RU2732610C1 - Vaccine strain of influenza a/17/kansas/2017/631 (h3n2) virus for production of live influenza intranasal vaccine for adults and children - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a vaccine strain A/17/Kansas/2017/631 (H3N2) - a reassortant produced by crossing epidemic virus A/Kansas/14/2017 (H3N2) with a cold-adapted temperature-sensitive virus A/Leningrad/134/17/57 (H2N2) - an attenuation donor, which is harmless for humans.

EFFECT: proposed strain can be used for preventing influenza in both adults and children from the age of three.

1 cl, 5 tbl, 1 dwg

Description

The invention relates to medical virology and can be used in health care for the prevention of the incidence of influenza among adults and children using a live influenza intranasal vaccine from the A / 17 / Kansas / 2017/631 (H3N2) strain of the influenza A virus, the Orthomyxoviridae family, the Influenzavirus A.

In February 2019, the World Health Organization (WHO) made recommendations to replace the 2019-20 Northern Hemisphere influenza vaccine composition. epidemic season, since most circulating H3N2 influenza viruses were antigenically different from those previously recommended for the preparation of influenza vaccines for the 2018-2019 epidemic season in the northern hemisphere and the 2019 epidemic season in the southern hemisphere. At the same time, since in the human population from September 2018 to February 2019, influenza viruses of the H3N2 subtype belonging to antigenically different clides 3C.2a1b and 3C.3a were simultaneously co-circulated, the decision on the choice of the influenza virus strain to replace the H3N2 component was postponed to March. In March, following a more detailed analysis by WHO for the preparation of the H3N2 component for influenza vaccines for the Northern Hemisphere 2019-20. the epidemic season were selected A / Kansas / 14/2017 (H3N2) -like viruses belonging to the 3C.3a [WHO. Recommended composition of influenza virus vaccines for use in the 2019-2020 northern hemisphere influenza season. 21 February 2019 (updated on 21 March 2019). https://www.who.int/influenza/vaccines/virus/recommendations/2019_20_north/en/].

As a result of the appearance in the circulation of a new epidemic strain of influenza A / Kansas / 14/2017 (H3N2), the well-known vaccine strain A / 17 / Singapore / 2016/3571 (H3N2) is a prototype [Larionova NV, Bazhenova Ye.A. , Krutikova E.V., Matyushenko V.A., Kiseleva I.V., Rudenko L.G. Vaccine strain of influenza A / 17 / Singapore / 2016/3571 (H3N2) virus for the production of live intranasal influenza vaccine for adults and children. RF patent No. 2683500 from 28.03.2019 - Publ. BI 2019. - No. 10] - has lost its antigenic relevance and, as a result, cannot cause a protective reaction during an epidemic caused by A / Kansas / 14/2017-like strains of the influenza virus.

The problem to be solved by the claimed invention is to obtain an antigenically relevant vaccine strain for adults and for children based on a cold-adapted attenuation donor A / Leningrad / 134/17/57 (H2N2) and a new epidemic strain A / Kansas / 14/2017 (H3N2 ).

Currently used strains for live influenza vaccines are obtained by the method of genetic reassortment of epidemically relevant viruses with cold-adapted strains - attenuation donors [Alexandrova G.I. 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 A / Leningrad / 134/17/57 (H2N2) - a cold-adapted (ca) and temperature-sensitive (ts) strain of the influenza virus - is allowed to obtain 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 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 donor attenuation.

Obtaining a vaccine strain. The vaccine strain A / 17 / Kansas / 2017/631 (H3N2) was obtained by the method of genetic reassortment of the epidemic virus A / Kansas / 14/2017 (H3N2) with the attenuation donor A / Leningrad / 134/17/57 (H2N2) by simultaneous infection of developing chickens embryos (ECE) with a mixture of parental viruses in equivalent infectious doses, followed by selection of clones with desired properties at an incubation temperature lowered to 26 ° C in the presence of rabbit antiserum to the attenuation donor. The clones were additionally purified by three sequential cloning by the limiting dilution method in the presence of antiserum to the donor at low (26 ° C) and optimal (32 ° C) incubation temperatures. A pure clone was tested for phenotypic characteristics (ts- and ca-phenotype) and according to the genome formula for compliance with the formula of the vaccine strain of a live influenza vaccine.

ANTIGENIC CHARACTERISTICS OF VACCINE STRAIN.

The study of the antigenic properties of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) showed that:

- the surface protein of the vaccine strain, hemagglutinin (HA), responsible for the antigenic specificity, is antigenically identical to the epidemic virus A / Kansas / 14/2017 (H3N2) in RTGA with homologous serum, since the indicators of interaction of the reassortant A / 17 / Kansas / 2017/631 (H3N2 ) and the parental strain A / Kansas / 14/2017 (H3N2) with the rat antiserum obtained against the A / Kansas / 14/2017 (H3N2) virus completely coincided (Table 1).

- the second surface protein of the vaccine strain responsible for antigenic specificity - neuraminidase (NA) - was verified by sequencing the sequence of the corresponding genome segment and corresponds to the neuraminidase of the virus A / Kansas / 14/2017 (H3N2).

To analyze the genome composition of the obtained reassortants, we used the method of pyrosequencing of DNA copies of genes [Stepanova EA, Krutikova EV, Kiseleva IV, Rudenko LG. Development of a pyrosequencing protocol for analyzing the origin of reassortant genes in the preparation of live influenza vaccine strains. // "Molecular Genetics, Microbiology and Virology" - №2. - 2018. - p. 101-107] using a set of highly specific oligonucleotides developed in the PSQ Assay Design program for genome analysis of modern strains of the H3N2 subtype and the attenuation donor A / Leningrad / 134/17/57 (H2N2).

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 A / Kansas / 14/2017 (H3N2), genes encoding internal proteins ( PB2, PB1, PA, NP, M, NS), belong to the attenuation donor A / Leningrad / 134/17/57 (H2N2). The results of the analysis of all genes of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) are presented in Table 2.

The data obtained during the initial selection of the vaccine candidate was confirmed by full sequencing of its genome and showed that the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) inherited all 6 genes encoding internal and non-structural proteins from the attenuation donor A / Leningrad / 134/17/57 (H2N2) (Table 2).

The genetic stability of the coding mutations of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) was studied by comparing the preservation of the coding mutations before and after fivefold passaging of the vaccine strain prepared on the basis of the attenuation donor A / Leningrad / 134/17/57 in chicken embryos with using the pyrosequencing method. For this analysis, specific primers were developed to assess the presence of these mutations using the PyroMark Q24 pyrosequenator. Primers were developed using the PSQ Assay Design computer program. An example of a pyrogram obtained for a PB1 gene fragment containing an attenuating mutation at position 1795 in an attenuation donor A / Leningrad / 134/17/57 (H2N2) is shown in Fig. 1, which shows the result of pyrosequencing of the PB1 gene fragment containing the attenuating mutation at position 1795 in the attenuation donor A / Leningrad / 134/17/57 (H2N2) for strains A / Leningrad / 134/57 (H2N2) (Fig.1a), A / Leningrad / 134/17/57 (H2N2) (fig.1b), A / 17 / Kansas / 2017/631 (H3N2) (fig.1c).

As a result of pyrosequencing of the A / Leningrad / 134/57 (H2N2) virus, an epidemic precursor of an attenuation donor that does not possess an attenuating mutation, there is no peak in the first position (T), the peak in the second position (C) has an increased height (Fig. 1, a ). The presence of the C → T attenuating mutation is characterized by peaks at the T and C positions, of the same height, which can be seen in the pyrogram obtained for the attenuation donor A / Leningrad / 134/17/57 (H2N2) (Fig. 1, b), black arrows are marked peaks confirming the presence of the mutation), as well as on the pyrogram obtained for the PB1 gene of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) (Fig. 1, c).

Table 3 shows the results of pyrosequencing of all genes of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) containing attenuating mutations corresponding to the attenuation donor A / Leningrad / 134/17/57 (H2N2), before and after five passaging in developing chicken embryos. All coding nucleotide substitutions characterized for the attenuation donor A / Leningrad / 134/17/57 (H2N2) as being responsible for its attenuation are present in the internal genes of the reassortant strain A / 17 / Kansas / 2017/631 (H3N2).

Thus, genome sequencing of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) showed the genetic stability of the attenuating mutations.

The assessment of the phenotypic properties of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) was carried out by means of its parallel titration in RCE at different temperatures. The vaccine strain A / 17 / Kansas / 2017/631 (H3N2), in developing chicken embryos, has a pronounced temperature-sensitive and cold-adapted phenotype identical to the phenotype of the attenuation donor A / Leningrad / 134/17/57 (H2N2). It was found that the vaccine virus is temperature-sensitive (ts phenotype) - its infectious activity at 40 ° C was ≤1.2 log ₁₀ EID ₅₀ / ml, and cold-adapted (ca phenotype) - infectious activity at an incubation temperature lowered to 26 ° C reached 5.0 log ₁₀ EID ₅₀ / ml, which indicates its harmlessness to humans, since by these indicators it is identical to the attenuation donor A / Leningrad / 134/17/57 (H2N2). The results of phenotypic analysis are presented in table 4.

Harmless to mice. Preclinical studies of acute toxicity of the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) 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 i K, 2012 .-- 536 p. ] and with the "Good Laboratory Practice" [Good Laboratory Practice. Order of the Ministry of Health of the Russian Federation No. 199n dated 01.04. 2016. https://rg.ru/2016/09/02/minzdrav-prikaz199-site-dok.html1

Mice were injected once with one human dose corresponding to 0.5 ml of the vaccine virus intraperitoneally at a dose of 7.0 lg EID ₅₀ / ml. The animals of the control group were injected intraperitoneally with physiological saline. 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 5) and did not lead to a change in their appearance, behavior, 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 A / 17 / Kansas / 2017/631 (H3N2) is characterized by a combination of useful traits required by the vaccine strain: antigenic specificity of the epidemic virus A / Kansas / 14/2017 (H3N2), genome structure 6: 2, optimal for reassortant vaccine strains, as well as characteristic for donor attenuation temperature sensitivity and cold adaptation and harmlessness for laboratory animals, which correlates with attenuation for humans. A sample passport for the vaccine strain A / 17 / Kansas / 2017/631 (H3N2) is attached.

Thus, the vaccine strain A / 17 / Kansas / 2017/631 (H3N2), in terms of the main biological properties studied in preclinical in vitro and in vivo experiments, meets the requirements for vaccine strains of the Pharmacopoeia Monograph (FSP: P N003224 / 01-270313 ) on Ultravac ^® , live allantoic influenza vaccine for intranasal use for adults and children.

The resulting strain A / 17 / Kansas / 2017/631 (H3N2) was deposited on July 12, 2019 in the State Collection of Viruses of the Institute of Virology named after I. DI. Ivanovsky Federal State Budgetary Institution “NITsEM named after N.F. Gamaleya "of the Ministry of Health of Russia under No. 2930.

CHARACTERISTIC OF THE STRAIN

The infectious activity of strain A / 17 / Kansas / 2017/631 (H3N2) during reproduction in developing chicken embryos at 32 ° C for 48 hours is 7.7 log ₁₀ EID ₅₀ / ml. Hemagglutinating activity - 1: 256.

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 influenza A / 17 / Kansas / 2017/631 (H3N2) virus is its suitability for the production of a live influenza vaccine. The vaccine strain of influenza A / 17 / Kansas / 2017/631 (H3N2) proposed according to the invention can be used for the prevention of influenza both in adults and in children from the age of three.

Claims (1)

Influenza virus strain A / 17 / Kansas / 2017/631 (H3N2) deposited in the State Collection of Viruses of the Institute of Virology named after DI. Ivanovsky FSBI “NITsEM named after N.F. Gamaleya "of the Ministry of Health of Russia under No. 2930, for the production of live influenza intranasal vaccine for adults and children.

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