RU2625024C1 - Reassortant strain of rn2/66-human a (h7n2) influenza virus for neuraminidase antibodies identification in case of influenza infection and vaccination - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: diagnostic strain of RN2/66-human A (H7N2) influenza virus was obtained by crossing the equine influenza A/horse/Prague/1/1956(H7N7) virus with the cold-adapted vaccine strain A/17/California/66/395(H2N2) based on the attenuation donor A/Leningrad/134/17/57(H2N2). The strain contains the neuraminidase of the influenza virus subtype N2 A/California/1/1966 (H2N2) and influenza A/horse/Prague/1/1956 (H7N7) hemagglutinin. The RN2/66-human A(H7N2) strain actively multiplies in developing chick embryos at the optimum temperature of 33°C, which allows to accumulate viral material for subsequent purification and concentration. The RN2/66-human A (H7N2) strain can be used for solid-phase inhibition of neuraminidase activity to analyse background values of serum antibodies to influenza neuraminidase N2 in the human population and their changes as a result of infection/vaccination.

EFFECT: improved strain properties.

2 dwg, 1 tbl, 2 ex

Description

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

Influenza A viruses of the H2N2 subtype have not been circulating in the human population since 1968, therefore, the majority of the population, primarily people over 45, are not immune to their main surface antigen, hemagglutinin (HA), and, accordingly, will be to some extent vulnerable to if the specified subtype of the virus returns to circulation. Modern epidemic strains of subtype A (H3N2), having significant antigenic differences in hemagglutinin with out-of-circulation strains of A (H2N2), represent one subtype of neuraminidase with them. Thus, the contingent of people vaccinated with the seasonal trivalent live influenza vaccine (VHF), or those who have had a natural infection caused by the epidemic strain of influenza A virus (H3N2), which constitutes an anti-influenza immune response aimed at the minor surface antigen of the virus (N2) may be decisive in reducing mortality and limiting the spread of a pandemic at its initial stage, until mass vaccination of the population with a strain with antigenically relevant HA is deployed.

One of the priorities of the World Health Organization is to prepare for the pandemic of influenza A (H2N2), which includes assessing the degree of susceptibility of the population to a potentially pandemic strain of the influenza A (H2N2) virus and creating international and national collections of vaccine strains with the goal of speedy start of production of pandemic vaccines and immunization in the event of the return of A (H2N2) viruses. The first stage of preparation involves the study of heterosubtypical influenza immunity mediated by antineuraminidase antibodies and other factors other than anti-hemagglutinating antibodies. The second stage includes a comprehensive, complete characterization of the immunogenicity and protective efficacy of vaccines of a potentially pandemic subtype, including using methods to detect a humoral immune response to the influenza NA.

Reassortant strains based on the influenza A / horse / Prague / 1/56 (H7N7) virus with hemagglutinin irrelevant to humans containing neuraminidase of the 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. Also known is the reassortant strain RN1 / 09-swine A (H7N1), which is used to detect antibodies to the 2009 pandemic strain neuraminidase subtype A (H1N1) in the solid-state reaction of inhibiting sialidase activity [RF Patent 2428476, claimed. 06/21/2010, publ. 09/10/2011, Bull. No. 25].

The task to which the invention is directed is the preparation of a diagnostic reassortant strain of influenza A virus for the specific detection in blood serum of antineuraminidase antibodies to out-of-circulation, potentially pandemic influenza viruses of subtype A (H2N2) in the solid-state reaction of inhibition of sialidase activity. For these purposes, classical genetic reassortment methods were used in developing chicken embryos (RCEs) of virus A / horse / Prague / 1/56 (H7N7) and vaccine strain A / 17 / California / 66/395 (H2N2) containing surface antigens from the influenza virus A / California / 1/1966 (H2N2), isolated at the end of the last period of circulation of strains A (H2N2). To select clones with the desired properties and genome composition, a series of selective passages were carried out at a temperature lowered to 25 ° C in the presence of rat immune serum to strain A / California / 1/1966 (H2N2) depleted in the content of anti-N2 antibodies.

Vaccine strain A / 17 / California / 66/395 (H2N2), which is a reassortant based on a cold-adapted attenuation donor A / Leningrad / 134/17/57 (H2N2) with a 6: 2 genome formula that inherits 6 genes of internal and non-structural proteins from the donor attenuation, and the HA and NA genes from the influenza virus A / California / 1/1966 (H2N2) [PloS one. - 2014 .-- Vol. 9.- No. 7. - e102339; PMID: 25058039], was obtained from the collection of the Virology Department of the Federal State Budget Scientific Institution "IEM".

Virus A / horse / Prague / 1/1956 (H7N7) was provided by the U.S. Centers for Disease Control and Prevention (Atlanta, Georgia).

The reassortant RN2 / 66-human A (H7N2) inherited the NA gene from strain A / California / 1/1966 (H2N2). In FIG. Figure 1 shows an example of the analysis of the NA gene using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for the neuraminidase of only one of the parent viruses: subtype N2 (lanes 2 through 4) or subtype N7 (lanes 7 through 9). Electrophoretic gel tracks have the following correspondence: 1 - DNA marker (100 bp + 1.5 Kb, NPO SibEnzyme), 2 - RT-PCR with primers for N2 and RNA of strain A / horse / Prague / 1/56 (H7N7) , 3 - RT-PCR with primers for N2 and RNA of strain A / 17 / California / 66/395 (H2N2), 4 - RT-PCR with primers for N2 and RNA of strain RN2 / 66-human A (H7N2), 5, 6 - DNA marker (100 bp + 1.5 Kb, NPO SibEnzyme), 7 - RT-PCR with primers for N7 and RNA strain A / horse / Prague / 1/56 (H7N7), 8 - RT-PCR with primers to N7 and RNA of strain A / 17 / California / 66/395 (H2N2), 9 - RT-PCR with primers to N7 and RNA of strain RN2 / 66-human A (H7N2). The presence of RN2 / 66-human A (H7N2) reassortant RNA amplification (lane 4) with N2 primers is similar to the parent strain A / 17 / California / 66/395 (H2N2) (lane 3) and the absence of a PCR product ( 9th lane) during polymerase chain reaction with primers complementary to regions of the NA gene of strain A / horse / Prague / 1/56 (H7N7), indicate the identity of the NA genes of the reassortant RN2 / 66-human A (H7N2) and strain A / California / 1/1966 (H2N2).

RT-PCR restriction analysis of six non-glycosylated protein genes [Journal of Virological Methods. - 1995. - No. 55. - P. 445-446] showed that 5 segments of RNA encoding the internal proteins of the virus (PB2, PB1, PA, NP, M) were inherited by the reassortant from the attenuation donor A / Leningrad / 134/17/57 (H2N2), and the RNA segment encoding non-structural proteins of the virus (NS) is from the horse influenza virus A / horse / Prague / 1/1956 (H71N7).

The identity of the hemagglutinin reassortant to the corresponding surface glycoprotein of the parent strain A / horse / Prague / 1/1956 (H7N7) was confirmed in the inhibition of hemagglutination (RTGA) with rat antisera.

The presence of influenza virus neuraminidase of a potentially pandemic subtype A (H2N2) makes it possible to use this strain to detect homologous and cross-reacting antineuraminidase antibodies in people who have been infected with A (H2N2) / A (H3N2) strains or have been vaccinated with the corresponding subtypes of influenza vaccines. Since the presented diagnostic reassortant strain has the antigenic specificity of hemagglutinin that is not relevant for human virus A / horse / Prague / 1/1956 (H71N7), its use 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 presumably pandemic strains.

The strain is deposited in the State virus collection Research Institute of Virology. DI. Ivanovo N.F. Gamalei ”of the Ministry of Health of Russia under the number of GKV 2771

CHARACTERISTIC OF THE RECEIVED STRAIN

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

Infectious activity of the reassortant strain RN2 / 66-human A (H7N2) during reproduction in developing chicken embryos at 33 ° C for 48 hours - 8.5 lg EID ₅₀ / 0.2 ml.

Hemagglutinating activity - 512 GAU / 50 μl.

A passport for the reassortant strain RN2 / 66-human A (H7N2) is attached.

PASSPORT STRAIN

influenza virus RN2 / 66-human A (H7N2)

1. Name of the virus, strain (family, taxonomic and antigenic group):

family Orthomyxoviridae, genus Influenzavirus A, subtype H7N2, strain RN2 / 66-human A (H7N2) [A / horse / Prague / 1/1956 (H7) × A / California / 1/1966 (N2)].

2. Isolated (year, place of isolation, from whom and from what material): the strain was obtained in October 2013 by classical genetic reassortment in developing chicken embryos. Characteristic of parent viruses:

a) horse flu virus - A / horse / Prague / 1/1956 (H7N7);

b) vaccine strain A / 17 / California / 66/395 (H2N2) containing the HA and NA genes of the epidemic influenza virus A / California / 1/1966 (H2N2), the remaining 6 segments of the RNA of the attenuation donor A / Leningrad / 134/17 / 1957 (H2N2).

3. Physico-chemical properties (cryptogram, size, filtration, RNA, DNA):

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

4. Genetic traits

The composition of the genome: ON from the horse influenza virus A / horse / Prague / 1/1956 (H7N7); NA from the epidemic influenza virus A / California / 1/1966 (H2N2); 5 RNA segments encoding the internal proteins of the virus (PB2, PB1, PA, NP, M), from the attenuation donor A / Leningrad / 134/17/1957 (H2N2); RNA segment encoding non-structural proteins of the virus (NS), from the horse influenza virus A / horse / Prague / 1/1956 (H7N7).

5. Antigenic properties

Antigenically identical to virus A hemagglutinin / horse / Prague / 1/1956 (H7N7) according to RTGA with rat antiserum.

6. Other properties

The strain RN2 / 66-human A (H7N2) has hemagglutinin that is irrelevant to humans and is intended for the specific detection of antineuraminidase antibodies in the blood serum.

7. Number of passages

7 in the process of reassortment; then 2 accumulative passages in the system of developing chicken embryos at an incubation temperature of 33 ° C.

8. Optimal titer

The optimal titer is achieved by culturing the strain in 10-11 day old developing chicken embryos at 33 ° C for 48 hours, while the infectious activity of the strain is 8.5 lg EID ₅₀ / 0.2 ml, hemagglutinating activity with 0.5% chicken suspension the erythrocyte count is 512 GAU / 50 μl.

9. Drying mode

The virus was lyophilized on October 21, 2013. The volume of material in the ampoule was 1.0 ml; the stabilizer was a solution of 13% peptone.

10. Storage conditions

The virus is stored in the form of a lyophilized suspension at a temperature not exceeding + 4 ° C. To restore the infectious properties, 1-2 passages on chicken embryos are needed. The infectious activity of the strain after lyophilization is 8.3 EID ₅₀ / 0.2 ml, the hemagglutinating activity is 256 GAE / 50 μl.

11. Pathogenicity for humans

Refers to microorganisms of the III group of pathogenicity.

12. Sensitivity to experimental infection (animals, embryos, arthropods and cell cultures)

13. The authors of the strain

Smolonogina T.A., Ph.D., Senior Researcher Department of Virology, Federal State Budget Scientific Institution "IEM";

Desheva Yu.A., doctor of medical sciences, senior researcher Department of Virology, Federal State Budget Scientific Institution "IEM";

Rudenko L.G., prof., Doctor of medical sciences, head. Department of Virology, Federal State Budget Scientific Institution "IEM".

The purpose of the obtained strain is a qualitative and quantitative determination of antibodies to NA of influenza viruses of a potentially pandemic subtype A (H2N2) in blood serum.

1. The strain can be used to detect the level of serum antineuraminidase antibodies in the population using 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 / 66-human A (H7N2). 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.

Venous blood samples are taken from the volunteers being examined. Serum obtained after centrifugation is stored at -20 ° C.

Experiment example

Antibodies to N2 in the blood serum of volunteers aged 18-40 years, examined in the fall of 2013, were determined using the solid-phase reaction of inhibition of neuraminidase activity and the diagnostic strain RN2 / 66-human A (H7N2).

The distribution of volunteers by the level of serum antineuraminidase antibodies to strain A / California / 1/66 (H2N2) is shown in FIG. 2. In 11 out of 36 examined volunteers (30.5%) born in 1973-1995, antibodies to the NA strain of subtype A (H2N2), which had gone out of circulation among the human population as early as 1968, were detected in solid-phase RINA in ranges above the value 1:40. The geometric mean titer of anti-NA antibodies for the indicated group of 11 seropositive individuals was 1:88. Due to the age of the volunteers, it is not possible to meet them directly with the epidemic viruses of the considered subtype, which is also confirmed by the RTGA data: none of the volunteers participating in the study was immune to HA of strain A / California / 1/66 (H2N2). It can be assumed that the presence of serum antineuraminidase antibodies to the 1966 strain among the considered age group is due to the cross-reaction of anti-N2 immunoglobulins formed as a result of the contact of the examined individuals with epidemic strains of subtype A (H3N2) that replaced the viruses A (H2N2) and representing the same subtype of neuraminidase with them.

2. The strain can be used to detect antineuraminidase antibodies in the blood serum of volunteers immunized with hepatitis B using the inhibition of neuraminidase activity.

Experiment example

After characterization of the initial immunological status, volunteers in clinical trials were administered intranasally twice with an interval of 28 days 7.9 lg EID ₅₀ / 0.5 ml of monovalent HBV based on the attenuated strain of the potentially pandemic subtype A / 17 / California / 66/395 (H2N2). Serum samples of vaccinated individuals and placebo volunteers were taken on day 28 after the first and second immunization, as well as 56 days after the second vaccination.

According to the solid-phase RINA and RTHA, the initial immunological status of the group of volunteers who received and received the placebo drug did not differ (Mann-Whitney U Test, p> 0.05). A statistically significant increase in titers of antineuraminidase antibodies to the vaccine strain was observed only on days 28 and 56 after double immunization with PHA (see table 1). A statistically significant increase in the titers of antihemagglutinating antibodies to the vaccine strain was observed already on the 28th day after a single injection of live hepatitis B virus, but revaccination was still required for a more pronounced immune response to the HA of the attenuated strain. In the group of people who received the placebo drug, there were no changes in the corresponding serological parameters.

The percentage of coincidence of the results of rtga and solid phase RINA in terms of detecting / not detecting seroconversions to surface antigens of strain A / 17 / California / 66/395 (H2N2) in the same pairs of blood serum of vaccinated volunteers was low - 52.0-74.1 % In 14.8-28.0% of vaccinees, significant increases in antibody titers were revealed only for HA vaccine strain; 7.4-29.6% accounted for individuals who responded positively with the production of antineuraminidase, but not anti-hemagglutinating antibodies. The two-sided version of Fisher’s exact test did not reveal a relationship between seroconversions of antibodies to two surface glycoproteins of the virus in the same individuals (p> 0.7).

The experimental results clearly demonstrate the suitability of the inventive reassortant strain for detecting antibodies to influenza virus N2 neuraminidase, due to which this strain can be used for diagnosis, studying the immunogenicity of the influenza vaccine, and for analyzing the background values of antineuraminidase antibodies in the human population as a whole.

Claims (1)

The strain of the influenza virus RN2 / 66-human A (H7N2), deposited in the State virus collection of the Research Institute of Virology. DI. Ivanovo N.F. Gamalei »Ministry of Health of Russia under the number of bills 2771, used to determine antibodies to neuraminidase N2 of the influenza virus.

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