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Sergeeva M.V., Kudrya K.S., Plotnikova M.A., Veretennikov V.V., Javadov E.D., Kraskov D.A., Ivanova A.A., Petrova P.A., Tarlavin N.V. A candidate vaccine strain of highly pathogenic avian influenza virus subtype A(H5N1) constructed by reverse genetics methods. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2025, Vol. 60, № 6, p. 1071-1084.
(how to cite this article)

doi: 10.15389/agrobiology.2025.6.1071eng

UDC: 619:578.2:577.2

Acknowledgements:
Supported financially by the Russian Science Foundation grant № 24-76-10044, https://rscf.ru/project/24-76-10044/

 

A CANDIDATE VACCINE STRAIN OF HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS SUBTYPE A(H5N1) CONSTRUCTED BY REVERSE GENETICS METHODS

M.V. Sergeeva1, K.S. Kudrya1, M.A. Plotnikova1,
V.V. Veretennikov2, E.D. Javadov2, D.A. Kraskov2, A.A. Ivanova1,
P.A. Petrova1, N.V. Tarlavin2 ✉

1Smorodintsev Research Institute of Influenza, the Ministry of Health of the Russian Federation, 15/17, ul. Professora Popova, St. Petersburg, 197022 Russia, e-mail mari.v.sergeeva@gmail.com, kira336@yandex.ru,
anna_e_svobodniy@mail.ru, biomalinka@mail.ru,
suddenkovapolina@gmail.com;
2Saint Petersburg State University of Veterinary Medicine, 5, ul. Chernigovskaya, St. Petersburg, 196084 Russia, e-mail tarlav1995@bk.ru (✉ corresponding author), vnivip1@mail.ru, vlad.veretennikov.96@mail.ru,kraskov-00@bk.ru

ORCID:
Sergeeva M.V. orcid.org/0000-0003-0411-9896
Kraskov D.A. orcid.org/0000-0002-2362-2641
Kudrya K.S. orcid.org/0000-0003-4774-4294
Ivanova A.A. orcid.org/0000-0002-3495-4393
Plotnikova M.A. orcid.org/0000-0001-8196-3156
Petrova P.A. orcid.org/0000-0001-8527-7946
Veretennikov V.V. orcid.org/0000-0001-9648-2259
Tarlavin N.V. orcid.org/0000-0002-6474-9171
Javadov E.J. orcid.org/0000-0002-1589-6300

Final revision received February 09, 2025
Accepted March 23, 2025

The highly pathogenic avian influenza virus subtype H5N1 actively circulates among domestic and wild poultry worldwide, causing widespread mortality. The primary methods of combating avian influenza include culling and vaccination. These measures have enabled control of the spread of the H5N1 influenza virus, reducing the number of new outbreaks and the viral load on the environment. Reverse genetics has become a widely used modern technology for creating vaccines against influenza A viruses. This study describes for the first time the creation of a recombinant strain of the H5N1 influenza virus subtype 2.3.4.4b with a modified hemagglutinin of an antigenically relevant virus isolated in Russia in 2023. We demonstrated that the constructed strain exhibits high production characteristics in the ECE (developing chicken embryos) system and in MDCK cell culture. We also used it for the first time in a vaccine with the addition of aluminum hydroxide as an adjuvant for immunization of target animals and found that vaccination stimulates antibody production in 1-day-old chickens. Our goal was to construct a candidate vaccine strain using reverse genetics that could be used to create a veterinary vaccine against modern highly pathogenic avian influenza viruses of the H5N1 subtype. The work was carried out in 2024 at the Smorodintsev Research Institute of Influenza of the Ministry of Health of the Russian Federation. The RNA source was a lysed sample containing the genetic material of the A/black-headed gull/Syktyvkar/19987/2023 (H5N1) virus. Amplification of full-length cDNA copies of the HA and NA viral gene segments was carried out using a set of universal primer pairs. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using the Extra Mix RT-PCR kit (Biolabmix LLC, Russia) and a Gentier 96E instrument (Xi'an Tianlong Science and Technology, Co., Ltd., China). The resulting products were cloned into the pHW2000 vector, designed for assembling influenza viruses by reverse genetics. Escherichia coli DH5-α bacterial cells were transformed with plasmids. The nucleotide sequence of the selected clones was verified by Sanger sequencing (Eurogen CJSC, Russia). To modify the HA cleavage site, a search for nucleotide sequences of closely related low-pathogenic influenza A/H5N1 viruses of clade 2.3.4.4b was performed using the EpiFlu database (GISAID, Germany). The nucleotide sequences of the cleavage sites of the selected low-pathogenicity viruses were used to modify the plasmid encoding the HA gene by site-directed mutagenesis (Eurogen, Russia). To assemble the recombinant virus by reverse genetics, a mixture of plasmids based on the pHW2000 vector was used: modified HA and NA of the H5N1 subtype (described above), as well as plasmids encoding the genes of internal and nonstructural proteins (PB2, PB1, PA, NP, M, NS) of the highly reproductive donor strain A/PR/8/34 (H1N1). The plasmid mixture was transfected into a coculture of HEK293FT/MDCK cells grown in DMEM/F12 medium. GenJect39 liposomes (Molecta, Russia) were used for transfection. In 1 day, the medium was changed to a serum-free medium with TPCK-trypsin (Sigma, USA). Then, every day, the presence of cytopathic effect (CPE) of the virus was checked visually using an AxioVert A1 light microscope (Carl Zeiss AG, Germany), and the appearance of the virus in the culture medium was monitored using the hemagglutination assay (HA). To accumulate the virus, 10- to 11-day-old ECEs were infected with virus-containing material in various dilutions in a volume of 0.2 ml into the allantoic cavity. To determine the infectious activity in the ECE system, a series of 10-fold dilutions were prepared from the virus-containing fluid in DPBS (OOO BioloT, Russia) added with an antibiotic-antimycotic. Calculation of 50 % embryonic infectious dose (EID50) was performed according to the method of L.J. Reed and H. Muench and expressed as lg EID50/ml. Whole-genome nucleotide sequences of the virus were obtained by next-generation sequencing using Illumina, Inc. (USA). The phenotype characteristic of low-pathogenic avian influenza viruses was confirmed by the absence of replication in a permissive MDCK cell culture lacking endogenous trypsin-like protease. The antigenic properties of the virus were studied in a hemagglutination inhibition assay (HI assay) with specific rat antisera to influenza A H5 subtype viruses of various years of isolation and corresponding viruses from the collection of the Smorodintsev Research Institute of Influenza. The ability of the strain to induce the formation of virus-specific antibodies was assessed in 10-day-old Dekalb White chickens. A recombinant vaccine strain A/Syktyvkar/PR8/6:2/HA20 (H5N1), a reassortant with a 6:2 genome composition, was assembled using reverse genetics, based on the highly reproductive donor A/PR/8/34 with surface antigens of the highly pathogenic avian influenza virus subtype H5N1. The strain was deposited to the State Collection of Viruses. To evaluate the strain, an antigen analysis was performed with antisera obtained to influenza viruses of the H5N1 subtype of various clades circulating in Russia between 2000 and 2025. The analysis showed that the constructed strain does not interact with antisera to clades 1 and 2.2, and the interaction with the antiserum to clades 2.3.4.4b was lower than with the homologous serum. When assessing the stability of the developed vaccine strain, high virus reproduction in the ECE system was noted over 5 consecutive passages. The appearance of additional mutations in the viral genome was excluded by the sequencing method. An inactivated vaccine was constructed using the developed strain with the addition of aluminum hydroxide as a sorbent adjuvant at a final concentration of 0.2 %. A trial on Dekalb White chickens demonstrated the suitability of the developed strain as an antigen in a vaccine. When the vaccine was administered to 1-day-old chickens at a dose of 0.5 ml/head (the vaccination dose was 8.4 lg/head), the antibody titer after 28 days was 1:388 in the HI assay. This antibody titer significantly exceeds that required for stable and long-term protection (1:32 in the HI assay). Thus, the resulting recombinant influenza virus strain A/Syktyvkar/PR8/6:2/HA20 (H5N1) is fully suitable for the production of biopreparations for veterinary use, including inactivated vaccines.

Keywords: highly pathogenic avian influenza, vaccine, recombinant vaccines, reverse genetics, avian infectious diseases, plasmid construction, influenza A virus of the H5N1 subtype.

 

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