RU2507256C2 - Strain of influenza virus a/17/mallard/netherlands/00/95(h7n3) for production of live and inactivated influenza vaccines - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: vaccine strain for influenza virus A/17/mallard/Netherlands/00/95(H7N3) is proposed. Strain A/17/mallard/Netherlands/00/95(H7N3) is harmless for laboratory animals. Strain A/17/mallard/Netherlands/00/95(H7M3) has antigenic actuality and attenuation thus meeting the requirements to vaccine strains included in influenza live vaccine, and may be used for production of influenza live and inactivated intranasal vaccine in health care service for influenza prevention.

EFFECT: temperature sensitivity and high degree of cold adaptability.

7 tbl, 3 ex

Description

The invention relates to medical virology and can be used in healthcare for the prevention of influenza caused by pandemic dangerous avian influenza viruses. For the production of intranasal live influenza vaccine (VHF) or inactivated influenza vaccine (IHV), a reassorted strain of influenza virus A / 17 / mallard / Netherlands / 00/95 (H7N3) has been proposed.

Despite the widespread spread of the pandemic influenza virus subtype A (H1N1), avian influenza viruses of subtypes A (H5N1), A (H7N1) and A (H9N2) continue to cause outbreaks in humans. The viruses of the H7 subtype can be widely distributed among poultry, acquiring the properties of increased pathogenicity as a result of additional adaptation and the recombination process, as was shown during outbreaks caused by A (H7N7), A (H7N1) and A (H7N4) in Europe and Australia. The need to develop appropriate vaccine preparations, including for the fight against avian influenza viruses, is reflected in the order of the Ministry of Health of the Russian Federation [Order No. 40 of the Ministry of Health of the Russian Federation of December 28, 2004].

Currently, vaccine strains for hepatitis B virus are obtained by reassorting modern epidemic viruses with cold-adapted donor strains, resulting in the formation of reassortants with a mixed genome. Attenuation donor A / Leningrad / 134/17/57 (H2N2) - a cold-adapted temperature-sensitive strain of influenza virus, approved for the production of harmless intranasal vaccines for adults and children [GI Aleksandrova New in the epidemiology and prevention of viral infections. L., 1986. - S. 66-83]. The genes encoding hemagglutinin (HA) and neuraminidase (NA) are inherited from the antigenically relevant epidemic strain, and six genes of internal and non-structural proteins (PB2, PB1, PA, NP, M, NS) from the harmless CA attenuation donor. From the obtained reassortants, a vaccine candidate with a genome formula of 6: 2 is selected that best meets the antigen specificity requirements that are characteristic of the parent virus of the "wild" type, and also meets the signs of cold adaptation, by its ability to reproduce at low temperature.

The vaccine strains of the H1N1 and H3N2 subtypes currently used for the prevention of epidemic influenza A cannot cause a defensive reaction in the event of a large-scale outbreak caused by influenza viruses with the H7 subtype hemagglutinin, to which the vast majority of the population are not immune.

Currently known reassortant strain A / 17 / duck / Potsdam / 86/92 (H5N2) [Patent No. 2318871, publ. 03/10/2008], intended for the production of both live and inactivated vaccines against bird flu subtype H5. But it cannot give a preventive effect in the case of the spread of influenza viruses of subtype H7.

Abroad, the highly pathogenic virus A / Netherlands / 219/03 (H7N7) [Min J., J. Virol. Was used to prepare a vaccine strain of the HBV subtype H7 based on the donor strain A / Ann Arbor / 6/60 (H2N2). 2010 Nov; 84 (22): 11950-60].

Also known is the temperature-sensitive reassortant strain A / 17 / wild duck / Netherlands / 00/84 (H7N3) obtained by classical genetic reassortment in developing chicken embryos (RKE) based on the donor strain Len / 17 [Desheva A., ZhMEI-2009. - No. 1. - S.31-36]. However, the indicated reassortant strain did not fully satisfy the requirements for vaccine strains included in the composition of the hepatitis B virus for immunization of people. First of all, the strain was not sufficiently cold adapted: the difference in reproductive activity at a temperature lowered to 25 ° C for strain A / 17 / wild duck / Netherlands / 00/84 (H7N3) was 4.2 Ig EID ₅₀ (rct ₂₅ sign) , while it was found that for cold-adapted vaccine strains, this difference should not exceed 3.0 Ig EID ₅₀ [Science, 1959. - Vol.129. - No. 3358. - R.1287-1288]. In addition, direct nucleotide sequencing showed the presence of a G → A nucleotide substitution at position 543 of the HA gene of the reassortant strain A / 17 / wild duck / Netherlands / 00/84 (H7N3) compared to the HA gene of the “wild” avian influenza virus. The indicated substitution was accompanied by amino acid substitution of glycine for glutamic acid at position 214, which led to a change in the polarity and charge of the amino acid near the receptor-binding site of HA. Such substitutions are undesirable for vaccine strains, as they can adversely affect their antigenic specificity, causing a decrease in the protective efficacy of vaccination.

Since the task to which the claimed invention is directed is to obtain a fully cold-adapted vaccine strain with hemagglutinin subtype H7 for immunization of people, additional cloning steps were carried out. For this, the so-called “short passages” method was chosen [Fazekas de St. Groth, Intervirology, 1975 .-- Vol. 5. - P.335-341] at a low temperature, when a mixture of viral clones is passaged in a sensitive system (chicken embryos, CE) in the presence of breeding factors with a shorter incubation period. A mixture of viral clones obtained after crossing the attenuation donor A / Leningrad / 134/17/57 (H2N2) and the pathogenic avian influenza virus A / mallard / Netherlands / 12/00 (H7N3) in the allantoic cavity of 10-11-day-old chicken embryos ), which passed 2 selective passages and one cloning in the presence of antiserum to the attenuation donor A / Leningrad / 134/17/57 (H2N2) at an incubation temperature lowered to 25 ° C, was additionally cloned 2 times at a temperature reduced to 30 ° C for a shortened up to 28 hours, increasing the final dilution to 1:10 ⁹ . By selecting precisely these conditions, it was possible to isolate clone A / 17 / mallard / Netherlands / 00/95 (H7N3), which differed in its characteristics from others, for example, in the most pronounced signs of cold adaptation, since the level of its reproduction at a temperature lowered to 25 ° C was 100 times higher than previously obtained reassortant viruses (Table 1).

Table 1 Reproduction at low temperature of clones A (H7N3) Reassortant (clone no.) Number of passages during reassortment Reproductive activity of EID ₅₀ / 0.2 ml RCT ₂₅ EID ₅₀ / 0.2 ml 34 ° C 34 ° C 8/4 6 8.1 ± 0.5 4.1 ± 0.4 4.0 8/6 6 8.7 ± 0.2 4.7 ± 0.2 4.0 8/10 6 8.3 ± 0.1 4.0 ± 0.1 4.3 9/5 AL 7 / mallard / Netherlands / 00/95 (H7N3) 7 (including 2 “shortened”) 8.7 ± 0.2 6.4 ± 0.2 2,3

The high level of cold adaptation of the reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) was substantiated by molecular genetic methods. Sequencing of the reassortant strain revealed the presence of an additional nucleotide substitution C-1066-A in the internal NP protein gene, which led to the amino acid substitution Leu-341-lie. This replacement was previously detected in an additional attenuated donor strain, A / Leningrad / 134/47/57 (H2N2), which was obtained after a series of passages of strain A / Leningrad / 134/17/57 (H2N2) in CE at a low temperature and in it was later widely used for several years in the preparation of PHA for immunization of young children [Klimov et al., 1992].

The analysis of the cloned vaccine strain A / 17 / mallard / Netherlands / 00/95 (H7N3) was also performed using restriction analysis of DNA copies of RNA segments obtained by reverse transcriptase polymerase chain reaction (RT-PCR) [Klimov AI, J. Virol. Method - 1995. - No. 55. - P. 445-446]. Obtained by RT-PCR DNA copies of segments PB2, PB1, PA, NP, M, and NS were treated with restriction endonucleases Tru9l, Hindlll, BamHI, Asnl, EcoRI, Pvul, Ncil, respectively. Additionally, copies of the PB1, PA, and M segments were digested with restriction enzymes BstXI, Asnl, and BciVI. Restriction analysis of DNA copies of RNA segments (Table 2) and direct nucleotide sequencing showed that reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) has a genome composition of 6: 2. All coding nucleotide substitutions characteristic of the attenuation donor A / Leningrad / 134/17/57 (H2N2) were found in the genes of internal and non-structural proteins of the reassortant strain A / 17 / mallard / Netherlands / 00/95 (H7N3).

The nucleotide sequencing of the HA gene of the reassortant virus A / 17 / mallard / Netherlands / 00/95 (H7N3) did not reveal differences in the nucleotide composition from the parent virus of the "wild" type. Sequencing of the NA gene of the reassortant virus A / 17 / mallard / Netherlands / 00/95 (H7N3) also did not reveal differences in the nucleotide composition from the parent virus of the "wild" type.

Thus, the obtained reassortant strain is characterized by a combination of the features required for vaccine strains of live hepatitis B virus: cold adaptation and the identity of surface antigens to the parent wild-type virus. At the same time, the obtained strain has high reproductivity in chicken embryos (> 10 ⁹ EID ₅₀ / ml), which allows it to be used in the production of inactivated influenza vaccine (IHV), where a large amount of viral material is required for its subsequent purification and concentration.

The affiliation of hemagglutinin to the parent wild-type strain A / mallard / Netherlands / 12/00 (H7N3) in the reassortant was confirmed in the hemagglutination inhibition reaction (RTHA). The study of antigenic properties showed that apatogenic avian virus A / mallard / Netherlands / 12/00 (H7N3) and its XA reassortant are antigenically identical (interaction with homologous type-specific serum according to TU 42-14-79-79). The interaction parameters of reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3), as well as the parent strain A / mallard / Netherlands / 12/00 (H7N3) with ferret antisera obtained from viruses isolated from humans during the outbreak in the Netherlands in 2003, including the case of a lethal infection (A / Netherlands / 219/03), did not differ from homologous titers of the corresponding antigens by more than 2 times (Table 2).

table 2 Antigenic specificity of apatogenic avian influenza virus A / mallard / Netherlands / 12/00 (H7N3) and its CA reassortant Viruses Subtype Ferret Antisera Mallard / nl / 00 NL / 219/03 NL / 230/03 NL / 231/03 Normal serum A / mallard / H Netherlands / 12/00 (Mallard / NL / 00) H7n3 320 640 160 40 5 A / 17 / mallard / Netherlands / 00/95 (6: 2 reassortant) H7n3 320 320 160 40 5 A / Netherlands / 219/2003 (NL / 219/03) H7n7 320 640 160 40 5 A / Netherlands / 230/2003 (NL / 230/03) H7n7 320 640 320 80 5 A / Netherlands / 231/2003 (NL / 231/03) H7n7 320 320 160 80 5

To assess the genetic stability of the reassortant strain A / 17 / mallard / Netherlands / 00/95 (H7N3), 5 serial passages were performed in 10-11-day CEs. After isolation of viral RNA, RT-PCR restriction analysis was performed. It was shown that nucleotide substitutions previously described for the genes of internal and non-structural proteins of the attenuation donor A / Leningrad / 134/17/57 (H2N2) are present in the genome of the vaccine strain after five passages in CE (Table 3).

Table 3 Nucleotide substitutions found in the genes of internal and non-structural proteins of the reassortant strain A / 17 / mallard / Netherlands / 00/95 (H7N3) before and after passages in chicken embryos Virus Characteristic Nucleotide positions in "internal" genes PB2 PB1 PA m NS 1459 819 1795 107 1045 68 969 798 A / Leningrad / 134/57 (H2N2) Epidemic virus G G G T G BUT G G A / Leningrad / 134/17/57 (H2N2) XA donor attenuation T T BUT FROM T G BUT BUT A / 17 / mallard / Netherlands / 00/95 (H7N3) to passages in CE Reassortant vaccine strain T T BUT C T G BUT BUT A / 17 / mallard / Netherlands / 00/95 (H7N3) after 5 passages in CE T T BUT C T G BUT BUT

Table 4 presents the reproduction rates in CE of the reassortant strain A / 17 / mallard / Netherlands / 00/95 (H7N3). It was shown that reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) showed high reproductive activity at an optimal incubation temperature of 34 ° C. Like HA, the parent strain A / Leningrad / 134/17/57 (H2N2), the reassortant strain A / 17 / mallard / Netherlands / 00/95 (H7N3) reproduced well at a temperature lowered to 25-26 ° C and practically lost the ability to reproduce at 40 ° С (ts-, ca- phenotype). The parent virus of the "wild" type A / mallard / Netherlands / 12/00 (H7N3), on the contrary, was characterized by temperature resistance and lack of reproduction at 25 ° C.

Table 4 Reproductive activity of reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) at different temperature incubation conditions in chicken embryos Virus (characteristic) Reproductive activity at a temperature of (Ig EID _50/1 ml) Phenotype 34 ° C 40 ° C 25 ° C 26 ° C A / 17 / mallard / Netherlands / 00/95 (H7N3) (6: 2 reassortant) 9.2 ± 0.5 1.8 ± 0.2 6.9 ± 0.5 7.5 ± 0.1 Ts, ca A / Leningrad / 134/17/57 (H2N2) (XA attenuation donor) 9.5 ± 0.3 1.6 ± 0.1 7.0 ± 0.1 8.7 ± 0.6 7s, ca A / mallard / Netherlands / 12/00 (H7N3) (apatogenic avian strain) 8.7 ± 0.2 8.0 ± 0.2 <1.5 1.6 ± 0.1 Non-ts, non-ca

Thus, the presented vaccine strain A / 17 / mallard / Netherlands / 00/95 (H7N3) is characterized by a combination of useful features necessary for the vaccine strain - antigenic specificity of hemagglutinin of the wild-type virus A / mallard / Netherlands / 12/00 (H7N3), genome structure required for reassortant vaccine strains, temperature sensitivity, good adaptation to low cultivation temperature, which correlates with attenuation characteristic of the attenuated donor strain. The characteristics of the new strain — cold adaptation and high reproductive activity in chicken embryos — make it possible to use it for the production of both live and inactivated influenza vaccines.

The strain is deposited in the collection of the Research Institute of Virology. DI. Ivanovo RAMS under No. 2717. The morphology of the strain is polymorphic, typical of the influenza virus.

CHARACTERISTIC OF THE RECEIVED STRAIN.

Infectious activity during reproduction in developing chicken embryos at 33 ° C for 48 hours - 9.2 Ig EID ₅₀ / 0.2 ml.

Hemagglutinating activity - 1: 256.

Passport for the vaccine strain A / 17 / mallard / Netherlands / 00/95 (H7N3)

attached (p. 7).

The results of preclinical studies of the claimed strain.

Example 1. The study of the safety of the reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) was carried out on chickens of the breed White Leghorn. Each of eight 5-week-old chickens was injected intravenously with 0.2 ml of virus-containing allantoic fluid with infectious activity of virus A / 17 / mallard / Netherlands / 00/95 (H7N3) 10 ^8.1 EID ₅₀ / 0.2 ml. Over the course of 10 days, the development of signs of infection was observed (respiratory infection, weakness, diarrhea, cyanosis in the area of inoculation, swelling of the head, neurological symptoms). The pathogenicity index was calculated as 0 - absence of symptoms, 1 - development of signs of infection, 2 - severe infection, 3 - fatal outcome.

Within 10 days after iv inoculation with the reassortant strain A / 17 / mallard / Netherlands / 00/95 (H7N3), no animals showed signs of infection (pathogenicity index 0). Thus, reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) was harmless when administered to hens.

On the third day after intranasal administration at a dose of 6 Ig, EID ₅₀ / ml reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) was not isolated from oropharyngeal and cloacal washings.

Isolation of virus A / 17 / mallard / Netherlands / 00/95 (H7N3) from swabs after intranasal administration to hens Animal number Oropharynx Cloaca 1001 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1002 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1003 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1004 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1005 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1006 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1007 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1008 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1009 ≤10 ^0.9 / ml ≤10 ^0.9 / ml 1010 ≤10 ^0.9 / ml ≤10 ^0.9 / ml

Within 14 days there were no signs of infection, the virus was not isolated from oropharyngeal and cloacal swabs and tissues of the lungs, kidneys, heart and brain. On the 14th day after intranasal administration, no seroconversions were recorded. Thus, the effect of administering the reassortant A / 17 / mallard / Netherlands / 00/95 (H7N3) virus to chickens was similar to seasonal influenza viruses with a low risk of infection.

Example 2. The inventive vaccine strain A / 17 / mallard / Netherlands / 00/95 (H7N3) is harmless to mice and guinea pigs.

Summary of acute toxicity in mice and guinea pigs Animal species Virus Dose Fallen / Survived Average weight change, g Mice A / 17 / mallard / Netherlands / 00/95 (H7N3) 0.5 ml intraperitoneally 0/10 +0.8 Guinea pigs A / 17 / mallard / Netherlands / 00/95 (H7N3) 5 ml subcutaneously 0/3 +11.7

Histological examination showed that the introduction of the vaccine strain A / 17 / mallard / Netherlands / 00/95 to laboratory animals - mice does not cause a protective reaction in the form of - inflammation, alterative manifestations - in the form of degenerative and destructive changes in the liver, kidneys, spleen, heart muscle - myocardium, stromal component of internal organs. In addition, there are no dystrophic changes in neurons, glial elements and circulatory disorders of the microvasculature of the brain. Thus, the use of the vaccine does not cause pathomorphological changes, which indicates the good tolerance and safety of the vaccine A / 17 / mallard / Netherlands / 00/95.

Example 3. The vaccine strain A / 17 / mallard / Netherlands / 00/95 (H7N3) is immunogenic for mice.

It was shown that the vaccine strain A / 17 / mallard / Netherlands / 00/95 (H7N3) exhibits immunogenic properties when administered intranasally to mice. Geometric mean titers of antihemagglutinating antibodies increase in the group of immunized mice, and double immunization is more effective.

Results of rtga with sera of mice immunized intranasally with 10 ⁷ Ig EID ₅₀ strain A / 17 / mallard / Netherlands / 00/95 (H7N3) 4 weeks after immunization Groups Ggt Before immunization Once Twice Ggv 5,0 8.7 40,0 Placebo (FB Solution) 5,0 5,0 5,0

Claims (1)

The strain of influenza virus A / 17 / mallard / Netherlands / 00/95 (H7N3), deposited in the State virus collection of the Virology Research Institute. DI. Ivanovo RAMS under No. 2717, for the production of live intranasal and the production of inactivated influenza vaccines.