RU2746274C2 - Vaccine strain of influenza b/60/colorado/2017/1 (victoria line) for the production of live influenza intranasal vaccine for adults and children - Google Patents

Abstract

FIELD: virology.

SUBSTANCE: proposed is influenza virus strain B / 60 / Colorado / 2017/1 (Victoria Line). It was obtained on the basis of the epidemic virus B / Colorado / 06/17 (Victoria line) and a cold-adapted attenuation donor B / USSR / 60/69, harmless to humans. The claimed strain is characterized by temperature sensitivity and cold adaptation, harmlessness to laboratory animals. The strain contains genes encoding the surface proteins of the influenza virus hemagglutinin (HA) and neuraminidase (NA), inherited from an antigenically relevant circulating epidemic strain, and the remaining six genes encoding internal and non-structural proteins (PB2, PB1, PA, NP, M, NS) from an attenuation donor harmless to humans.

EFFECT: strain B / 60 / Colorado / 2017/1 (Victoria Line) can be used in medicine to prevent the incidence of influenza among adults and children as part of a live influenza intranasal vaccine.

1 cl, 4 tbl

Description

The invention relates to medical virology and can be used in practical health care for the prevention of influenza among adults and children using a live influenza intranasal vaccine from a strain of influenza type B, the Orthomyxoviridae family, the genus Influenzavirus B, Victorian line, B / 60 / Colorado / 2017 / one.

As a result of the emergence and widespread circulation in the circulation of a new epidemic strain of the influenza virus belonging to the antigenic line B / Victoria - B / Colorado / 06/2017, the World Health Organization decided to replace the composition of influenza vaccines in the Southern and then in the Northern Hemispheres. for the 2019 and 2019/2020 epidemic seasons, respectively [World Health Organization. WHO Recommendations on the composition of influenza virus vaccines. Recommended composition of influenza virus vaccines for use in the 2019 southern hemisphere influenza season. URL: https://wvvw.who.int/influenza/vaccines/virus/recommendations/2019_south/en/. World Health Organization. WHO Recommendations on the composition of influenza virus vaccines. Recommended composition of influenza virus vaccines for use in the 2019-2020 northern hemisphere influenza season. URL: https://www.who.int/influenza/vaccines/virus/recommen dations / 2019_20_north / en /). In this regard, the well-known vaccine strain B / 60 / Phuket / 2013/26 antigenic line B / Yamagata is a prototype [Kiseleva I.V., Krutikova E.V., Dubrovina I.A., Kuznetsova V.A., Bazhenova E.A., Larionova N.V., Rudenko L.G. - RF patent No. 2605926. Publ. 12/27/2016, Bull. No. 36] - has lost its antigenic relevance, since it belongs to a different antigenic lineage, and as a result cannot cause a protective reaction during an epidemic caused by B / Colorado / 06/17-like strains of the influenza virus.

The problem to be solved by the claimed invention is to obtain a vaccine strain for a live influenza vaccine of the current antigenic variety based on the epidemic virus B / Colorado / 06/17 (Victoria line) and a cold-adapted attenuation donor B / USSR / 60/69, harmless to humans.

Currently used strains for live influenza vaccines are obtained by the method of genetic reassortment (recombination) of epidemically relevant viruses with cold-adapted attenuation donors [GI Aleksandrova. Application of the method of genetic recombination to obtain vaccine strains of the influenza virus. Vopr. virusol. 1977. No. 4. S. 387-395].

Attenuation donor B / USSR / 60/69 - cold-adapted (ca) and temperature-sensitive (ts) strain of the influenza virus, approved for the production of harmless live intranasal vaccines for adults and children [Alexandrova G.I. New in epidemiology and prevention of viral infections. L., 1968. S. 66-83]. The attenuation donor B / USSR / 60/69 was obtained from the epidemic influenza virus B / USSR / 69 by the method of cold adaptation [GI Alexandrova, AI Klimov. Live flu vaccine. SPb .: Science. 1994. 151 s.], As a result of which it became harmless to humans, ie. acquired the ability to multiply asymptomatically in the upper respiratory tract of vaccinated persons, causing the development of full-fledged antiviral immunity.

The purpose of reassortment is to obtain a strain with a vaccine genome formula of 6: 2. The genes encoding the surface proteins of the influenza virus hemagglutinin (HA) and neuraminidase (NA) are inherited from an antigenically relevant circulating epidemic strain, and six genes encoding internal and non-structural proteins (PB2, PB1, PA, NP, M, NS) are inherited from a harmless for human donor attenuation.

Getting a vaccine strain. The vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) was obtained by the method of genetic reassortment of the epidemic virus B / Colorado / 06/2017 (Victoria line) with the attenuation donor B / USSR / 60/69 by simultaneous infection of developing chicken embryos (ECE ) a mixture of parental viruses in equivalent infectious doses, followed by three-fold selection of clones with the desired properties at an incubation temperature lowered to 26 ° C in the presence of antiserum to the attenuation donor. The clones were additionally purified by three consecutive clonings by the limiting dilution method in the presence of antisera to the attenuation donor at low (26 ° C) and optimal (32 ° C) incubation temperatures. A pure clone was tested for phenotypic characteristics (ts, ca-phenotype) and according to the genome formula for compliance with the requirements for vaccine strains.

Antigenic characteristics of the vaccine strain.

The study of the antigenic properties of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) obtained by the authors showed that:

- Responsible for the antigenic specificity of the surface protein of the vaccine strain - hemagglutinin (HA) - is antigenically identical to the epidemic virus B / Colorado / 06/2017 (Victoria line) according to RTGA with homologous serum, since the indicators of interaction of the reassortant B / 60 / Colorado / 2017/1 and the parental strain B / Colorado / 06/2017, with the rat antiserum obtained against the epidemic virus B / Colorado / 06/2017, completely coincided (Table 1), which was also confirmed by full genome sequencing;

- the second surface protein of the vaccine strain responsible for antigenic specificity - neuraminidase (NA) - was determined by partial sequencing, as well as by full genome sequencing and is identical to the B / Colorado / 06/2017 virus (Victoria line).

Genome formula. Partial DNA sequencing of gene copies was used to analyze the genome composition of the obtained reassortants [Matyushenko V. et. al. Genotyping assay for differentiation of wild-type and vaccine viruses in subjects immunized with live attenuated influenza vaccine. PLoS One. 2017. Vol. 12 No. 7. P.e0180497. doi: 10.1371 / journal.pone.0180497].

The 6: 2 genome formula meets the requirements for live influenza vaccine strains: the genes encoding the surface proteins hemagglutinin (HA) and neuraminidase (NA) belong to the epidemic parental virus B / Colorado / 06/2017 (Victoria lineage), and the genes encoding internal and non-structural proteins (PB2, PB1, PA, NP, M, NS) belong to the attenuation donor B / USSR / 60/69. The results of the analysis of all genes of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) are presented in table. 2.

The data obtained during the initial selection of the vaccine candidate was confirmed by its genome-wide sequencing and showed that the vaccine strain B / 60 / Colorado / 2017/1 (Victoria lineage) inherited all 6 genes encoding internal and non-structural proteins from the attenuation donor B / USSR / 60/69 (Table 2).

All coding nucleotide and amino acid substitutions characterized for the attenuation donor B / USSR / 60/69 as responsible for its attenuation are present in the internal genes of the reassortant strain B / 60 / Colorado / 2017/1 (Victoria line).

Thus, whole genome sequencing of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria lineage) showed that it inherited a full set of attenuating mutations from donor B / USSR / 60/69.

The genetic stability of the coding mutations of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) was studied by comparing the persistence of the coding mutations before and after five-fold passaging of the vaccine strain in chicken embryos using the method of partial sequencing. It was shown that the nucleotide sequence of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) before and after its five passages remained unchanged (Table 3).

The assessment of the phenotypic properties of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) was carried out by its parallel titration in ECE at different temperatures. The vaccine strain B / 60 / Colorado / 2017/1 has a pronounced temperature-sensitive and cold-adapted phenotype, identical to the phenotype of the attenuation donor B / USSR / 60/69. It was found that the vaccine virus is temperature-sensitive (ts phenotype) - its infectious activity at 38 ° C was ≤1.2 lg EID ₅₀ / ml, and cold-adapted (ca phenotype) - infectious activity at an incubation temperature lowered to 26 ° C was 7 , 0 lg EID ₅₀ / ml, which indicates its harmlessness to humans, since according to these parameters it is identical to the attenuation donor B / USSR / 60/69.

The phenotypic properties of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) did not change after its fivefold passage in developing chick embryos when using large infectious doses, which indicates the genetic stability of the drug (Table 3).

Harmless to mice. Preclinical studies of acute toxicity of the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line) were carried out on white outbred mice in accordance with the Guidelines for conducting preclinical studies of drugs [Guidelines for conducting preclinical studies of drugs (Immunobiological drugs); Part two. - M .: Grif and K, 2012. - 536 s] and with the "Rules of Good Laboratory Practice" [Rules of Good Laboratory Practice. Order of the Ministry of Health of the Russian Federation No. 199n dated 04/01/2016. https://rg.ru/2016/09/02/minzdrav-prikaz199-site-dok.html].

Mice were injected with a single human dose corresponding to 0.5 ml of the vaccine virus intraperitoneally at a titer of 7.0 lg EID ₅₀ / ml. The animals of the control group were injected intraperitoneally with phosphate buffered saline (PBS). Every day during the entire study (7 days), the general condition of each animal was monitored.

Physiological research data (mobility, behavioral reactions, kinetics of body weight) showed that intraperitoneal administration of the vaccine strain did not cause death of experimental animals (Table 4) and did not lead to a change in their appearance, behavior, and did not affect their food and water consumption. which indicates the harmlessness of the vaccine preparation.

CONCLUSION

As a result of the preclinical studies, it was established that the claimed vaccine strain of live influenza vaccine B / 60 / Colorado / 2017/1 (Victoria line) is characterized by a combination of useful traits required for the vaccine strain: antigenic specificity of the B / Colorado / 06/2017 epidemic virus (Victoria line ), genome structure 6: 2, optimal for reassortant vaccine strains, as well as temperature sensitivity and cold adaptation characteristic of the donor, which correlates with attenuation for humans. The strain is attenuated and harmless to mice.

All this is reflected in the Passport of the strain attached below.

Thus, the vaccine strain B / 60 / Colorado / 2017/1 (Victoria line), in terms of the main biological properties studied in preclinical in vitro and in vivo experiments, meets the requirements for vaccine strains by the Pharmacopoeia Monograph (FSP: P No. 003224/01 -270313) on Ultravac®, live allantoic influenza vaccine for intranasal use for adults and children.

The resulting strain B / 60 / Colorado / 2017/1 (Victoria line) was deposited on 02.28.2019 in the State Collection of Viruses of the Institute of Virology named after V.I. DI. Ivanovsky Federal State Budgetary Institution “NITsEM named after N.F. Gamalea "of the Ministry of Health of Russia under No. 2901 and has the characteristics presented in the strain passport.

CHARACTERISTIC OF THE STRAIN

The infectious activity of strain B / 60 / Colorado / 2017/1 (Victoria line) during reproduction in developing chicken embryos at 32 ° C for 72 hours is 9.2 log ₁₀ EID ₅₀ / ml.

Hemagglutinating activity - 1: 512.

The strain shows genetic stability of phenotypic traits after 5 passages in chick embryos (when using large infectious doses).

A useful property of the vaccine strain of the influenza B / 60 / Colorado / 2017/1 virus (Victoria line) is its suitability for the production of a live influenza vaccine. The proposed according to the invention vaccine strain of influenza virus B / 60 / Colorado / 2017/1 (Victoria line) can be used for the prevention of influenza both in adults and in children from the age of three.

VACCINE STRAIN PASSPORT

1 Strain name: B / 60 / Colorado / 2017/1

2 IEM identification no .: Col / 1

Series 3: No. 1

4 Serosubtype: Victoria lineage, clyde V1A.1 (2 Del)

5 Preparation method: classical reassortment in chick embryos

6 Parental viruses:

6.1 Epidemic virus: B / Colorado / 06/2017 (Victoria lineage), CDC (Atlanta, USA) ID # 3025629447

6.2 Attenuation donor: B / USSR / 60/69

7 Number of passages in the process

E8 reassortment (co-infection, two selective passages, one blind passage, three cloning and one accumulation)

8 Genome composition: genes PB2, PB1, PA, NP, M and NS from the attenuation donor, HA and NA from the epidemic virus (genome formula 6: 2)

9 a) Characterization of the strain before freeze drying:

9.1 b) Optimal conditions for reproduction: 32 ° C, 48 hours

9.2 c) Hemagglutinating activity: 1: 512 with 1.0% chicken erythrocytes

9.3 d) Infectious activity at an optimum temperature of 32 ° C: 9.2 log ₁₀ EID ₅₀ / ml

9.4 f) ts marker (temperature sensitivity of reproduction; infectious activity at 38 ° C): ≤1.2 lg EID ₅₀ / ml (ts phenotype)

9.5 f) ca marker (cold resistance of reproduction; infectious activity at a temperature of 26 ° C): 7.0 lg EID ₅₀ / ml (ca phenotype)

10 g) Characterization of the strain after freeze-drying:

10.1 Date of lyophilization: 04/26/2018

10.2 a) Volume of material in ampoule: 0.5 ml

10.3 b) Infectious activity at an optimum temperature of 32 ° C: 8.2 lg EID ₅₀ / ml

10.4 s) Hemagglutinating activity: 1: 256 with 1.0% chicken erythrocytes

11 d) Antigenic specificity:

11.1 a) Hemagglutinin: identical to the epidemic virus B / Colorado / 06/2017 (Victoria lineage) according to sequencing and RTGA

11.2 b) Neuraminidase: Identical to epidemic virus B / Colorado / 06/2017 (Victoria lineage) by sequencing

12 Toxicological

in vivo study

(acute toxicity): non-toxic after intraperitoneal injection in mice

13 Sterility control: sterile

Claims (1)

Influenza virus strain B / 60 / Colorado / 2017/1 (Victoria Line) deposited in the State Collection of Viruses of the Institute of Virology named after DI. Ivanovsky Federal State Budgetary Institution “NITsEM named after N.F. Gamaleya "of the Ministry of Health of Russia under No. 2901, for the production of live influenza intranasal vaccine for adults and children.