RU2465006C2 - Method for improving immunogenicity of cold-adapted live influenza vaccine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medical industry and concerns production of a live influenza xa vaccine. The method for improving immunogenicity of the cold-adapted influenza vaccine is characterised by the fact that the vaccine is added by an adjuvant presented by chitosonium glutamate of final concentration 0.5% having a degree of deacetylation within 66-99% and molecular weight within 70-500 kDa.

EFFECT: method is easy to implement and enables effectively improving immunogenicity of the cold-adapted influenza vaccine.

5 tbl, 5 ex

Description

The invention relates to the medical industry and relates to increasing the immunogenicity of attenuation donors used to produce live influenza vaccines.

Preventive vaccination is the most effective way to prevent influenza infection. Along with inactivated influenza vaccines, live cold-adapted (, ha) influenza vaccines have recently been widely used, which, in contrast to inactivated influenza vaccines, not only induce neutralizing serum IgG and IgA antibodies, but also are good stimulators of the cellular immune response. These vaccines are obtained by reassortment of the attenuation donor strain and antigenically relevant virulent influenza virus strains. They are applied intranasally, have a long protective effect and cause immune defense against drifting NS variants. However, the existing attenuation donor strains have a reduced reproduction potential in the upper respiratory tract, which leads to a reduced prophylactic activity of live influenza vaccines.

One of the possible ways to increase the immunogenicity of influenza attenuation donor strains is the use of adjuvants. Recently, a group of American researchers achieved an increase in the immunogenicity of a strain of influenza A virus, an attenuation donor with a truncated NS gene, using alpha-C-galactosylceramide as an adjuvant. Alpha-C-galactosylceramide has been shown to increase the immunogenicity of this influenza strain-donor attenuation tenfold (Kopezky-Bromberg et al., 2009). We consider this source of information as the closest analogue.

Recently, it was shown that, to increase immunogenicity, chitosan aminopolysaccharide chitosan can be used as adjuvants for influenza inactivated vaccines, whose salts enhance the immunogenicity of inactivated influenza vaccines both intranasally (Illum et al., 2001; Bacon et al., 2000) and with parenteral administration of these vaccines (Zaharoff et al. 2007). A known method (US 6534065) for producing an intranasal mucosal influenza vaccine using chitosonium salts obtained from 80-90% deacetylated chitosan with a molecular weight of 10 to 100 kDa as mucosoadhesive adjuvant. The concentration of chitosonium salts in the vaccine was up to 0.5%. However, to date, the use of chitosan preparations as adjuvants to increase the immunogenicity of Ha-strains of the influenza virus, donors of attenuation for live influenza vaccines administered intranasally, has not been carried out.

The technical result of the claimed invention is a method for increasing the immunogenicity of a cold-adapted (ha) influenza attenuation donor strain, characterized in that chitosonium glutamate preparations with a molecular weight of 70-500 kDa and a degree of deacetylation are simultaneously added to a preparation of this strain 66-99%.

The ha-strain A / Krasnodar / 101/35/59 was used as a model. This strain was obtained by serial passages in chicken embryos and MDCK cell culture at a gradually decreasing incubation temperature, followed by three-time plaque cloning in MDCK cell culture, which ensures the genetic homogeneity of the strain. The resulting strain has good productive activity in chicken embryos and MDCK cells at low temperatures and does not reproduce at elevated temperatures. The resulting version of the virus has unique nucleotide mutations in the genes encoding the proteins PB2, PB1, PA, NP and M1 / M2. The strain is registered in the collection of the depository of the Research Institute of Virology. D.I. Ivanovsky RAMS under number 2439 from 06/09/2008. The patent “Attenuated cold-adapted strain of the influenza virus A / Krasnodar / 101/35/59 (H2N2) for obtaining strains of live intranasal influenza vaccine” was obtained. Patent for invention No. 2354695.

It is known that preparations of chitosan salts (CX), when ingested on the mucous membrane of the nasopharynx, cause pronounced biological effects. In particular, CX preparations slow down the activity of the cilia of the ciliated epithelium, weaken the intercellular contacts and, as a result, significantly increase the permeability of the mucous membranes. There is also evidence that CX in combination with plasmid DNA increase the efficiency of transfection of eukaryotic cells. In sum, these biological effects can contribute to the faster entry of viral material during increased virus reproduction into regional nasopharyngeal lymph nodes and stimulate the contact of virus-specific molecules with antigen-presenting cells, thereby increasing both the local secretory and systemic immune responses.

Chitosan is not soluble at physiological pH values. With a decrease in pH below 6.5 (pK protonation of the amino groups of chitosan), swelling begins, and at pH 4-4.5, the initial preparation is completely dissolved, which turns into a chitosonium salt with the acid used,

However, both Bacon et al. And Illum et al. Observed the adjuvant effect of CX when applied to the mucous membrane of the nasopharynx of laboratory animals and humans, which is characterized by physiological pH values (7.0–7.2). Data from another group of researchers report high adjuvant properties of chitosonium tripolyphosphate nanoparticles in intranasal inactivated microbial and viral preparations (van der Lubben et al., 2003; Amidi et al., 2006).

Research conducted at the Research Institute of Vaccines and Serums named after I.I. Mechnikov RAMS showed high adjuvant properties of the high-polymer preparation of chitosonium glutamate (M.V. 200-300 kDa, degree of deacetylation of the initial chitosan = 85%), administered parenterally with inactivated influenza vaccines (Ghendon et al., 2008; Ghendon et al., 2009).

On the other hand, it was shown that contact of CX with lymphoid tissues in the absence of a protein antigen during mucosal administration stimulated the production of regulatory cytokines (IL-12, IL-10, IL-4, TGF-beta), and increased production of OX62 + dendritic cells and activation of CD3 + T cells (Porporatto et al., 2004;

Porporatto et al., 2005).

Examples of the method

Example 1

To tenfold dilutions of Ha attenuation donor strain A / Krasnodar / 101/35/59 (H2N2), a 1% solution of chitosonium glutamate (GC) was added to obtain a 0.5% final GC concentration. The final mixture also contained 0.9% sodium chloride. Balb / c mice weighing 10-12 g under mild ether anesthesia were immunized intranasally twice with different dilutions of the attenuation donor strain strain with GC preparations in a volume of 50 μl. The second dose of the GC vaccine virus was administered 21 days after the first immunization and 10 days later the blood was taken from mice. In control experiments, mice were injected with viral material without GC. In experiment we observed an increase in the formation of serum antibodies to delay hemagglutination ha-donor strain is 4 times during vaccination virus dose of 10 ^5.0 EID _50/50 ul and 8 times at a virus vaccination dose of 10 ^3.0 EID _50/50 ul as compared to the control. Based on these facts, it can be concluded that the addition of GC to the ha donor strain A / Krasnodar / 101/35/59 (H2N2) during intranasal immunization of mice increased antibody titers delaying hemagglutination by 4-8 times (table 1).

Example 2

Tenfold dilutions of the native attenuation donor strain strain

A / Krasnodar / 101/35/5 9 (H2N2) was added 1% GC solution to obtain a 0.5% final concentration of GC. The final mixture also contained 0.9% sodium chloride. Mice were immunized intranasally twice by injecting 50 ul of various doses ha donor strain of 10 ^6.0 EID ₅₀ /0.05 mL to 10 ^2.0 EID ₅₀ / ml with 0.05 GC into the nostrils of each animal under light ether anesthesia. In control experiments, mice were administered intranasally similar doses of viral material without GC. The second immunization was carried out 21 days after the first immunization. 10 days after the second immunization, mice were infected intranasally with virulent A / Krasnodar / 101/59 (H2N2) a dose of 10 ^5.5 EID _50/50 .mu.l under light ether anesthesia. After 3-4 days, the mice were sacrificed and the lungs recovered. A 10% suspension was prepared from the lungs. After centrifugation of the suspension in a low speed centrifuge, the supernatant was titrated by infection of 10-day-old chicken embryos. The study of the protective effect in mice immunized intranasally with strain A / Krasnodar / 101/35/59 (H2N2) with chitosan revealed no reproduction of the virulent virus in the lungs of mice immunized with doses of the virus 10 ^6.0 , 10 ^5.0 , 10 ^4.0 , 10 ^3.0 , 10 ^2.0 EID ₅₀ / 0.05 ml ha vaccine variant. Propagation of the virulent virus in the lungs of mice was observed only in the group immunized with 10 ^2.0 EIDs of ₅₀ / 0.05 ml of the vaccine variant (see Table 2).

In the control group of mice immunized intranasally with appropriate doses of Ha strain A / Krasnodar / 101/35/59 without GC, active propagation of the virulent variant was observed in doses of 10 ^5.0 , 10 ^4.0 , 10 ^3.0 EID ₅₀ /0.05 ml. Only in the group of mice immunized with a dose of 10 ^6.0 EID of ₅₀ / 0.01 ml ha strain, the reproduction of the virulent variant was suppressed (see table 2).

The results obtained indicate a significant increase in the protective effect of intranasal vaccination with live virus when GC is introduced into vaccine material.

Example 3

Mice were infected intranasally with different doses of attenuation A / Krasnodar / 101/35/59 (H2N2) attenuation donor strain containing GC at a final concentration of 0.5%, or various doses of virulent strain A / Krasnodar / 101/59 (H2N2) without GC. Mice were infected with a dose of 50 μl under light ether anesthesia. The infected mice were sacrificed on the 3rd day or on the 4th day after infection, the lungs were removed from the mice and a 10% suspension was prepared from the lungs. The supernatant of suspensions after centrifugation in a low speed centrifuge was titrated by infection of 10-day-old chicken embryos. It was shown that the virulent variant A / Krasnodar / 101/59 proliferated actively in the lungs of mice infected with various doses of this virus (Table 3), both 3 and 4 days after infection. However, in the lungs of mice infected with intranasally different doses of Ha strain A / Krasnodar / 101/35/5 9 in combination with GC, the virus did not multiply in the lungs on the 3rd or on the 4th day after infection (Table 3). Xa donor strain A / Krasnodar / 101/35/59, when administered intranasally to mice, does not multiply in the lungs of mice, in contrast to the virulent strain A / Krasnodar / 101/59. As can be seen from the obtained data, the addition of GC to the ha strain donor did not lead to the propagation of the ha strain in the lungs of mice.

Example 4

Mice were immunized intranasally with different doses of the attenuation strain A / Krasnodar / 101/35/59 (H2N2) ha strain containing 0.5% GC. A control group of mice was immunized intranasally with similar doses containing GC of the same virus inactivated by UV radiation. 21 days after the first immunization, they were re-immunized with the corresponding native virus or virus inactivated with UV radiation. After 10 days in each group in some of the mice were bled to determine the serum antibodies and another part intranasally infected with a virulent strain of A / Krasnodar / 101/59 (H2N2) at a dose of 10 ^5.5 EID _50/50 l in order to determine the protective effect of immunization. The results of a study of the induction of serum antibodies in mice immunized with various doses of native Ha strain A / Krasnodar / 101/35/5 9 (H2N2) showed the presence of antibodies in the titer of 1:80 when immunized with doses of 10 ^4.0 and 10 ^3.0 EID ₅₀ / 0.05 ml and in a titer of 1:20 for immunization with 10 ^2.0 EID ₅₀ /0.05 ml. In the blood serum of mice immunized with similar dilutions of the virus, inactivated by UV radiation, antibodies to the virus were not detected. A study of the protective efficacy of immunization with a native virus and a virus inactivated by UV irradiation with GC followed by infection with a virulent virus showed that in the lungs of mice immunized with various doses of a UV-inactivated vaccine variant, active propagation of the virulent variant was observed. Moreover, the infectious titer in some cases exceeded 10 ^5.5 EID ₅₀ /0.2 ml. However, in the lungs of mice immunized with dilutions of the Ha strain of the donor strain with GC, when infected with a virulent virus, the titers of the virulent virus did not exceed 10 ^2.0 EIDs of ₅₀ / 0.2 ml. These data indicate that the inactivated preparation of donor strain A / Krasnodar / 101/35/59 with GC during intranasal immunization of mice at doses of 10 ^4.0 , 10 ^3.0 , 10 ^2.0 EID _{50 /} 0.05 ml does not protect against infection with virulent virus A / Krasnodar / 101/59 (table 4), in contrast to the same doses of intact ha donor strain with GC.

Example 5

Mice weighing 10-12 g were injected intranasally with a donor strain A / Krasnodar / 101/35/59 (H2N2) at a dose of 10 ^7.0 EID ₅₀ / 0.05 ml containing 0.5% GC under light ether anesthesia. A control group of mice was injected intranasally with a similar dose of the same virus without GC. After 2 hours, 24 hours and 48 hours after intranasal administration of the virus, part of the mice were sacrificed and 10% suspension was prepared from the nasal cavities of mice. These suspensions were centrifuged in a low speed centrifuge and the supernatant titrated by infection of 10-day-old chicken embryos. 2 hours after the introduction of the virus, the titers of the infectious virus exceeded 10 ^4.0 EID ₅₀ / 0.2 ml both with the introduction of the virus with GC, and without it. 24 hours and 48 hours after the intranasal injection of the virus, the infectious titers of the virus were also the same both with the introduction of the virus with GC and without it, which indicated that GC did not stimulate the increase of virus reproduction in the nasopharynx of animals (table 5).

General conclusion

The addition of GC to the ha donor strain A / Krasnodar / 101/35/59, administered intranasally, significantly increases the formation of humoral antibodies and protection against subsequent infection of mice with a virulent strain of the influenza virus. The data obtained allow us to conclude that the addition of GC to the influenza virus strain, an attenuation donor for live influenza vaccines, can significantly increase the immunogenicity of this drug.

Table 1 The effect of chitosan on increasing titers of serum antibodies during intranasal immunization of mice with the donor strain A / Krasnodar / 101/35/59 (H2N2) Infectious virus titer in a dose of immunizing material Antibody titers inhibiting hemagglutination in the blood serum of mice immunized intranasally without gc with the addition of GC 10 ^5.5 EID _50/50 .mu.l 1: 640 1: 2560 10 ^3.5 EID _50/50 .mu.l 1:80 1: 640

To the attenuation donor strain A / Krasnodar / 101/35/59 (H2N2) at doses of 10 ^3.5 and 10 ^5.5 EID _{50 /} 0.05 ml, a 1% GC solution (pH 6.5) was added to obtain a 0.5% final concentration GC. The final mixture also contained 0.9% sodium chloride. Mice weighing 10-12 g under mild ether anesthesia were intranasally immunized with twice different dilutions of the Ha strain with or without GC, injecting 25 μl into each nostril. The second immunization was carried out 21 days after the first immunization and 10 days after the second immunization, blood was taken from the mice. The sera of mice were treated with a receptor-degrading enzyme before rTGA. For this purpose, 3 volumes of the Denka Seiken CO., LTD receptor-destroying enzyme were added to 1 volume of serum and incubated in an incubator for 16-18 hours at 37 ° C. The mixture was then heated at 56 ° C for 30 minutes. and 6 volumes of phosphate buffer were added, after which antibody titers were analyzed.

table 2 The effect of GC on increasing the protective effect during intranasal immunization of mice with a donor strain A / Krasnodar / 101/35/59 Doses of the strain of the donor strain A / Krasnodar / 101/35/59 (H2N2) used for intranasal immunization (EID ₅₀ /0.05 ml) Propagation of the virulent strain A / Krasnodar / 101/59 (H2N2) in the lungs of mice immunized intranasally with the donor strain A / Krasnodar / 101/35/59 (H2N2) with and without GC chitosan immunization with the addition of GC-free immunization 10 ^6.0 <10 ^1.0 * 10 ^1.5 * 10 ^5.0 <10 ^1.0 10 ^2.5 10 ^4.0 <10 ^1.0 10 ^3.0 10 ^3.0 <10 ^1.0 10 ^3.0 10 ^2.0 10 ^3.0 10 ^3.0 *) EID ₅₀ /0.2 ml

Mice weighing 10-12 g were immunized intranasally twice with different doses of the attenuation donor strain A / Krasnodar / 101/35/59 (H2N2) containing 0.5% GC or without GC. Virus-containing material was injected in a volume of 50 μl. The second immunization was performed 21 days after the first immunization. 10 days after the second immunization, mice were infected with an intranasally virulent strain A / Krasnodar / 101/59 (H2N2) at a dose of 10 ^5.5 EID ₅₀ / 0.2 ml. After 3 or 4 days, the mice were sacrificed, the lungs were removed and a 10% suspension was prepared from them. The suspension was centrifuged in a low speed centrifuge and the supernatant titrated by infection of 10-day-old chicken embryos.

Table 3 Comparative study of the reproduction of Ha donor strain A / Krasnodar / 101/35/59 and the virulent strain A / Krasnodar / 101/59 (H2N2) in the lungs of mice with intranasal administration the studied strains and doses of infection (EID ₅₀ / 0.05 ml)
………………………………
Ha strain A / Krasnodar / 101/35 / 59.0 Reproduction of viruses in the lungs of mice virus without GC virus with GC 3rd day after infection 4th day after infection 3rd day after infection 4th day after infection 10 ^7.0 <10 ^1.0 * <10 ^1.0 * <10 ^1.0 * <10 ^1.0 * 10 ^6.0 <10 ^1.0 <10 ^1.0 <10 ^1.0 <10 ^1.0 10 ^5.0 <10 ^1.0 <10 ^1.0 <10 ^1.0 <10 ^1.0 virulent strain
A / Krasnodar / 101/59
10 ^7.0 10 ^3.5 10 ^3.5 n.d. n.d. 10 ^6.0 10 ^3.0 10 ^3.0 n.d. n.d. 10 ^5.0 10 ^3.0 10 ^2.5 n.d. n.d. *) EID ₅₀ /0.2 ml

Used various doses of the Ha strain of the donor strain A / Krasnodar / 101/35/59 (H2N2) containing 0.5% GC and without GC. Mice weighing 10-12 g were infected intranasally with different strains of this strain. As a control, various doses of virulent strain A / Krasnodar / 101/59 without chitosan were used. 3 or 4 days after infection, the mice were sacrificed and the lungs were removed, from which a 10% suspension was prepared. The supernatant of the suspensions after centrifugation in a low speed centrifuge was titrated by infection of 10 day old chicken embryos.

Mice were immunized intranasally with different doses of the donor strain A / Krasnodar / 101/35/59 or the same strain inactivated by UV radiation. Virus dilutions contained 0.5% GC. 21 days after the first immunization, re-immunization was done with an intact virus, or a virus inactivated by UV radiation. 10 days after the second immunization, mice were bled part to determine serum antibody in RZGA and another portion mice were infected intranasally with virulent A / Krasnodar / 101/59 (H2N2) at a dose of 10 ^5.5 EID _50/50 microliters. 3 days after infection, the mice were sacrificed, the lungs were removed, and 10% suspension was made from the lungs. After centrifuging the suspension in a low speed centrifuge with ten-fold dilutions of the supernatant by infection of 10-day-old chicken embryos. When analyzing the titer of serum antibodies, mouse sera were treated with a receptor-degrading enzyme.

Table 5 Reproduction of the Ha strain A / Krasnodar / 101/59/35 (H2N2) in the nasopharynx of mice by intranasal administration without chitosan and with the addition of GC Time after intranasal administration of the virus Propagation titers of Ha strain A / Krasnodar / 101/59/35 in the nasopharynx of mice with intranasal administration (EID ₅₀ / 0.2 ml) No gc With the addition of GC 2.0 hours 10 ^4.0 10 ^4.0 24 hours 10 ^3.0 10 ^3.0 48 hours 10 ^3.0 10 ^3.0

Mice weighing 10-12 g were injected intranasally with a donor strain A / Krasnodar / 101/35/59 (H2N2) at a dose of 10 ^7.0 EID ₅₀ / 0.05 ml containing 0.5% GC under light ether anesthesia. A control group of mice was injected intranasally with a similar dose of the same virus without GC. After 2 hours, 24 hours and 48 hours after intranasal administration of the virus, part of the mice were sacrificed and 10% suspension was prepared from the nasal cavities of mice. These suspensions were centrifuged in a low speed centrifuge and the supernatant titrated by infection of 10-day-old chicken embryos.

Claims (1)

A method for increasing the immunogenicity of a cold-adapted influenza attenuation donor strain A / Krasnodar / 101/35/59 for live influenza vaccines, characterized in that chitosonium glutamate is added to the virus preparation as an adjuvant to a final concentration of 0 to enhance the immune response achieved during immunization , 5% in the vaccine used.