for preventing the influenza rate in adults and children.

1 dwg, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. An influenza virus contains 6 internal genome segments of one or more virus donors and genome segments coding an NA polypeptide, and the NA polypeptide. The NA polypeptide comprises the amino acid sequence SEQ ID NO:6. There are also described immunogenic composition and vaccine containing the above virus, as well as using it for preparing a therapeutic agent. The invention can be used in medicine.

EFFECT: what is described is an influenza reassortant virus.

16 cl, 4 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: chicken embryos are infected; a virus-containing allantoic fluid (VAF) is collected, and the VAF is purified by microfiltration. The VAF is purified with the use of polyethylene glycol of the molecular weight of 6000 in the concentration of 1.5% and microfiltered on depth filters having pore diameters of 1.0 mcm and 10.0 mcm. Further, the purified VAF is concentrated by ultrafiltration on membranes with the molecular weight cut 100 kD, and the prepared concentrate is purified in a sucrose gradient by ultracentrifugation.

EFFECT: using the method enables the higher quality of virion purification, as well as the higher number of viral antigens recovered from one chicken embryo.

1 tbl, 1 dwg, 6 ex

FIELD: biotechnology.

SUBSTANCE: strain A/17/Victoria/2011/89 (H3N2) propagates actively in developing chicken embryos at the optimum temperature of 32°C. It is characterised by temperature-sensitivity and cold-adaptiveness. The reassortant inherited genes encoding the surface antigens of virus of haemagglutinin (HA) and neuraminidase (NA), from the epidemic parental virus and other six genes encoding internal non-glycated proteins from attenuation donor. The strain is deposited in the State Collection of viruses FSBI "Research Institute of Virology n.a. DI Ivanovsky" under the number 2724.

EFFECT: invention can be used in practical healthcare for prevention of seasonal influenza morbidity among adults and children, using live influenza intranasal vaccine from the said strain.

1 dwg, 6 tbl

FIELD: biotechnology, medicine, pediatrics.

SUBSTANCE: invention proposes the vaccine strain A/47/Panama/99/234 (H3N2) that represents reassortant prepared by breeding the epidemic virus A/Panama/2007/99/ (H3N2) with the cold adopted temperature-sensitive virus A/Leningrad/134/47/57 (H2N2) as attenuation donor that is harmless for children. The strain A/47/Panama/99/234 (H3N2) multiplies actively in developing chicken embryos at optimal temperature 33-34oC and characterized by sensitivity to temperature and adaptation to cold. From epidemic virus the reassortant inherited two genes encoding surface proteins (hemagglutinin and neuraminidase) and six genes encoding non-glycosylated proteins from the attenuation donor. The strain A/47/Panama/99/234 (H3N2) is areactogenic for children in its intranasal administration. The vaccine strain A/47/Panama/99/234 (H3N2) corresponds by its biological properties and reactogenic indices to requirements for vaccine strains in accordance to Pharmacopoeia article 42-3353-97 with respect to the live anti-influenza vaccine for intranasal applying for children of 3-14 years old.

EFFECT: valuable properties of strain.

2 tbl, 1 dwg, 1 ex

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