RU2702834C1 - Strain of influenza virus a/unl/hk/2:6/2017 (h5n8) for producing inactivated and live influenza vaccines - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Influenzavirus A, subtype H5N8, strain A/UNL/HK/2:6/2017 (H5N8), deposited in the State collection of viruses FGBU FNIZEM of N. F. Gamalea of Ministry of Health of Russia under No. 2885, for production of inactivated and live influenza vaccines against potentially pandemic influenza virus A/H5N8.

EFFECT: invention provides enhanced protective action of vaccines against pandemic influenza virus A subtype H5.

1 cl, 6 dwg, 5 tbl, 5 ex

Description

The invention relates to the field of medical biotechnology and is intended for the production of mono- or polyvalent inactivated (IHV) and live (IHV) influenza vaccines in order to immunize the population against potentially pandemic influenza A viruses of subtype H5N8. The resulting vaccine strain belongs to the family Orthomyxoviridae, genus Influenza virus A.

Influenza viruses circulating among animals pose a threat to human health if they are directly infected with various subtypes of avian or swine virus, and even more so when reassorted with human viruses.

A potentially pandemic subtype of avian influenza virus is A / H5N8. There are 2 groups of influenza virus subtype A / H5N8 - a (Buan-like) and b (Gochang-like) [Lee D.H. et al., 2017]. The influenza virus subtype A / H5N8 was first detected in 2013 in China, then in 2014 in South Korea and Northeast Siberia. Currently, due to the seasonal migration of birds, Group A viruses have spread throughout Eurasia and North America and are designated icA (from intercontinental - intercontinental). Moreover, they caused great mortality in different species of wild and poultry [Kapczynski D.R. et al., 2017; Kleyheeg E. et al., 2017].

The A / H5N8 subtype virus is recognized as highly pathogenic [Woo C. et al., 2017]. Currently, no cases of human infection with this virus have been detected [Hui D. S. C., Lee N., Chan R. K. S., 2017]. However, this virus is avian, for which the ability to cross the interspecific barrier and infect mammals has been proven [Leymarie O. et al., 2017]. So, for a related virus of the A / H5N6 subtype, belonging to the same clyde, cases of infection and death in humans have been reported [Grund C. et al., 2018]. In strains of the influenza virus subtype A / H5N8 isolated in Russia, mutations were found that were responsible for virulence, changes in host specificity and for the adaptation of viruses for transmission from person to person [Marchenko V.Yu. et al., 2017].

The migration routes of many species of migratory birds intersect in the area of Lake Uvs-Nuur, located on the Russian-Mongolian border. It is a permanent habitat for 46 species of waterfowl and a stopping place for 215 species of birds migrating south from Siberia. During the widespread spread of highly pathogenic influenza viruses of the A / H5N1 subtype in 2006 and 2009, these viruses were found in wild waterfowl on Lake Uvs Nuur, which is why this zone is a significant place for tracking outscopic pathogenic bird viruses. In June 2016, 3 strains belonging to group B of influenza viruses subtype A / H5N8 of Clyde 2.3.4.4 were isolated and characterized in this area: A / common tern / Uvs-Nuur Lake / 26/2016, A / great crested grebe / Uvs -Nuur Lake / 341/2016 and A / gray heron / UVs-Nuur Lake / 20/2016 [Lee DH et al., 2017].

The need to develop anti-pandemic measures, including for combating bird flu viruses, is reflected in the order of the Russian Federal Service for Supervision of Human Welfare [Order No. 40 of 12.28.2004]. Prior to a pandemic, national authorities recommend testing and licensing pandemic vaccines, as well as supporting the development of production and exchange of prototypes of seasonal and pandemic vaccines, for possible targeted use, in the WHO Global Influenza Preparedness Plan. In this regard, studies on the preparation of vaccines against influenza viruses of the A / H5N8 subtypes, as well as tests of these vaccines on animal models, are relevant.

There are currently no data on clinical trials of vaccines against influenza virus subtype A / H5N8 [Tables on clinical evaluation of influenza vaccines, URL: http://www.who.int/inmiraization/diseases/influenza/clinical_evaluation_tables/en/#] .

As a donor of internal protein genes for obtaining a reassortant based on the A / H5N8 virus, we used the universal donor A / Hong Kong / 1/68/162/35 (H3N2) developed at the Research Institute of Influenza of the Ministry of Health of Russia [Patent No. 2511431 of 07.25.2011 ], which is focused on obtaining reassortant strains for both live and inactivated influenza vaccines.

The problem to which the invention is directed, was to obtain a reassortant based on the influenza A / H5N8 virus and the donor of the internal protein genes A / Hong Kong / 1/68/162/35 (H3H2) by classical genetic reassortment, for the production of inactivated and live influenza vaccines .

Obtaining a strain of influenza virus RA-54 (A / UNL / HK / 2: 6/2017 (H5N8))

The strain of influenza virus RA-54 (A / UNL / HK / 2: 6/2017 (H5N8)) was obtained by classical genetic reassortment of the donor strain A / Hong Kong / 1/68/162/35 (H3N2) and a potentially pandemic virus A / Common tern / Uvs-Nuur Lake / 26/2016 (H5N8). Upon receipt of the strain, 7 passages were performed in 10-12 day-old developing chicken embryos. Selective factors for the selection of the necessary clones were low temperature (26 ° C) and the presence of hyperimmune serum to strain A / Hong Kong / 1/68/162/35 (H3N2).

The strain is a reassortant with a 2: 6 genome formula. Genome composition: 2 genes (HA and NA) from the wild-type virus and 6 genes of internal and non-structural proteins (PB2, PB1, PA, NP, M and NS) from the donor virus A / Hong Kong / 1/68/162/35 ( H3N2).

Confirmation of the genome composition was performed using restriction analysis of DNA copies of RNA segments obtained by reverse transcriptase polymerase chain reaction (RT-PCR). Obtained by RT-PCR DNA copies of segments PB2, PB1, PA, NP, NS and M of viruses A / Hong Kong / 1/68/162/35 and A / Common tern / Uvs-Nuur Lake / 26/2016 (marked on Fig. 1 as NK and UNL) were treated with restriction endonucleases Acc36I, MhlI, BsmBl, Acc36I, Bse3D1 and Bse3D1, respectively. The affiliation of NA and HA was determined by RT-PCR with type-specific primers for various subtypes. Analysis of DNA copies of RNA segments (Fig. 1) showed that the reassortant RA-54 has a genome composition of 2: 6.

The antigenic correspondence of hemagglutinin to the parent wild-type strain in the RA-54 reassortant was confirmed in the hemagglutination inhibition reaction (RTHA) (Table 1).

The vaccine candidate RA-54 was shown to be antigenically identical to the parent wild-type strain.

Full genome sequencing showed the presence of several nucleotide and amino acid substitutions in the reassortant RA-54, compared with the parental strains (Table 2-4). The nucleotide sequences of the RA-54 reassortant segments are published in the GISAID database under the number EPI_ISL_321098.

Note: Bold mutations leading to amino acid substitution

Characterization of the obtained strain

Example 1. Reproductive properties of strain A / UNL / HK / 2: 6/2017 (H5N8) (RA-54).

The RA-54 influenza virus strain was tested for infectious activity by infection of 10-day-old developing chicken embryos in accordance with Methodological Instructions 3.3.2.1758-03 “Methods for Determining the Quality Indicators of Immunobiological Preparations for the Prevention and Diagnosis of Influenza”. Ten-fold falling dilutions of virus-containing allantoic fluid were prepared. Each dilution starting from 10 ^-4 to 10 ^-10 infected 3 chicken embryos. Incubated 48 hours at a temperature of 32 ° C. The presence of the virus was determined in the RGA, the infectious activity of the virus was calculated by the method of Reed and Mench.

The influenza virus strain A / UNL / HK / 2: 6/2017 (H5N8) showed a high reproductive activity in the RCE. The hemagglutinating activity of the reassortant was RA-54 16-64 GAU / 50 μl with 0.5% suspension of chicken red blood cells. Infectious activity in the RCA was 7.75 lgEID ₅₀ / 0.2 ml.

Example 2. The vaccine strain RA-54 has the properties of temperature sensitivity and cold adaptation.

Donor A / Hong Kong / 1/68/162/35 is universal, that is, it has both high reproducibility and markers of attenuation - it has a temperature-sensitive (ts) and a cold-adapted (ca) phenotype. Reassortants based on donor A / Hong Kong / 1/68/162/35 also inherit the ts-, ca - phenotype.

The resulting reassortant inherited from the donor strain A / Hong Kong / 1/68/162/35 ts-, the phenotype is the ability to reproduce well at low temperatures and an almost complete loss of reproducibility at elevated temperatures (Table 5).

RCT (Reproductive capacity at different temperatures) - reproductive ability at different temperatures

ts - temperature sensitive phenotype (RCT ₃₉ over 5 lgEID ₅₀ / 0.2 ml)

CA - cold-adapted phenotype (RCT ₂₆ no more than 3 lgEID ₅₀ / 0.2 ml)

The stability of the reassortant was checked by 5-fold passaging at a temperature of 32 ° C in the RCE system. The degree of temperature sensitivity and cold adaptation, as well as the level of reproduction of the reassortant strain, did not change. Thus, the reassortant strain satisfies the requirements of temperature sensitivity and cold adaptation for vaccine strains for live hepatitis B virus, and the requirements of high reproductive performance for the hepatitis B virus.

Example 3. The inventive vaccine strain RA-54 is safe for mice.

Testing the strain of the influenza virus RA-54 for specific safety was carried out on linear white Balb / c mice, females, weighing 18-20 g (16 pcs.). The studies used healthy animals that had not previously been tested. The tests were carried out in accordance with the guidelines of MUK 4.1 / 4.2.588-96.

Mice were infected intranasally at a dose of 6.5 lgEID ₅₀ per mouse, in a volume of 50 μl. During the observation period (14 days) all animals remained alive and not one of them showed visible signs of the disease. On the 3rd and 6th day after infection, organs were collected from three mice (lungs, nasal passages and brain) in order to indicate antigen and its accumulation. Virus reproduction was determined by titration of an organ suspension in an MDCK cell culture. The results are shown in table. 6.

In mice infected with the wild-type strain, the virus is reproduced in the nasal passages and lungs on the 3rd day. By the 6th day, the virus shows a high level of reproduction in all considered organs of mice. In mice infected with the reassortant strain, the presence of the virus was observed only in the nasal passages on the 3rd and 6th day. The virus does not replicate in the lungs and brain, which confirms its attenuated phenotype. The data obtained prove the specific safety of the reassortant strain RA-54.

Example 4. The vaccine strain RA-54 is immunogenic for mice.

On the basis of reassortant strains, candidate vaccine preparations of live and inactivated influenza vaccines (PGV and IGV, respectively) were prepared. For LIV accumulated viral material reassortant virus was diluted with phosphate-buffered saline (PBS) to a concentration of 6.5 lgEID _50/50 microliters. PHA was administered intranasally, in a volume of 50 μl. To obtain IHV, viral material was inactivated with 0.02% formalin, then purified and concentrated by isopycnic centrifugation in a sucrose density gradient. The hemagglutinin content in the vaccine preparation was evaluated by polyacrylamide gel electrophoresis followed by densitometry. The dose of IVG (250 μl) contained 10 μg of HA. As an adjuvant in one of the groups used polyoxidonium (PO) - 200 μg per dose. IHV was administered intramuscularly (in the upper third of the thigh of the hind limbs).

Preparations of PGV and IVH were used to immunize mice and study immunogenicity. Immunization was carried out twice with a two-week interval. Data on the content of anti-hemagglutinating antibodies to influenza viruses in the blood serum of mice immunized with the preparations of HBV and IGV RA-54 demonstrate a significant (p <0.05) increase in the level of anti-hemagglutinating antibodies compared to control animals. The geometric mean titer (CHT) of anti-hemagglutinating antibodies in sera after the 2nd immunization with HBV and IGV was 285.3 and 205.8, respectively (Fig. 2). The serum hypertrophy of neutralizing antibodies in serum after the 2nd immunization with HBV and IGV was 7240.8 and 2560.0, respectively (Fig. 3).

The data obtained show that, after immunization of mice with mono-vaccines, hepatitis B and hepatitis B virus, both hemagglutinating and neutralizing antibodies are formed in the titers exceeding the protective titers based on the RA-54 reassortant (protective titer in RTGA≥1: 40; protective titer in PH≥1: 80 ) Thus, both live and inactivated mono-vaccines based on the obtained RA-54 reassortant are highly immunogenic.

Example 5. The vaccine strain RA-54 has a protective effect for mice.

The degree of protection provided by the mono-vaccines of HBV and IGV based on the RA-54 reassortant, in comparison with the control group, was evaluated on female Balb / c mice using a lethal infection model. Mice (10 in the group) 2 weeks after the last immunization were infected intranasally with wild-type virus A / Common tern / Uvs-Nuur Lake / 26/2016 at a dose of 10LD ₅₀ . Mice were monitored for 14 days after infection, daily changes in body weight (an indicator of the severity of infection) and the death of animals were evaluated.

In mice immunized with the HBV monovaccine, the maximum weight loss was 2.7% on the 5th day after infection, and in mice immunized with the HBV monovirus, there was no decrease in body weight (Fig. 4). In the control group of mice, a sharp decrease in the body weight of mice was observed starting from 5 days after infection (the maximum decrease in body weight was 21%), and the clinical signs of the disease were manifested: frequent, shallow breathing occurred, the hair became disheveled, motor activity decreased, and motor activity decreased feed intake.

Immunization of mice with mono-vaccines of hepatitis B and hepatitis B virus provided complete protection (Fig. 5) from lethal infection with the A / H5N8 influenza virus at a dose of 10LD ₅₀ (100% survival rate). All mice in the control group died.

The data obtained indicate that the mono-vaccines of HBV and IGV have a pronounced protective effect against the influenza virus A / Common tern / Uvs-Nuur Lake / 26/2016. In mice immunized with mono-vaccines of live hepatitis B and hepatitis B virus based on RA-54 reassortant, after infection with the influenza A / H5N8 virus at a dose of 10LD _50, there was no decrease in body weight, the development of signs of influenza infection, and animal death, which indicates the formation of protective sterile immunity.

Claims (1)

Influenzavirus A, subtype H5N8, strain A / UNL / HK / 2: 6/2017 (H5N8), deposited in the State collection of viruses Federal State Budgetary Institution Scientific Center named after N.F. Gamalei ”of the Ministry of Health of Russia under No. 2885, for the production of inactivated and live influenza vaccines against the potentially pandemic influenza virus A / H5N8.

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