Influenza virus strain RN2 / 18-human a(H6N2) for determination of antibodies to neuraminidase in influenza infection and vaccination

Abstract

FIELD: medical virology.SUBSTANCE: used in healthcare for diagnostic purposes in influenza infection, as well as for assessing immunogenicity during vaccination with influenza vaccines. The diagnostic strain of the influenza virus RN2 / 18-human A(H6N2) was obtained by reverse genetics methods based on the influenza viruses A / herring gull / Sarma / 51c / 2006 (H6N1), A / Brisbane / 34 / 2018 (H3N2) and the donor of attenuation A / Leningrad / 134 / 17 / 57 (H2N2). The RN2 / 18-human A(H6N2) virus contains the gene encoding the surface antigen hemagglutinin (HA) from the A / herring gull / Sarma / 51c / 2006 (H6N1) virus; the gene encoding the surface antigen neuraminidase (NA) from the influenza A / Brisbane / 34 / 2018(H3N2) virus; genes for the internal and non-structural proteins PB2, PB1, PA, NP, M, NS from the attenuated donor strain A / Leningrad / 134 / 17 / 57 (H2N2). The RN2 / 18-human A(H6N2) strain exhibits high reproductive activity in developing chicken embryos at an optimal temperature of 33 °C, which allows the accumulation of viral material for subsequent purification and concentration.EFFECT: RN2 / 18-human A(H6N2) strain can be applied to detect antibodies to influenza virus N2 neuraminidase in blood serum using a solid-phase reaction of inhibition of neuraminidase activity.1 cl, 2 dwg, 1 ex

Description

[0001] The invention relates to medical virology and can be used in healthcare for diagnostic purposes in influenza infection, as well as for assessing immunogenicity during vaccination with influenza vaccines.

[0002] The current influenza epidemic situation is characterized by the simultaneous circulation of several serosubtypes of the Orthomyxoviridae family, Influenzavirus genus: A(H1N1), A(H3N2) and B. As new influenza vaccines are developed, there is a need for a comprehensive evaluation of the immunogenicity of the prepared preparations. Traditionally, the immunogenicity of influenza vaccines is assessed by the formation of antibodies to hemagglutinin (HA) in the blood serum after vaccination [Guidelines for the Conduct of Clinical Trials of Drugs (Immunobiological Drugs). Part Two / Ed. A.N. Mironov. - Moscow: Grif i K, 2012. - 212 p.]. However, with regard to live influenza vaccines, a direct relationship has not yet been shown between serological efficacy, which is assessed by the increase in antihemagglutinating antibodies, and a high level of protective efficacy.Antibodies to the second surface antigen of the influenza virus, neuraminidase (NA), are also one of the factors of protection against infection.

[0003] To detect antibodies to neuraminidase subtype N2 in a solid-phase reaction of inhibition of sialidase activity, a diagnostic strain of influenza virus RN2 / 14-human A(H6N2) was used. The strain was obtained by the method of classical genetic reassortment in developing chicken embryos (DCE) of two vaccine strains obtained on the basis of the attenuation donor A / Leningrad / 134 / 17 / 57 (H2N2): A / 17 / herring gull / Sarma / 06 / 887 (H6N1) and A / 17 / Hong Kong / 2014 / 8296 (H3N2), containing surface antigens from the epidemic virus subtype A / Hong Kong / 4801 / 2014 (H3N2) [Patent for invention of the Russian Federation 2716416 C1, Published: 11.03.2020 Bulletin No. 8. Application 2019130130, 24.09.2019.]. This strain is currently not relevant, since after 2016, the replacement of the N2 glycosylation site by amino acid substitutions S245N and S247T led to significant antigenic drift of N2 neuraminidase [Wan H. et al.The neuraminidase of A (H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A / Hong Kong / 4801 / 2014 vaccine strain / / Nature microbiology. - 2019. - Vol. 4. - No. 12. - Pp. 2216-2225.]. It was found that due to its proximity to the active site of the enzyme, the attached glycan at position 245 sterically blocks the attachment of NA-inhibitory antibodies [Powell H., Pekosz A. Neuraminidase antigenic drift of H3N2 clade 3c. 2a viruses alters virus replication, enzymatic activity, and inhibitory antibody binding / / PLoS pathogens. - 2020. - Vol. 16. - No. 6. - Pp. e1008411]. This may lead to a decrease in the specificity of the results of serological tests aimed at detecting antineuraminidase antibodies.

[0004] The problem to be solved by the claimed invention is to obtain a recombinant strain for determining antibodies to neuraminidase subtype N2 of modern seasonal influenza A(H3N2) viruses in a solid-phase reaction for inhibiting neuraminidase activity.

[0005] Obtaining a recombinant diagnostic strain. To determine antibodies that inhibit the activity of neuraminidase (anti-NA antibodies) of A / H3N2 viruses, a diagnostic strain of the RN2 / 18-human A(H6N2) influenza virus was prepared. The virus was obtained by electroporation of the Vero cell line with a set of 8 plasmids encoding the influenza virus genes [Smolonogina T.A., Isakova-Sivak I.N., Kotomina T.S., Evsina A.S., Stepanova E.A., Prokopenko P.I., Leontyeva G.F., Suvorov A.N., Rudenko L.G. Construction of a vector vaccine based on cold-adapted influenza virus to protect against bacterial infection caused by group B streptococci. / / Molecular Genetics, Microbiology and Virology. - 2019. - V. 37. - No. 1. - pp. 25-34.]. To assemble the H6N2 virus, plasmids encoding HA of the A / herring gull / Sarma / 51c / 2006 (H6N1) virus, NA of the influenza A / Brisbane / 34 / 2018 (3c) virus were used.3a, A / Kansas / 14 / 2017-like, recommended for use in the 2019-2020 northern hemisphere vaccine), 6 plasmids encoding the attenuation donor genes A / Leningrad / 17 / 134 / 57 (H2N2) PB2, PB1, PA, NP, M, and NS, respectively. Electroporation was performed using two pulses of 1150 V, 20 ms. After transfection, the cells were incubated for 72 hours at 33°C in 5% CO2, after which ECEs were infected with the transfection material.

[0006] Sequencing of the viral stock was performed using an ABI Prism 3031x1 Genetic Analyzer automated capillary sequencer according to the manufacturer's protocol. The HA sequence contains the following substitutions compared to the HA of the A / herring gull / Sarma / 51c / 2006 (H6N1) virus (EPI942364): G381A (leads to the G106R substitution); A387G (R108G); T1001C (non-coding); T1696C (M544T); A1707G (1548V). The NA sequence of the H6N2 strain corresponds to the NA sequence of the influenza A / Brisbane / 34 / 2018 (E2) virus (EPI1255304).

[0007] Thus, the presented recombinant strain possesses the hemagglutinin of the A / Herring Gull / Sarma / 51c / 2006 (H6N1) virus, eliminating the possibility of distorting the results of serological tests aimed at detecting anti-neuraminidase antibodies due to the presence of antibodies to the hemagglutinin of modern epidemic and potentially pandemic influenza virus strains in the blood serum samples being tested. The presence of influenza N2 neuraminidase makes it possible to use this strain to detect antibodies to neuraminidase of this subtype in individuals who have had an infection or been vaccinated with influenza vaccines.

[0008] Study of the neuraminidase activity of the A RN2 / 18-human (H6N2) virus

[0009] Fig. 1 shows that the RN2 / 18-human (H6N2) virus containing the NA of A / Brisbane / 34 / 2018(H3N2) was significantly inferior in enzymatic activity to the RN2 / 14-human (H6N2) virus containing the NA of A / 17 / Hong Kong / 2014 / 8296.

[0010] Detection of antineuraminidase antibodies in a solid-phase reaction of inhibition of neuraminidase activity (SPAR) with patients' blood sera.

[0011] This detection method is based on determining the titer of anti-neuraminidase inhibitory antibodies, which is the reciprocal of the test serum dilution that demonstrates a 50% reduction in the sialidase activity of the NA diagnostic strain RN2 / 18-human A(H6N2). NA sialidase activity is determined against a high-molecular-weight natural substrate (fetuin) adsorbed on a polymer carrier using a peroxidase-labeled lectin that specifically binds unmasked polysaccharides. A twofold or greater increase in antibody titers in the convalescent serum compared to serum obtained during the acute phase of the disease or before and after vaccination is considered diagnostically reliable.

[0012] Experiment example:

[0013] The RN2 / 18-human A(H6N2) diagnostic strain was used to determine the titer of anti-neuraminidase antibodies in archival patient sera collected in 2019-2020. Work with human sera was approved by the local ethics committee of the Institute of Experimental Medicine. Protocol 3 / 23 dated September 20, 2023.

[0014] Figure 2 shows the results of detection of antibodies to NA of A / H3N2 viruses in archival sera of patients (n=65). It was shown that despite a significant decrease in the enzymatic activity of NA of the A / Brisbane / 34 / 2018 (H3N2) virus compared to the A / Hong Kong / 2014 / 8296 (H3N2) virus, a significant drop in the inhibitory activity of antibodies specific to the A / Hong Kong / 2014 / 8296 (H3N2) virus against the A / Brisbane / 34 / 2018 (H3N2) virus was observed in the same samples. It was shown that there was a decrease in the level of collective immunity to the new antigenic variant of the A / H3N2 virus.

[0015] Conclusion

[0016] As a result of the conducted studies, it was established that the proposed diagnostic strain RN2 / 18-human A(H6N2) is characterized by the necessary parameters for a diagnostic strain and can be used to detect antineuraminidase antibodies in individuals who have had an infection with influenza virus strains carrying the N2 subtype NA or who have been vaccinated with influenza vaccines of the corresponding subtypes. Since the presented diagnostic reassortant strain has the antigenic specificity of the hemagglutinin of the A / 17 / herring gull / Sarma / 06 / 887 (H6N1) virus, which is irrelevant to humans, its use eliminates the distortion of the results of serological tests aimed at detecting antineuraminidase antibodies due to the presence of antibodies to the hemagglutinin of modern epidemic and potentially pandemic influenza virus strains in the studied blood serum samples.

[0017] CHARACTERISTICS OF THE OBTAINED STRAIN.

[0018] The strain morphology is polymorphic, typical for the influenza virus.

[0019] Infectious activity of the reassortant strain RN2 / 18-human A(H6N2) during reproduction in developing chicken embryos at 33°C for 48 hours - 7.0 lg EID 50 / 0.2 ml.

[0020] Hemagglutinating activity - 1:256.

[0021] Passport for the reassortant strain RN2 / 18-human A(H6N2) is attached.

[0022]

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