RU2529772C1 - Cold-adapted influenza virus strain b-b/victoria/2/63/87 intended as strain-donor of attenuation for obtaining reassortants of cold-adapted strains for live influenza vaccine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: cold-adapted strain of influenza virus B/Victoria/2/63/87 has unique mutations in the genes encoding proteins PB2, PB1, PA, NP, M1, NS1, and is intended for obtaining cold-adapted reassortant candidate strains for obtaining live influenza seasonal vaccine.

EFFECT: improvement of the strain properties.

2 tbl, 5 ex

Description

The invention relates to medical virology and can be used in the production of live cold-adapted attenuated influenza vaccines.

It is known that a live influenza vaccine is made from attenuated virus strains that are harmless to humans and have the ability to stimulate influenza immunity.

Due to the fact that the antigenic specificity of influenza viruses is very variable, vaccine strains of this virus for influenza vaccines have to be replaced with new almost every year in accordance with the recommendations of the World Health Organization. To obtain such new vaccine strains for live cold-adapted attenuated influenza vaccine and to obtain new reassorted cold-adapted vaccine strains in Russia, cold-adapted strains are used - attenuation donors B / USSR / 60/69 and B / Leningrad / 14/55, also obtained by passages at low temperature (RU 2084525, 1997; RU 2068271, 1996).

However, these strains have a serious drawback - the authors do not have data on which mutations in certain regions of the genes of this virus are present in these strains, which does not allow determining which genes of the virus may be related to strain attenuation, which makes it possible to characterize the genetic homogeneity of the strains , and also to determine their genetic stability during reproduction in the body of vaccinated people. Both strains were cloned by limiting dilution in chicken embryos, which leaves the genetic homogeneity of these strains in great doubt.

In the United States, strain B / Ann Arbor / 1/66, obtained by passage at low temperature (Maassab H, De Borde B. Development and characterization of cold-adapted virus for use as), is used as an attenuation strain donor for preparing a live cold-adapted influenza vaccine. live virus vaccines. Vaccine, 1983 vaccines. Questions of virology, 2005, No. 2: 43-46).

This strain has eight coding mutations in three viral proteins (PA, NP and M1) (see table 2).

Closest to the claimed invention is an attenuated cold-adapted strain of influenza virus B / Leningrad / 14/17/55 to obtain live influenza vaccine strains (RU 2068271, 1996).

The objective of the study is to obtain a genetically homogeneous strain-donor strain attenuating strain of influenza B virus, which has unique mutations in the genes encoding proteins PB2, PB1, PA, NP, M1, NS1 and is intended to produce cold-adapted reassortant candidate strains of a live seasonal influenza vaccine.

The technical result of the claimed invention is the creation of an attenuated cold-adapted strain - an influenza virus donor with genetic homogeneity, characterized by the number and distribution of amino acid mutations in each of the internal proteins of the virus, and capable of multiplying at low temperatures and temperatures of 34 ° C both on chicken embryos and in cell culture, in particular in the MDCK cell line, approved in Russia for the production of viral vaccines for humans (4, 5).

The inventive cold-adapted strain of influenza virus B / Victoria / 2/63/87 obtained by serial passages in chicken embryos and MDCK cells at a gradually decreasing incubation temperature. The resulting strain passed 63 passages at a reduced temperature (of which 29 passages on chicken embryos and 34 passages on MDCK cells), followed by triple cloning by plaque method on an MDCK cell culture. The latter provides complete genetic homogeneity of the ha strain. The resulting strain has good reproductive activity in chicken embryos and on MDCK cells at low temperatures (26 ° C) and does not reproduce at elevated temperatures (38 ° and 40 ° C). The resulting strain of influenza B virus has 8 mutations in all internal genes that distinguish it from the parent wild strain, and 6 out of 8 mutations encode amino acid substitution in proteins PB2, PB1, PA, NP, M1, NS1. 5 out of 6 coding mutations are unique to this strain compared to all the epidemic strains of influenza B currently sequenced.

Strain B / Victoria / 2/63/87 of the influenza B virus is registered in the State collection of the depository of the Research Institute of Virology named after DI. Ivanovsky at number 2615 from 12/13/2010.

Strain B / Victoria / 2/63/87 was fully genetically characterized and cloned three times in MDCK culture by plaque assay. The method of sequencing individual RNA segments of its genome proved its genetic homogeneity.

Examples of obtaining a cold-adapted (ha) variant of the influenza virus B / Victoria / 2/63/87.

Example 1. Obtaining a cold-adapted version. The epidemic parent strain of influenza virus B / Victoria / 2/87 proliferated well at an elevated temperature of 38.5 ° C (ts ₃₈ + sign) and did not breed at a low temperature of 26 ° C (ca ₂₆ - sign). This strain of the virus passed in chicken embryos for 2 passages at 30 ° C, then 3 passages at 28 ° C, then 6 passages at 26 ° C, then 34 passages at 26 ° C in MDCK cell culture and then 18 passages at 26 ° C in chicken embryos.

The obtained variant of the virus, in contrast to the parental epidemic strain, proliferated well at 26 ° C (ca ₂₆ + trait), and also proliferated well at 34 ° C, but did not proliferate at 38.5 ° C (ts ₃₈ - trait).

To obtain a genetically homogeneous strain of the virus, the obtained variant was cloned three times by plaque on an MDCK cell line at 34 ° C and one clone was selected that proliferated well at 26 ° C (ca ₂₆ + trait) and did not proliferate at 38.5 ° C (ts _38.5 - sign). A number of passages of the obtained ha variant on chicken embryos at 34 ° C did not change its ca ₂₆ + and ts _38.5 are signs, which testified to the genetic homogeneity of the cloned ha variant of this virus.

Example 2. Reproduction of a cold-adapted variant in chicken embryos.

When propagated in chicken embryos at 34 ° C of the parent strain, virus titers were 128 GAU / ml and 10 ^8.0 EID ₅₀ / 0.1 ml, at 38.5 ° C - 256 GAU / ml and 10 ^8.5 EID ₅₀ / 0.1 ml , and at 26 ° C the virus did not multiply, i.e. possessed ts _38.5 + and ca ₂₆ - signs. When chicken embryos were infected with the virus variant, the titers at 26 ° C were 64 GAU / ml and 10 ^6.0 EID ₅₀ / 0.1 ml, at 34 ° C - 256 GAU / ml and 10 ^8.5 EID ₅₀ / 0.1 ml, and at 38.5 ° C this variant did not breed, i.e. possessed ca ₂₆ + and ts ₃₈ signs.

Example 3. Reproduction of the xa variant in the MDCK cell line.

Xa variant of virus B / Victoria / 2/63/87 was well propagated in the MDCK cell line at 33 ° C (titers were 128-256 GAU / ml and 10 ^8.0 EID ₅₀ /0.1 ml) and did not breed at 26 ° C.

Example 4. Reproduction of the xa variant and the parent strain of the virus upon infection of mice.

In case of intranasal infection of female Balb / c mice (18.0-20.0 g) with the parent virus strain (50 μl in two nostrils) at a dose of 10 ^7.5 EID ₅₀ / 0.2 ml on the 3rd day after infection, the virus titers in nasopharynx amounted to 10 ^4.5 EID ₅₀ / 0.2 ml, and in the lungs 10 ^3.9 EID ₅₀ /0.2 ml.

For intranasal infection of mice with the same dose of the x-variant, virus titers on the 3rd day after infection the virus titers in the nasopharynx were 10 ^2.0 EID of ₀₅ / 0.2 ml, and in the lungs <10 ^1.0 EID of ₅₀ / 0.2 ml. These data indicate a good attenuation of the ha variant, which lost the ability to reproduce upon infection of mice.

Example 5. Analysis of mutations in the genome of the x variant.

A comparative analysis of the nucleotide sequence in the genes of the parent strain and virus variant B / Victoria / 2/63/87 revealed 8 mutations in 6 genes encoding the internal proteins PB2, PB1, PA, NP, M1, NS1, of which 6 mutations led to the replacement amino acids in the corresponding proteins (see table 1). In RNA segment 1 encoding PB2 protein, at position 555, a G-A nucleotide substitution was observed, which was accompanied by the Arg-Lys amino acid substitution at position 185 of the PB2 protein. The A-G nucleotide substitution in RNA segment 2 at position 366 was accompanied by the Asp-Gly amino acid substitution at position 120 of the PB1 protein. In RNA segment 3, 2 mutations were observed, one of which at position 972 (AT) turned out to be non-coding, and the second at position 1885 (G-A) led to the amino acid replacement of Val-Ile at position 625 of the PA protein. The unique nucleotide substitution of C-T at position 156 in the RNA segment 5 encoding the NP protein resulted in the amino acid substitution Pro-Ser at position 40 of the NP protein. In the RNA segment of the 7th variant, 2 nucleotide substitutions were observed. The first substitution (G-A) at position 70 was followed by the amino acid substitution of Glu-Lys at position 23 of the M1 protein. The second nucleotide substitution of C-T at position 387 of this RNA segment was non-coding. The last G-A nucleotide substitution at position 62 of RNA segment 8 resulted in the Gly-Ser amino acid substitution in the NS1 protein.

A comparison of mutations found in the genome of the xa variant strain B / Victoria / 2/63/87 and mutations in the genome of the American xa variant of the virus B / Ann Arbor / 1/60 (see table 2) showed that the mutations identified by us in the genome The variants B / Victoria / 2/63/87 are unique and differ from the mutations found in the B / Ann Arbor strain 1/60 (5).

Based on the obtained donor strain, reassortants with relevant epidemic strains of influenza B virus were obtained.

LITERATURE

1. Patent No. 2084525 "Attenuated cold-adapted strain of influenza virus B / USSR (69) E for live intranasal influenza vaccine." from 07/20/1997.

2. Patent No. 2068271. An attenuated cold-adapted strain of the influenza virus B / Leningrad / 14/55 to obtain live influenza vaccine strains from 10/27/1996.

3. Maassab H, De Borde B. Development and characterization of cold-adapted virus for use as live virus vaccines. Vaccine, 1983 vaccine. Questions of Virology, 2005, N2: 43-46.

4. Radaeva I.F., Kolokoltseva T.D., Nechaeva E.D. et al. "Creation and certification of banks of transplantable MDCK cell culture for the production of influenza vaccine." Questions of Virology, 2005, No. 2: 43-46.

5. Extracts from Protocol No. 6 of the Committee of Medical Immunobiological Preparations of the Ministry of Health of the Russian Federation of 06.26.2003

6. Hoffmann E., Mahmood K., Chen Z. et al. Multiple gene segments control the temperature sensitivity and attenuation phenotypes of ca B / Ann Arbor / 1/66. J. Virol. 2005.79: 11014-11021.

Table 1 Mutations in the internal genes of cold-adapted variants of strain B / Victoria / 2/63/87 RNA segment Length (nt) Mutations in the nucleotide sequence Protein Length (AK) Amino Acid Substitutions Position Uniqueness* Vic (Dt) Vic (ha) Position Vic (dt) Vic (ha) one 2396 555 Yes AGG Aag PB2 770 185 Arg Lys 2 2386 366 Yes Gac Ggc PB1 752 120 Asp Gly 3 2304 972 No ASA ACT PA 726 - - - 1885 No GTA ATA 625 Val Ile 5 1841 156 Yes SSA TSA NP 560 40 Pro Ser 7 1191 70 Yes GAA AAA M1 248 23 Glu Lys 387 No STS CTT - - - - - - - BM2 109 - - - 8 1096 62 Yes Ggt AGT NS1 281 13 Gly Ser NS2 122 * Uniqueness compared to influenza B viruses from the GenBank database

table 2 Amino acid substitutions unique to the internal genes of strain B / Ann Arbor / 1/66 compared to wild strains of influenza B virus according to Hoffman et al. (6) RNA segment Length (nt) Nucleotide sequence Protein Amino Acid Substitutions Position Uniqueness* Consensus (dt) W / Ann Arbor / 1/66 (ha) Position Consensus (dt) W / Ann Arbor / 1/66 (ha) one 2396 1913 No * ¹ AGC AGA PB2 770 630 Ser Arg 2 2386 PB1 752 3 2304 1320 Yes GTG A PA 726 431 Val Met 1518 Yes Tat CAT 497 Tyr His 5 1841 221 No * ² ACC Gcc NP 560 55 Thr Ala 399 Yes GTG Gcg 114 Val Ala 1287 Yes CCT CAT 410 Pro His 1583 Yes Gct ACT 509 Ala Thr 7 1191 501 No * ² CAT CAA M1 248 759 His Gin 571 Yes ATC GTG 183 Met Val - - - - BM2 109 - - - 8 1096 NS1 281 NS2 122 * Uniqueness in comparison with wild strains of influenza B virus from the GenBank database, including with wild strain ancestor of XA virus V / Ann Arbor / 1/66

* ¹ Mutation was present in only one wild strain of influenza B virus from all studied (B / Harbin / 7/94 (AAF89735)) * ² These mutations were present in the wild strain B / Ann Arbor / 1/66 (wt5).

Claims (1)

The cold-adapted strain of the influenza virus B / Victoria / 2/63/87, deposited in the State virus collection of the FSBI Research Institute of Virology named after D.I. Ivanovskogo of the Ministry of Health and Social Development of Russia under number 2615, which has unique mutations in the genes encoding the proteins PB2, PB1, PA, NP, M1, NS1, and is intended to produce cold-adapted reassortant candidate strains for the production of a live seasonal influenza vaccine.