RU2266329C1 - Strain for production of living influenza intranasal vaccine for adults and infants - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: claimed strain is obtained by hybridization of epidemic virus with cold adapted and temperature sensitive virus, which represents attenuation donor and is harmless for adults, and infants of 3-14 years old. Said virus makes it possible to obtain reassortant vaccine strains from new epidemic viruses. Strain is effectively cultivated in germinative hen embryos at 32°C, and has temperature sensitivity and cold adaptation. Reassortant has two genes derived from epidemic virus encoding surface proteins (hemagglutinin and neuroamidinase) and six genes derived from attenuation donor encoding non-glycosilated proteins. Strain has no reactogenicity in relation to adults and infants of 3-14 years old at intranasal application.

EFFECT: strain for living influenza intranasal vaccine with good biological properties and reactogenicity.

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Description

The invention relates to medical virology and can be used in healthcare for the prevention of epidemic influenza among adults with a live intranasal influenza vaccine from strain B / 60 / Hong Kong / 01/22. The known vaccine strain of influenza virus type B - B / 60 / Johannesburg / 99/50 - prototype (RF Patent No. 2215786 dated 10.11.03, BI No. 31, 2003) - lost its antigenic relevance and therefore cannot cause a protective reaction during an epidemic caused by modern strains of influenza virus type B.

The problem to which the invention is directed, is to obtain a vaccine strain of the actual antigenic variety based on the epidemic influenza virus B / Hong Kong / 330/01.

The vaccine strain of influenza virus B / 60 / Hong Kong / 01/22 was obtained in developing chicken embryos by the method of genetic reassortment of the epidemic virus B / Hong Kong / 330/01 with a cold-adapted strain B / USSR / 60/69 - an attenuation donor - followed by selection in the presence of antiserum to the virus B / USSR / 60/69 at a decrease in incubation temperature to 25 ° C. Attenuation donor is a cold-adapted temperature-sensitive strain of influenza virus B / USSR / 60/69, approved for the production of harmless live intranasal vaccines for adults and children 3-14 years old (Alexandrova G.I., Klimov A.I. Live vaccine against influenza. - St. Petersburg .: Science. - 1994. - 151 p.).

The composition of the vaccine strain genome was determined by comparative analysis of the electrophoretic mobility of single-stranded RNA fragments of the parent viruses and the reassortant clone in a 2.8% polyacrylamide gel (Gaal E., Medieshi G., Veretskei L. Electrophoresis in the separation of biological macromolecules. - M .: Mir. - 1982. - 446 p.) In the presence of 4.5 Mole of urea. Vertical electrophoresis was carried out for 20 hours at + 4 ° C at a constant voltage of 100 V. It was found that the vaccine strain B / 60 / Hong Kong / 01/22 inherited six genes encoding internal non-glycosylated proteins (PB1, PB2, PA, NP, M , NS) from an attenuation donor, and two genes encoding surface proteins - HA (hemagglutinin) and NA (neuraminidase) - from epidemic virus B / Hong Kong / 330/01. The belonging of the gene encoding the surface HA protein to the epidemiologically relevant virus B / Hong Kong / 330/01 was also confirmed in the hemagglutination inhibition reaction (RTGA) with 1% suspension of chicken erythrocytes.

The vaccine strain is temperature sensitive (the difference in the rate of infectious activity at an incubation temperature of 32 ° C and 38 ° C is 7.7 Ig EID ₅₀ / ml) and cold-adapted (the difference in the rate of infectious activity at an incubation temperature of 32 ° C and 25 ° C is 3 6 Ig EID ₅₀ / ml).

The results of the analysis of the genome composition of the vaccine strain B / 60 / Hong Kong / 01/22 are presented in table 1.

Thus, the presented vaccine strain B / 60 / Hong Kong / 01/22 is characterized by a combination of useful features necessary for the vaccine strain: antigenic specificity of the surface proteins of the epidemic virus B / Hong Kong / 330/01, the genome structure optimal for reassorted vaccine strains, temperature sensitivity and cold adaptation , which correlates with attenuation for a person characteristic of an attenuation donor.

The strain was deposited in the collection of the State Institute of Standardization and Control of Medical and Biological Preparations named after L.A. Tarasevich under No. 142.

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

Table 1. The genome composition of the reassortant vaccine strain of influenza virus B / 60 / Hong Kong / 01/22 (according to electrophoresis of single-stranded RNA in a polyacrylamide gel) Gene Parent viruses Reassortant vaccine strain B / 60 / Hong Kong / 01/22 Epidemic virus B / Hong Kong / 330/01 Attenuation donor B / USSR / 60/69 PB2 GK ¹ B60 ² B60 PB1 GK B60 B60 RA GK B60 B60 ON THE GK B60 GK NP GK B60 B60 NA GK B60 GK M GK B60 B60 NS GK B60 B60 ¹ The gene belongs to the epidemic influenza virus B / Hong Kong / 330/01. ² The gene belongs to the attenuation donor B / USSR / 60/69. Table 2. Reactogenicity for adults of the vaccine strain B / 60 / Hong Kong / 01/22 with intranasal administration A drug The number of subjects Indicator Temperature reactions (in ° С) Reactogenicity Index Weak (≤37.5 ° C) Medium (37.6-38.5 ° C) strong (≥38.5 ° C) Vaccine 51 abs.% 5
9.8 0
0 0
0 0 Placebo 31 abs.% 4
12.9 0
0 0
0 Table 3. Reactogenicity for children 3-14 years of vaccine strain B / 60 / Hong Kong / 01/22 with intranasal administration A drug The number of subjects Indicator Temperature reactions (in ° С) Reactogenicity Index Weak (≥ 37.5 ° C) Medium (37.6-38.5 ° C) strong (≥38.5 ° C) Vaccine 58 abs.% 4
6.9 0
0 0
0 0 Placebo 29th abs.% 2
6.5 0
0 0
0

PASSPORT STRAIN

1. The name of the strain is B / 60 / Hong Kong / 01/22

2. Series - Series 1

3. The method of receipt - reassortment; characteristic of parent viruses:

a) epidemic virus - B / Hong Kong / 330/01

b) donor of attenuation - B / USSR / 60/69

4. The number of passages - 6 in the process of reassortment

5. Characterization of the strain:

a) optimal conditions for reproduction - 32 ° C, 72 hours;

b) hemagglutinating activity -1: 512;

c) infectious activity - 8.4 Ig EID ₅₀ / ml;

d) sensitivity to inhibitors - inhibitory according to rtga with horse serum;

d) the difference in the indicators of infectious activity at 32 ° C and 38 ° C is 7.7 Ig EID ₅₀ / ml;

f) the difference in the indicators of infectious activity at 32 ° C and 25 ° C is 3.6 Ig EID ₅₀ / ml.

g) the structure of the genome of the reassortant:

- genes from an epidemic virus - ON, NA

- genes from an attenuation donor - PB2, PB1, PA, NP, M, NS

6. Characterization of the strain after lyophilization:

a) date of lyophilization - January 28, 2004;

b) the volume of material in the ampoule is 1 ml;

c) the number of doses in a series is 4;

d) infectious activity - 7.7 Ig EID ₅₀ / ml;

d) hemagglutinating activity - 1: 256.

7. The recommended dilution for vaccination is 1: 2.

8. Antigenic specificity

a) hemagglutinin in rtga - identical to virus B / Hong Kong / 330/01, the ferret antiserum to which is completely neutralized;

b) neuraminidase - identical to virus B / Hong Kong / 330/01 according to polyacrylamide gel electrophoresis.

9. Harmless to mice with subcutaneous and intraperitoneal administration - harmless.

10. Bacteriological control of lyophilized material:

Date - January 12, 2004. The result is sterile.

11. Control for the absence of extraneous viruses - extraneous viruses are absent.

Head of the laboratory (department)

STRAIN CHARACTERISTIC

Infectious activity during reproduction in developing chicken embryos at 32 ° C for 72 hours - 8.4 Ig EID ₅₀ / ml. Hemagglutinating activity - 1: 512. The strain exhibits genetic stability of biological traits after 5 passages on chicken embryos (when using large infectious doses).

A sample passport for the vaccine strain B / 60 / Hong Kong / 01/22 is attached.

STRAIN B / 60 / HONG KONG / 01/22 AREACTOGEN FOR ADULTS CONTINGENT

Example 1. When vaccinating 51 adults with an intranasal reassortant vaccine strain B / 60 / Hong Kong / 01/22 with an infectious activity of 7.7 Ig EID ₅₀ / ml in a dilution of 1: 2 in a volume of 0.5 ml of strong temperature reactions with increasing temperature above 38.5 ° C and average temperature reactions with increasing temperature in the range of 37.6-38.5 ° C were not observed. Weak temperature reactions (up to 37.5 ° C) were observed in 9.8% of cases. The reactogenicity index (the difference in the percentage of average reactions in vaccinated vaccines and received placebo) was 0%. The survey results are shown in table 2.

In the group receiving a placebo of 31 persons, moderate and strong temperature reactions were not observed, weak reactions were observed in 12.9% of cases.

Thus, the vaccine strain B / 60 / Hong Kong / 01/22 in terms of reactogenicity complies with the requirements for vaccine strains Pharmacopeia article FSP 42-0417-4097-03 for live dry influenza allantoic vaccine for intranasal use.

STRAIN B / 60 / HONG KONG / 01/22 AREACTOGEN FOR CHILDREN 3-14 YEARS

Example 2. A group of 58 children aged 3-14 years old were vaccinated intranasally in a volume of 0.5 ml with a reassortant vaccine strain B / 60 / Hong Kong / 01/22 with an infectious activity of 7.7 Ig EID ₅₀ / ml in a 1: 2 dilution. A group of children of the same age of 29 received a placebo. The research results are shown in table 3.

In the group of vaccinated children, strong temperature reactions with fever above 38.5 ° C were not observed. Average reactions in the range of 37.6-38.5 ° C were also not observed. The reactogenicity index was 0%. Weak temperature reactions with a short-term increase in temperature to 37.5 ° C were noted in 6.9% of cases.

In the group of children who received a placebo, strong temperature reactions with a temperature increase of over 38.5 ° C and average temperature reactions in the range of 37.6-38.5 ° C were not observed. Weak temperature reactions were noted in 6.5% of cases.

Thus, the vaccine strain B / 60 / Hong Kong / 01/22 in terms of reactogenicity complies with the requirements for vaccine strains Pharmacopeia article FSP 42-0417-4097-03 for live dry influenza allantoic vaccine for intranasal use.

Claims (1)

The strain of influenza virus B / 60 / Hong Kong / 01/22, GISK No. 142 (State Institute of Standardization and Control of Medical and Biological Preparations named after L.A. Tarasevich) used to obtain live influenza intranasal vaccine for adults and for children 3-14 years old .