RU2444570C1 - Method of producing recombinant vaccine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of producing a recombinant vaccine provides producing chimeric proteins or DNA expressing genes of these chimeric proteins consisting of three or more functional parts. These parts are active material of the vaccine and presented by a pathogenic microorganism antigen, a TLR-receptor ligand and ligants for receptors transferring signal through ITAM-motives. The chimeric proteins are directly used as vaccines for human and animal immunisation. For the vaccines representing DNA coding the chimeric protein, recombinant adenovirus particles and/or plasmid DNA are used as a carrier.

EFFECT: vaccination accompanying preventive treatment of infectious diseases.

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Description

The invention relates to the field of biotechnology, genetic engineering and immunology. It relates to methods for creating vaccine preparations that are chimeric proteins or DNA containing the gene sequences of these chimeric proteins.

Many pathogens pose a threat to public health. In particular, such microorganisms include viruses (Influenza virus, HIV, Rabies vims), bacteria (M. tuberculosis, B.anthracis, C.trachomatis, S.enterica, L. pneumophila) and protozoa (Leishmania, Plasmodium). To date, vaccines against some pathogens (HIV, C.trachomatis) have not been developed at all, and existing vaccines against others (M. tuberculosis: B.anthracis) are not effective enough. As shown by numerous studies (Kaufman SH. 1995 Immun Infekt., Nadia R. 2008 Cellular Microbiology, Annie L. 2004 Cellular Microbiology, Balaram P.2009 Int. J. Med Microbiol.), The formation of specific T is required for vaccination against all these pathogens. lymphocytes to the corresponding antigens.

So, at present, pulmonary tuberculosis (TB) continues to be one of the most common infectious diseases. According to WHO, around 2 million people die from TB every year around the world, and about a third of the world's population are infected. TB incidence and mortality in Russia remain one of the highest in the world. The BCG vaccine remains the only approved human vaccine against TB. This vaccine effectively reduces the incidence of disseminated TB and tuberculous meningitis in children, but it is not effective against adult pulmonary TB. This implies the need for new vaccines.

There are several directions for the development of new TB vaccines, alternative to BCG: subunit vaccines, DNA vaccines and live vaccines with improved properties. Subunit vaccines are most fully characterized in experimental animal models, including preparations consisting of several secreted Mycobacterium tuberculosis proteins / peptides (Doherty T.M. 2004 J Infect Dis.). Many M. tuberculosis antigens have been identified, potentially important as components of new vaccines. These include, in particular, immunodominant secreted antigens present in the culture filtrate of M. tuberculosis. Among the secreted M. tuberculosis antigens, ESAT-6 (Early Secret Antigen Target-6), CFP-10 (culture-filtrate protein-10) and complex 85 proteins (Ag85A, B, C) are best studied.

The ESAT-6 and CFP-10 proteins are translated as a single reading frame, RD1, present in different strains of virulent mycobacteria, but lost by the BCG vaccine strain. Both native and recombinant ESAT-6 and CFP-10 proteins induce the synthesis of interferon-gamma (IFN-gamma, IFN-γ) by peripheral blood T lymphocytes of M. tuberculosis infected individuals (Johnson PD 1999 Clin. Diagn. Lab. Immunol, Berthet PX 1998 Microbiology), therefore, both proteins are used to develop various serological tests for the diagnosis of tuberculosis (Arend SM 2000 J. Infect. Dis., Van Pinxteren LA, 2000 Clin. Diagn. Lab. Immunol.).

The closely related Ag85A, B, C are encoded by three separate genes present in many strains of mycobacteria, including the BCG vaccine strain (Wiker H.G. 1992 Microbiol. Reviews). Antigens 85 are also involved in the formation of a T-cell response in individuals infected with M. tuberculosis (Launois P, 1994 Infect. Immun.).

Another serious problem is sexually transmitted diseases. For example, Chlamydia trachomatis infects more than 90 million people a year. Despite numerous attempts to create new chlamydial vaccines, it has not yet been possible to develop a vaccine that provides long-term protection (Hafner L 2008 Mucosal Immunol.). It is believed that a protective immune response against chlamydia depends on a population of CD4 ⁺ Th lymphocytes, which, by activating macrophages, increase their phagocytic and antimicrobial activity (Nadia R 2008 Cellular Microbiology).

Influenza virus is a dangerous infectious agent that can cause global pandemics (Deeva E.G. 2008 GEOTAR-Media). To date, attenuated and inactivated vaccines are used to vaccinate animals and humans against the influenza virus, which are aimed at the formation of specific antibodies. However, the narrow specificity of antibodies makes them ineffective against other subtypes of viruses with different antigenic structures. It seems promising to create influenza vaccines that stimulate the formation of a specific population of T-lymphocytes that recognize influenza antigens. Such T lymphocytes can activate B cells producing antibodies against various virus subtypes. The combination of highly specific “B-cell” vaccines and “T-vaccines” vaccination with broad specificity can increase the effectiveness of vaccination.

T-lymphocytes of the two main subclasses, CD4 ⁺ and CD8 ⁺ , provide immunological recognition and the immune response on their own. In particular, activated T cells, especially CD8 ⁺ and NKLT, are able to exert a direct cytotoxic effect on infected cells. However, the ability of T cells to activate activating interactions with B-lymphocytes (humoral immunity), neutrophils and macrophages (innate immunity) is even more important for protection against pathogens. One of the main elements of intercellular interactions in the immune system is the secretion of a number of cytokines by T cells and expression of bound ligands to various receptors on the membrane. In particular, T-cell cytokines INF-γ and TNFα have the following properties:

- INF-γ activates the antibacterial properties of macrophages, increasing the production of reactive oxygen and nitrogen;

- INF-γ secreted by T cells, triggers the process of autophagy and, thus, leads to cleansing of intracellular parasites;

- INF-γ induces apoptosis in infected cells;

- activated macrophages increase the secretion of TNF-α, which, through activation of the vascular endothelium, attracts cells of the immune system to the point of infection.

Activation of T cells requires the presentation of antigens to antigen-presenting cells, leading to immunological recognition, as well as a second signal, which is provided by a complex system of auxiliary receptors and regulatory cascades. It is known that TLR receptors, a group of receptors that recognize individual groups of prokaryotic parasite molecules, play a large role in the activation of immune cells. When activated, they transmit signals to transcription factors such as NF-kB, AP-1 and IRF, which increase the expression of co-stimulatory molecules (CD40, CD80, CD86), interferons and interleukins, thereby introducing the cell into an activated state. The creation of artificial or genetic engineering use of natural ligands for TLR is a very promising direction in the design of adjuvants for vaccines. In particular, work is underway with CpG oligonucleotides that activate the TLR9 receptor, a bacterial flagellin protein that can activate the TLR5 receptor, etc. (McDonald WF 2007 J Infect Dis., Huleatt JW 2007, 2008 Vaccine.).

To present antigenic peptides to T cells, processing and delivery of intracellular antigenic molecules to the surface of antigen-presenting cells is necessary. These processes are associated with a number of receptors, which, on the one hand, have motives for internalizing receptor-antigen complexes, and, on the other hand, are capable of conducting a specific signal upon activation. Such motives include the immunoreceptor tyrosine activation motif (ITAM motif). For example, Fc receptors are able to bind immunoglobulin-antigen complexes, and with the help of dectin-1 cells are able to phagocytose the components of the fungal cell wall.

On the other hand, it has been proven that the activation of TLR receptors and receptors containing ITAM motifs, individually or in different parts of the membrane, does not lead to the secretion of key cytokines necessary for the formation of an adaptive immune response (Ivashkiv LB. 2008, 2009 Nat Rev Immunol, Colonna M 2003 Nat Rev Immunol., Novak N 2010 Int Arch Allergy Immunol.). For example, activation of TLR2 or dectin-1 alone does not induce an increase in IL-12p40. However, the simultaneous stimulation of both receptors greatly increases the production of this cytokine (Gantner BN 2003 J Exp Med.).

One approach to creating vaccines leading to the induction of T lymphocytes has been implemented in the creation of a chimeric protein consisting of an antigen and / or adjuvant based on the Fc fragment of an antibody (Seok YJ 2010 Appi Biochem Biotechnol., Wang M 2008 Scand J Immunol. ) Such vaccines have been proposed to be used either in the form of a subunit vaccine (purified protein) or in the form of a vector carrying the gene of the protein. This technical solution, as the closest to the claimed one, was selected for the prototype (Patent No. US 7,067,110 B1).

The disadvantages of the prototype:

1) As the base molecule, to which antigen, adjuvant, or both are added to one fusion protein, only the Fc fragment of antibodies is used.

2) When constructing a protein consisting of three parts in which the Fc fragment is located either in the center or in the N-terminal part, the functional activity of the Fc fragment is lost.

3) Fc-adjuvant fusion proteins act as adjuvants, in which cytokines such as INFγ, IL-2, IL-4, IL-12, IL-18, TNF, GMCSF and CD40 ligand act as adjuvant. Activation of the innate immune response (which leads to an adaptive immune response) depends on TLR receptors, and cytokines can only modulate this response.

4) When constructing vaccines consisting of a mixture of two proteins - Fc-antigen and Fc-adjuvant, the adjuvant properties of the Fc-adjuvant protein are reduced, because according to current immunological concepts, the antigen with adjuvant should be physically related (Gantner BN 2003 J Exp Med.).

The objective of the invention is to obtain an effective and versatile vaccine against various pathogens, consisting of recombinant chimeric proteins and nucleic acids encoding them, a single injection of which leads to the formation of specific T-lymphocytes.

The essence of the invention is to increase the immunogenicity of chimeric proteins, due to the introduction of two ligands in the composition of the chimeric protein of the ligand to TLR receptors and the ligand to receptors that signal through the ITAM motif. Activation of TLR receptors by a ligand directly associated with the antigen leads to the induction of expression of co-stimulatory molecules and the maturation of antigen-presenting cells. Receptors such as Dectin-1, Fc receptors, and KIR transmit a signal through ITAM motifs and promote antigen phagocytosis, which ultimately leads to the presentation and recognition of antigens by T cells. Thus, activation via TLR receptors and modulation through the ITAM motive of the immune response during vaccination allows the formation of an effective adaptive immune response.

The inventive method has the following advantages compared with analogues and prototype:

1) In contrast to the prototype method, the claimed invention does not have a fixed base sequence, the vaccine consists of the antigen of the pathogenic organism, any ligand to the TLR receptor and the ligand to the receptor that conducts the signal through the ITAM motif.

2) The antigen, the ligand to the TLR receptor and the ligand of the receptor that conducts the signal through the ITAM motif can be located in any part of the chimeric protein. The choice of the arrangement order is determined taking into account the functional activity of each component and options are selected without loss of activity of each component.

3) Any ligand to the TLR receptor acts as an adjuvant in our invention.

4) To modulate the immune response in the claimed invention uses ligands that interact with a receptor that transmits a signal through an ITAM motif. Such ligands are the domains of MICA molecules, Fc fragments of antibodies and the domain of the ULBP1 molecule.

Example 1. The creation of a nucleotide sequence encoding a chimeric protein against a pathogen on the example of M. tuberculosis.

Recombinant adenoviral vector was selected as the carrier of the active substance of the vaccine. By chemical synthesis, the nucleotide sequence of LP-Ag85B-Flagellin-FcIgG2a is obtained, consisting of the leader peptide of tissue plasminogen activator (tpa), M. tuberculosis Ag85B antigen, S.enterica flagellin and Mus musculus IgG2a Fc fragment (Figure 1). The Western blot method evaluates the expression and secretion of this chimeric antigen (figure 2). The biological activity of flagellin is measured as part of a chimeric protein on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR5 gene (Figure 3). The measurement of the biological activity of flagellin in the composition of the chimeric protein expressed from the Ad-Ag85B-Fl-FcIgG2a adenoviral vector is evaluated on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR5 gene. Measurement of β-galactosidase activity is performed 24 hours after adding the test drug to the cells, the whole medium is removed from them and the buffer with β-galactosidase substrate is poured (1 mM MgCl ₂ ; 0.25 M Tris-HCl, pH 7.4; 0 , 02% NP ₄ O; 2 g / l o-nitrophenyl-β-D-galactopyranoside). The level of activity of β-galactosidase is determined spectrophotometrically (414 nm) by the conversion of o-nitrophenyl-β-D-galactopyranoside.

.In the lymphoproliferative assay, the ability of a chimeric protein to possess immunogenic properties is evaluated (Figure 4). The specific antigen-proliferative response of primed T cells is measured on the cells of the popliteal and inguinal lymph nodes. Lymph node cells were cultured in 96-well flat-bottomed plates (Costar), at a concentration of 2.5 × 10 ⁵ cells / well in 0.2 ml of culture medium containing 5% ETS, incubated with 10 μg / ml of antigen for 72 hours at 37 ° C in 5% CO ₂ . For the last 18 hours, 0.5 µCi of methyl- [3H] -thymidine was added to the wells. The contents of the wells are transferred to the filters using a semi-automatic harvester (Scatron, Norway), and the inclusion of the radioactive label in the nuclei of dividing cells on a liquid scintillation counter (Wallac, Finland) is evaluated. Results are presented in pulses per minute (cpm) or in stimulation indices

.

Further investigate the ability of triple chimeric protein to provide protective properties against mycobacterial infection (figure 5). Groups of four C57BL / 6 mice were immunized twice with recombinant adenoviruses at two-week intervals. Immunization is performed subcutaneously, in a volume of 300 μl of 6 · 10 ⁹ PFU / mouse. Five weeks after the second immunization, mice were challenged with iv 10 · ^{6 6} CFU / mouse M. tuberculosis H37Rv. The results are evaluated by seeding mycobacteria from organs.

Vaccination with this construct, which encodes, in addition to the mycobacterial antigen, flagellin and the Fc fragment of immunoglobulin, significantly reduces the number of mycobacteria in the lungs (A) and spleen (B) compared with the control groups. Thus, a recombinant chimeric antigen capable of providing protective properties against M. tuberculosis infection was obtained.

Example 2. The creation of a nucleotide sequence encoding a chimeric protein against a pathogen by the example of B.anthracis.

Recombinant adenoviral vector and plasmid pUC19 were selected as the carrier of the active substance of the vaccine. By chemical synthesis, the nucleotide sequence of LP-PA-Flagellin-FcIgG2a is obtained, consisting of the leader peptide of the tissue plasminogen activator (tpa), the protective antigen B.anthracis, S.enterica flagellin and the Muscus musculus IgG2a Fc fragment (Figure 6). The Western blot method assesses the expression and secretion of this chimeric antigen (figure 7). The biological activity of flagellin is measured as part of a chimeric protein on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR5 gene (Figure 8). The measurement of the biological activity of flagellin in the composition of the chimeric protein expressed from the adenoviral vector Ad-PA-Fl-FdgG2a was evaluated on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expressing the TLR5 gene. Measurement of β-galactosidase activity is performed 24 hours after adding the test drug to the cells, the whole medium is removed from them and the buffer with β-galactosidase substrate is poured (1 mM MgCl ₂ ; 0.25 M Tris-HCl, pH 7.4; 0 , 02% NP ₄ O; 2 g / l o-nitrophenyl-β-D-galactopyranoside). The level of activity of β-galactosidase is determined spectrophotometrically (414 nm) by the conversion of o-nitrophenyl-β-D-galactopyranoside.

.In the lymphoproliferative assay, the ability of a chimeric protein to possess immunogenic properties is evaluated (Figure 9). The specific antigen-proliferative response of primed T cells is measured on the cells of the popliteal and inguinal lymph nodes. Lymph node cells were cultured in 96-well flat-bottomed plates (Costar), at a concentration of 2.5 × 10 ⁵ cells / well in 0.2 ml of culture medium containing 5% ETS, incubated with 10 μg / ml of antigen for 72 hours at 37 ° C in 5% CO ₂ . For the last 18 hours, 0.5 µCi of methyl- [3H] -thymidine was added to the wells. The contents of the wells are transferred to the filters using a semi-automatic harvester (Scatron, Norway), and the inclusion of the radioactive label in the nuclei of dividing cells on a liquid scintillation counter (Wallac, Finland) is evaluated. Results are presented in pulses per minute (cpm) or in stimulation indices

.

Further investigate the ability of triple chimeric protein to provide protective properties against the introduction of lethal toxin B.anthracis. Groups of ten Balb / c mice are immunized with the recombinant Ad-null and Ad-LP-PA-Flagellin-FcIgG2a adenoviruses. Immunization is performed intramuscularly in a volume of 50 μl of 10 ⁹ PFU / mouse. Plasmid DNA is administered intramuscularly in a volume of 50 μl of 100 μg / mouse. Four weeks after immunization, 85 ml of a lethal B.anthracis toxin is administered intravenously in a volume of 100 μl. The results are evaluated by the survival of mice (figure 10).

Vaccination with recombinant adenovirus encoding the triple chimeric protein gene protects 80% of mice from death. Plasmid DNA vaccination protects 50% of mice. Thus, a recombinant chimeric antigen capable of providing protective properties against B.anthracis infection was obtained.

Example 3. The creation of a nucleotide sequence encoding a chimeric protein against a pathogen on the example of C.trachomatis.

Recombinant adenoviral vector was selected as the carrier of the active substance of the vaccine. By chemical synthesis, the nucleotide sequence of LP-OmcB-Flagellin-FcIgG2a is obtained, consisting of the leader peptide of tissue plasminogen activator (tpa), OmcB C. trachomatis antigen, flagenterin S.enterica, and Muscus musculus IgG2a Fc fragment (Figure 11). The Western blot method evaluates the expression and secretion of this chimeric antigen (figure 12). The biological activity of flagellin is measured as part of a chimeric protein on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR5 gene (Figure 13). The measurement of the biological activity of flagellin in the composition of the chimeric protein expressed from the adenoviral vector Ad-PA-Fl-FcIgG2a was evaluated on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expressing the TLR5 gene. Measurement of β-galactosidase activity is performed 24 hours after adding the test drug to the cells, the whole medium is removed from them and the buffer with β-galactosidase substrate is poured (1 mM MgCl ₂ ; 0.25 M Tris-HCl, pH 7.4; 0 , 02% NP ₄ O; 2 g / l o-nitrophenyl-β-D-galactopyranoside). The level of activity of β-galactosidase is determined spectrophotometrically (414 nm) by the conversion of o-nitrophenyl-β-D-galactopyranoside.

.In the lymphoproliferative assay, the ability of a chimeric protein to possess immunogenic properties is evaluated (FIG. 14). The specific antigen-proliferative response of primed T cells is measured on the cells of the popliteal and inguinal lymph nodes. Lymph node cells were cultured in 96-well flat-bottomed plates (Costar), at a concentration of 2.5 × 10 ⁵ cells / well in 0.2 ml of culture medium containing 5% ETS, incubated with 10 μg / ml of antigen for 72 hours at 37 ° C in 5% CO ₂ . For the last 18 hours, 0.5 µCi of methyl- [3H] -thymidine was added to the wells. The contents of the wells are transferred to the filters using a semi-automatic harvester (Scatron, Norway), and the inclusion of the radioactive label in the nuclei of dividing cells on a liquid scintillation counter (Wallac, Finland) is evaluated. Results are presented in pulses per minute (cpm) or in stimulation indices

.

The ability of a triple chimeric protein to induce and synthesize specific antibodies against C.trachomatis is further investigated. Groups of six Balb / c mice are immunized twice with recombinant adenoviruses Ad-null and Ad-OmcB-PA-Flagellm-FcIgG2a. Immunization is carried out intranosally, in a volume of 50 μl of 6.8 · 10 ⁹ PFU / mouse. Two weeks after immunization, a blood sampling was taken from experimental animals and the titers of specific antibodies to C. trachomatis OmcB antigen were determined by the culture method. Immunogenicity is assessed by antibody titer: diagnostic titer from 1:32 and above (figure 15-16).

Vaccination with a recombinant adenovirus encoding the triple chimeric protein gene induces the synthesis of specific antibodies against the C. trachomatis OmcB antigen. Thus, a recombinant adenovirus capable of secreting a triple chimeric antigen was obtained, on which specific antibodies are generated that interact with the surface antigen of C.trachomatis.

Example 4. The creation of a nucleotide sequence encoding a chimeric protein against a pathogen on the example of Influenza virus.

Recombinant adenoviral vector was selected as the carrier of the active substance of the vaccine. By chemical synthesis, the nucleotide sequence of LP-NP-Flagellin-FcIgG2a is obtained, consisting of the leader peptide of the tissue plasminogen activator (tpa), NP antigen Influenza vims, S.enterica flagellin and Mus musculus IgG2a Fc fragment (Figure 17). The Western blot method assesses the expression and secretion of this chimeric antigen (Figure 18). The biological activity of flagellin in the composition of the chimeric protein is measured on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR5 gene (Figure 19). The measurement of the biological activity of flagellin in the composition of the chimeric protein expressed from the adenoviral vector Ad-NP-Fl-FcIgG2a was evaluated on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expressing the TLR5 gene. Measurement of β-galactosidase activity is carried out 24 hours after adding the test drug to the cells, the whole medium is removed from them and the buffer with β-galactosidase substrate is poured (1 mM MgCl ₂ ; 0.25 M Tris-HCl, pH 7.4; 0 , 02% NP ₄ O; 2 g / l o-nitrophenyl-β-D-galactopyranoside). The activity level of β-galactosidase is determined spectrophotometrically (414 nm) from the conversion of o-nitrophenyl-β-D-galactopyranoside.

.In the lymphoproliferative assay, the ability of a chimeric protein to possess immunogenic properties is evaluated (FIG. 20). The specific antigen-proliferative response of primed T cells is measured on the cells of the popliteal and inguinal lymph nodes. Lymph node cells were cultured in 96-well flat-bottomed plates (Costar), at a concentration of 2.5 × 10 ⁵ cells / well in 0.2 ml of culture medium containing 5% ETS, incubated with 10 μg / ml of antigen for 72 hours at 37 ° C in 5% CO ₂ . For the last 18 hours, 0.5 µCi of methyl- [3H] -thymidine was added to the wells. The contents of the wells are transferred to the filters using a semi-automatic harvester (Scatron, Norway), and the incorporation of the radioactive label into the nuclei of the dividing cells on a liquid scintillation counter (Wallac, Finland) is evaluated. Results are presented in pulses per minute (cpm) or in stimulation indices

.

Further investigate the ability of triple chimeric protein to provide protective properties against Influenza virus. Groups of ten Balb / c mice were immunized twice at two-week intervals with the recombinant adenoviruses Ad-null and Ad-LP-NP-Flagellin-FcIgG2a. Immunization is performed intranosally, in a volume of 50 μl of 10 ⁹ PFU / mouse. Four weeks after immunization, mice are infected with Influenza virus A / USSR / 90/1977 (H1N1), intranosally in a volume of 100 μl. The results are evaluated by the survival of mice (figure 21).

Vaccination with a recombinant adenovirus encoding the triple chimeric protein gene protects 60% of mice from death. Thus, a recombinant chimeric antigen capable of providing protective properties against Influenza virus infection was obtained.

Example 5. The creation of a nucleotide sequence encoding a chimeric protein against a pathogen on the example of Influenza virus.

Recombinant adenoviral vector was selected as the carrier of the active substance of the vaccine. By chemical synthesis, the nucleotide sequence of LP-M1-Tc52-MICA is obtained, consisting of the leader peptide of plasminogen tissue activator (tpa), M1 antigen Influenza virus, Tc52 Trypanosoma cruzi and MICA Homo sapiens (figure 22). The Western blot method evaluates the expression and secretion of this chimeric antigen (figure 23). The biological activity of flagellin is measured as part of a chimeric protein on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR2 / 6 gene (Figure 24). The measurement of the biological activity of flagellin in the composition of the chimeric protein expressed from the adenoviral vector Ad-M1-Tc52-MICA was evaluated on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expressing the TLR2 / 6 gene. Measurement of β-galactosidase activity is performed 24 hours after adding the test drug to the cells, the whole medium is removed from them and the buffer with β-galactosidase substrate is poured (1 mM MgCl ₂ ; 0.25 M Tris-HCl, pH 7.4; 0 , 02% NP ₄ O; 2 g / l o-nitrophenyl-β-D-galactopyranoside). The level of activity of β-galactosidase is determined spectrophotometrically (414 nm) by the conversion of o-nitrophenyl-β-D-galactopyranoside.

.In the lymphoproliferative assay, the ability of a chimeric protein to possess immunogenic properties is evaluated (FIG. 25). The specific antigen-proliferative response of primed T cells is measured on the cells of the popliteal and inguinal lymph nodes. Lymph node cells were cultured in 96-well flat-bottomed plates (Costar), at a concentration of 2.5 × 10 ⁵ cells / well in 0.2 ml of culture medium containing 5% ETS, incubated with 10 μg / ml of antigen for 72 hours at 37 ° C in 5% CO ₂ . For the last 18 hours, 0.5 µCi of methyl- [3H] -thymidine was added to the wells. The contents of the wells are transferred to the filters using a semi-automatic harvester (Scatron, Norway), and the inclusion of the radioactive label in the nuclei of dividing cells on a liquid scintillation counter (Wallac, Finland) is evaluated. Results are presented in pulses per minute (cpm) or in stimulation indices

.

Thus, a recombinant adenovirus expressing the chimeric antigen M1-Tc52-MISA capable of possessing immunogenic properties was obtained.

Example 6. The creation of the nucleotide sequence encoding a chimeric protein against a pathogen on the example of Influenza virus.

By chemical synthesis, the nucleotide sequence of LP-CSP-EDN-ULBP1 is obtained, consisting of the plasminogen tissue activator leader peptide (tpa), CSP Plasmodium malariae antigen, eosinophil neurotoxin - EDN Homo sapiens and ULBP1 Homo sapiens (Figure 26). The Western blot method evaluates the expression and secretion of this chimeric antigen (figure 27). The biological activity of flagellin is measured as part of a chimeric protein on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR2 / 6 gene (Figure 28). The measurement of the biological activity of EDN in the composition of the chimeric protein is evaluated on the HEK293 cell line, which carries the β-galactosidase gene under the NF-kB promoter and expresses the TLR2 / 6 gene. Measurement of β-galactosidase activity is performed 24 hours after adding the test drug to the cells, the whole medium is removed from them and the buffer with β-galactosidase substrate is poured (1 mM MgCl ₂ ; 0.25 M Tris-HCl, pH 7.4; 0 , 02% NP ₄ O; 2 g / l o-nitrophenyl-β-D-galactopyranoside). The level of activity of β-galactosidase is determined spectrophotometrically (414 nm) by the conversion of o-nitrophenyl-β-D-galactopyranoside.

.In the lymphoproliferative assay, the ability of a chimeric protein to possess immunogenic properties is evaluated (Figure 29). The specific antigen-proliferative response of primed T cells is measured on the cells of the popliteal and inguinal lymph nodes. Lymph node cells were cultured in 96-well flat-bottomed plates (Costar), at a concentration of 2.5 × 10 ⁵ cells / well in 0.2 ml of culture medium containing 5% ETS, incubated with 10 μg / ml of antigen for 72 hours at 37 ° C in 5% CO ₂ . For the last 18 hours, 0.5 µCi of methyl- [3H] -thymidine was added to the wells. The contents of the wells are transferred to the filters using a semi-automatic harvester (Scatron, Norway), and the inclusion of the radioactive label in the nuclei of dividing cells on a liquid scintillation counter (Wallac, Finland) is evaluated. Results are presented in pulses per minute (cpm) or in stimulation indices

.

Thus, a recombinant chimeric protein LP-CSP-EDN-ULBP1 was obtained that contained the CSP Plasmodium malariae antigen with immunogenic properties.

Thus, the claimed method allows to obtain vaccine preparations against various pathogens with immunogenic and protective properties.

Claims (1)

A method for producing a recombinant vaccine, comprising constructing a nucleotide sequence encoding a chimeric protein, or an amino acid sequence characterizing such a chimeric protein, where the chimeric protein consists of an antigen of a pathogenic organism, a ligand to receptors that conducts a signal through an ITAM motif — an Fc fragment of antibodies, characterized in that the nucleotide sequence encoding a chimeric protein includes a sequence encoding a ligand for TLR receptors, or an amino acid sequence The characteristics characterizing such a chimeric protein include the amino acid sequence of the ligand to TLR receptors, and where MICA or ULBP1 domains are used as ligands that conduct the signal through the ITAM motif, the nucleotide sequence being attached to a carrier that is plasmid DNA or recombinant adenoviral particle.

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