REASSORTANT STRAIN OF INFLUENZA VIRUS A / 17 / SYDNEY / 2021 / 2312 (H1N1)pdm09 FOR PRODUCTION OF LIVE INFLUENZA INTRANASAL VACCINE FOR ADULTS AND CHILDREN

Abstract

FIELD: medical virology.SUBSTANCE: influenza virus strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 has been proposed. The strain was deposited in the State Collection of Viruses named after D.I. Ivanovsky of FSBI National Research Center for Epidemiology and Microbiology named after the honorary academician N. F. Gamaleya of the Ministry of Health of the Russian Federation under No. 3019, for the production of a live intranasal influenza vaccine for adults and children.EFFECT: obtaining a live intranasal influenza vaccine for the prevention of influenza among adults and children.1 cl, 5 tbl

Description

[0001] The invention relates to medical virology and can be used in healthcare for the prevention of influenza among adults and children using a live influenza intranasal vaccine from the A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 strain of the influenza A virus, Orthomyxoviridae family, Influenzavirus A genus.

[0002] According to the World Health Organization (WHO), from February to September 2022, A / Sydney / 5 / 2021 (H1N1)pdm09-like influenza viruses belonging to subclade 6B.1A.5a.2 were predominant in circulation in the countries of the Southern Hemisphere. In this regard, in September 2022, WHO recommended replacing the A / H1N1 component in the composition of influenza vaccines for the Southern Hemisphere for the 2023 epidemic season with A / Sydney / 5 / 2021 (H1N1)pdm09-like viruses [WHO. Recommended composition of influenza virus vaccines for use in the 2023 southern hemisphere influenza season. 23 September 2022. https: / / www.who.int / publications / m / item / recommended-composition-of-influenza-virus-vaccines-for-use-in-the-2023-southern-hemisphere-influenza-season].

[0003] As a result of the emergence in circulation of a new epidemic strain of influenza virus A / Sydney / 5 / 2021 (H1N1)pdm09, the known vaccine strain A / 17 / Victoria / 2019 / 276 (H1N1)pdm09 - prototype [Stepanova E.A., Bazhenova E.A., Krutikova E.V., Matyushenko V.A., Larionova N.V., Kiseleva I.V., Isakova-Sivak I.N., Rudenko L.G. Reassortant strain of influenza virus A / 17 / Victoria / 2019 / 276 (H1N1)pdm09 for the production of live influenza intranasal vaccine for adults and children. Russian Federation Patent No. 2783878. Published: 21.11.2022 Bulletin No. 33] - has lost its relevance and, as a result, cannot effectively protect the population during the epidemic caused by A / Sydney / 5 / 2021 (H1N1)pdm09-like strains of the influenza virus.

[0004] The problem to be solved by the claimed invention is to obtain an antigenically relevant vaccine strain for adults and children based on the cold-adapted donor of attenuation A / Leningrad / 134 / 17 / 57 (H2N2) and the new epidemic virus A / Sydney / 5 / 2021 (H1N1)pdm09.

[0005] Currently used strains for live influenza vaccines are obtained by genetic reassortment of epidemically relevant viruses with cold-adapted strains harmless to humans - attenuation donors [Shcherbik, S.; Pearce, N.; Kiseleva, I.; Larionova, N.; Rudenko, L.; Xu, X.; Wentworth, DE; Bousse, T. Implementation of new approaches for generating conventional reassortants for live attenuated influenza vaccine based on Russian master donor viruses. J Virol Methods 2016, 227, 33-39, DOI: 10.1016 / j.jviromet.2015.10.009].

[0006] Attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2) - cold-adapted (ca) and temperature-sensitive (ts) strain of influenza virus - approved for the production of live intranasal vaccines for adults and children [Aleksandrova G.I. New in epidemiology and prevention of viral infections. L., 1968. - P. 66-83].

[0007] The purpose of reassortment is to obtain a strain with a 6:2 genome vaccine formula - two 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 internal and non-structural proteins (PB2, PB1, PA, NP, M, NS) are inherited from the attenuation donor.

[0008] Obtaining a vaccine strain. The vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 was obtained by genetic reassortment of the epidemic virus A / Sydney / 5 / 2021 (H1N1)pdm09 with the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2) by simultaneously infecting developing chicken embryos (CE) with a mixture of parental viruses at equivalent infectious doses, followed by selection at an incubation temperature reduced to 26°C in the presence of rabbit antiserum to the attenuation donor. The clones were further purified by three sequential limiting dilution clonings in the presence of antiserum to the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2) at reduced (26°C) and optimal (32°C) incubation temperatures. The selected pure clone was tested for phenotypic characteristics (ts- and ca-phenotypes) and genome conformity with the vaccine strain.

[0009] Antigenic characteristics of the vaccine strain.

[0010] The surface protein of the vaccine strain, HA, responsible for the antigen specificity, was studied in a hemagglutination inhibition cross-reaction (HI) with sera obtained against the attenuation donor A / Leningrad / 134 / 17 / 57 and the epidemic virus A / Sydney / 5 / 2021 (H1N1)pdm09. It was shown that the epidemic virus A / Sydney / 5 / 2021 (H1N1)pdm09 and the vaccine reassortant A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 do not have antigenic affinity with the attenuation donor A / Leningrad / 134 / 17 / 57, since the titers at which hemagglutination inhibition by the antiserum occurs were below the detection threshold for the viruses studied.

[0011] At the same time, the hemagglutination inhibition indices of the reassortant A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 and the parental strain A / Sydney / 5 / 2021 (H1N1)pdm09 in the cross-RTGA with homologous rat antisera obtained to the epidemic virus A / Sydney / 5 / 2021 (H1N1)pdm09 and the vaccine reassortant A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 completely coincided (Table 1), which indicates the antigenic identity of the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 to the epidemic virus

[0012] A / Sydney / 5 / 2021 (H1N1)pdm09. Sequencing of the fourth segment of the genome (the gene encoding HA) also showed that the sequence of the translated protein of the vaccine reassortant A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 and the parental virus A / Sydney / 5 / 2021 (H1N1)pdm09 matched.

[0013] The second surface protein of the vaccine strain responsible for antigen specificity, NA, was verified by the method of complete sequencing of the corresponding gene sequence, during which the identity of the sequence of this gene to the corresponding gene of the epidemic parent virus A / Sydney / 5 / 2021 (H1N1)pdm09 was confirmed, which obviously implies its complete antigenic identity.

[0014]

[0015] 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] using specific primers developed for genome analysis of modern strains of the A / H1N1pdm09 subtype and the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2).

[0016] The results of the analysis of all genes of the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 are presented in Table 2. It was found that the genome formula of the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 is 6:2, which meets the requirements for live influenza vaccine strains: the genes encoding the surface proteins HA and NA are inherited from the epidemic parent virus A / Sydney / 5 / 2021 (H1N1)pdm09, and the genes encoding the internal and non-structural proteins (PB2, PB1, PA, NP, M, NS) are inherited from the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2).

[0017]

[0018] The data obtained during the selection of the vaccine candidate were confirmed by complete genome sequencing. It was shown that the sequence of genes encoding internal and non-structural proteins is completely consistent with the sequence of the corresponding genes of the attenuation donor virus A / Leningrad / 134 / 17 / 57 (H2N2) (Table 2), and the sequence of genes encoding HA and NA is completely consistent with the sequence of the corresponding genes of the A / Sydney / 5 / 2021 (H1N1)pdm09 virus.

[0019] The genetic stability of coding mutations associated with the attenuated phenotype of the A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 vaccine strain was studied by comparing the preservation of coding mutations before and after five-fold passaging in the recurrent viral load (RVE) of the vaccine strain prepared on the basis of the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2). The nucleotide sequence in the region of interest was determined by partial sequencing using specific primers that allow assessing the presence of these mutations [Rudenko L., Kiseleva L., Naykhin A. N., et. al. Assessment of human immune responses to H7 avian influenza virus of pandemic potential: results from a placebo-controlled, randomized double-blind phase I study of live attenuated H7N3 influenza vaccine. PLoS One. 2014 Feb 12; 9(2):e87962. doi: 10.1371 / journal.pone.0087962].

[0020] Table 3 shows the results of partial sequencing of all gene fragments of the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 containing attenuating mutations corresponding to the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2), before and after five-fold passaging in the ECE. 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 / Sydney / 2021 / 2312 (H1N1)pdm09 and are not lost during passaging.

[0021] Thus, the analysis of the preservation of attenuating mutations of the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 showed the genetic stability of its attenuating mutations and the absence of their reversions during passaging.

[0022]

[0023] The phenotypic properties of the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 were assessed by titrating it in parallel in the ECE at different temperatures. The vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 in the ECE has a phenotype identical to that of the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2). The vaccine virus was found to be temperature-sensitive (ts phenotype) - its infectious activity at 40°C was ≤1.2 lg EID 50 / ML, and cold-adapted (ca phenotype) - infectious activity at an incubation temperature reduced to 26°C reached 6.0 lg EID 50 / ML, which indicates its harmlessness to humans, since in these respects it is identical to the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2). The results of the phenotypic analysis are presented in Table 4.

[0024]

[0025] Safe for mice. Preclinical studies of acute toxicity of the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 were conducted on white outbred mice in accordance with the Guidelines for Preclinical Trials of New Immunobiological Drugs [Guidelines "Preclinical Trials of the Efficacy and Safety of New Immunobiological Medicinal Products." Moscow: 2010. 39 p.], the Guidelines for Conducting Preclinical Trials of Medicinal Products [Guidelines for Conducting Preclinical Trials of Medicinal Products (Immunobiological Medicinal Products); Part Two. - Moscow: Grif i K, 2012. - 536 p.], and the Rules of Good Laboratory Practice of the Eurasian Economic Union in the Field of Circulation of Medicines [Decision of the Council of the Eurasian Economic Commission dated 03.11.2016.No. 81 “On approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in the Sphere of Circulation of Medicines”] and with the Guidelines for preclinical safety studies for the purpose of conducting clinical trials and registration of medicinal products [Decision of the Board of the Eurasian Economic Commission dated November 26, 2019 No. 202 “On approval of the Guidelines for preclinical safety studies for the purpose of conducting clinical trials and registration of medicinal products”].

[0026] Mice were injected intraperitoneally with a single dose of the vaccine virus with a titer of 7.0 lg EID. 50 / ML in 0.5 ml, which corresponded to one dose of the human vaccine. Animals in the control group were injected intraperitoneally with saline. The general condition of each animal was monitored daily throughout the study (7 days).

[0027] Physiological study data (mobility, behavioral reactions, body weight kinetics) showed that intraperitoneal administration of the vaccine strain did not cause death of the experimental animals (Table 5) and did not lead to changes in their appearance, behavior, or affect their consumption of food and water, which indicates the harmlessness of the vaccine preparation.

[0028]

[0029] Preclinical studies have shown that the proposed live influenza vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 is characterized by a combination of beneficial properties required for a vaccine strain: antigenic specificity of the current epidemic virus A / Sydney / 5 / 2021 (H1N1)pdm09, a 6:2 genome structure optimal for reassortant vaccine strains, as well as temperature sensitivity characteristic of the attenuation donor, cold adaptation, and harmlessness to laboratory animals, which correlates with attenuation for humans. A sample passport for the vaccine strain is attached.

[0030] Thus, the vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09, in terms of its main biological properties studied in preclinical experiments in ovo and in vivo, meets the requirements for vaccine strains in the Pharmacopoeial Article (FSP: P N003224 / 01-270313) for Ultravac ®, live allantoic influenza vaccine for intranasal use for adults and children.

[0031] The obtained strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 was deposited on July 5, 2023, in the State Collection of Viruses of the D.I. Ivanovsky Institute of Virology of the Gamaleya National Research Center for Epidemiology and Microbiology of the Ministry of Health of the Russian Federation under No. 3019 and has the characteristics presented in the sample strain passport.

[0032] STRAIN CHARACTERISTICS

[0033] The infectious activity of the A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 strain during reproduction in the RCE at 32°C for 48 hours is 8.5 log 10 EID 50 / ML. Hemagglutinating activity - 1:128.

[0034] The strain exhibits genetic stability of phenotypic traits after 5 passages in the RCE (when using large infecting doses).

[0035] A useful property of the influenza virus vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 is its suitability for the production of a live influenza vaccine. The influenza virus vaccine strain A / 17 / Sydney / 2021 / 2312 (H1N1)pdm09 according to the invention can be used to prevent influenza in both adults and children aged three years and older.

[0036]

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