RU2606019C1 - Vaccine influenza virus strain a/17/hongkong/2014/8296 (h3n2) for preparing live influenza intranasal vaccine for adults and children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medical virology and concerns influenza virus strain. Disclosed a vaccine strain A/17/Hongkong/2014/8296 (H3N2)-reassortant, produced by crossing "wild" virus A/Hongkong/4801/2014 (H3N2) with cold-adaptive heat-sensitive virus A/Leningrad/134/17/57 (H2N2) – attenuation donor, safe for humans. Presented strain A/17/Hongkong/2014/8296 (H3N2) actively propagated in chicken embryos at optimal temperature of 32 °C, characterized by temperature sensitivity and cold adaptation and safety for laboratory animals. Strain is deposited in the State collection of viruses FGBU "FNICEM nam. N.F. Gamalei" of the Ministry of Health of Russia, Institute of virology nam. D.I. Ivanovskogo under № 2818.

EFFECT: invention can be used in practical health services for preventing influenza incidence in adults and children by a live influenza intranasal vaccine.

1 cl, 1 dwg, 5 tbl

Description

The invention relates to medical virology and can be used in healthcare for the prevention of influenza in adults and children using a live influenza intranasal vaccine from strain A / 17 / Hong Kong / 2014/8296 (H3N2) of influenza A virus, the family of Orthomyxoviridae, genus Influenzavirus A.

As a result of the emergence in circulation of a new epidemic strain of influenza virus A / Hong Kong / 4801/2014 (H3N2), the known vaccine strain A / 17 / Texas / 2012/30 (H3N2) is a prototype [RF Patent No. 2566352 of 09/20/15. - Publ. BI 2015. - No. 26] - lost antigenic relevance and therefore cannot cause a defensive reaction during the epidemic caused by A / Hong Kong / 4801/2014-like strains of the influenza virus.

The problem to which the invention is directed, 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 the new virus A / Hong Kong / 4801/2014 (H3N2) .

The currently used strains for live influenza vaccines are obtained by genetic reassortment of epidemiologically relevant viruses with cold-adapted strains - donors of attenuation [Alexandrova G.I. Application of the genetic recombination method 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 approved for the production of harmless live intranasal vaccines for adults and children [G. Alexandrova New in the epidemiology and prevention of viral infections. L., 1968. - S. 66-83].

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

Obtaining a vaccine strain. The vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) was obtained by the genetic reassortment of the epidemic virus A / Hong Kong / 4801/2014 (H3N2) with the attenuation donor A / Leningrad / 134/17/57 (H2N2) by simultaneous infection of developing chicken embryos (RCE) with a mixture of parent viruses in equivalent infectious doses, followed by selection of clones with desired properties at a temperature of incubation lowered to 26 ° C in the presence of a rabbit antiserum to an attenuation donor. Clones were further purified by three consecutive cloning by the method of limiting dilutions in the presence of antiserum to the donor at low (26 ° C) and optimal (32 ° C) incubation temperatures. The pure clone was tested according to phenotypic characteristics (ts- and sa-phenotype) and according to the genome formula for compliance with the vaccine strain.

Antigenic characteristics of the vaccine strain.

The study of the antigenic properties of the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) showed that:

- the surface protein of the vaccine strain, hemagglutinin (HA), which is responsible for antigenic specificity, in the rtga with homologous serum is antigenically identical to the epidemic virus A / Hong Kong / 4801/2014 (H3N2), since the reassortant interaction parameters A / 17 / Hong Kong / 2014/8296 (H3N2 ) and the parent strain A / Hong Kong / 4801/2014 (H3N2) with the rat antiserum obtained to the virus A / Hong Kong / 4801/2014 (H3N2) completely coincided (Table 1).

- the second surface protein of the vaccine strain, neuraminidase (NA), responsible for antigenic specificity, was tested by the method of partial sequencing, as well as by genome-wide sequencing and is identical to virus A / Hong Kong / 4801/2014 (H3N2).

To analyze the genome composition of the obtained reassortants, partial DNA sequencing of gene copies was used [Isakova-Sivak I. et. al. Development and pre-clinical evaluation of two LAIV strains against potentially pandemic H2N2 influenza virus. Plos one. 2014. Vol. 9. No. 7. P. e102339].

Genome formula 6: 2 meets the requirements for live strains

influenza vaccine: genes encoding the hemagglutinin (HA) and neuraminidase (NA) surface proteins belong to the epidemic parent virus A / Hong Kong / 4801/2014 (H3N2), genes encoding the 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 / Hong Kong / 2014/8296 (H3N2) are presented in table. 2.

The data obtained during the initial selection of the vaccine candidate were confirmed by complete sequencing of its genome and showed that the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) inherited all 6 genes encoding internal and non-structural proteins from the attenuation donor A / Leningrad / 134/17/57 (H2N2) (Table 2).

All coding nucleotide substitutions characterized for the attenuation donor A / Leningrad / 134/17/57 (H2N2) as responsible for its attenuation (Table 3) are present in the internal genes of the reassortant strain A / 17 / Hong Kong / 2014/8296 (H3N2) . Thus, complete sequencing of the genome of the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) showed the genetic stability of the attenuating mutations.

The genetic stability of the coding mutations of the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) was studied by comparing the safety of the coding mutations before and after five-fold passage 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. Specific primers have been developed for this analysis, which allow us to evaluate the presence of these mutations using the PyroMark Q24 sequencer. Primers are developed using the PSQ Assay Design computer program.

Pyrosequencing showed that the nucleotide sequence of vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) before and after its five-fold passage remained unchanged, including in the polymerase complex genes (PB2, PB1, RA) responsible for the attenuating phenotype, as evidence of this, the results of pyrosequencing are shown in Table 3 and in FIG. one.

In FIG. 1 as an example, data confirming the presence of an attenuating mutation at position 1459 of the PB2 gene of the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) by pyrosequencing are presented. Replacing G with T at position 1459 leads to the replacement of Val with Leu at position 478. The 1st sequence corresponds to the virus A / Leningrad / 134/57, which does not have an attenuating mutation in this position, the 2nd sequence corresponds to the donor A / Leningrad / 134/17/57, which has an attenuating mutation in this position, and the 3rd sequence belongs to the fifth passage of the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2), which retains an attenuating mutation in this position, which indicates genetic stability of the vaccine strain A / 17 / Hong Kong / 2014/8 296 (H3N2) at position 1459 of the PB2 gene.

The phenotypic characteristics of the passage variant also did not undergo any changes (Table 4), which confirms the high genetic stability of the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2).

The phenotypic properties of the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) were evaluated by parallel titration in RCE at different temperatures. The vaccine strain A / 17 / Hong Kong / 2014/8296 (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 established that the vaccine virus is temperature-sensitive (ts phenotype) - its infectious activity at 40 ° C was ≤0.7 log ₁₀ EID ₅₀ / ml and cold-adapted (sa phenotype) - its infectious activity at incubation temperature lowered to 26 ° C reached 6.2 log ₁₀ EID ₅₀ / ml, which indicates its harmlessness to humans, because according to 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 guinea pigs. Preclinical studies of acute toxicity of vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) were carried out on guinea pigs of both sexes in accordance with the Methodological recommendations for preclinical trials of new immunobiological drugs [Methodical recommendations "Preclinical trials of the efficacy and safety of new immunobiological drugs." M .: 2010. 39 pp.] And with the "Rules of laboratory practice" [Rules of laboratory practice. Order of the Ministry of Health of the Russian Federation No. 708n of 08/23/20010. http://www.rg.ru/2010/10/22/laboratornaya-praktika-dok.html]. Guinea pigs were injected once with 5 ml of the vaccine virus subcutaneously at a dose of 7.0 lg EID ₅₀ /0.2 ml. The animals of the control group were injected subcutaneously with saline. Every day throughout the study (7 days), the general condition of each animal was monitored.

The safety of the vaccine strain for laboratory animals with its subcutaneous administration was shown (Table 5).

Physiological data showed that subcutaneous administration of the vaccine strain did not cause the death of experimental animals 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 preclinical studies, it was found that the inventive vaccine strain of live influenza vaccine A / 17 / Hong Kong / 2014/8296 (H3N2) is characterized by a combination of useful features necessary for the vaccine strain: antigenic specificity of the epidemic virus A / Hong Kong / 4801/2014 (H3N2), genome structure 6: 2, optimal for reassortant vaccine strains, as well as temperature sensitivity and cold adaptation and harmlessness for laboratory animals characteristic of an attenuation donor, which correlates with attenuation for person.

A sample passport for the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) is attached.

Thus, the vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) according to the main biological properties studied in preclinical experiments in vitro and in vivo, meets the requirements for vaccine strains Pharmacopeia article (FSP: P N003224 / 01-270313 ) Ultravac ^® , a live allantoic influenza vaccine for intranasal use for adults and children.

The resulting strain A / 17 / Hong Kong / 2014/8296 (H3N2) was deposited on December 14, 2015 into the State Virus Collection of the Federal State Budgetary Institution of Health and Economics. N.F. Gamalei Ministry of Health of Russia Institute of Virology named after DI. Ivanovsky (Russia RU, 123098, Moscow, 16 Gamalei Street) under No. 2818 and has the characteristics presented in the sample passport strain.

STRAIN CHARACTERISTIC

Infectious activity of strain A / 17 / Hong Kong / 2014/8296 (H3N2) during reproduction in developing chicken embryos at 32 ° C for 48 hours - 8.7 log ₁₀ EID ₅₀ / ml. Hemagglutinating activity - 1: 256

The strain shows the genetic stability of phenotypic characters after 5 passages on chicken embryos (when using large infectious doses).

A useful property of the influenza virus vaccine strain A / 17 / Hong Kong / 2014/8296 (H3N2) is its suitability for producing a live influenza vaccine. Proposed according to the invention, the vaccine strain of influenza virus A / 17 / Hong Kong / 2014/8296 (H3N2) can be used for the prevention of influenza in both adults and children from the age of three.

Claims (1)

The strain of the influenza virus A / 17 / Hong Kong / 2014/8296 (H3N2), deposited in the State collection of viruses FSBI "FNITsEM im. N.F. Gamalei »Ministry of Health of Russia, Institute of Virology named after DI. Ivanovsky under No. 2818, used to obtain live influenza intranasal influenza vaccine for adults and for children.

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