RU2716416C1 - Reassortant strain of influenza virus rn2/14-human a(h6n2) for detecting neuraminidase antibodies in influenza infection and vaccination - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention represents RN2/14-human A(H6N2) strain actively propagating in developing chick embryos at the optimum temperature of 33 °C, which enables to accumulate viral material for further purification and concentration. Reassortant inherited a gene which codes the surface antigen of the haemagglutinin virus (HA) and six genes coding internal and non-structural proteins from A/17/silver gull/Sarma/06/887 (H6N1), and the gene coding the surface antigen of the virus neuraminidase (NA), from A/17/Hong Kong/2014/8296 (H3N2).

EFFECT: strain RN2/14-human A(H6N2) can be used for detecting antibodies to neuraminidase N2 of influenza virus in blood serum using solid-phase inhibition reaction of neuraminidase activity.

1 cl, 4 dwg

Description

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

The current influenza epidemic situation is characterized by the simultaneous circulation of several seropod types of the virus of the Orthomyxoviridae family of the genus Influenzavirus: A (H1N1), A (H3N2) and B. As new flu vaccines are created, a comprehensive assessment of the immunogenicity of the prepared drugs becomes necessary. Traditionally, the immunogenicity of influenza vaccines is assessed by the formation of serum antibodies to hemagglutinin (HA) after vaccination [Guidelines for clinical trials of drugs (immunobiological drugs). Part Two / Ed. A.N. Mironova. - M .: Grif and K, 2012. - 212 p.]. However, in relation to live influenza vaccines, there has still not been a direct relationship between serological effectiveness, which is evaluated by the growth of anti-hemagglutinating antibodies, with a high level of protective effectiveness. Antibodies to the second surface antigen of the influenza virus - neuraminidase (NA) - are also one of the protective factors against infection. Reassortant strains based on the influenza A / horse / Prague / 1/56 (H7N7) virus with hemagglutinin irrelevant to humans containing neuraminidase of human epidemic viruses A (H1N1) and A (H3N2) were previously used to determine antibodies to influenza neuraminidase [Vopr. virology. - 1985. - No. 1. - p. 35-39]. In this case, the reaction of inhibiting the elution of the virus from red blood cells was used, based on the blocking of the enzymatic activity of NA by antibodies, which prevented the destruction of red blood cell agglutination caused by influenza HA.

Reassortant strain RN2 / 57-human A (H7N2), used to detect antibodies to neuraminidase of subtype N2 in the solid-state reaction of inhibition of sialidase activity [RF Patent 2464312 stated. 06/16/2011; publ. 10.20.2012, Bull. No. 29], became irrelevant in connection with the appearance in 2013 in the human population of viruses that carry HA subtype H7 [Tu C. et al. The first case of avian influenza A (H7N9) virus occurring in the autumn season, China // American journal of infection control. - 2014. - T. 42. - No. 2. - C. 212-213.], Which can lead to a distortion of the results of serological tests aimed at detecting antineuraminidase antibodies, due to the presence of potentially pandemic antibodies to hemagglutinin in the studied blood serum samples.

The problem to which the invention is directed, is to obtain a reassortant strain for determining antibodies to neuraminidase of subtype N2 in the solid-state reaction of inhibition of neuraminidase activity.

The purpose of reassortment is to obtain a strain carrying neuraminidase of subtype N2 for the qualitative and quantitative determination of antibodies to NA subtype N2 in serum.

Obtaining reassortant strain. The reassortant RN2 / 14-human A (H6N2) was obtained by classical genetic reassortment in developing chicken embryos (RKE) of two vaccine strains obtained from the attenuation donor A / Leningrad / 134/17/57 (Н2Ы2), A / 17 / silver 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) by simultaneously infecting developing chicken embryos (RCE) a mixture of parent viruses in equivalent infectious doses, followed by selection of clones with desired properties you at a temperature of incubation lowered to 26 ° C in the presence of monoclonal antibodies to anti-H3 (H3N2) mAb, clone InA246. Clones were further purified by two serial cloning by the method of limiting dilutions in the presence of monoclonal antibodies to anti-H3 (H3N2) mAb, clone InA246 (26 ° C) and optimal (32 ° C) incubation temperatures.

A study of the antigenic properties of the reassortant RN2 / 14-human A (H6N2) showed that: - the surface glycoprotein of the strain responsible for antigenic specificity - hemagglutinin (HA) - in rtga with homologous serum is antigenically identical to virus A / 17 / silver gull / Sarma / 06 / 887 (H6N1), because the interaction indicators of the reassortant RN2 / 14-human A (H6N2) and the parent strain A / 17 / silver gull / Sarma / 06/887 (H6N1) with the rat antiserum obtained for virus A / 17 / silver gull / Sarma / 06/887 (H6N1) completely coincided (Table 1). Sequencing of the reassortant RN2 / 14-human A (H6N2) was also performed, which showed the identity of the HA reassortant and strain A / 17 / silver gull / Sarma / 06/887 (H6N1).

- the second surface glycoprotein of the strain, neuraminidase (NA), responsible for antigenic specificity, was tested by restriction analysis of DNA copies of genes, as well as by sequencing and is identical to virus A / 17 / Hong Kong / 2014/8296 (H3N2).

To analyze the composition of surface glycoproteins of the obtained reassortants, the method of restriction analysis of DNA copies of genes was used. In FIG. Figure 1 shows an example of a restriction analysis of NA gene DNA using the BstMBI restriction enzyme. Electrophoretic gel tracks have the following correspondence: 1 - DNA marker (100 bp + 1.5 Kb, NPO SibEnzyme), 2 - reassortant RN2 / 14-human A (H6N2), 3 - strain A / 17 / Hong Kong / 2014 / 8296 (H3N2).

The presence of two DNA fragments of the reassortant RN2 / 14-human A (H6N2) coincides with the parent strain A / 17 / Hong Kong / 2014/8296 (H3N2), which indicates the identity of the NA genes of the reassortant RN2 / 14-human A (H6N2) and strain A / 17 / Hong Kong / 2014/8296 (H3N2).

The data obtained during the selection of reassortants were confirmed by sequencing and showed that the reassortant RN2 / 14-human A (H6N2) inherited the HA gene from A / 17 / silver gull / Sarma / 06/887 (H6N1), and NA from A / 17 / Hong Kong / 2014/8296 (H3N2).

Thus, the presented reassortant strain has the antigenic specificity of hemagglutinin virus A / 17 / silver gull / Sarma / 06/887 (H6N1), which eliminates the distortion of the results of serological tests aimed at identifying antineuraminidase antibodies, due to the presence of blood serum in the studied samples antibodies to hemagglutinin of modern epidemic and potentially pandemic strains of the influenza virus.

The presence of the N2 influenza virus neuraminidase makes it possible to use this strain to detect antibodies to this subtype neuraminidase in people who have been infected or have been vaccinated with influenza vaccines.

Identification of antineuraminidase antibodies in the solid-phase reaction of inhibition of neuraminidase activity (RINA).

The methodology for this detection is based on determining the titer of antineuraminidase inhibitory antibodies, which is the reciprocal of the dilution of the test serum, in which there is a 50% decrease in sialidase activity of the NA diagnostic strain RN2 / 14-human A (H6N2). The sialidase activity of NA is determined with respect to a high molecular weight natural substrate (fetuin) adsorbed on a polymer carrier using specific binding of unmasked peroxidase-labeled lectin polysaccharides. A reliable and reliable diagnosis is a twofold or more increase in antibody titers in the serum of convalescence in comparison with serum obtained in the acute phase of the disease, or before and after vaccination. Experiment Example:

The diagnostic strain RN2 / 14-human A (H6N2) was used to determine the titer of antineuraminidase antibodies in human serum after immunization with the Ultravac® seasonal live influenza vaccine (NPO Microgen, JSC). 17 pairs of human serum were examined before immunization and 28 days after immunization. The titer of antibodies to neuraminidase was determined using the solid-phase reaction of inhibition of neuraminidase activity (RINA), the titer of antibodies to hemagglutinin was determined using the reaction of inhibition of hemagglutination (RTGA). Work with human sera is approved by the local ethics committee of the Federal State Budget Scientific Institution "IEM". Minutes 3/17 dated 11/30/2017.

As a result of the study, a statistically significant (p = 0.01) increase in the titer of antibodies to neuraminidase of subtype N2 after vaccination with live hepatitis B was found (Fig. 2).

7 out of 17 (41%) people who responded to vaccination with live hepatitis B showed a twofold or more increase in titers of antibodies to neuraminidase of subtype N2 (Fig. 3). The increase is statistically significant - p = 0.0001.

The determination of hemagglutinin-inhibiting antibodies to hemagglutinin of the H3 subtype in human sera was carried out using rtga to the vaccine strain A / 17 / Hong Kong / 14/8296 (H3N2). A slight increase in titers of hemagglutinin-inhibiting antibodies after vaccination with live hepatitis B was detected (Fig. 4). Antibody growth was not statistically significant - p = 0.055. Only 1 person out of 17 (5.6%) showed a fourfold or more increase in titers of antibodies to hemagglutinin of subtype H3.

Conclusion

As a result of the studies, it was found that the claimed diagnostic strain RN2 / 14-human A (H6N2) is characterized by the necessary parameters for the diagnostic strain and can be used to detect antineuraminidase antibodies in people who have been infected with influenza virus strains carrying the N2 subtype or who have been vaccinated with influenza vaccines of the corresponding subtypes. Since the presented diagnostic reassortant strain has the antigenic specificity of hemagglutinin of the human A / 17 virus / silver gull / Sarma / 06/887 (H6N1) irrelevant hemagglutinin, its use eliminates the distortion of the results of serological tests aimed at detecting antineuraminidase antibodies, due to the presence in the studied samples serum antibodies to hemagglutinin of modern epidemic and potentially pandemic strains of the influenza virus.

The strain was deposited 02/28/2019 in the State collection of viruses of the Institute of Virology. DI. Ivanovo Federal State Budgetary Institution Scientific Center N.F. Gamalei ”of the Ministry of Health of Russia under No. 2905 and has the characteristics presented in the sample passport strain.

CHARACTERISTIC OF THE RECEIVED STRAIN.

The morphology of the strain is polymorphic, typical of the influenza virus.

Infectious activity of the reassortant strain RN2 / 14-human A (H6N2) during reproduction in developing chicken embryos at 33 ° C for 48 hours - 8.0 lg EID ₅₀ / 0.2 ml.

Hemagglutinating activity - 1: 256.

A passport for the reassortant strain RN2 / 14-human A (H6N2) is attached.

Claims (1)

The strain of the influenza virus RN2 / 14-human A (H6N2), deposited in the State collection of viruses of the Institute of Virology. DI. Ivanovo Federal State Budgetary Institution Scientific Center N.F. Gamalei "of the Ministry of Health of Russia under No. 2905, for the determination of antibodies to neuraminidase N2 of the influenza virus.

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