Reassortant rn a9-swine a(h7n1) influenza virus strain for neuraminidase antibody assay in influenza infection and vaccination

FIELD: medicine.

SUBSTANCE: strain is produced by cross breeding of sterile equine influenza virus A/horse/Prague/1/1956(H7N1) and cold-adapted vaccine strain A/17/California/09/38(N1) on the basis of an attenuation donor A/Leningrad/134/17/57(H2N2) and contains neuraminidase of pandemic influenza virus A//California/07/09(H1N1) and hemagglutinin of equine influenza virus A/horse/Prague/1/1956(H7N7). The strain RN1/09-swine A(H7N1) is deposited in the State collection of viruses of Institution of Russian Academy of Medical sciences of D.I. Ivanovsky Institute of virology of Russian Academy of Medical Science, No. GKV 2473 and can be applied for influenza virus neuraminidase N1 antibody assay.

EFFECT: higher assay accuracy.

2 dwg, 4 tbl, 1 ex

 

The invention relates to medical Virology and can be used in healthcare for diagnostic purposes during influenza infection, and to assess the immunogenicity of vaccination with pandemic influenza vaccines.

Modern epidemiology of influenza is characterized by the simultaneous circulation of several variants of the virus family Orthomyxoviridae, genus Influenzavirus A(H1N1), A(H3N2) and B. while it is also activation of influenza viruses of animals and birds, to which the immune system in most people is absent. In such conditions, the presence of antibodies to minor surface antigen of the virus (the neuraminidase N1) of cohort vaccinated with seasonal pandemic influenza vaccines, or in people who have had natural infection epidemic strains of influenza virus And may be crucial in reducing mortality and limit the spread of the pandemic [PLoS Med. - 2007. - Jun; 4(6). -e 212]. Traditionally, the immunogenicity of flu vaccines is estimated on the formation after vaccination, antibodies to the hemagglutinin (ON) in the serum [order No. 156/29 health Ministry 07.05.1998]. However, in the case of live vaccines is still not shown a direct link serological efficiency, which is appreciated in increments antihemagglutinin antibodies with a high level of protective efficacy.

As new flu vacc the n against pandemic strains you will need to use more effective and accessible criteria for assessing the immunogenicity of vaccine strains.

For the detection of antibodies to the neuraminidase of influenza virus previously used reassortant strains on the basis of influenza virus a/horse/Prague/1/56(7N7)containing the neuraminidase epidemic of human viruses A(H1N1) and A(H3N2) [Matters. Virology. - 1985. - No. 1 - p.35-39]. We used the test of inhibition of elution based on blocking antibodies sialidase activity of neuraminidase, which was estimated by the delay elution pre-agglutinated influenza virus erythrocytes.

Currently in circulation, new variants of influenza viruses such as the virus of swine origin A(H1N1) and avian viruses A(H5N1), in connection with which it was necessary to develop new diagnostic strains containing the appropriate neuraminidase.

The task, which is aimed by the invention, is getting reassortant strain containing the neuraminidase N1 pandemic virus of swine origin A/California/07/09(H1N1). Reassortant strain -RN1/09-swineA(H7N1)obtained by the method of classical genetic reasontly in developing chicken embryos (CE) virus And/horses/Prague/1/1956(7N7) and vaccine strain A/17/California/09/38(N1N1) followed by selection with reduced to 25°C temperature in the presence of polyclonal antibodies to the hemagglutinin of influenza virus H1.

Vaccine ø the AMM A/17/California/09/38(N1N1), obtained from the collection of the Department of Virology, epidemiology and Microbiology NWB RAMS, was reassortants with the formula genome 6:2 on the basis of holodnodeformirovannogo donor of attenuation A/Leningrad/134/17/57(H2N2) and contained surface antigens ON and NA from the pandemic virus A/California/07/09(H1N1).

The virus And/horses/Prague/1/1956(7N7) was obtained from the center for control and prevention of diseases (Atlanta, GA).

ReassortantRN1/09-swine(7N1)inherited the NA gene from strain A/California/07/09(H1N1). Figure 1 presents an example of the analysis of the NA gene using polymerase chain reaction (PCR) with primer sets specific for neuraminidase subtypes N7 and N1. Tracks have the following correspondence: 1 - DNA marker (100 bp), 2/horse/Prague/1/1956(7N7), 3 - A/17/California/09/38(N1N1), 4 - RN1/09-swine A(H7N1), 5 a/horse/Prague/1/1956(7N7), 6 - a/17/California/09/38(N1N1), 7 - RN1/09-swine A(H7N1). The presence of amplification reassortant RN1/09-swine (4th track) (H7N1) with primers N1 like the parent strain A/17/California/09/38 (3rd track) and no amplification with primers N7 (7th track) testify to the identity of the NA gene reassortant RN1/09-swine and strain A/California/07/09(H1N1).

PCR-restriction analysis of six genes deglycosylated proteins [J. Virol. Method. - 1995. No. 55. - R-446] showed that all of them were inherited from the attenuated donor strains A/Leningrad/134/17/57(H2N2). Membership of the hemagglutinin reas is ortant parent strain A/horse/Prague/1/56(H7N7) was confirmed in the response inhibition of haemagglutination (rtga).

No cross reactions reassortant strain A(H7N1) epidemic viruses A(H1N1) and A(H3N2) demonstrated in rtga with sera from volunteers vaccinated with seasonal trivalent live influenza vaccine (live flu vaccine). It is shown that the components of the vaccine A(H1N1) and A(H3N2) after vaccination, we observed the growth of antibodies, while at the same volunteers cross-reacting antibodies to equine virus A(H7N7) are absent (see table 1).

Table 1
Antihemagglutinin antibodies to viruses A(H1N1), A(H3N2) and A(H7N1) in the sera of volunteers after vaccination with seasonal trivalent live flu vaccine
GroupA(H1N1)A(H3N2)A(H7N7)
% seroconvertedThe GTS-% seroconvertedThe GTS-% seroconvertedThe GTS-
Live flu vaccine3013,84021,605,0
Placebo09,1511,705,0

It is also shown that reassortants the virus does not show cross-reaction with avian influenza virus A/duck/Potsdam/1402-6/86 (H5N2) (see table 2).

Table 2
Titles antihemagglutinin of antibodies to the virus A(H5N2) and A(H7N1) in the sera of volunteers vaccinated with monovalent live flu vaccine from strain A/17/duck/Potsdam/86/92(5N2)
The GTS antihemagglutinin antibodies to viruses
A(H5N2)A(H7N7)
Before inoculation21 days42nd dayBefore inoculation21 days42nd day
Live flu vaccine4,87,014,05,05,0Placebo5,05,25,35,05,05,0

Thus, presents reassortant strain antigenic specificity of the hemagglutinin of the virus And/horses/Prague/1/56(7N7)that there is no cross-reaction with hemagglutinin circulating seasonal viruses A(H1N1) and A(H3N2), and hemagglutinin of avian viruses of subtype H5.

The presence of the neuraminidase of the influenza birds N1 makes possible the use of this strain for the detection of cross-reacting antibodies to the neuraminidase in people who have had the infection or previous vaccination with pandemic influenza vaccines.

Reproductive activity of the strain in chicken embryos is 8.8 Ig EID50/ 0,2 ml

The strain is deposited in the State collection of viruses institutions of the Russian Academy of Medical Sciences Institute of Virology. Dijanoveckog RAMS # GKV No. 2473. Morphology of strain - polymorphic typical of influenza virus.

CHARACTERISTICS OF THE OBTAINED STRAIN.

Infectious activity of reproduction in developing chicken embryos at 33°C for 36 hours - 8,8 Ig EID50/ 0,2 ml

Hemagglutinin activity - 1:512.

the Passport on vaccinal strain RN1/09-swine A(H7N1) is attached.

PASSPORT STRAIN

influenza RN1/09-swine(7N1)

1. The name of the strain -RN1/09-swine A(H7N1)[A/the horse/Prague/1/1956(H7)-A/17/California/09/38(N1)]

2. Series - series 1

3. A method of obtaining reassurace

4. Characteristics of the parent viruses:

a) influenza virus horses/horse/Prague/1/1956(7N7);

b) vaccine strain a/17/California/09/38(H1N1) on the basis of the donor attenuatio A/Leningrad/134/17/1957(H2N2)

5. The number of passages 6 in the recombination process; 3 cumulative passage system of the developing chick embryo

6. Characterization of strain prior to lyophilization:

a) optimal conditions for reproduction - 33°C, 48 hours;

b) hemagglutinin activity 1:512;

C) infectious activity of 8.8 Ig EID50/ 0.2 ml;

g) sensitivity to inhibitors: inhibitorresistant;

d) the structure of the genome reassortant:

- genes from influenza virus horses: BY;

- genes from the vaccine strain - RW, RV, PA, NP, M, NS, and NA

7. The characteristic strain after lyophilization:

a) the date lyophilization: 24.12.2009;

b) amount of material in the vial: 1 ml;

C) infectious activity - 6,05 Ig EID50/ 0.2 ml;

d) hemagglutinin activity - 1:160

8. Antigenic specificity:

a) hemagglutinin - identical to the virus And/horses/Prague/1/1956(7N7) according to rtga with rat anticorodal and PCR restricting analysis of DNA copies of the gene;

b) neuraminidase - the IDA is part of the virus A/17/California/09/38(H1N1) according to PCR restricting analysis of DNA copies of the gene

9. Bacteriological control of lyophilized material: date - 13.01.2010 - sterile.

The purpose of the obtained strain - qualitative and quantitative determination of antibodies to NA strain A/California/07/09(H1N1) pandemic influenza virus in the blood sera.

1. Strain can be used to identify antineuraminidase antibodies in the test of inhibition sialidase activity of NA, as well as enzyme-linked immunosorbent assay (ELISA) in the sera of laboratory animals after inoculation with influenza virus.

The methods of such identification is based on the determination of the titer antineuraminidase inhibiting antibodies, which represents the reciprocal of the last dilution of the test serum, which is still observed blocking sialidase activity of NA diagnostic strain RN1/09-swine A(H7N1). Sialidase activity of NA is determined with respect to the natural substrate (fetuin) with subsequent recognition zamaskirovannyj polysaccharides peroxidase-labeled lectins.

When conducting a solid phase ELISA diagnostic strain RN1/09-swine A(H7N1) can be used as a substrate for sorption on 96-well tablets, which later are falling dilution of the test serum and peroxidase-labeled conjugate specific to an identifiable class of immunoglobulins. Titles antineuraminidase antibodies is allocated in decimal logarithms of the values, reverse the last dilution of the test serum, in which there is a two-fold excess over the average optical density of the wells of negative control.

For obtaining immune sera of mice subjected to immunization alive or killed influenza virus in different ways (intranasally, intramuscularly, administered intraperitoneally). Euthanasia is conducted according to the Rules of work with the use of experimental animals". The blood sample is from the subclavian artery, blood serum are selected after centrifugation and stored at -20°C.

An example run of the experiment.

The mice CBA was administered intranasally once reassortant strain a/17/California/09/38(H1N1) at a dose of 106EID50/0,05 ml of the blood Serum of immunized mice were obtained 14 days after immunization. It is shown that in the sera of immunized mice were determined by specific antibodies to the hemagglutinin and the neuraminidase strain A/California/07/09(H1N1) (see table 3). In the control group of intact animals, specific antibodies to the virus A/California/07/09(H1N1) cases have not been identified.

Table 3
The growth of antibodies in the sera of mice 14 days after odnokratno the nd vaccine strain A/17/California/09/38(H1N1)
GroupAntibodiesAntibodies to NA
Rtga (BHT)Petrinovic test (BHT)ELISA(Ig)
Vaccine17,431,7(p<0,02)1,7±0,3
Control7,610,61,0±0,0

A comparison was made between ELISA results obtained using the substrate cleaned reassortant virus RN1/09-swine A(H7N1) (100 GAI) or purified neuraminidase of influenza virus a/California/07/09(H1N1) (1 μg/ml). Figure 2 presents significant correlation for both types of antigen (Kendall Tau 0,538; p<0,05). On the x-axis presents the titers of serum immunoglobulin G (IgG) in sera of mice by purified NA pandemic strain A/California/07/09(H1N1), expressed in log10. Y-axis presents the titers of serum IgG to the whole reassortant strain RN1/09-swine A(H7N1), also expressed in log10.

2. Strain can be used to identify antineuraminidase antibodies in the test of inhibition sialidase activity of NA and ELISA in the serum in itih and sick persons.

From the surveyed volunteers are selected blood samples from the finger before and after vaccination. Obtained after centrifugation the serum stored at

-20°C.

An example run of the experiment.

The purpose of a clinical study monovalent live flu vaccine A/17/California/09/38(H1N1) volunteers were administered the drug intranasally twice with an interval of 21 days at a dose of not less than 107EID50/0,5 ml of Serum were collected before vaccination and after the first vaccination and revaccination. Antibodies to the neuraminidase was determined in feminova test and ELISA. Shows an increase after two vaccinations average titers of antibodies to the neuraminidase of influenza virus according to the two tests (see table 4). The most pronounced increases in antibodies to the neuraminidase detected in sick persons.

Table 4
The formation of serum antibodies to the neuraminidase in volunteers inoculated with live flu vaccine monovalent A/17/California/09/38(H1N1)
GroupThe number in the groupFetuin-leckenby test, the GTS patients with seroconversion (61%)ELISA(Log10)
Before inoculation After vaccinationBefore inoculationAfter vaccination
Live flu vaccine twice3112,422,32,2±0,32,5±0,3
Recover5n/a177,7n/a3,4±0,3
Placebo1012,212,22,4±0,52,4±0,4

The experimental results clearly demonstrate the suitability of the claimed reassortant strain for the detection of antibodies to the neuraminidase N1 influenza virus of swine origin, making this strain can be used to diagnose, to study the immunogenicity of influenza vaccine for the analysis of the background values antineuraminidase antibodies in the population as a whole in terms of the circulation of the pandemic influenza A(H1N1).

The strain of influenza virus deposited in the State collection of viruses institutions of the Russian Academy of Medical Sciences Institute of Virology RAMS them. Dijanoveckog # GKV 243, used for the detection of antibodies to the neuraminidase N1 influenza virus of swine origin.



 

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1 dwg, 3 tbl

FIELD: biotechnology, medicine, pediatrics.

SUBSTANCE: invention proposes the vaccine strain A/47/Panama/99/234 (H3N2) that represents reassortant prepared by breeding the epidemic virus A/Panama/2007/99/ (H3N2) with the cold adopted temperature-sensitive virus A/Leningrad/134/47/57 (H2N2) as attenuation donor that is harmless for children. The strain A/47/Panama/99/234 (H3N2) multiplies actively in developing chicken embryos at optimal temperature 33-34oC and characterized by sensitivity to temperature and adaptation to cold. From epidemic virus the reassortant inherited two genes encoding surface proteins (hemagglutinin and neuraminidase) and six genes encoding non-glycosylated proteins from the attenuation donor. The strain A/47/Panama/99/234 (H3N2) is areactogenic for children in its intranasal administration. The vaccine strain A/47/Panama/99/234 (H3N2) corresponds by its biological properties and reactogenic indices to requirements for vaccine strains in accordance to Pharmacopoeia article 42-3353-97 with respect to the live anti-influenza vaccine for intranasal applying for children of 3-14 years old.

EFFECT: valuable properties of strain.

2 tbl, 1 dwg, 1 ex

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