RU2307161C2 - Influenza virus strain for living influenza intranasal vaccine for infants and adults - Google Patents

Abstract

FIELD: virology, immunology.

SUBSTANCE: vaccine strain reassortant is obtained by specific epidemic virus with cold-adapted temperature-sensitive virus as attenuation donor. Strain actively multiplies in developing hen embryos at optimal temperature of 32-33°C and is characterized with cold-adaptation and temperature-sensitivity. Strain has two genes from epidemic virus encoding surface proteins (hemagglutinin and neuroaminidase) and six genes from attenuation donor encoding internal non-glycosylated proteins.

EFFECT: new strain for living influenza intranasal vaccine for infants and adults.

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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 and children with a live intranasal influenza vaccine from strain B / 60 / Dzhilin / 03/1.

Known vaccine strain of influenza virus - In / 60 / Johannesburg / 99/50 - prototype [Patent No. 2215786, state. reg. 11/10/2003, Bull. Number 3. The influenza virus strain B / 60 / Johannesburg / 99/50 for the production of live influenza intranasal vaccine for adults and children] has lost its antigenic relevance and therefore will not be able to provoke a protective reaction during an influenza epidemic caused by a strain of influenza virus similar to the B / Jilin virus / 20/03.

The problem to which the invention is directed, is to obtain a vaccine strain of the actual antigenic variety based on the epidemic virus B / Dzhilin / 20/03.

The vaccine strain B / 60 / Dzhilin / 03/1 was obtained by genetic reassortment of the epidemic virus B / Dzhilin / 20/03 with a cold-adapted strain B / USSR / 60/69 - an attenuation donor - followed by selection in the presence of antiserum for virus B / USSR / 60/69. 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 [Aleksandrova GI, Klimov AI Live flu vaccine. SPb .: Science. - 1994 - 151 p.].

The vaccine strain is temperature sensitive (the difference in the rate of infectious activity at an incubation temperature of 32 ° C and 37 ° C is 7.0 lg EID ₅₀ / 0.2 ml) and cold-adapted (the difference in the rate of infectious activity at an incubation temperature of 32 ° C and 25 ° C is 3.0 lg EID ₅₀ / 0.2 ml).

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., Vereckey L. Electrophoresis in the separation of biological macromolecules. M .: Mir: Mir . - 1982 - 446 S.) in the presence of 4.5 M urea. Vertical electrophoresis was performed for 20 hours at + 4 ° C at a constant voltage of 100 V. It was found that the vaccine strain B / 60 / Dzhilin / 03/1 inherited six genes encoding internal non-glycosylated proteins (PB1, PB2, PA, NP, M , NS), from an attenuation donor and two genes encoding the surface proteins HA (hemagglutinin) and NA (neuraminidase), from the epidemic virus B / Dzhilin / 20/03. The belonging of the gene encoding the surface HA protein to the epidemiologically relevant virus B / Dzhilin / 20/03 was also confirmed in the hemagglutination inhibition reaction (RTGA) with 1% suspension of chicken erythrocytes.

The results of the analysis of the genome composition of the vaccine strain B / 60 / Dzhilin / 03/1 are presented in Table 1 and in the drawing, where electrophoresis of single-stranded RNA in a 2.8% polyacrylamide gel in the presence of 4.5 M urea is schematically shown. 1st track - epidemic influenza virus B / Jilin / 20/03; 2nd track - reassortant vaccine strain B / 60 / Dzhilin / 03/1; 3rd track - attenuation donor B / USSR / 60/69.

Table 1. The genome composition of the reassortant vaccine strain of influenza virus B / 60 / Dzhilin / 03/1 (according to electrophoresis of single-stranded RNA in a polyacrylamide gel) Gene Parent viruses Reassortant vaccine strain B / 60 / Dzhilin / 03/1 Epidemic virus B / Jilin / 20/03 Attenuation donor B / USSR / 60/69 PB2 J ¹ B60 ² B60 PB1 J B60 B60 RA J B60 B60 ON J B60 J NP J B60 B60 NA J B60 J M J B60 B60 NS J B60 B60 ¹ The gene belongs to the influenza B virus / Jilin / 20/03. ² The gene belongs to the attenuation donor B / USSR / 60/69. Table 2. Reactogenicity for adults of the vaccine strain B / 60 / Dzhilin / 03/1 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 53 abs. four 0 0 0 % 7.55 0 0 Placebo 42 abs. 3 one one % 7.14 2,38 2,38 Table 3. Reactogenicity for children 3-10 years of vaccine strain B / 60 / Dzhilin / 03/1 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 62 abs. 5 0 0 0 % 8.1 0 0 Placebo 44 abs. 3 2 one % 6.8 4,5 2,3

Thus, the presented vaccine strain B / 60 / Dzhilin / 03/1 is characterized by a combination of useful features necessary for the vaccine strain: antigenic specificity of the surface proteins of the epidemic virus B / Dzhilin / 20/03, the genome structure optimal for reassortant 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. 144.

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

STRAIN CHARACTERISTIC

Infectious activity during reproduction in developing chicken embryos at 32-33 ° C for 72 hours - 7.5 lg EID ₆₀ / 0.2 ml.

Hemagglutinating activity - 1: 512.

The strain exhibits genetic stability of biological traits after 5 passages on chicken embryos (when using large infectious doses).

Characteristics of the vaccine strain B / 60 / Dzhilin / 03/1 are presented in the attached passport. Based on their strain used in the preparation of the vaccine and tested the vaccine on volunteers.

PASSPORT STRAIN

1. The name of the strain is B / 60 / Dzhilin / 03/1

2. Series - Series 1

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

a) epidemic virus - B / Jilin / 20/03

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

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

5. Characterization of the strain before lyophilization:

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

b) hemagglutinating activity - 1: 512;

c) infectious activity - 7.5 lg EID ₅₀ / 0.2 ml;

d) sensitivity to inhibitors - inhibitory-sensitive in rtga with normal horse serum;

d) the difference in the indicators of infectious activity at 32 ° C and 37 ° C is 7.0 lg EID ₅₀ / 0.2 ml;

f) the difference in the indicators of infectious activity at 32 ° C and 25 ° C is 3.0 lg EID ₅₀ / 0.2 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 - July 7, 2004

b) the volume of material in the ampoule is 1.0 ml;

c) the number of doses in the ampoule is 4;

d) infectious activity - 7.0 lg EID ₅₀ /0.2 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 / Dzhilin / 20/03, the rat antiserum to which is completely neutralized;

b) neuraminidase - identical to the virus B / Dzhilin / 20/03 according to electrophoresis of single-stranded virion RNA in 2.8% PAAG in the presence of 4.5 M urea.

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

10. Bacteriological control:

Date: July 9, 2004

the result is sterile.

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

STRAIN - B / 60 / JILIN / 03/1 AREACTOGEN FOR ADULTS

Example 1. When vaccinating 53 adults with an intranasally reassorted vaccine strain B / 60 / Dzhilin / 03/1 with an infectious activity of 7.5 lg EID ₆₀ / 0.2 ml in a dilution of 1: 2 in a volume of 0.5 ml of strong temperature reactions with an increase in temperature above 38.5 ° С and average temperature reactions with an increase in temperature in the range of 37.6-38.5 ° С were not observed. Weak temperature reactions (up to 37.5 ° C) were observed in 7.55% of cases. The survey results are shown in table 2.

In a group of 42 individuals who received a placebo, mild reactions were observed in 7.14% of individuals, average temperature reactions in 2.38%, and strong temperature reactions in 2.38%.

The reactogenicity index (the difference in the percentage of average reactions in vaccinated vaccines and received placebo) was 0%.

Thus, the vaccine strain B / 60 / Dzhilin / 03/1 in terms of reactogenicity meets the requirements for vaccine strains Pharmacopeia article FSP 42-0417-4097-03 for live dry influenza allantoic vaccine for intranasal use.

STRAIN B / 60 / JILIN / 03/1 AREACTOGEN FOR CHILDREN 3-10 YEARS

Example 2. A group of 62 children aged 3-10 years were vaccinated intranasally in a volume of 0.5 ml with a reassortant vaccine strain B / 60 / Dzhilin / 03/1 with an infectious activity of 7.5 lg EID ₆₀ / ml in a 1: 2 dilution. A group of children of the same age of 44 people 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. Weak temperature reactions with a short-term increase in temperature to 37.5 ° C were observed in 8.1% of cases.

In the group of children who received a placebo, there were strong temperature reactions with a rise in temperature over 38.5 ° C and average temperature reactions in the range of 37.6-38.5 ° C in 2.3% and 4.5% of cases, respectively. Weak temperature reactions were noted in 6.8% of cases.

The reactogenicity index (the difference in the percentage of average reactions in vaccinated vaccines and received placebo) was 0%.

Tests on volunteers confirmed that the vaccine strain B / 60 / Dzhilin / 03/1 in terms of reactogenicity meets the requirements for vaccine strains Pharmacopoeia article FSP 42-0417-4097-03 for live dry influenza allantoic vaccine for intranasal use.

Based on the foregoing, the vaccine strain of the influenza virus according to the invention can be used for the prevention of influenza in both adults and children from the age of three, which is confirmed by the study of its reactogenicity for different age groups.

Claims (1)

The strain of influenza virus B / 60 / Dzhilin / 03/1 GISK No. 143 (State Institute of Standardization and Control named after L. A. Tarasevich), used to obtain live influenza intranasal vaccine for adults and for children 3-14 years old.

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