RU2670514C1 - Method for obtaining attenuated strains of influenza a virus - candidates for live influenza vaccines - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: present invention relates to method for producing attenuated strains of the influenza A virus candidate for live influenza vaccines. Method consists in inserting an attenuated amino acid substitution F658A simultaneously into the conserved portion of the COOH domain of the PA gene of the epidemic virulent strain A/WSN/33 of the influenza A virus, simultaneously with the inclusion of site-specific mutations in the PB1 gene and the PB2 gene, PB1 gene of strain A/WSN/33 includes a complex of ts mutations from the genome XA of the donor attenuation donor A/Ann Arbor/6/60 (H2N2) : K391E, E581G, E457D or one ts-mutation I147T from genome XA of attenuation donor A/Krasnodar/101/35/59 (H2N2), and as a site-specific mutation included in the PB2 gene of strain A/WSN/33, ts-mutation of V290L from the genome XA of the donor attenuation A/Krasnodar/101/35/59 (H2N2) gene is used.EFFECT: invention allows to obtain attenuated strains of the influenza A virus that provide better immune protection than preparation of reassortants based on the standard XA donor attenuation and can be used in the medical industry.1 cl, 5 tbl, 7 ex, 1 dwg

Description

The invention relates to the medical industry and relates to obtaining attenuated strains of influenza A virus - candidates for live influenza vaccines. Preventive vaccination is the most effective way to prevent influenza infection. Along with inactivated influenza vaccines, lively influenza cold-adapted (HA) vaccines, which, unlike inactivated influenza vaccines, have not only become neutralizing serum IgG and IgA antibodies, but also are good stimulators of the cellular immune response, have recently become widely spread. These vaccines are used intranasally, have a long term protective action.

Experience to date, the use of XA live influenza vaccine in Russia and the United States has shown that this drug is most effective in mass vaccination against influenza, especially children. It should be noted that, unlike inactivated influenza vaccines, a live vaccine can protect against infection with antigenic variants of the influenza virus.

Currently, the practice of obtaining production reassortants for live influenza vaccine in our country is limited to the outdated method of reassorting parental variants in chicken embryos, followed by selection of the resulting reassortants and their selection after genomic analysis. Introduction to the practice of obtaining influenza live vaccines of genetically engineered approaches allows to significantly optimize the individual stages of this process. There are a number of patents (US 8093033 B2, US 20090175907 A1, US 7465456, US 20020164770, WO 2003091401 A2, WO 2005062820 A2, WO 2005115448 A2, US 7744901, US 7504109), where the multiplasmid system in tissue culture is used to obtain candidate reassortants live influenza vaccines that have genes encoding internal proteins from HA strain A / Ann Arbor / 6/60 / or HA strain B / Ann Arbor / 1/60 and genes encoding surface glycoproteins from antigen-relevant epidemic influenza A strains of influenza A and Q. Recently, a genetically engineered approach has been of great interest among researchers. This problem involves the direct inclusion of previously known and studied ts mutations taken from the genome of the donor strain of attenuation into the genome of the virulent strain of influenza virus (Subbarao et al., 1993; Subbarao et al., 1995; Parkin et al., 1997; Hickman et al., 2008; Song et al., 2007; Pena et al., 2011, Solorzano and Perez, 2010; Zhou et al., 2012).

The literature data for the last period suggests that the use of site-specific mutation technology can significantly advance the development of live influenza vaccines in both medicine and veterinary medicine. At the same time, progress in this area has marked the presence of serious unsolved problems. As can be seen from the literature, the main source of site-specific ts mutations in all countries is the XA strain A / Ann Arbor / 6/60, obtained by Dr. Maassab X. more than half a century ago (Maassab, 1967). Three mutations from the PB1 gene of this strain (K391E, E581G, A661T), one mutation from the PB2 gene (N265G) and one of the NP gene (D34G) make up the “gentlemen’s kit” for modifying the genome of virulent strains of human, animal and the birds. However, the use of this set of mutations does not completely attenuate not only the pandemic strain, but also some virulent seasonal variants of the human influenza virus. To fully attenuate a pandemic strain, 7-8 mutations are required if mutations from the genome of strain A / Ann Arbor / 6/60 are used (Zhou et al., 2012). At the same time, for many years in Russia, XA strain A / Leningrad / 134/17/57 (H2N2) has been used as a donor of attenuation for live influenza vaccines (Alexandrova, Klimov, 1994). Genetic determinants responsible for the attenuation of this strain are concentrated in the PB1 gene (K265N, V591I) and the PB2 gene (V478L). In another recently obtained domestic CA strain A / Krasnodar / 101/35/59 (H2N2) (Gendon et al., 2013), genetic determinants responsible for the attenuation of this strain are localized in the PB1 gene (I147 T) and the NS gene.

The disadvantages of the above methods is the lack of an integrated approach to the development of attenuated strains of influenza A virus - candidates for live influenza vaccines.

To eliminate the above drawbacks, we propose a method for obtaining attenuated strains of influenza A virus - candidates for live influenza vaccines, consisting in that they include attenuating substitutions of the virulent strain in the conservative region of the COOH-domain of the RA-gene simultaneously with the inclusion of site-specific mutations from PB1- and PB2 genes. In the particular case, the following strains are used as virulent strains: A / Leningrad / 134/17/57 (H2N2), A / Krasnodar / 101/35/59 (H2N2) and A / Ann Arbor / 6/60 (H2N2).

In this regard, for a more efficient use of the aforementioned genetic engineering approach, it is planned to use new alternative sources of attenuating mutations. First, it is proposed to use the inclusion of site-specific substitutions in conservative regions of virus-specific proteins, in particular, proteins of the polymerase complex. An example is the replacement of F658A, the inclusion of which in the terminal part of the COOH-domain of the RA gene of the virulent strain A / WSN / 33 leads to the attenuation of this strain. Secondly, along with ts mutations from strain A / Ann Arbor / 6/60, ts mutations from the genome of native HA strains A / Leningrad / 134/17/57 (H2N2) and A / Krasnodar / 101/35 / can be used 59. A significant effect can be obtained by using a combination of attenuating mutations from new alternative sources.

The technical result of the claimed invention is the inclusion of site-specific mutations that do not violate the antigenic structure of proteins of the virulent strain. This strategy provides better immune protection than obtaining reassortants based on standard XA donor attenuation. This is explained by the fact that immune epitopes of 8 or 9 proteins encoded by 6 “internal” genes differ in HA of reassortants and antigenically relevant epidemic strains. Direct inclusion of combinations of site-specific mutations in the genome of a virulent strain allows you to change the phenotypic characteristics of this strain and modify it in an attenuated version.

Examples of the method

Example 1

Modifying the ts and att phenotype of a virulent strain A / WSN / 33 by including a specific attenuating mutation at a functionally important site in the conserved sequence of the COOH domain of the PA gene of this strain

A comparative study of the nucleotide sequence of the PA-genes XA strain A / Krasnodar / 101/35/59 (H2N2) and the original wild-type strain A / Krasnodar / 101/59 (H2N2) revealed a mutation in the COOH-domain of the RA gene XA strain: F707L. This mutation is located in the hydrophobic core of the COOH domain and probably destabilizes it. Assuming that there is a π-π (T-stacking) interaction between the two amino acid residues F707 and F658, a site-specific replacement F658A was constructed using two-step PCR, and this replacement was included in the conservative sequence of the COOH-domain of the RA-gene virulent strain.

The example is illustrated in Table 1, where the column marked * reflects the activity of reproduction of influenza viruses at the optimal and non-resolving incubation temperature, which was estimated from the results of titration in chicken embryos incubated at 34 ° C and 39 ° C and expressed in RCT (reproductive capacity at different temperatures). RCT ₃₉ = (lg EID ₅₀ / 0.2 ml at 34 ° C - lg EID ₅₀ / 0.2 ml at 39 ° C). Viruses were considered temperature sensitive (ts phenotype) if RCT ₃₉ was more than 5.0 lg EID ₅₀ / 0.2 ml.

In table 1, the column marked ** reflects the definition of virus reproduction in the lungs of mice; for this, animals were infected intranasally in an infectious titer of 10 ^6.0 EID ₅₀ / 0.2 ml (50 μl per mouse). 72 hours after infection, the lungs were removed from the mice. Infectious titer was determined in chicken embryos and expressed in an EID of ₅₀ / 0.2 ml.

As can be seen from table 1, the inclusion of the F658A substitution in this conservative region of the genome of strain A / WSN / 33 led to a noticeable change in the ts-phenotype of the strain, which sharply increased its temperature sensitivity. The replacement also reduced the ability of the virus to multiply in the lungs of mice. It can be concluded that the inclusion of the F658A substitution in the genome of the virulent strain A / WSN / 33 contributed to its attenuation.

Example 2

Study of the ts-phenotype of variants of the A / WSN / 33 strain of influenza virus with site-specific mutations in the genes encoding polymerase complex proteins

Ts-marker is the most important marker of attenuation for candidate strains for live influenza vaccines. As can be seen from the previous example, the inclusion of the attenuating replacement of F658A in the terminal part of the COOH-domain of the PA-gene of the virulent strain A / WSN / 33 causes a noticeable, but limited, change in the ts-phenotype of this strain. In order to further attenuate the virulent strain, the individual ts mutations from the PB1 and PB2 genes of the CAA strains A / Leningrad, 134/17/57 (H2N2), A / Krasnodar / 101/35/59 (H2N2 ) and A / Ann Arbor / 6/60 (H2N2). As can be seen from table 2, the combined inclusion of additional ts mutations from the PB1 and PB2 genes of XA strains leads in some cases to the formation of a pronounced ts phenotype in the recipient strain. Thus, the further sequential insertion into the genome of the strain A / WSN / 33, containing the F658A substitution, the additional substitution V290L from the PB2 gene of the CAA strain A / Krasnodar / 101/35/59, and the substitutions K391E, E581G and E457D from the PB1 gene XA- strain A / Ann Arbor / 6/60 resulted in the appearance of a variant that completely lost its ability to multiply in chicken embryos and at a non-resolving temperature (39 ° C). An example is illustrated in Table 2, where A / AA means A / Ann Arbor / 6/60; A / Kr35 - A / Krasnodar / 101/35/59, A / Len 17 - A / Leningrad / 134/17/57.

Similarly, the inclusion of the attenuating F658A substitution in the terminal part of the COOH domain of the RA gene of the virulent strain A / WSN / 33 in combination with the inclusion of the ts mutation I147T from the PB1 gene XA of strain A / Krasnodar / 101/35/59 also leads to variants with a pronounced ts-phenotype.

Example 3

The study of the weight characteristics of mice infected with intranasal variants of the strain A / WSN / 33 of influenza A virus, containing site-specific mutations in the genes encoding polymerase complex proteins.

As can be seen from fig. 1, mice infected with the virulent strain A / WSN / 33 were characterized by rapid and significant weight loss. At 8-9 days after infection, the weight loss of mice reached 35%. At the same time some of the animals died. In mice infected with intranasal variants of strain A / WSN / 33, containing site-specific mutations in the genes encoding the proteins of the polymerase complex, significantly less weight loss (10-15%) and a complete absence of lethal cases were observed.

Example 4

Study of the att-phenotype of variants of A / WSN / 33 strain containing site-specific mutations in the genes encoding the polymerase complex proteins.

White purebred mice weighing 10-12 grams. (females) were intranasally infected with variants of strain A / WSN / 33, containing various combinations of ts mutations in the genes encoding the polymerase complex proteins in the genome. The dose of infection (0.05 ml) contained the virus at a concentration of 10 ^6.0 EID ₅₀ / 0.2 ml. 72 hours after infection, the lungs were removed from the mice. From the lungs, a 10% suspension was prepared, which was titrated in 9-day chicken embryos. Infectious titer was determined in chicken embryos and expressed in EID50 / 1 g of the lungs. The results are presented in Table 3.

From table 3 it can be seen that variants of strain A / WSN / 33, were characterized by varying degrees of reproduction in the lungs of mice. Thus, the inclusion of the I147T ts mutation from the PB1 gene XA strain A / Krasnodar / 101/35/59 and the replacement of F658A in the terminal part of the COOH domain of the RA gene of the virulent strain completely suppressed this strain in the genome of the virulent strain A / WSN / 33 in the lungs of mice. A similar result was observed when a series of ts mutations from the PB1 gene XA strain A / Ann Arbor / 6/60 (K391E, E581G, E457D), ts mutations V290L from PB2 gene XA strain was inserted into the genome of the virulent strain A / WSN / 33 A / Krasnodar / 101/35/59 and the replacement of F658A in the PA-gene of the recipient strain. On the other hand, the inclusion in the genome of the virulent strain A / WSN / 33 ts-mutations V591I from the PB1 gene XA strain A / Leningrad / 134/17/57, ts-mutations V290L from PB2 gene XA strain A / Krasnodar / 101 / 35/59 and the replacement of F658A in the COOH domain of the RA gene of the virulent strain only reduced the replication of this variant in the lungs of mice compared to the replication of the original virulent strain A / WSN / 33. The findings suggest that by incorporating specially selected ts mutations of different origin into the genome of the virulent strain, it is possible to obtain a deeply attenuated variant of the influenza virus.

Example 5

The study of the immunogenicity of variants of the strain A / WSN / 33, containing site-specific mutations in the genes encoding polymerase complex proteins

Outbred mice weighing 10-12 grams. (females) under light ether anesthesia were immunized intranasally with two variants of strain A / WSN / 33, containing various combinations of ts mutations in the genes encoding the proteins of the polymerase complex. The injected dose of virus contained 10 ^5.5 EID ₅₀ / 0.05 ml. The second dose of the virus was administered on day 21 after the first immunization and 10 days after the second immunization, blood was taken from the mice. A study of the humoral response was conducted to assess the potential of these variants as live influenza vaccines. As can be seen from table 4, a moderate titer of humoral antibodies (1: 160-1: 320) was detected in mice immunized with a variant of strain A / WSN / 33 containing mutations from the PB1 gene XA strain A / Ann Arbor / 6 / in the genome. 60 (K391E, E581G, E457D) and the replacement of F658A in the terminal part of the COOH-domain of the PA-gene of the virulent strain. A similar titer of humoral antibodies was observed in mice immunized with a variant of strain A / WSN / 33, containing in the genome a ts-mutation of V591I from the PB1-XA gene of strain A / Leningrad / 134/17/57 and the replacement of F658A in the terminal part of the COOH-domain of PA- gene virulent strain. It should be noted that the noticeable level of humoral response induced by the aforementioned variants could be due to their insufficient degree of attenuation, which allowed them to multiply not only in the upper respiratory tract, but also in the lungs of mice. The variant strain A / WSN / 33, containing in the genome a ts mutation I147T from the PB1 gene XA strain A / Krasnodar / 101/35/59 and the substitution of F658A in the COOH domain of the RA gene of the virulent strain, induced a low level of the humoral immune response ( 1:80).

Example 6

Study of the protective efficacy of A / WSN / 33 strain variants containing site-specific mutations in genes encoding polymerase complex proteins

Outbred mice weighing 10–12 g (females) under light ether anesthesia were twice intranasally immunized with variants of strain A / WSN / 33 containing combinations of ts mutations in the genes encoding the proteins of the polymerase complex. The injected dose of virus contained 10 ^5.5 EID ₅₀ / 0.05 ml. A second immunization was performed 21 days after the first immunization. 10 days after the second immunization, mice were infected intranasally with the virulent strain A / WSN / 33 at a dose of 10 ^2.5 EID ₅₀ / 0.05 ml. 72 hours after infection, the lungs were removed from the mice. From the lungs, a 10% suspension was prepared, which was titrated in 9-day embryos. As can be seen from table 5, all variants of strain A / WSN / 33 used for immunization (containing the F658A substitution in the COOH domain of the RA gene and ts mutations from the PB1 gene of various HA strains of influenza virus) completely blocked the reproduction of the virulent strain of the virulent strain in the lungs of mice. It is important to note that the variant strain A / WSN / 33, which contains the ts mutation I147T from the PB1 gene HA strain A / Krasnodar / 101/35/59 in the genome and the replacement of F658A in the PA gene, which induces a low humoral immune response (1 : 80), however, provided full protective efficacy in immunization, which can be explained by a significant activation in this case of cellular immunity.

Example 7

Studying the genetic stability of variants of the A / WSN / 33 strain containing site-specific mutations in the genes encoding polymerase complex proteins

Live virus vaccines should have a significant margin of genetic stability. In this regard, two variants of the strain A / WSN / 33, which showed satisfactory phenotypic characteristics, investigated the safety of ts mutations in the PA and PB1 genes and the ts phenotype after 5 consecutive passages in the MDCK cell culture at 37 ° C. A variant of the A / WSN / 33 strain containing mutations from the PB1 gene XA strain A / Ann Arbor / 6/60 (K391E, E581G, E457D) and the F658A substitution in the PA gene, as well as another variant containing a mutation in the genome from PB1-XA gene of strain A / Krasnodar / 101/35/59 I147T and the replacement of F658A in the PA-gene were passed through in the MDCK cell culture and after 5 passages analyzed for the presence of attenuating mutations in the PB1-gene and the RA-gene. Sequencing of these genes revealed the safety of attenuating mutations in the studied variants (not shown). At the next stage of work, the ts-phenotype was studied in the passive variants, which did not undergo any changes. The presence of unchanged ts-phenotype indirectly testifies in favor of the absence of arisen suppressor mutations in the genome of passive variants, which allows us to conclude about their genetic stability.

General conclusion

genome modification of the virulent strain of influenza virus by incorporating specific attenuating mutations at functionally important sites in the conserved gene sequences of the virulent strain in combination with the inclusion in the genome of a virulent strain ts-mutation of the HA of influenza virus strains allows to obtain attenuated strains of influenza A virus - candidates for live influenza vaccines .

BIBLIOGRAPHY

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Claims (1)

The method of obtaining attenuated influenza A virus strains - candidates for live influenza vaccines, which consists in the fact that in the conservative region of the COOH - domain of the RA gene of the influenza A virus epidemic strain A / WSN / 33, they include an attenuating amino acid substitution F658A simultaneously with the inclusion of the site specific mutations in the PB1 gene and the PB2 gene, while the PB1 gene of the A / WSN / 33 strain includes a complex of ts mutations from the XA genome of the A / Ann Arbor / 6/60 (H2N2) strain donor strain: K391E, E581G , E457D or one I147T ts mutation from the XA genome of the A / Krasno donor strain of the donor strain ar / 101/35/59 (H2N2), and the ts-mutation V290L from the XA genome of the attenuation donor strain A / Krasnodar / 101/35 / is used as site-specific mutations included in the PB2 gene of the strain A / WSN / 33. 59 (H2N2).

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