RU2676768C2 - Chlamydial vaccine and method for its preparation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to immunology, and relates to a combination of chlamydial vaccine. Vaccine consists of a recombinant adenoviral vector based on human fifth-type adenovirus serotype expressing the gene for the full-length structural protein CdsF of the chlamydia type III secretion system, and an emulsion containing recombinant structural protein rCdsF secretion system type III chlamydia and a combined adjuvant. Methods for producing a vaccine and methods for producing recombinant proteins for a combination vaccine is also proposed.

EFFECT: group of inventions provides for the induction of a protective immune response against a chlamydial infection in a subject.

14 cl, 30 dwg, 9 ex

Description

The invention relates to biotechnology and medicine and relates to a chlamydial vaccine and a method for its preparation. The vaccine proposed for the prevention of chlamydia is able to protect against urogenital chlamydia and venereal lymphogranuloma.

State of the art

As you know, chlamydia are intracellular pathogenic bacteria responsible for a wide range of important infections of humans and animals. Chlamydia is a serious public health problem in both developed and developing countries. Urogenital chlamydia caused by Chlamydia trachomatis is a socially significant, most commonly reported, sexually transmitted bacterial infectious disease. According to the World Health Organization (WHO), more than 100 million cases of C. trachomatis infection are diagnosed annually (WHO, 2012). A significant number of asymptomatic infections also contributes to the spread of urogenital chlamydia. Most often, chlamydia is clinically manifested as urethritis and cervicitis. Acute inflammation usually subsides within a few weeks. However, a significant number of patients experience long-term complications, including inflammatory processes in the pelvic organs, ectopic pregnancy and infertility. In developing countries, trachoma and lymphogranuloma caused by C. trachomatis are also often recorded.

A scientifically based method for the prevention of chlamydia is vaccination. To date, not a single commercial vaccine aimed at the prevention and treatment of diseases caused by C. trachomatis has been registered. This is due to the characteristics of the immune response to chlamydia. Thus, it was shown that the immune response to chlamydia is often accompanied by damage to the tissues of the reproductive organs [41], which is mediated by the production of inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNFα) [25, 26, 41]. Thus, the main task in the development of a chlamydial vaccine is the formation of an immune response aimed at resolving inflammatory processes in the reproductive organs and not causing the development of pathology. In addition, when developing a vaccine preparation composition, it is preferable to use conservative antigens capable of inducing a protective immune response against various serotypes of C. trachomatis.

It is important to form an immune response effective at various stages of the life cycle of chlamydia, both in acute and chronic infections. Currently, it is believed that the creation of an effective vaccine against chlamydial infection involves the formation of a response of specific T-helpers of the first type (T × 1), which can efficiently reach the tissues and organs of localization of the pathogen, and the formation of specific systemic and local antibodies [5].

The development of genetic vaccines is primarily based on the selection of antigens with the highest immunogenicity and protective activity. Currently, a number of C. trachomatis antigens have been studied with a view to their potential use in the development of vaccine preparations. Chlamydial proteins of the outer membrane, such as the main outer membrane protein MOMP (Major Outer Membrane Protein), polymorphic proteins POMPs (Polymorphic Outer Membrane Proteins), cysteine rich proteins CrpA (Cysteine richp rote A), OmcB (Outer membrane complex protein B) [24, 30, 38, 42].

The most studied can be attributed to the main protein of the outer membrane, MOMP, which has high immunogenicity while maintaining its native conformation [10]. A known composition based on MOMP antigens obtained from various serovars of C. trachomatis and / or other types of chlamydia, as well as the clinically approved adjuvant E6020, CAS Number 287180-63-6 (WO 2012065263 A1). This development is registered as a method of inducing an immune response to various types of chlamydia. The advantage of this technical solution is the wide spectrum of action of the drug based on the recombinant MOMP protein and E6020 adjuvant in relation to various types of chlamydia and the use of an adjuvant approved for use in a clinic in the United States.

The disadvantages of development are:

1. The lack of data regarding the effectiveness of the proposed vaccine preparation, in relation to the pathology developing in models other than urogenital chlamydia caused by C. trachomatis.

2. The need for a 3-fold administration of a vaccine preparation, which complicates the immunization procedure.

3. An insufficiently characterized T-cell response, its orientation and duration.

4. In this work, ligands of toll-like receptors of types 7 and 8 were used. However, there was no evidence of the effect of the used natural resistance activators on the effectiveness of a vaccine preparation containing a composition of MOMP antigens.

The need to maintain the native conformation of antigen in the composition of the vaccine creates significant technological difficulties and makes the production process of such a vaccine unnecessarily expensive [34, 38]. In addition, this antigen is variable, which also significantly limits the effectiveness of the immune response to it against various serotypes of C. trachomatis [27, 32, 46, 47].

A technical solution is known in which a chimeric vaccine against hepatitis B and C. trachomatis is created in which peptides of the recombinant MOMP are included in the hepatitis B core antigen (HBcAg). It has been shown that such a chimeric protein is able to induce a cellular and humoral immune response against C. trachomatis. The immunogenicity and immunoprotective properties of various variants of chimeric proteins have been demonstrated [50].

The disadvantages of this technical solution can be considered:

1. The universality of the studied vaccine preparation for all C. trachomatis serotypes was not confirmed.

2. The absence in the chimeric protein of any additional components that stimulate the innate immune response, which may limit the ability of the vaccine to suppress intracellular infection.

3. The need for multiple injections of the vaccine preparation, which complicates the immunization procedure.

4. The anti-inflammatory potential of the proposed vaccine has not been characterized.

Other candidate vaccine target proteins are secreted proteins. It is known to use the secreted chlamydial protein CPAF (Chlamydial Protease / proteasome-like Activity Factor), which is a protease / factor with proteasome-like activity [22]. Intranasal immunization with recombinant CPAF protein in combination with IL-12 induced a pronounced production of interferon-γ (IFγ) and led to a significant decrease in pathogen accumulation in the genital tract of mice, as well as limited the development of complications in the upper parts of the reproductive system [28, 48].

The creation of a vaccine based on an adenovirus vector expressing CPAF protein (Ad-CPAF) can be considered an analogue [9]. Immunization of Ad-CPAF with boosting with recombinant CPAF (rCPAF) in combination with CpG oligodeoxynucleotide adjuvants and the synthetic immunomodulating peptide HH2 (CpG / HH2) led to the formation of a humoral protective response in mice with urogenital infection of C. trach.

The disadvantages of this development are:

1. Such immunization caused the formation of an insignificant T-cell response with a predominance of Tx1 and Tx17. It should be noted that Tx17 production is associated with the development of inflammatory reactions in the reproductive organs with urogenital chlamydia, leading to severe pathology.

2. The adjuvant used in the study is not approved for use in clinics in Russia and abroad.

A new approach is the use of type III secretion system effector protein (CCTT), TARP (Translocated Actin-Recruiting Phosphoprotein) as the target protein for the vaccine [42]. The induction of an immune response to the TARP CCTT chlamydia protein may enhance the defense mechanisms against the pathogen. The use of TARP for immunization led to the formation of protective Tx1 immunity, reduced productive infection in the lower reproductive tract, and the development of pathology of the fallopian tubes [42]. It should be noted that TARP is not an antigen constantly expressed by chlamydia, and perhaps the immune response to it will not be effective at the stage of chronic infection. In addition, in this study [42], CpG and Freund's incomplete adjuvant (NAF) were used as an adjuvant, which were not approved for use in clinics in Russia and abroad.

It is known to develop a vaccine for cross-protection against a wide range of Chlamydia trachomatis serovars based on polypeptide compositions containing one chlamydial antigen or several chlamydial proteins [51]. A variant of such a vaccine may contain the Ct-089 protein, also known as CopN, the alleged exported regulator of type III secretion system [49]. In the case of using only this protein, the disadvantage is its presence in the cell wall of chlamydia not at all stages of the development of the pathogen.

A technical solution is known in which highly immunogenic B-cell epitopes of the effector protein of the third transport system TARP C. trachomatis are proposed for the vaccine [50]. A strong point of this invention is the immunization with epigones of this chlamydial CTTT protein, which is aimed at enhancing the protective potential of the host organism.

The disadvantages of this technical solution are:

1. Lack of developed antigen and adjuvant delivery systems allowed in clinics in Russia and abroad.

2. Immunization is aimed at the B-cell response, which is not enough to create a long-term protective immunity.

3. The use of peptide epitopes of the effector protein of the third transport system of TARP C. trachomatis requires multiple immunizations.

4. Since TARP is not an antigen constantly expressed by chlamydia, the immune response to it may not be effective at the stage of chronic infection.

One of the promising targets for the creation of antichlamydia vaccines can be considered a type III secretion system, which is the dominant factor in the pathogenicity of chlamydia. This structure is characterized by a high degree of conservatism for all members of the Chlamydiaceae family. It provides transport of effector molecules to the host cell, which allows to suppress the host's defense and maintain the infectious process in both acute and chronic infections. This secretion system is characterized by the presence of more than 20 proteins that make up the apparatus (“molecular syringe”) for transporting pathogenicity factors into the host cell, interacting with the membrane or penetrating directly into the cytoplasm of the host cell and changing its normal physiological state, promoting invasion and intracellular reproduction of the pathogen [1 ]. An annular protein structure is located in the inner membrane of bacteria, which plays the main role in the recognition of secreted molecules, in the initiation of the secretion process and its energy supply. This protein structure consists of a basal body and surrounding exporting proteins, which ensure active transport of proteins into the periplasmic space. Directly connected to this basal structure is a protein channel passing through the peptidoglycan and the outer membrane of the bacterial cell. In the outer membrane, the channel is fixed by ring protein structures (secreton). Secreton consists of a number of bacteria outer membrane proteins involved in the transport of effector proteins and translocon components through the outer membrane of the bacterium. Above the surface of the microbial cell stands the protein structure — the “needle” and the translocon, which forms the pore in the membrane of the eukaryotic cell [8]. The CCTT needle has dimensions of about 60-80 nm in length and 8 nm in diameter and is formed by structural proteins present on the surface of the outer membrane of the cell wall. The diameter of the hole in the needle is approximately 3 nm.

It is known that a single bacterium can have several hundred structures of CTST. Chlamydia at all stages of the life cycle in acute infection, as well as in chronic infection, are completely dependent on the functioning of CTST. This secretory apparatus provides the pathogen with invasion, blocking the immune response, and preventing fusion with the lysosome; regulates the maturation of inclusion and its migration in the cytoplasm towards the center of microtubule organization; determines the intake of nutrients and lipids; allows you to control the cell cycle and signaling pathways that induce inflammation, and also provides a new generation of infectious particles after the end of the intracellular cycle. Thus, the suppression of the functional activity of the CCTT of chlamydia leads to the suppression of the multiplication of the pathogen and the blocking of the infectious process, which indicates the promising choice of CCTT proteins as targets for creating vaccines.

Chlamydia structural protein CdsF (Chlamydia needle filament protein of the injectisome) CTTT, proposed as the main component of the candidate vaccine in our invention, is the main injectosome protein of the third Chlamydia transport system. CdsF protein concentrates on the outer membrane of elementary bodies (ET) and reticular bodies (RT) and forms a surface structure, referred to as a “needle” [13]. During contact with a eukaryotic cell, the CdsF protein polymerizes and, as a result, the injection (assembly) of the injectosome [6]. The formation of an immune response against this key component of CCT chlamydia seems extremely promising. Being a structural protein of CTST, CdsF is expressed on the chlamydia membrane at all stages of the pathogen development, including both in acute and chronic infections. In addition, the CdsF CCTT “needle” chlamydial protein contains cysteine residues that are unique to the CCTT “needle” proteins [6]. Disulfide bonds in the polymerized “needle” of CCTT are associated with the oxidation state of the chlamydial membrane and the stages of pathogen development [7]. It is suggested that disulfide bonds found in CdsF localized in elementary bodies (ETs) can be involved in the functioning of the chlamydial secretory apparatus.

Since antigens with the greatest protective potential are not always highly immunogenic, the development of effective and safe delivery systems and the rational use of adjuvants that are allowed in clinical practice is extremely important.

One of the most effective approaches to creating safe and effective new generation vaccines today is the use of genetic vaccines, including those based on recombinant adenoviral vectors [45]. When such vaccines are introduced into the body, genetic material enters the cells and the genes of the target pathogen proteins are expressed in them. As a result, antigens of the corresponding pathogens are recognized by the immune system, which leads to the induction of both a humoral and a cellular immune response. Currently, the most promising and often used to create genetic vaccines are recombinant adenoviral vectors created on the basis of human adenovirus of the fifth serotype [20].

Vaccines based on recombinant adenoviral vectors have several advantages:

- recombinant adenoviral vectors are replicatively defective and are not capable of causing disease. The safety of human adenoviruses of the fifth serotype with deleted E1 and E3 regions of the genome is confirmed by clinical trials of various vaccine and therapeutic drugs based on them.

- do not require multiple additional introductions, since the expression of the target antigen (fusion protein) occurs directly in the body of the immunized subject;

- recombinant adenoviral vectors allow for intranasal immunization, and, as a result, induce the formation of a mucosal immune response;

- recombinant adenoviral vectors compared with recombinant proteins obtained in eukaryotic culture are cheaper and highly immunogenic, since their small dose allows significant amounts of protein to be produced in the body.

- At present, fast and flexible technologies for the production of recombinant adenoviral vectors have been developed that allow the large-scale production of various candidate vaccines based on them to be implemented on the same production line without re-equipment and changing the schedule. Of the genetic vaccines undergoing clinical trials, recombinant adenovirus-based vaccines account for about 24% (clinicaltrials.gov).

The above properties make recombinant adenoviral vectors a good technological platform for creating a wide range of vaccines against various pathogens [2, 12, 29, 36, 37, 44].

In addition to choosing highly immunogenic antigens and effective delivery systems, the successful selection of adjuvants plays an important role in vaccine development. One of the first adjuvants developed was oil emulsions. They are combinations of two immiscible components, usually water and oil, one of which is dispersed (dispersed) in the other in the form of drops. Since incompatible substances are combined in the emulsion, stabilizers are needed, which include surfactants and emulsifiers that cover the droplets on the outside. As adjuvants, so-called "nanoemulsions" are used - such emulsions, the diameter of which drops of oil or water is 10-1000 nm (an average of 20-600 nm). There are two main types of emulsions: oil-in-water (oil-in-water) and water-in-oil (water-in-oil). Both types have adjuvant activity and enhance the production of antibodies to vaccine antigens (this is an important indicator of the effectiveness of stimulation of immunity), but water-oil adjuvants are more reactogenic and toxic. For this reason, oil-in-water adjuvants are widely used in licensed human vaccines [16].

Most oil-water adjuvants of modern vaccines contain squalene as an oil component, the structural formula of which is presented below:

Squalene - an organic polymer from the group of carotenoids - a component of animal and plant cells, is an intermediate in the biological synthesis of steroids, and is also synthesized by a number of microorganisms. These factors determine the biocompatibility and biodegradability of squalene. Until recently, its main source was shark liver. However, after the discovery of squalene in plants, it began to be obtained from vegetable oils, since the technology for isolating squalene from shark liver is very laborious. Unlike mineral oils, which are part of many water-in-oil emulsions, squalene is much more metabolized (broken down) in the body, which means it is more biodegradable and biocompatible, a nanoemulsion

When choosing adjuvants to create a chlamydial vaccine, the following circumstance must be considered. The adjuvant used, in addition to solving the problem of increasing the immunogenicity of the vaccine preparation, should neutralize the pathological symptoms accompanying the infection. Some ligands of toll-like receptors possess such properties. Toll-like receptors are transmembrane proteins localized on the surface of the plasma membrane of cells, as well as in intracellular compartments (endosomes). Till-like receptor ligands can be molecules of various chemical nature and structure, such as lipopolysaccharides (LPS), flagellin, bacterial lipopeptides, bacterial and viral DNA, and others. Numerous data have been obtained characterizing the ability of toll-like receptor ligands to activate NF-kB (transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells) both in vitro and in vivo [11]. The NF-kB-mediated ability of toll-like receptor ligands to trigger various reactions of the immune system allows the use of ligands as immediate protection against pathogens, molecular adjuvants, etc. [19, 43].

One of these ligands approved for clinical use is monophosphoryl lipid A (monophosphoryl lipid A, MPLA) [12], the structural formula of which is presented below. MPLA is widely used as an adjuvant to vaccines, and its safety has been confirmed by many years of research [18]. MPLA is an endotoxin derivative that is used as an adjuvant for vaccines for humans, such as Angerix B, to form an immune response in patients with immunodeficiency status [17] and against hepatitis B virus [40], Cervarix is against human papillomavirus [fourteen]; and in the development of a vaccine against tuberculosis [15]. MPLA is produced by hydrolysis of native diphosphoryl lipid A, a component of lipopolysaccharide (LPS), which is recognized by type 4 toll-like receptor, resulting in the removal of all phosphate groups except one and of varying degrees of deacylation [31]. These structural changes can reduce systemic toxicity by more than 99% compared to native lipid A, resulting in an immunomodulating agent with a higher potential for clinical use [3, 39]. In the absence of toxicity, MPLA reduces the induction of proinflammatory cytokines such as TNFα, IL-1, and gamma interferon (IFγ) during the initial exposure [33]. After lethal doses of LPS are administered to mice, the use of MPLA enhances survival [3, 23]. Thus, the use of MPLA as an adjuvant to vaccines is promising.

In the development of a vaccine, the choice of the optimal immunization method is significant. When comparing different methods of immunization, it was found that the use of intranasal immunization, which provides the induction of a mucosal immune response, which is especially important for urogenital chlamydia, is promising. In addition, it is known that the mucosal route of administration of vaccines, as a rule, is not associated with the development of inflammatory post-vaccination complications [21].

Thus, there is a need to search for new candidate Chlamydia proteins, to select an effective delivery method, to increase their immunogenicity due to adjuvants, in an optimal method of immunization and to enhance protective properties that prevent the development of ascending infection and pathology in the reproductive organs.

Disclosure of invention

The purpose of the present invention is the creation of a combined antichlamydia vaccine with protective activity by inducing both humoral and cellular immune responses against various serotypes of C. trachomatis.

The term “combined antichlamydia vaccine” for the purposes of the present invention means a vaccine capable of inducing a protective immune response against chlamydial infection by sequential immunization, wherein the priming is carried out by a component based on a recombinant adenovirus vector expressing type III full-length structural secretion system protein (CCTT) C. trachomatis, while boosting is carried out by a component based on the recombinant structural protein CSTT C. trachomatis, monophosph aryl lipid A (MPLA) and squalene.

The term “priming component” for the purposes of the present invention means a component consisting of a recombinant adenoviral vector expressing the full-length structural protein of C. trachomatis CCTT, which is administered once intranasally.

The term “boosting component” for the purposes of the present invention means a component that is injected subcutaneously two weeks after the prime component and which consists of an emulsion containing the recombinant structural protein CCTT of C. Trachomatis and a combination of adjuvants: MPLA - ligand of the toll-like receptor type 4 and squalene .

The essence of the invention lies in the development of a technology (production method) of a new original combined anti-chlamydia vaccine containing a priming component - a recombinant adenovirus carrying a chimeric gene, consisting of the N-terminal part of the mannose-binding lectin, spacer and chlamydia CdsF antigen gene, which are fused into a single frame read and expressed as a single protein product; and containing a boosting component consisting of the recombinant structural protein rCdsF CCTT of chlamydia, a combined adjuvant containing monophosphoryl lipid A (MPLA), a toll-like receptor of type 4 ligand and squalene.

By the CdsF antigen gene in the present invention, it is to be understood the chlamydial gene encoding a protein forming the CTTT injectosome. In different strains, this protein has different designations, for example: CT666 - Chlamydia trachomatis (strain D / UW-3 / Cx), TC_0037 in Chlamydia trachomatis MoPn (strain Nigg).

The technical result of the present invention is that a combined chlamydia vaccine, created on the basis of the full-sized structural protein CdsF of the chlamydia type III secretion system, in in vivo experiments in preclinical studies induces pronounced protective activity by inducing a specific humoral and specific T-cell immune response.

The claimed combination antichlamydia vaccine contains a dose of:

- a recombinant adenoviral vector expressing a gene of a full-sized structural protein of the chlamydia type III secretion system - 108-109 PFU in a pharmaceutically acceptable buffer with a volume of 0.5 ml.

- recombinant structural protein of the chlamydia type III secretion system 10.0-20.0 μg, MPLA 50.0 μg, squalene 10.0 mg in 0.5% Tween 80, in a volume of 0.5-1 ml.

By “pharmaceutically acceptable buffer” is meant a solution containing substances or compositions that do not cause an allergic or similar adverse reaction when administered to humans.

17 For a clearer understanding of the present invention, which is reflected in the claims, as well as to demonstrate its features and advantages, the following is a detailed description of the preparation and action of a combination vaccine with examples as the target gene for the chlamydia CdsF protein and a reference to the drawings.

Because Since it was necessary to show that the proposed vaccine provides protection against chlamydial infection in vivo, a model of acute and ascending urogenital infection in mice was developed. To simulate the infection process in mice, a serotype of C. trachomatis MoPn, which is a natural mouse pathogen, was selected. This model of urogenital chlamydial infection imitates many aspects of urogenital infection in women caused by human C. trachomatis strains. In urogenital infection caused by C. trachomatis MoPn, mice develop an infection in the lower parts of the urogenital tract within a few weeks, which then goes up, leading to the development of reproductive organs pathology and infertility, similarly to urogenital chlamydial infection in women.

Brief Description of the Drawings

In FIG. 1 shows the structure of C. trachomatis MoPn CCTT and the conservativeness of the CdsF protein forming the CTCT injectosome in representatives of the Chlamydiaceae family

1A. Scheme of the structure of the third type secretion system (CCT) C. trachomatis MoPn, in which

1 - CdsF CTTT injection protein.

2 is a side view of an injectosome.

3 is a top view of an injectosome.

4 - CdsF protein monomer.

1B. Conservativeness of the CdsF injecting SSTT protein in members of the Chlamydiaceae family. A conserved B-cell epitope is marked above the amino acid sequence of the protein.

In FIG. Figure 2 shows the epitope map of the CdsF antigen for MHC molecules of BALB / c mice with the H-2d haplotype (2A) and the most common HLA molecules in the Russian population (2B). Epitopes of MHC class I are marked above the amino acid sequence of the protein, sub - epitopes of MHC class II. Epitopes for various alleles of MHC II are represented by different shades of gray. The wavy line marks the B-cell epitope.

In FIG. Figure 3 shows the nucleotide sequence of the gene encoding the full-length protein TC_0037 C. trachomatis MoPn.

In FIG. 4 shows the amino acid sequence of the full-length protein TC_0037 C. trachomatis MoPn.

In FIG. 5 shows the nucleotide sequence of a glycine-serine spacer (GS spacer).

In FIG. Figure 6 shows the nucleotide sequence of the mannose binding lectin (MBL) protein gene.

In FIG. 7 shows the amino acid sequence of a glycine-serine spacer.

In FIG. 8 shows the amino acid sequence of MBL.

In FIG. 9 shows the complete nucleotide and amino acid

the sequence of the genetic construct MBL-TC_0037.

In FIG. 10 is a diagram of an expression cassette of recombinant human adenovirus 5 serotype under the control of the human cytomegalovirus promoter (CMV promoter). ΔE3-deleted genes of the E3 region.

At the site of the removed E1 region (ΔE1) of the adenoviral genome, an expression cassette is inserted that contains:

CMV - promoter of human cytomegalovirus,

pA - polyadenylation signal,

CdsF - C. trachomatis antigen,

MBL is the mannose-binding lectin of Mus musculus.

In FIG. 11 shows the nucleotide sequence of the gene encoding the C. trachomatis MoPn TC_0037 protein with six histidine codons at the C-terminus (TC_0037-6 * His).

In FIG. 12 shows the amino acid sequence of the protein TC_0037-6 * His.

In FIG. 13 shows a scheme for producing a recombinant protein for a boosting component using the example of the TC_0037 protein gene C. trachomatis MoPn, in which

tc_0037 - gene of the full-sized protein TC_0037 C. trachomatis;

pET29b is a plasmid;

E. coli BL21 (DE3) -E. coli strain BL21 (DE3);

IPTG - isopropyl-β-D-1-thiogalactopyranoside;

rTC_0037 - recombinant protein TC_0037.

In FIG. 14 shows a Western blot of protein TC_0037-6 * His purified from E. coli cells in which

1 - control with 6 * His.

2-5 - dilution of the insoluble fraction of the protein obtained under denaturing conditions (8 M urea):

1 - control with 6 * His;

2-1: 50;

3-1: 100;

4-1: 200;

5 - 1: 250.

In FIG. 15 (A, B, C) are graphs for the determination of specific antibodies to the recombinant protein rTC_0037 C. trachomatis MoPn.

15A. Determination of specific antibodies to the recombinant protein rTC_0037 C. trachomatis MoPn in rabbit blood serum (1: 500 dilution) immunized with rTC_0037, where

- rTC_0037

- rTC_0037

15B. Determination of specific antibodies to the recombinant protein rTC_0037 C. trachomatis MoPn in the blood serum of a rabbit immunized with C. trachomatis L2, where

- сыворотка к С. trachomatis L2;

- serum to C. trachomatis L2;

- отрицательная сыворотка.

- negative serum.

15B. Determination of specific antibodies to the recombinant protein rTC_0037 C. trachomatis MoPn in blood serum taken from a patient with chlamydia caused by C. trachomatis, where

сыворотка к С. trachomatis L2;

serum to C. trachomatis L2;

- отрицательная сыворотка.

- negative serum.

In FIG. 16 shows a specific lymphoproliferative response to the rTC_0037 antigen, where

- контроль;

- the control;

- rTC_0037.

- rTC_0037.

In FIG. 17 shows a design of an experiment on adaptive T-cell transfer, where

rTC_0037 - recombinant protein rTC_0037 C. trachomatis MoPn;

DC - dendritic cells;

s / c - subcutaneous injection;

iv - intravenous injection;

UGT - urogenital tract.

In FIG. Figure 18 shows a graph for the study of the rTC_0037 antigen protectiveness of C. trachomatis MoPn after adaptive transfer on a model of acute urogenital infection in mice, in which

rTC_0037 - группа мышей, которым перенесли Т-клетки от мышей иммунизированных rTC_0037;

rTC_0037 - a group of mice that received T cells from mice immunized with rTC_0037;

С. trachomatis - группа мышей, которым не производили перенос Т-клеток (контроль заражения С.trachomatis MoPn), где

C. trachomatis - a group of mice that did not produce T-cell transfer (C. trachomatis MoPn infection control), where

In FIG. 19 (A, B, C) shows a scheme for obtaining a genetic construct based on an adenoviral vector carrying the C. trachomatis MoPn CdsF protein gene, including:

19A. Synthesis of the MBL-TC_0037 gene into pAL-TA plasmid;

19B. Obtaining recombinant adenovirus Ad-MBL-TC_0037;

19B. Schematic representation of a recombinant adenovirus carrying the gene of antigen TC_0037 C. trachomatis MoPn (Ad-MBL-TC_0037), where:

TC_0037 C. trachomatis MoPn - protective antigen;

MBL — N-terminal fragment of a mannose binding lectin;

leader peptide - N-terminal sequence of the secretory protein TC_0037, consisting of 15 amino acids, which is cleaved during the post-translational change of such a prepeptide, as a result of which it turns into a mature secreted peptide;

spacer - glycine-serine spacer.

In FIG. 20 shows the expression of the recombinant protein MBL-TC_0037 (24 kDa) from an adenoviral vector.

In FIG. 21 is a schematic for preparing the boosting component of rTC_0037-MPLA combination vaccine.

In FIG. 22 shows a design of an experiment for immunization with a combination vaccine, where

0С (С - day) - intranasal immunization with the priming component Ad-MBL-TC_0037; Intranasal administration to Ad-null mice.

14C - subcutaneous immunization with the rTC_0037-MPLA boosting component in the squalene emulsion; subcutaneous administration of MPLA as part of squalene emulsion.

28C - assessment of the humoral immune response.

58C - assessment of the T-cell immune response.

In FIG. 23 shows the level of specific antibodies of the IgG class to the antigen TC_0037 C.trachomatis MoPn:

23A. IgG2a isotype;

23B. IgG1 isotype.

Ad-MBL-TC_0037 + rTC_0037-MPLA - экспериментальная группа;

Ad-MBL-TC_0037 + rTC_0037-MPLA - experimental group;

Ad-null + MPLA - контрольная группа;

Ad-null + MPLA - control group;

Ad-null - контрольная группа;

Ad-null - control group;

ФПБ - контрольная группа.

FPB - control group.

In FIG. 24 shows a design of an experiment for studying the induction of a T-cell immune response in mice.

In FIG. 25 shows the number of T cells corresponding to IF secretion in response to the antigen TC_0037 of C. trachomatis MoPn in the lower parts of the urogenital tract after infection with C. trachomatis MoPn.

Groups:

получивших ФПБ;

received FPB;

иммунизированных Ad-MBL-TC_0037+rTC_0037-MPLА;

immunized Ad-MBL-TC_0037 + rTC_0037-MPLA;

получивших Ad-null+MPLA;

Received Ad-Null + MPLA;

получивших Ad-null.

received Ad-null.

In FIG. 26 shows a design of an experiment for studying the protective activity of a combination vaccine, in which:

0C - intranasal immunization with the priming component of Ad-MBL-TC_0037; Intranasal administration to Ad-null mice.

14C - subcutaneous immunization with the rTC_0037-MPLA boosting component in the squalene emulsion; subcutaneous administration of MPLA as part of squalene emulsion.

24C - mice sexual cycle synchronization with progesterone analogue - Cavinan preparation.

28C - assessment of the T-cell immune response.

31C-48C - taking vaginal swabs.

58C - assessment of the accumulation of chlamydia in the reproductive organs, the study of the pathology of the reproductive organs.

Ad-MBL-TC_0037 + rTC_0037-MPLA - experimental group;

Ad-null + MPLA - control group;

Ad-null - control group;

FPB - control group.

C. trachomatis MoPn - infection control.

UGT - urogenital tract.

In FIG. 27 is a table showing the neutralizing activity of antibodies obtained from the blood sera of mice immunized with a combination vaccine in a culture of cells infected with C. trachomatis MoPn, where

Ad-MBL-TC_0037 - intranasal immunization of mice with the priming component of Ad-MBL-TC_0037;

Ad-null administration once intranasally to mice Ad-null (negative control);

rTC_0037-MPLA - subcutaneous immunization of mice with the boosting component rTC_0037-MPLA in the squalene emulsion;

MPLA - subcutaneous administration of MPLA in squalene emulsion.

In FIG. 28 (A, B, C, D, D) graphs show the suppression of chlamydial infection in the lower parts of the urogenital tract after infection with C. trachomatis MoPn: A. on day 3; B. on the 6th day; B. on the 9th day; G. on the 15th day; D. on the 20th day.

Ad-MBL-TC_0037 - a single intranasal immunization of mice with the priming component of Ad-MBL-TC_0037;

Ad-null - the introduction of a single intranasal mouse Ad-null (negative control);

rTC_0037-MPLA - subcutaneous immunization of mice with the boosting component rTC_0037-MPLA in the squalene emulsion;

MPLA - subcutaneous administration of MPLA in squalene emulsion;

C. trachomatis MoPn - intravaginal infection of mice C. trachomatis MoPn.

Ad-MBL-TC_0037/rTC_0037-MPLA;

Ad-MBL-TC_0037 / rTC_0037-MPLA;

Ad-null/MPLA;

Ad-null / MPLA;

Ad-null;

Ad-null

С. trachomatis.

C. trachomatis.

In FIG. 29 presents tables showing the determination of C. trachomatis MoPn DNA in reproductive organs, where:

Ad-MBL-TC_0037 - a single intranasal immunization of mice with the priming component of Ad-MBL-TC_0037, where

Ad-null - the introduction of a single intranasal mouse Ad-null (negative control);

rTC_0037-MPLA - subcutaneous immunization of mice with the boosting component rTC_0037-MPLA in the squalene emulsion;

MPLA - subcutaneous administration of MPLA in squalene emulsion;

C. trachomatis MoPn - intravaginal infection of mice C. trachomatis MoPn.

In FIG. 30 (A, B) are photographs of the uterus and ovaries of mice, which show a comparison of pathological changes in the reproductive organs after infection with C. trachomatis MoPn in the control group and the absence of pathology in the immunized group:

30A. The absence of pathological changes.

30B. Pathological changes.

Ad-MBL-TC_0037 + rTC_0037-MPLA - experimental group;

Ad-null + MPLA - control group;

Ad-null - control group;

FPB - control group.

C. trachomatis MoPn - infection control.

Examples of the claimed invention

The following examples are provided below for information and for a more detailed illustration of the present invention and do not limit the scope of the invention.

Example 1. CdsF protein sequence comparisons.

Example 2. A method of obtaining a recombinant chlamydial CdsF protein.

Example 3. Immunogenic properties of the recombinant chlamydia rCdsF protein.

Example 4. The creation of the priming component of the proposed vaccine.

Example 5. A method of obtaining a boosting component of the proposed vaccine.

Example 6. Immunization of mice with a combination vaccine.

Example 7. A humoral immune response to a combination vaccine.

Example 8. T-cell immune response to a combination vaccine.

Example 9. The protective activity of the resulting combination vaccine.

Example 1. Comparison of CdsF protein sequences.

In our invention, the structural protein CdsF CTST C. trachomatis MoPn was proposed as the main component of the candidate vaccine. It concentrates on the outer membrane of elementary Taurus (ET) and reticular Taurus (RT) and forms a surface structure, referred to as a “needle” (Fig. 1. A), which has dimensions of about 60-80 nm in length and 8 nm in diameter. The diameter of the hole in the needle is approximately 3 nm.

The CdsF protein is expressed on the membrane of chlamydia and plays an important role in the development of infection at all stages of its life cycle. In addition, the CdsF CCTT “needle” chlamydial protein contains cysteine residues that are unique to it, and the disulfide bonds found in CdsF can participate in the functioning of the chlamydial secretory apparatus. They are also associated with the oxidation state of the chlamydial membrane and the stages of development of the pathogen.

To assess the possibility of using CdsF protein as a target protein (antigen) to create a vaccine preparation that is effective against all serotypes of C. trachomatis, we analyzed the presence of B- and T-cell epitopes in the amino acid sequence, as well as their main characteristics, using available bioinformation databases (https://tools.iedb.org/bcell/; https://www.cbs.dtu.dk/services/BepiPred/; https://www.cbs.dtu.dk/services/NetMHC/ ; https://www.cbs.dtu.dk/services/NetMHCII;). Amino acid sequences were obtained from the OMA phylogenetic database (https://omabrowser.org), (OMA Group 877704). Alignments are shown in FIG. 1. B (Note: amino acid sequences were obtained from the OMA phylogenetic database (https://omabrowser.org), (OMA Group 877704)).

It was found that both B-cell epitopes and T-cell epitopes for histocompatibility molecules of class I and II, both in humans and in mice, were located in highly conserved regions of the CdsF antigen, which indicates the prospects of this protein for use as a target antigen candidate vaccine preparation for humans (Fig. 1B, 2).

Epitope mapping showed that both B-cell and T-cell epitopes for CD8 + and CD4 + T-lymphocytes are present in the CdsF antigen. Further, in order to prove their conservatism in other strains of chlamydia, all known sequences of the CdsF CCTT protein were taken and aligned with each other for maximum compliance within a given area (Fig. 1B). Presented in FIG. 1B, the amino acid sequences of the CdsF protein of different serotypes and types of chlamydia are “substantially identical” (have more than 85% homology).

We confirmed the data obtained using bioinformation methods on the presence of conservative B-cell epitopes in the CdsF antigen, experimentally. The presence of antibodies to the CdsF protein in the sera of patients with chlamydial infection was shown. These data indicate the promise of the conservative CdsF protein for use as the target antigen of the candidate vaccine preparation for different serotypes, because it has a high degree of sequence identity among various Chlamydia trachomatis serovars.

The selected CdsF protein is the main component of the vaccine preparation, effective against all C. trachomatis serotypes, due to its important role in the functioning of chlamydia CTST, which provides pathogen pathogenicity in both acute and chronic infections, the conservativeness of its B and T cell epitopes and the immunogenicity of this protein for humans revealed by us. Thus, cross reactivity can be seen between antigens of different Chlamydia serotypes and, therefore, it can be assumed that this conserved chlamydial antigen CCT can provide a protective immune response against serovar different from the one from which the antigen was obtained.

Example 2. A method of obtaining a recombinant protein CdsF.

Because it was necessary to show that the proposed vaccine provides protection against chlamydial infection in vivo; a model of acute and chronic urogenital infection in mice was developed. For this purpose, as an option, a vaccine was created based on the use of Chlamydia trachomatis MoPn proteins

To simulate the infection process in mice, a serotype of C. trachomatis MoPn, which is a natural mouse pathogen, was selected. This model of urogenital chlamydial infection imitates many aspects of urogenital infection in women caused by human C. trachomatis strains. In urogenital infection caused by C. trachomatis MoPn, mice develop an infection in the lower parts of the urogenital tract within a few weeks, which then goes up, leading to the development of reproductive organs pathology and infertility, similarly to urogenital chlamydial infection in women.

The proposed method for producing a recombinant structural protein CdsF will be shown on an example of creating a structural protein CCTT C. trachomatis MoPn, namely: rTC_0037 (Fig. 3, 4).

To this end, a gene encoding the full-length protein TC_0037 containing a label of six histidips (6 * His) (Figs. 11, 12) was cloned into the plasmid vector pET29b (Novagen, USA) at Ndel / Xhol restriction sites. The obtained plasmid was transformed with E. coli strain B1.21 (DE3) cells, after which expression was induced by addition of 1 M isopropyl-β-D-1-thiogalactopyraposide (IPTG) (Fig. 13, 14). To purify rTC_0037 isolated from E. coli cells, a total bacterial lysate prepared using 8M urea was used. Purification was performed using a Co ²⁺ affinity sorbent under denaturing conditions. Protein expression was determined by Western blot using monoclonal antibodies to a label consisting of six histidines. As a result, a specific fragment with a size of ~ 13 kDa corresponding to the calculated molecular weight of rTC_0037 protein fused with 6 histidine molecules equal to 10134.51 Da was detected. Recombinant protein rTC_0037 was obtained at a concentration of 200 mg / ml in 50 mM sodium phosphate buffer, pH 6.0 with 4 M urea.

The proposed method for producing recombinant CdsF protein using C. trachomatis MoPn as an example is universal and identical for different serotypes of Chlamydia trachomatis.

Example 3. Immunogenic properties of the recombinant protein rCdsF.

Studies of the immunogenic properties of rCdsF CCTT are given on the variant of C. trachomatis MoPn protein - rTC_0037.

To assess the immunogenic properties of the protein rTC_0037 C. trachomatis MoPn:

1) determined specific antibodies to the recombinant protein rTC_0037;

2) analysis of specific lymphoproliferation;

3) conducted studies on the adaptive transfer of T cells.

1) Determination of specific antibodies to the recombinant protein rTC_0037 C. trachomatis MoPn

Specific antibodies to the recombinant protein rTC_0037 C. trachomatis MoPn were determined by indirect enzyme-linked immunosorbent assay (ELISA). (Fig. 15A, B, C)

A. Determination of specific antibodies to the TC_0037 protein in rabbit serum immunized with rTC_0037 protein.

For these purposes, we used the serum obtained by immunizing the rabbit with the recombinant protein rTC_0037 in combination with Freund's adjuvant, at a dose of 2.612 mg / rabbit. The rabbit was immunized 4 times. The first immunization: rTC_0037 protein with Freund's complete adjuvant (1: 1) - in the amount of 500 mcg, the second immunization: rTC_0037 protein with Freund's complete adjuvant (1: 1) - in the amount of 600.0 mcg (4 weeks after the first) ; the third immunization: protein rTC_0037 in the amount of 720.0 μg (two weeks after the second) and the fourth immunization was carried out a week after the third in the amount of 792.0 μg. Blood sampling was performed 14 days after the last immunization. As a negative control, serum obtained from an intact rabbit was used.

ELISA was performed by the conventional method. Protein rTC_0037 was used as antigen. The resulting serum from the immunized rabbit was diluted 1: 500. To determine specific IgG antibodies, goat monoclonal anti-rabbit IgG antibodies (Sigma-Aldrich, USA) at a 1: 1000 dilution labeled with horseradish peroxidase were used as conjugate; tetramethylbenzidine (TMB) (Biolegend, USA) was used as a substrate . The reaction was taken into account at a wavelength of 450 nm.

The study revealed specific IgG antibodies to the rTC_0037 protein at a dilution of 1: 500, which is more than 6 times higher than in the blood serum from an intact rabbit in the control group (negative control), which indicates a high immunogenicity of this protein (Fig. 15A )

B. Determination of specific antibodies to the rTC_0037 protein in rabbit serum immunized with a human inactivated strain of C. trachomatis L2 / Bu434 (hereinafter - L2).

For these purposes, we used the serum obtained by immunizing the rabbit C. trachomatis L2 at a dose of 10 ⁷ VOE / rabbit. The rabbit was immunized three times with an interval of 2 weeks: the first - C. trachomatis L2 with PAF (1: 1) - in the amount of 500.0 μg, the second and third - C. trachomatis L2 with NAF (1: 1) - in the amount of 500 , 0 mcg. Blood sampling was performed 14 days after the last immunization. Hyperimmune rabbit serum was obtained from blood for C. trachomatis L2 with a titer in ELISA of 1: 2000 for the complete complex of C. trachomatis L2 antigens. An intact rabbit serum was obtained as a negative control.

ELISA was performed by the conventional method. As the antigen used protein rTC_0037. The serum was diluted 1:32, 1:64 and 1: 128. To determine specific IgG class antibodies, goat monoclonal anti-rabbit IgG class antibodies (Sigma-Aldrich, USA) at a 1: 1000 dilution labeled with horseradish peroxidase were used as conjugate. Tetramethylbenzidine (TMB) (Biolegend, USA) was used as a substrate. The reaction was taken into account at a wavelength of 450 nm.

In the study in serum obtained by immunizing a rabbit with a human inactivated strain of C. trachomatis L2, antibodies with a dilution of more than 1: 128, specifically binding to rTC_0037 CCTT of C. trachomatis MoPn, were detected. This once again confirms the conservativeness of the structural CdsF protein among different serotypes of Chlamydia trachomatis (Fig. 15B).

B. Determination of specific antibodies to the protein TC_0037 in the blood serum of a person with a chlamydia patient caused by C. trachomatis.

For these purposes, the blood serum of a person suffering from chlamydia was used, at a dilution of 1:32, 1:64 and 1: 128. ELISA was performed by the conventional method. As a negative control, human serum without chlamydial infections was used. To determine specific IgG class antibodies, goat monoclonal anti-human IgG class antibodies (Sigma-Aldrich, USA) at a 1: 1000 dilution labeled with horseradish peroxidase were used as conjugate. Tetramethylbenzidine (TMB) (Biolegend, USA) was used as a substrate. The reaction was taken into account at a wavelength of 450 nm.

It was found that in the serum obtained from a patient with chlamydia, there are antibodies in a dilution of more than 1: 128, specifically binding to rTC_0037 C. trachomatis MoPn (Fig. 15B).

The interaction of this antigen with antibodies of the blood serum of a patient with chlamydia infection once again confirms the conservativeness of the CdsF protein in relation to different chlamydial serotypes.

2) Analysis of specific lymphoproliferation in response to the rTC_0037 antigen C. trachomatis MoPn

To assess the immunogenicity of the rCdsF protein, rTC_0037 exemplified antigen-specific proliferation in response to the recombinant rTC_0037 protein of splenocytes isolated from spleens of BALB / c mice (4 mice / group) pre-immunized subcutaneously: rTC_0037 at a concentration of 10 μg / ml with complete adjuvant Freind's (PAF) (in the ratio 1: 1) in a volume of 100.0 μl or killed by heating (60 minutes at 60 ° C) C. trachomatis MoPn at a dose of 10 ⁴ VOU / 100 μl in PAF (1: 1). The controls were non-immunized intact mice.

The resulting splenocytes were adjusted to a concentration of 2.5 × 10 ⁶ / ml in culture medium (RPMI-1640, 5% FBS) and incubated in 200.0 μl in the presence of 10.0 μg of rTC_0037 protein. Cells not stimulated with rTC_0037 were used as a control. After 36 hours, 0.5 μN ³ -thymidine was added to the culture and incubated for 12 hours. A scintillation liquid counter (Wallac Laboratory, Turku, Finland) was used to analyze the amount of radioactive label bound to DNA. The results are presented in pulses per minute. (Fig. 16).

It was shown that splenocytes of mice immunized with the recombinant protein rTC_0037 and C. trachomatis MoPn proliferated in response to the recombinant protein rTC_0037, which indicates the immunogenicity of this protein for T cells. Cells obtained from intact mice did not proliferate in response to the rTC_0037 antigen.

3) Adaptive transfer of T cells from mice immunized with rTC_0037 protein.

The experimental design of the adaptive transfer of T cells immunized with the rTC_0037 protein of C. trachomatis MoPn is shown in FIG. 17.

For the adaptive transfer experiment, BALB / c mice (5 mice / group) were used, which were immunized subcutaneously with the recombinant protein rTC_0037 at a concentration of 10.0 μg / mouse with Freund's complete adjuvant (PAF) - 500.0 μl / mouse (in the ratio 1: 1). After 14 days. mice were boosted intravenously with dendritic cells (DCs) loaded with rTC_0037 antigen. One week after the boost, cells from regional lymph nodes and spleen from immunized mice were isolated. Isolated cells were incubated in a 24-well plate at a concentration of 4 × 10 ⁶ / ml in the presence of rTC_0037 antigen (10 μg / ml). In 7 days T cells were isolated according to the manufacturer's protocol (Cedarlane, Canada).

The obtained T cell lines were used for adoptive transfer in order to determine their protective potential in vivo. After T-cells were transferred to recipient mice (administered intravenously) (5 mice / group), the next day they were infected intravaginally with C. trachomatis MoPn 10 ⁶ VOU / ml. The control was intact mice.

The suppression of infection in the lower parts of the urogenital tract was evaluated by cultural analysis. Cultural isolation of C. trachomatis MoPn was performed on a McCoy cell line according to a standard procedure [35]. For this, vaginal swabs were taken from mice (5 mice / group) and placed in 500.0 μl of RPMI medium containing 5% fetal serum, glucose, amphotericin B, gentamicin. The McCoy cell line was infected with the resulting material in a 24 well plate. After infection, the plate was centrifuged at 2000 rpm at t = + 25 ° C for 1 hour. After incubation in a thermostat (37 ° C, 5% CO ₂ ) for 2 hours, the medium was removed and 1.0 ml of medium with cycloheximide was added. The plate was incubated at 37 ° C, 5% CO ₂ for 48 hours. Then the cells were fixed and stained with chlamydial LPS-specific (lipopolysaccharide) monoclonal antibodies labeled with FITC. The identification and quantification of the content of chlamydia in infected cultures was carried out by immunofluorescence microscopy. To calculate the chlamydial inclusions of the forming units (BOE), the standard formula was used [35].

A culture study of vaginal swabs from the lower parts of the urogenital tract revealed a decrease in the level of chlamydial infection in the group with T-cell transfer obtained from mice immunized with rTC_0037 protein, compared with the control with mice infected with C. trachomatis MoPn (Fig. 18).

Thus, a model of acute urogenital infection showed that immunization with the recombinant protein rTC_0037 promotes the formation of memory T cells. The transfer of these cells to intact recipients protects them upon subsequent infection with C. trachomatis MoPn (Fig. 18).

Example 4. Creating a priming component of the proposed vaccine

The creation of an adenoviral genetic construct, which is the prime component of the vaccine, was carried out using the protein example ТС_0037 C. trachomatis MoPn. The technology for preparing the prime component of the vaccine is shown in FIG. 19A, B, C.

The nucleotide sequence of the gene for the full-sized protein TC_0037 C. trachomatis MoPn was obtained from the UniProtKB database (Q9PLQ8). The tc_0037 gene was analyzed in silico for the presence of a bacterial signal sequence (https://www.cbs.dtu.dk/services/SignalP/) that was not found in the protein. The codons encoding the tc_0037 gene have been modified for better expression in Mus musculus cells. The most common codons of Chlamydia trachomatis MoPn and Mus musculus are determined according to the database of used codons https://www.kazusa.or.jp/codon/. Since, firstly, the TC_0037 antigen is in high density on the surface of bacterial cells, and secondly, the antigen is small in size (82 amino acids, 9 kDa), it was decided to add the N-terminal part of mannose to the N-terminal part of the antigen binding lectin (MBL) Mus musculus. MBL is able to hexamerize an antigen due to a collagen-like domain. The nucleotide sequence of the MBL gene was obtained from the UniProtKB database (P41317). A flexible glycine-serine spacer was inserted between the TC_0037 and MBL antigen sequences. The modified MBL-TC_0037 gene was synthesized, and then the plasmid pAL-TA-MBL-TC_0037 for Chlamydia trachomatis MoPn was obtained (Fig. 19A). PAL-TA-MBL-CT666 for Chlamydia trachomatis was created in the same way

Then, the MBL-TC_0037 gene was subcloned into the pShuttle-CMV shuttle vector, which was further used to obtain plasmid constructs of recombinant adenoviruses by homologous recombination in Escherichia coli cells using the AdEasy Adenovir Vector System (Stratagene, United States). Recombinant adenoviruses constructed on the basis of the genome of human adenovirus of the 5th serotype were obtained as a result of transfection of HEK293 cell line with the corresponding plasmid construct digested with PacI restriction enzyme. As a result of hydrolysis, a fragment containing the Ori replication initiation site and the antibiotic resistance gene ampicillin was cut to form a linear structure consisting of the full-sized adenovirus genome and expression cassette. Transfection was performed with Lipofectamin reagent (Invitrogen, USA) according to the attached manual in a 24-well plate on HEK293 cells with 90% confluency. Ten days later. after transfection, the cytopathic effect (JPC) of the virus was observed. (Fig. 19B). The resulting viral samples, in the form of a suspension of infected cells with a titer of PFU / ml 10 ⁸ , were stored at a temperature of minus 70 ° C.

The expression of the protein MBL-TC_0037 was determined by the Western blot method using hyperimmune mouse serum. For this, cells of the HEK293 line were transduced with Ad-MBL-TC_0037, after 48 hours, the culture medium was selected and concentrated 10 times. As a result, specific fragments, ~ 24 kDa, corresponding to the calculated molecular weight of 23742.76 Da were detected (Fig. 20).

Similarly, the Ad-MBL-CT666 construct was created on the basis of a recombinant adenovirus carrying the CdsF CSTT gene of C. trachomatis gene, which indicates the compliance of this construct with the requirements of the invention claimed.

In the anti-chlamydia combination vaccine, in the component priming the immune response consisting of a recombinant adenovirus, the nucleotide sequence of the gene encoding the optimized full-length structural protein TC_0037 C. trachomatis MoPn is represented by SEQ ID NO: 1 (Fig. 3). The amino acid sequence of the optimized full-length structural protein TC_0037 C. trachomatis MoPn is represented by SEQ ID NO: 4 (Fig. 4). The nucleotide sequence of the gene encoding the glycine-serine spacer is shown in SEQ ID NO: 2 (FIG. 5) and the nucleotide sequence of the gene for the MBL protein is shown in SEQ ID NO: 3 (FIG. 6). The polypeptides encoding these nucleotide sequences are shown in SEQ ID NO: 5 (FIG. 7) and SEQ ID NO: 6 (FIG. 8). The above nucleotide sequences are interconnected in the following order: MBL - spacer - TC_0037 and form a single reading frame, designated as MBL - TC_0037 (Fig. 9). The gene of the chimeric, hyperimmunogenic antigen is inserted into the expression cassette of the recombinant adenovirus under the control of the CMV promoter (Fig. 10).

Getting the proposed vaccine takes place in several stages. The developed technology should ensure the production of the prime component of the vaccine of the following quality:

- dose of 10 ⁶ -10 ⁹ PFU / ml of recombinant pseudo-adenovirus particles;

- dose volume of 0.5 ml (adjusted with a pharmaceutically acceptable buffer solution).

For this, the obtained cell suspension containing recombinant adenoviral particles with a titer of 10 ⁸ PFU / ml was used for growth in order to prepare a vaccine with a given content of recombinant adenoviral particles.

Thus, to produce the necessary titers of recombinant adenovirus particles, a wave bioreactor with 4,500.0 ml of a suspension of a permissive cell culture 293НЕК was seeded with a 500.0 ml cell suspension containing recombinant adenovirus particles with a titer of 10 ⁸ PFU / ml.

To build up recombinant adenoviral particles and achieve a titer of 2 × 10 ⁸ PFU / ml, they were cultured for 48 hours. Then, the cell mass was purified in several stages:

1) First, the cell mass was sedimented by centrifugation at 6000 g for 15 min, the solid part containing cells and recombinant adenoviral particles was submitted for further purification.

2) The extraction of recombinant adenoviral particles from the cell culture was performed by disrupting the cells by freezing at pH 8.0 (5 mM Tris HCl buffer solution, 0.075 MNaCl, 1 mM MgCl ₂ , 5% sucrose, 1% polysorbate 80) in liquid nitrogen.

3) For further removal of genomic cellular DNA, an additional treatment with benzonase (Merk Millipore, Germany) (150.0 U / ml) was carried out and put on gentle stirring for 3 hours at room temperature (21-23 ° C).

4) Cell debris was removed by centrifugation at 9000 g in order to obtain a supernatant containing recombinant adenoviral particles.

5) The resulting supernatant was diluted with buffer (50 mM TrisHCl pH 7.5, 1 M NaCl, 2 mM MgCl ₂ , 5% sucrose, pH 7.5) to a volume of at least 200 ml and was ultrafiltered.

6) Further purification was carried out by anion exchange chromatography.

Retentate was applied to a column (AxiChrom 70/300 with a volume of 400.0 ml) containing an anion exchange sorbent Q Sepharose virus licenced (GE Healthcare, Sweden). Chromatography conditions: flow 193.0 ml / min, buffer A (40.0 mM TrisHCl, 0.27 M NaCl, 2.0 mM MgCl ₂ , 5% Sucrose, 0.1% Polysorbate 80, pH 7.5), conductivity ~ 28-30 mS / cm; buffer B (40.0 mM TrisHCl, 0.5 M NaCl, 2.0 mM MgCl ₂ , 5% sucrose, 0.1% polysorbate 80, pH 7.5) conductivity ~ 50 mS / cm.

7) Size exclusion chromatography.

The eluate obtained in the previous step in a volume of 200.0 ml was applied to a column (AxiChrom 100/300 with a volume of 800.0 ml) containing a Q Sepharose 4 FastFlow sorbent (GE Healthcare, Sweden). High-molecular substances that are not included in the pores of the sorbent were eluted with the first peak (these include recombinant adenoviral particles), and impurities were eluted after the peak of non-replicating nanoparticles exited. Chromatography conditions: flow 130.0 ml / min, buffer (10 mM TrisHCl, 75 mM NaCl, 1 mM MgCl ₂ , 5% sucrose, 0.05% polysorbate 80, pH 8.0).

The eluate was then stabilized by adding ethanol to a concentration of 0.5% and ethylenediaminetetraacetic acid (EDTA) to a concentration of 100 μM.

8) Normal filtering.

For sterilization, the resulting preparation was filtered through a 0.22 μM filter system and diluted with a sterile pharmaceutical acceptable buffer solution, for example: 10 mM TrisHCl, 75 mM NaCl, 1 mM MgCl ₂ , 5% sucrose, 0.05% polysorbate 80, 0 , 5% ethanol, 100 μm EDTA, pH 8.0, to obtain the active component, taking into account the required final activity of 10 ⁸ -10 ⁹ PFU of recombinant adenoviral particles. The resulting volume of the drug is poured into 0.5 ml vials in a single dose.

Thus, the task of preparing the priming component of the vaccine according to the invention is completed. It is possible to obtain a prime component of a vaccine containing:

- recombinant adenoviral particles expressing the gene for the CdsF protein of chlamydia, - 10 ⁸ -10 ⁹ PFU / ml

- pharmaceutically acceptable buffer solution - up to 0.5 ml.

Example 5. Obtaining a boosting component of the proposed vaccine.

This example describes a method for producing a boosting component of the vaccine — squalene emulsion (oil in water) containing the recombinant chlamydial CCT protein — rTC_0037 C. trachomatis MoPn and monophosphoryl lipid A (MPLA) (FIG. 21).

To obtain one therapeutic dose of the boosting component, it is necessary to prepare the polar phase — an aqueous solution containing MPLA (50.0 μg), as well as a recombinant structural protein of the chlamydia type III secretion system 10.0–20.0 μg. To prepare the non-polar phase, add 0.5% Tween 80 to squalene (10.0 mg). Mix the polar and non-polar phases, bring the final volume to 0.5-1.0 ml with an isotonic buffer solution. To obtain a nanoscale emulsion, the sample is treated with ultrasound.

Thus, the task of preparing the boosting component of the vaccine according to the invention is completed. It is possible to obtain a boosting component containing: recombinant protein rCdsF - 10.0 μg, monophosphoryl lipid A - 50.0 μg, squalene 10.0 mg, 0.5% Tween 80, in a volume of 0.5 ml.

Example 6. Immunogenicity of a chlamydial combination vaccine

A study of the immunogenicity of a vaccine consisting of a priming component and a boosting component was carried out by its ability to induce the production of specific antibodies and a specific T-cell immune response in mice.

In FIG. Figure 22 shows the immunization schedule for Balb / c mice, females, weighing 14-16 g. To do this, we used the developed variant of a combination vaccine adapted for mice and consisting of a prime component designated Ad-MBL-TC_0037 and a boost component designated rTC_0037-MPLA . Immunization with the priming component was carried out intranasally with a dose of 10 ⁹ PFU. After 14 days. immunized animals were subcutaneously boosted with 0.5 ml of an emulsion containing 10.0 μg squalene, 10.0 μg rTC_0037 protein and 50.0 μg MPLA.

Example 7. A humoral immune response to a combination vaccine.

The resulting combination vaccine containing Ad-MBL-TC_0037 and rTC_0037-MPLA was used to study the humoral immune response.

All work was carried out in accordance with the "Rules for the use of experimental animals" (order of the Ministry of Health No. 755 of 08/12/1977). The study used BALB / c mice (females), 6-7 weeks (weighing 14-16 g). There were 4 groups of animals of 10 animals each. The first group of mice was immunized with Ad-MBL-TC_0037 at a dose of 10 ⁹ PFU / ml followed by boosting rTC_0037-MPLA, the remaining groups were used as controls — the group receiving Adnull with MPLA boosting; the group receiving Ad-null at a dose of 10 ⁹ PFU / ml; and a group of mice that received a pharmaceutically acceptable buffer (FPB) -50.0 μl.

Mice were injected once intranasally with 50.0 μl of the proposed drug, either Ad-null or FPB. Then, two weeks after intranasal immunization, rTC_0037-MPLA and MPLA were subcutaneously boosted. Blood sampling in animals in all groups was performed 14 days after boosting. In blood serum titers of specific antibodies of the IgG class of IgG2a and IgG1 isotypes were determined for the TC_0037 protein in an enzyme-linked immunosorbent assay (ELISA) (Fig. 23A, B).

Recombinant protein rTC_0037 C. trachomatis MoPn at a concentration of 10.0 μg / ml was used as an antigen to evaluate antibodies to the TC_0037 protein.

ELISA was performed by the conventional method. Used 96-well tablets with high sorption ability (Costar, USA). To determine specific antibodies of the IgG class, goat monoclonal anti-mouse antibodies of the IgG2a and IgG1 isotypes (Sigma-Aldrich, USA) at a 1: 1000 dilution labeled with horseradish peroxidase were used as conjugate. Tetramethylbenzidine (TMB) (Biolegend, USA) was used as a substrate. The reaction was taken into account at a wavelength of 450 nm. The titer was taken as the largest dilution of serum, which gives an optical density of at least 2 times more than the serum taken from control groups.

The study revealed the production of specific antibodies of the IgG-IgG2a and IgG1 class of isotypes in response to immunization with Ad-MBL-TC_0037 with boosting rTC_0037-MPLA, which indicates the immunogenicity of this drug, which induces protective immunity of the Th1 type. The titer of antibodies to the TC_0037 antigen in the blood sera of mice immunized with Ad-MBL-TC_0037 with rTC_0037-MPLA boosting averaged 1: 400, which is higher than in mice of the control groups (Fig. 23A, B).

Thus, specific antibodies to the TC_0037 protein were detected in the blood sera of mice immunized once intranasally with the Ad-MBL-TC_0037 candidate vaccine with rTC_0037-MPLA boosting. The presence of specific activity - immunogenicity means the possibility of using the Ad-MBL-TC_0037 construct with this immunization scheme, followed by boosting rTC_0037-MPLA as a combined antichlamydia vaccine.

Example 8. T-cell immune response to a combination vaccine.

The resulting combination vaccine containing Ad-MBL-TC_0037 with boosting rTC_0037-MPLA was also used to study the T-cell immune response in mice. The experimental design is shown in FIG. 24.

For this purpose, the ELISPOT method was used, which makes it possible to detect T lymphocytes specific for TC_0037 protein by their ability to secrete IFγ in response to stimulation by antigen-presenting cells loaded with rTC_0037 antigen.

All work was carried out in accordance with the "Rules for the use of experimental animals" (MZ order No. 755 of 08/12/1977). The study used 4 groups of Balb / c mice (females), 6-7 weeks (weighing 14-16 g), 5 animals each.

The experiment was performed on animals after a single intranasal injection of 50.0 μl of a preparation containing Ad-MBL-TC_0037 followed by subcutaneous boosting rTC_0037-MPLA (see immunization schedule of Fig. 22). As negative controls, mice were used, which once received intranasally Ad-null at a dose of 10 ⁹ PFU, or a pharmaceutically acceptable buffer, or Ad-null at a dose of 10 ⁹ PFU with MPLA boosting. After 44 days. after booster immunization, mice were sacrificed by the bloodless method and splenocytes were isolated. Splenocytes were reactivated in vitro by dendritic cells presenting TC_0037 protein, and the number of splenocytes responding by IFγ secretion in response to a specific reactivation was determined.

During specific reactivation of splenocytes by antigen-presenting cells loaded with TC_0037 antigen, an average of 144 IF-secreting cells were detected per 1 million splenocytes in the group of animals immunized with Ad-MBL-TC_0037 with rTC_0037-MPLA boosting. It was shown that the level of IFγ-secreting cells in immunized Ad-MBL-TC_0037 with boosting rTC_0037-MPLA mice was significantly higher than in the control groups. The experimental results are shown in FIG. 25.

Thus, the ELISPOT method was used to demonstrate the induction of a significant level of the T-cell immune response to the TC_0037 antigen in mice that were single-shot intranasally immunized with Ad-MBL-TC_0037 followed by subcutaneous boosting of rTC_0037-MPLA.

It was concluded that the study of the T-cell response in animals immunized with the vaccine according to the invention showed the presence of a pronounced T-cell immune response, which corresponds to modern requirements for vaccines.

Example 9. The protective activity of the resulting combination vaccine.

This example demonstrates the possibility of inducing a protective immune response by immunization with a candidate vaccine containing Ad-MBL-TC_0037 with boosting rTC_0037-MPLA (see immunization and infection schedule of Fig. 26).

To this end, experiments were carried out to study protective activity on a model of urogenital chlamydial infection in mice. Studies of protective properties were carried out using C. trachomatis MoPn, because this model of urogenital chlamydial infection in mice imitates many aspects of urogenital infection in women caused by other strains of C. trachomatis.

1) Neutralizing activity of specific antibodies upon immunization with the obtained vaccine containing Ad-MBL-TC_0037 with rTC_0037-MPLA boosting

The most important studied immune reactions include the neutralization reaction with specific antibodies. For the neutralization reaction, McCoy cells were used. The obtained sera from immunized animals were incubated with C. trachomatis MoPn in various dilutions (1: 8; 1:16; 1:32; 1:64; 1: 128; 1: 256) for 30 minutes at t = 37 ° C. Then, the resulting mixture was added to the cells and centrifuged at 2000 rpm, t = 25 ° C for 45 min. Incubation was carried out in a CO ₂ atmosphere at a temperature of 37 ° C for 48 hours. The neutralization effect was quantified by immunofluorescence by counting chlamydial inclusions in a McCoy cell culture.

It was shown that immunization with Ad-MBL-TC_0037, followed by boosting rTC_0037-MPLA, led to the production of specific antibodies with 100% neutralizing activity against C. trachomatis MoPn infection in vitro at a dilution of 1: 128 (Fig. 27, table. 1 )

2) Evaluation of the development of infection in the lower parts of the urogenital tract in mice during immunization with a vaccine containing Ad-MBL-TC_0037 with boosting rTC_0037-MPLA.

To simulate the infection process in mice, a serotype of C. trachomatis MoPn, which is a natural mouse pathogen, was selected. This model of urogenital chlamydial infection imitates many aspects of urogenital infection in women caused by human C. trachomatis strains. In urogenital infection caused by C. trachomatis MoPn, mice develop an infection in the lower parts of the urogenital tract within a few weeks, which then goes up, leading to the development of reproductive organs pathology and infertility, similarly to urogenital chlamydial infection in women.

From 3 to 20 days. after infection with C. trachomatis MoPn, vaginal swabs were collected in mice. The development of chlamydial infection was determined using the cultural method of research.

Cultural isolation of C. trachomatis MoPn was performed on a McCoy cell line according to a standard procedure [35]. For this, vaginal swabs were taken from mice and placed in 500.0 μl of culture medium (RPMI supplemented with 5% fetal serum, glucose, amphotericin B, gentamicin). The McCoy cell line was infected with the resulting material in a 24 well plate. After infection, the plate was centrifuged at 2000 rpm at t = + 25 ° C for 1 hour. After incubation in a thermostat (37 ° C, 5% CO ₂ ) for 2 hours, the medium was removed and 1.0 ml of medium with cycloheximide was added. The plate was incubated at 37 ° C, 5% CO ₂ for 48 hours. Then, the fixed cells were stained with chlamydial LPS-specific monoclonal antibodies labeled with FITC. The identification and quantification of the content of chlamydia in infected cultures was carried out by immunofluorescence microscopy. To calculate the chlamydial inclusion of forming units (BOE), the standard formula was used [35].

A study of the dynamics of the development of infection in the lower parts of the urogenital tract showed that immunization with Ad-MBL-TC_0037 with boosting rTC_0037-MPLA led to an almost complete suppression of chlamydial infection on the 20th day after infection, while in the control group the infection was detected in the amount of 10 ⁴ VEU / ml (Fig. 28).

3) Evaluation of the suppression of ascending chlamydial infection in the upper parts of the urogenital tract during immunization with a vaccine containing Ad-MBL-TC_0037 with boosting rTC_0037-MPLA.

a) Isolation of DNA from tissues

, Нидерланды). Суспензию в объеме 1000,0 мкл лизировали при температуре 65°C в течение 1 часа в 1,0 мл лизирующего буфера с протеиназой K («Синтол», Россия) в количества 40,0 мкл.Samples of organs (uterus, ovaries) from BALB / c mice (5 mice / group) (see experimental design of Fig. 26) were homogenized in 1 ml of a 1% PBS solution. DNA was isolated on a NucliSENS® easyMAG® automated nucleic acid extractor (

, The Netherlands). A suspension in a volume of 1000.0 μl was lysed at 65 ° C for 1 hour in 1.0 ml of lysing buffer with proteinase K (Syntol, Russia) in an amount of 40.0 μl.

b) PCR

In order to detect C. trachomatis MoPn in the test material from mice, using primers 3 and Oligo38, primers and a TaqMan probe were selected for the region of cryptic plasmid (C. trachomatis plasmid pMoPn) for PCR (Table 2).

The volume of the reaction mixture was 25.0 μl: 2.5 μl 10 × PCR buffer, 2.5 mm MgCl ₂ , 0.25 mm DNTP, 5 pmol primers, 2.5 pmol probe, 2.5 units of thermostable polymerase, 5 μl of DNA. The temperature-time profile is shown in table 3.

The sensitivity of DNA detection by PCR was shown to be 100 copies / sample.

It was revealed that immunization with a vaccine containing Ad-MBL-TC_0037 with rTC_0037-MPLA boosting completely suppresses chlamydial infection in the upper parts of the urogenital tract in all the mice examined when compared with the control groups (see experimental design of Fig. 26), in which all mice were infected (Fig. 29, table. 4).

4) Assessment of pathological changes in the reproductive organs during immunization with a vaccine containing Ad-MBL-TC_0037 with boosting rTC_0037-MPLA.

After 30 days. after infection with C. trachomatis MoPn, pathological changes were evaluated visually. For this, mice were killed, autopsy and removal of reproductive organs (uterus, ovaries) was performed. Macro photographs of organs and a comparative analysis of pathological changes between the experimental and control groups of animals were made (Fig. 30, Table 5).

Autopsy of the ovaries and uterus in the group of animals immunized with a combination vaccine containing Ad-MBL-TC_0037 and rTC_0037-MPLA revealed no pathological changes. Adhesion process was not observed. Hydrosalpinx was not detected (Fig. 30A).

At autopsy of the ovaries and uterus in non-immunized mice infected with C. trachomatis MoPn, the pathology of the reproductive organs was pronounced, especially in the form of hydrosalpinx, characteristic of a chlamydial infection, the presence of serous hemorrhagic exudate in small quantities and severe hyperemia of the uterine horns were also recorded. ovaries (Fig. 30B).

The above examples confirm the industrial applicability of the claimed vaccine.

Industrial applicability

A new original combined anti-chlamydia vaccine has been created containing a priming component with a genetic construct based on the recombinant fifth serotype adenovirus encoding a chimeric gene consisting of the N-terminal part of the mannose-binding lectin, spacer and chlamydia CdsF antigen gene, which are fused and expressed in a single reading frame as a single protein product; and also containing a boosting component, consisting of the recombinant structural protein rCdsF CCTT of chlamydia, an adjuvant of monophosphoryl lipid A (MPLA), a toll-like receptor of type 4 receptor and squalene. A production technology (method for producing) such a vaccine is proposed. Tests of this vaccine have been carried out, it is shown that it provides protection against chlamydial infection in mouse models.

The developed anti-chlamydia vaccine, which has the ability to induce the production of specific antibodies and a specific reaction of the T-cell immune response, and consisting of a recombinant adenovirus vector expressing the hyperimmune full-size structural protein CdsF CCT chlamydia, and an emulsion for boosting, which includes the recombinant CTCT rCds protein adjuvant containing MPLA and squalene.

The proposed vaccine can be considered as a candidate drug for preclinical studies as a chlamydia vaccine against urogenital chlamydia and venereal lymphogranuloma.

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IPC: A61K 39/118

Chlamydia vaccine and method for its preparation

Information in electronic form prepared in accordance with WIPO Standard ST. 25 and is identical to the list of amino acid sequences contained in the application:

SEQ ID NO: 4 is the amino acid sequence of the full length TC_0037 protein of C. trachomatis MoPn.

MetAlaLysSerCysSerAlaLeuAsnPheAsnGluMetSerGluGlyValCysLysTyrValLeuGlyValGlnGlnTyrLeuThrGluLeuGluThrSerThrGlnGlyThrValAspLeuGlyThrMetPheAsnLeuGlnTyrArgThrGlnIleLeuCysGlnTyrMetGluAlaSerSerAsnIleLeuThrAlaValHisThrGluMetIleThrMetAlaArgSerAlaLysGlySer

SEQ ID NO: 5 is the amino acid sequence of a glycine-serine spacer.

GlySerProGlySerGlySerGlySerGlySerGlySerArgSer

SEQ ID NO: 6 is the amino acid sequence of MBL.

MetSerIlePheThrSerPheLeuLeuLeuCysValValThrValValTyrAlaGluThrLeuThrGluGlyValGlnAsnSerCysProValValThrCysSerSerProGlyLeuAsnGlyPheProGlyLysAspGlyArgAspGlyAlaLysGlyGluLysGlyGluProGlyGlnGlyLeuArgGlyLeuGlnGlyProProGlyLysValGlyProThrGlyProProGlyAsnProGlyLeuLysGlyAlaValGlyProLysGlyAspArgGlyAspArgAlaGluPheAspThrSerGluIleAspSerGluIleAlaAlaLeuArgSerGluLeuArgAlaLeuArgAsnTrpValLeuPheSerLeuSerGlu

Фиг. 9. Полная нуклеотидная и аминокислотная последовательность генетической конструкции MBL-TC_0037.

FIG. 9. The complete nucleotide and amino acid sequence of the genetic construct MBL-TC_0037.

SEQ ID NO: 8 is the amino acid sequence of TC_0037-6 * His protein.

MetAlaLysSerCysSerAlaLeuAsnPheAsnGluMetSerGluGlyValCysLysTyrValLeuGlyValGlnGlnTyrLeuThrGluLeuGluThrSerThrGlnGlyThrValAspLeuGlyThrMetPheAsnLeuGlnTyrArgThrGlnIleLeuCysGlnTyrMetGluAlaSerSerAsnIleLeuThrAlaValHisThrGluMetIleThrMetAlaArgSerAlaLysGlySerLeuGluHisHisHisHisHisHis

IPC: A61K 39/118

Chlamydia vaccine and method for its preparation

Information in electronic form prepared in accordance with WIPO Standard ST. 25 and is identical to the list of nucleotide sequences contained in the application:

SEQ ID NO: 1 represents the nucleotide sequence of the gene for the full-length protein TC_0037 C. trachomatis MoPn.

atggccaagagctgctccgctctgaacttcaatgagatgagcgaaggcgtgtgtaagtacgtcctcggagtgcagcaatatctgaccgagctggaaacatccacccagggcacagtggacctcggaaccatgttcaacctgcagtacaggacacaaatcctctgccagtacatggaggcctccagcaacattctgaccgctgtgcacacagagatgatcaccatggccaggtctgctaagggcagctaa

SEQ ID NO: 2 is the nucleotide sequence of a glycine-serine spacer.

ggatccccaggcagcgggtctggatctggcagcggaagcagatct

SEQ ID NO: 3 represents the nucleotide sequence of the gene for the MBL protein.

atgtccattttcacatccttccttctgctgtgtgtggtgacagtggtttatgcagagaccttaaccgaaggtgttcaaaattcctgccctgtggttacctgcagttctccaggcctgaatggcttcccaggcaaagatggacgtgacggtgccaagggagaaaagggagaaccaggtcaagggctcagaggcttgcaaggccctcctggaaaagtaggacctacaggacccccagggaatccggggttaaaaggagcagtgggaccgaaaggagaccgtggggacagagcagaatttgatactagcgaaattgattcagaaattgcagccctacgatcagagctgagagccctgagaaactgggtgctcttctctctgagtgaa

Фиг. 9. Полная нуклеотидная и аминокислотная последовательность генетической конструкции MBL-TC_0037.

FIG. 9. The complete nucleotide and amino acid sequence of the genetic construct MBL-TC_0037.

SEQ ID NO: 7 represents the nucleotide sequence of the gene for the protein TC_0037-6 * His.

atggctaaatcctgctccgctctgaacttcaacgaaatgtccgaaggtgtttgcaaatacgttctgggtgttcagcagtacctgaccgaactggaaacctccacccagggtaccgttgacctgggtaccatgttcaacctgcagtaccgtacccagatcctgtgccagtacatggaagcttcctccaacatcctgaccgctgttcacaccgaaatgatcaccatggctcgttccgctaaaggttccctcgagcaccaccaccaccaccactga

Claims (16)

1. A combined chlamydia vaccine inducing a protective immune response against chlamydial infection, consisting of a recombinant human adenovirus vector of the fifth serotype expressing the gene for the full-sized structural protein CdsF of the chlamydia type III secretion system and an emulsion containing recombinant structural type C protein R chlamydia and combined adjuvant. 2. The vaccine according to claim 1, which is based on the use of Chlamydia trachomatis proteins. 3. The vaccine according to claim 1, which is based on the use of Chlamydia trachomatis MoPn proteins. 4. The vaccine according to claim 1, used as a composition for the induction of specific T-cell and specific humoral immunity through the introduction of a recombinant adenovirus vector based on human fifth adenovirus expressing the gene for the full-sized structural protein CdsF of Chlamydia type III secretion system, intranasally and subsequent administration with an interval of 2 weeks of the emulsion containing the recombinant structural protein rCdsF secretion system type III chlamydia and a combined adjuvant, subcutaneously. 5. A method for creating a vaccine according to claim 1, which comprises producing in a culture of cells cells of recombinant adenovirus particles constructed on the basis of human adenovirus of the fifth serotype with two deleted regions ΔЕ1 / ΔЕ3 and containing an expression cassette with an insert of a chimeric gene controlled by an inducible promoter and consisting of from the N-terminal fragment of a mannose-binding lectin, a glycine-serine spacer and a gene for the full-sized chlamydia CdsF antigen, which are fused into a single reading frame for expression in as a single protein product; as well as providing for the production of a recombinant structural protein rCdsF CCTT chlamydia by creating a plasmid by cloning into a plasmid vector a gene encoding a full-length CdsF protein gene containing a label of six histidine codons, transforming the E. coli cell plasmid obtained and inducing expression of the recombinant protein in them and purifying it using an affinity sorbent under denaturing conditions, adjust the pharmaceutical acceptable buffer to the desired concentration and prepare an emulsion consisting of recombinantly rCdsF structural protein and a combined adjuvant containing monophosphoryl lipid A and squalene. 6. The method according to p. 5, involving the creation of a vaccine according to p. 2, the construction of an expression cassette based on the gene of the full-size antigen CT666 Chlamydia trachomatis. 7. The method according to p. 5, involving the creation of a vaccine according to p. 3, the construction of an expression cassette based on the gene of the full-size antigen TC_0037 Chlamydia trachomatis MoPn. 8. The method according to any one of paragraphs. 5-7, which uses a glycine-serine spacer with the nucleotide sequence of SEQ ID NO: 2, wherein the amino acid sequence is represented by SEQ ID NO: 5. 9. The method of claim 7, wherein the TC_0037 full-sized antigen gene has the nucleotide sequence of SEQ ID NO: 1, wherein the amino acid sequence of the protein is represented by SEQ ID NO: 4. 10. The method according to any one of paragraphs. 5-7, in which the mannose-binding lectin has the nucleotide sequence of SEQ ID NO: 3, wherein the amino acid sequence of the protein is represented by SEQ ID NO: 6. 11. A method of creating a recombinant protein CT_666 - Chlamydia trachomatis for the vaccine according to claim 2, which provides for obtaining the plasmid ct666-pET29b by cloning into the plasmid vector pET29b of the gene encoding the full-length CT666 protein containing a label of six histidines, transforming the obtained plasmid with E. col cells induction of expression of a recombinant protein in them and purification thereof. 12. The method of creating a recombinant protein TC_0037 Chlamydia trachomatis MoPn for the vaccine according to claim 3, which provides for obtaining the plasmid tc_0037-pET29b by cloning into the plasmid vector pET29b of the gene encoding the full-length protein TC_0037 containing a label of six histidines, transforming E. coli cells obtained induction of expression of a recombinant protein in them and purification thereof. 13. The method according to p. 12, in which the gene containing the recombinant structural protein TC_0037 of Chlamydia trachomatis MoPn with six histidine codons at the C-terminus is represented by the nucleotide sequence of SEQ ID NO: 7 with the amino acid sequence of SEQ ID NO: 8. 14. The vaccine according to claim 1, containing at a dose: - a recombinant adenovirus vector based on human adenovirus of the fifth serotype, expressing a gene of the full-sized structural protein CdsF of the chlamydia type III secretion system in an amount of 108-109 PFU, in a pharmaceutically acceptable buffer, having the following composition: 10.0 mM TrisHCl, 75.0 mM MgCl2, 5% sucrose, 0.05% polysorbate 80, 0.5% ethanol, 100 μm EDTA, pH 8.0, 0.5 ml in volume. - recombinant rCdsF protein - 10-20 μg, monophosphoryl lipid A - 50.0 μg, squalene - 10.0 mg, 0.5% Tween 80, in a volume of 0.5-1.0 ml.

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