RU2724896C1 - Polyantigenic vaccine for preventing and adjunctive treatment of tuberculosis - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: invention relates to medicine and pharmaceutics and represents a recombinant tuberculosis vaccine comprising an effective amount of recombinant protein ESAT6-CFP10-Ag85a-Rv2660c, which is fused mycobacterial proteins ESAT-6, CFP10, Ag85a, Rv2660c with histidine tag with length of 8 residues, and an adjuvant represented by a CPG oligonucleotide and a muramyl dipeptide, wherein the recombinant protein and the adjuvant are immobilized on carrier particles from a lactic and glycolic acid copolymer PLGA.EFFECT: increased immunogenic action of recombinant protein-antigen ESAT6-CFP10-Ag85a-Rv2660c in combination with adjuvant presented by CpG-ODN of class A and N-acetylmuramyl-L-alanyl-D-isoglutamine.5 cl, 13 dwg, 10 tbl, 7 ex

Description

The invention relates to medicine, namely to immunology, and can be used to obtain a vaccine that acts on immunodominant antigens of M. tuberculosis, expressed at an early stage of infection and latent phase of infection for the prevention and auxiliary treatment of tuberculosis.

Tuberculosis (TB) remains one of the most dangerous and common infectious diseases. This is a socially significant contact-transmitted disease caused by infection with Mycobacterium tuberculosis mycobacteria (also M. bovis, M. Africanum). In total, according to epidemiological estimates, over one billion people have died from tuberculosis over the past 200 years [1]. This is more than the number of people who died in the same period from smallpox, malaria, plague, flu, cholera and AIDS combined. By the end of the XIX century. one in five deaths from any cause was associated with tuberculosis [1]. To date, according to WHO [2], an average of about 9 million new cases and about 2 million deaths per year are recorded in the world on average (in 2015, 10.4 million new cases of active TB; 1.8 million people died from the disease). In total, up to 2 billion people worldwide are infected with the causative agent of tuberculosis, and WHO experts predict that the number of new infections by 2020 may increase by another 1 billion [3].

The urgent problem of tuberculosis is also in our country. Over the past six years, the incidence of tuberculosis has been decreasing, however, both today and in the future, tuberculosis remains a public health problem that requires constant attention, supervision and control [2].

Despite the presence of the BCG vaccine, and the fact that tuberculosis is a rather successfully treatable disease, it is still one of the three main causes of death from infectious diseases [4].

The other side of the problem of tuberculosis is an increase in the incidence of its multi-drug resistant forms. It is known that in 2015, about half a million new cases of multidrug-resistant tuberculosis (MDR-TB) were recorded [4]. The main problem of MDR-TB is the very high cost of treatment (more than $ 10,000 per course) with a prognosis of recovery in only 50% of cases. Already now, according to experts, about 50 million people around the world are latently infected with MDR-TB [4]. Meanwhile, the probability of transition of the latent form to the active one can be more than 10% during the course of life [5]. The problem of the spread of MDR-tuberculosis is particularly acute in Russia. So, in the total global incidence of MDR-tuberculosis, the combined contribution of India, China and Russia is 45% [4].

It is already obvious that global control of tuberculosis can be achieved only by creating effective preventive and therapeutic vaccines, developing diagnostics and optimizing treatment regimens [6]. According to experts, while maintaining the existing research trends, the reduction in morbidity and mortality will reach only 1.5% of the existing [6]. Optimization of existing methods of prevention and treatment will reduce the incidence by 10% annually by 2025, and the development of new approaches will reduce the incidence to the expected level of 90% [7].

Currently, Calmett-Guerin Bacillus (BCG) is the most widely used vaccine in the world and the only tuberculosis vaccine approved for clinical use. Over the nearly 100-year period of its existence, more than 3 billion people have been vaccinated with this vaccine, and now around the world the share of vaccinees has reached 80% [8]. But, despite its widespread use, BCG provides only minimal protection against TB and is not able to deal with an existing infection.

The protective effect provided by BCG is short-lived and lasts only a few years. BCG does not protect against pulmonary TB in adolescence and adulthood, and it is these cases of TB that are most common in our time [9]. Although BCG is considered safe, it poses a certain risk to immunodeficient HIV-infected patients. Therefore, WHO does not recommend BCG vaccination with HIV-positive infants [10].

BCG elicits an immune response that can prevent the development of miliary or meningeal tuberculosis, but cannot prevent and eradicate tuberculosis infection [11]. The effectiveness of BCG varies over a wide range in different vaccination programs: in some it reaches 80%, in others it tends to zero [12, 11]. The effectiveness of the vaccine depends on many factors, including the characteristics of the BCG strain, the genetic characteristics of the individual’s immune system, his age, preliminary infection of M. tuberculosis, the form of TB disease, and preliminary sensitization to BCG of the immune system with saprophytic Mycobacterium species [9]. An obvious drawback of BCG is its inability to cause long-term protection against TB: individuals vaccinated shortly after birth lose their protection already in adolescence and adulthood, and repeated injections of BCG no longer cause the expected immune response [13].

To date, there are at least 11 different BCG strains in the world sent to different laboratories at the beginning of the 20th century [14]. At that time, there were no standardized procedures for storing, growing and replanting bacteria, and until the 1960s, until methods for lyophilization of maternal bacterial lines were developed, each laboratory used its own methods. This led to the accumulation of many different mutations in different strains and to significant genetic divergence of the strains [15, 14]. The genetic differences of the strains can affect their effectiveness in protecting against infection, since they carry differing surface antigen proteins that induce an immune response [15]. Nevertheless, all available BCG strains remained non-virulent, and all of them lacked the ESX-1 secretion system (groups of genes expressing type VII virulence factors, which, according to modern concepts, determine the pathogenicity of M. tuberculosis), the components of which (products of ESAT6, CFP10 et al.) Are potent antigens of M. tuberculosis.

Thus, mainly BCG provides protection only against extrapulmonary forms of tuberculosis, and only in early childhood. The relatively low effectiveness of BCG vaccination in the adult population has led to an active search for alternative solutions to protect against various forms of tuberculosis in adults. Attempts to prolong or restore the action of BCG with the help of booster vaccines did not lead to noticeable success [13].

In this regard, dozens of laboratories around the world are currently developing new TB vaccines that can not only replace BCG for vaccination of newborns, but also can effectively protect against tuberculosis in adolescence and adulthood, prevent the reactivation of latent forms of tuberculosis and, ideally giving the opportunity to fight TB in the latent phase, completely destroying M. tuberculosis in the body.

Almost all effective vaccines against infectious pathogens currently in use are aimed at developing a humoral immune response, that is, they lead to the production of specific antibodies [16]. Since M. tuberculosis is an intracellular pathogen and is located inside macrophages in special vacuoles (phagosomes) that carry special markers of early phagosomes and do not combine with lysosomes, the humoral immune response to fight M. tuberculosis is not enough. To kill M. tuberculosis, cellular immunity is required, provided by immunocompetent M. tuberculosis-specific T cells capable of recognizing and eliminating infected macrophages without significant harm to the host. Cellular immunity against M. tuberculosis is provided primarily by Th1 type CD4 + T cells. These cells produce IFN-γ, which causes an increase in the antimycobacterial activity of macrophages. Although the central role of CD4 + T cells in immunity against M. tuberculosis is not in doubt, it has recently become clear that CD4 + T cells producing IFN-γ alone are not enough [17, 18]. Successful protection against M. tuberculosis requires a simultaneous coordinated response of CD4 + and CD8 + T cells, producing not only INF-γ, but also TNF-α, and even more than 10 types of interleukins. An important role in protecting against M. tuberculosis is played by other types of T cells and cells of long-term immune memory [19, 20]. In addition to intracellular localization, M. tuberculosis has a number of features that complicate the creation of a vaccine. This is, first of all, the ability to persistently persist in the host organism (causing latent infection, LTP) and suppress macrophage presentation of its antigens to T cells, which is necessary for recognition of M. tuberculosis infected cells and the formation of immune memory, as well as the ability to control macrophage apoptosis and necrosis processes , which provides M. tuberculosis with favorable conditions for spreading over host tissues [21]. Therefore, when creating new effective vaccines, one must strive to activate the immune system in such a way that it overcomes all ways of protecting M. tuberculosis and can recognize and destroy cells infected with mycobacteria.

As already noted, human tuberculosis has four stages of development: infection, LTP, reactivation of infection, and the active form of the disease, during which infection of other individuals occurs [22]. Protein antigens expressed by M. tuberculosis differ significantly at different stages of the disease [23], so a vaccine effective in one of the stages may be useless in others. At the initial infection of M. tuberculosis of an immunologically healthy individual, the infection becomes latent without manifesting itself externally. As experience shows, the complete elimination of M. tuberculosis from the body is extremely rare, and, as a rule, the activity of the immune system is reduced to maintaining M. tuberculosis in a dormant state and preventing the transition of the disease from the latent phase to the active one. Therefore, if the immune system of infected individuals does not weaken, M. tuberculosis in a persistent state persists for their whole life. An individual with LTP does not have external signs of the disease and does not infect others, but he has memory T cells specific for M. tuberculosis antigens [16]. Thus, the human immune system, by itself, is apparently unable to completely eliminate M. tuberculosis in the body, and it is not yet clear whether we can achieve this in the near future with the help of new vaccines. Most likely, different vaccines will be used at different stages of TB and in various cases, but a vaccine capable of killing M. tuberculosis at all stages of the development of the disease would be ideal [24].

According to their purpose, new anti-tuberculosis vaccines can be divided into three types - primary, booster (auxiliary, reinforcing) and therapeutic. Primary vaccines are being developed to replace BCG, while booster vaccines are used to enhance its effect after vaccination. Therapeutic vaccines are primarily aimed at combating active TB in HIV-infected patients. Of particular interest are vaccines that suppress M. tuberculosis in the latent phase of infection and are ideally able to completely remove M. tuberculosis from the body [26].

Almost all vaccines currently under development could be used after infection with M. tuberculosis. By their origin, recombinant vaccines based on transgenic BCG strains carrying some individual M. tuberculosis antigens (these are usually live non-virulent strains) or inactivated cells and full-cell extracts of some M. tuberculosis or other Mycobacterium strains: M. smegmatis, M. vaccae are distinguished. M. indicus. In some cases, their genetically modified forms are used, having M. tuberculosis antigens capable of enhancing immune memory [27]. In addition, two more types of vaccines are being developed that have the greatest prospects to date: these are subunit DNA vaccines and subunit protein vaccines. Subunit DNA vaccines are a genetic construct comprising a gene for one or more M. tuberculosis antigens under the control of a eukaryotic promoter. DNA vaccines are delivered as part of a DNA vector or virus. For this, transgenic forms of adenoviruses and poxviruses incapable of replication in humans are used that carry genes encoding M. tuberculosis antigens transcribed in human cells [18]. Subunit protein vaccines are artificially created protein molecules, including protective antigens of the pathogen, synthesized in various expression systems.

To date, several hundred M. tuberculosis antigens are known that are promising for the construction of new subunit vaccines [28]. Only antigens specific for the latent stage of TB revealed more than 60 [23]. Most of the subunit vaccines currently being tested contain M. tuberculosis antigens Ag85a, Ag85b and ESAT-6. The first two are surface proteins of M. tuberculosis and are involved in the formation of the bacterial cell wall. ESAT6 is a secretory protein necessary for the manifestation of virulence of M. tuberculosis, which is absent in BCG. ESAT-6 interacts closely with another promising M. tuberculosis antigen, CFP10, which is part of the bacterial type VII secretion system [29].

Recombinant, attenuated, and “classic” vaccines are created primarily on the basis of live, non-virulent strains of genetically modified mycobacteria of various species, or their lysates and whole-cell extracts. Table 1 provides a list of vaccines that are currently at various stages of clinical and preclinical studies. New TB vaccines are being developed mainly in three directions. The first is to replace BCG with its improved recombinant forms; the second and third - in the creation of vaccines based on either genetically attenuated strains of mycobacteria or transgenic strains of saprophytic species of mycobacteria carrying genes of the main antigenic determinants of M. tuberculosis. Genetically improved BCG should have the following characteristics: they should be safer, more immunogenic, able to provide long-term residual protection against tuberculosis, especially against extremely virulent clinical isolates (such as Beijing, MDR, XDR, TDR strains, etc.). One way to improve BCG is to introduce immunodominant antigens of M. tuberculosis in its genome, which are absent in the BCG genome, such as antigens encoded by the RDl locus (ESAT-6, CFP10), or to increase the production of antigens already found in BCG (for example, the complex Ag85), but not actively expressed. Another way to improve BCG is through its genetic modifications, which would activate such important immune processes as cross-presentation of CD8 + antigens to T cells. All these “improvements” in BCG can be combined in one new vaccine strain [30].

The second direction in the creation of new live vaccines against TB is the genetic attenuation of M. tuberculosis. This strategy includes a deletion of some important metabolic genes leading to the appearance of auxotrophic mutants that are unable to synthesize certain compounds necessary for the manifestation of “normal” virulence, or a deletion of the main virulence genes (or their regulators). To prevent the return of virulence, at least two independent loci on which this property depends on the M. tuberculosis genome must be removed. The first auxotrophic strains of M. tuberculosis were obtained back in the mid-1990s. [31]. Later, strains of M. tuberculosis, which are very promising for testing as a vaccine, were obtained, including the ARDlApanCD strain having deletions in RD1, the main gene cluster of M. tuberculosis responsible for virulence, and in the gene participating in the pantothenate biosynthesis pathway. The use of this strain causes effective protection against TB in normal and immunocompromising mice, and is quite safe to use [32]. Another promising attenuated strain of M. tuberculosis as a vaccine is APhoPAfadD26. The two-component regulatory system PhoP / PhoR at the transcriptional level regulates the expression of a large number of genes, including the main virulence genes of M. tuberculosis. Thus, an attenuated laboratory strain of H37Ra contains a mutation that inactivates the phoP gene [33]. The fadD26 gene, also inactivated in the APhoPAfadD26 strain, is involved in the biosynthesis of PDIM glycolipid, an important lipid component of the M. tuberculosis cell wall [34].

The third area is the creation of new vaccines based on other types of mycobacteria. These are, first of all, the moderately virulent and avirulent for humans species M. vaccae, M. microti, M. smegmatis, as well as the saprophytic species M. indicuspranii. Inactivated cells of M. vaccae [35] and M. indicuspranii, as well as full-cell extract of M. smegmatis [36] are now at different stages of clinical trials as therapeutic vaccines. In animal models, the effectiveness of the recombinant strain of M. microti expressing the antigens RD-1 of the M. tuberculosis locus has been shown to protect against infection with tuberculosis [37]. A live vaccine is being developed based on a recombinant attenuated strain of M. smegmatis, from the genome of which its own locus esx-3 has been removed [one of the virulence factors of M. tuberculosis] and a homologous locus esx-3 from M. tuberculosis has been added instead. Such a replacement does not lead to the restoration of virulence, but in mouse models causes a sharp increase in the number of memory T cells and generates a strong immune response to M. tuberculosis, which in some cases leads to the complete elimination of the pathogen [38].

The range of vaccines successfully developed (CI stage 1 or higher) over the past 10 years has expanded significantly, but mainly due to the composition of protein antigens. Noteworthy is the number of products that contain the same antigens, mainly humoral immunodominants and the same or similar adjuvants from the TLR (Toll-Like Receptors) agonist group.

The use of viral vectors for recombinant subunit TB vaccines was the first attempt to find a replacement for BCG, or at least to increase its effectiveness by subsequent boost vaccination. The first disappointment in the trials of new vaccines was the results of clinical trials (CI) of MVA85A (No. 7 in Table 1) developed by Oxford University (based on vaccinia virus, Ankara strain), which did not reveal significant differences in prophylactic efficacy between this candidate and control (only BCG) in children. Nevertheless, the vaccine is still being tested, but already as a therapeutic one, for adults.

Another viral, adeno-recombinant (Adeno-5) TB vaccine Ad5Ag85A (No. 2 in Table 1) was developed at McMaster University (Canada) and entered Phase I clinical trials, which were terminated in 2013 after the acquisition of all rights for the vaccine by the Chinese company Kansino. At present, KanSino is developing a new Ad5Ag85A project in conjunction with the non-profit company Aeros (USA), which finances the majority of modern developments of new TB vaccines.

A similar adeno-recombinant (Adeno-35) TB vaccine Ad35 / Aeras 402 (No. 6 in the table) (manufactured by Krucell and Aeras) showed safety and a broad immune response according to the type of TM in phase 1 CT, with the exception of one but an important marker is interleukin-17 (IL-17). Phase 2 CIs are currently undergoing. It is noteworthy that the same Ag85a antigen expressed in another viral vector (Ankara) as part of MVA85A induces IL-17. Based on these data, Aeros decided to finance another project - the use of Ad35 / Aeras 402 and then MVA85A as a booster.

The use of adjuvants is one of the ways to increase the immunogenicity and thus the effectiveness of vaccines. A number of candidates for TB vaccines use for this purpose a commercially available substance based on DNA oligonucleotides of the class CpG - IC31® (Intersel, Austria), which is an agonist of TLR9 receptors. The Danish Institute of Serum (Statens Serum Institut), together with Aeros, is testing various combinations of recombinant (in the expression system of E. coli) mycobacterial antigens and this adjuvant.

The first candidate was Hybrid-1 (ESAT6 / Ag85B + IC31), which generally showed good results in CI, but did not reveal the expected response to key ESAT-6 antigen (No. 5 in the table). In addition, studies have shown that the presence of ESAT-6 in a vaccine formulation casts doubt on the results of the IGRA (interferon-gamma release assay) test used in evaluating efficacy.

To overcome this drawback, ESAT-6 was replaced by functionally similar TB-10.4 antigen in the new experimental TB vaccine Hybrid 4 / Aeras-404 (or HyVac4 / Aeras404), developed jointly with Sanofi Pasteur, Canada (No. 3 in the table) . One phase 1 CI with this candidate was terminated without explanation, but another attempt is being made to conduct a combined phase CI 1/2.

The last candidate from this series to date is Hybrid 56 / Aeras-456 (or H56 / IC31), in addition to the original version (ESAT6 / Ag85B + adjuvant IC31) includes the third antigen (latent-associated) Rv2660c (No. 4 in the table) . The results of preclinical studies (DCI) on lower primates showed the effectiveness of this drug in both preventing primary infection and blocking the reactivation of latent infection. Clinical studies of phase 1/2 are ongoing.

Another adjuvant, although also a TLR agonist, uses GlaxoSmithKlein-AS02 (MPL ™ + QS-21) (TLR4 agonist) in its development, in combination with two mycobacterial antigens Rv1196, Rv0125, which are expressed immunodominant (No. 8 in the table ) Candidate M72 successfully completed a Phase 1 clinical trial and, following MVA85A, entered Phase IIb performance study.

A similar adjuvant (TLR4 agonist), but synthesized (GLA-SE) was used by IDRI (Research Institute of Infectious Diseases, USA) in conjunction with Aeros immediately with four latently associated mycobacterial antigens: Rv2608, Rv3619, Rv3620, Rv1913 (No. 2 table) Candidate ID-93 has been shown to be highly effective in DCI, including protection against resistant strains of MTB in lower primates.

The following solutions are the most relevant in relation to the subject under study, from among the identified patent documents.

The invention according to the application US 2011/0020384 A1 relates to the use of different antigens ESAT-6, ESAT-6-Ag85B, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rvl036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59 , Ag85C, 19 kDa lipoprotein, MPT32 Mycobacterium tuberculosis for the development of a multivalent anti-TB vaccine.

The invention of US 7968105 B2 relates to an immunogenic composition or drug consisting of one antigen ESAT-6, Ag85A, Ag85B, TB 10.4 Mycobacterium tuberculosis; combinations of various chimeric (fused) proteins based on these antigens; the use of fusion proteins in different combinations with BCG vaccine.

The invention according to the application US 2011/0027349 B2 relates to an immunogenic composition comprising one of the antigens Ag85A, Ag85B, MPT-64, Pst-S1, Ara, GroES, GroEL, Dnak, CFP-10, Rv083 1c, Rv1324 Mycobacterium tuberculosis, or various combinations thereof ; the use of polypeptides as components for vaccination according to the prime boost scheme in combination with the BCG vaccine; the use of various adjuvants.

The invention described in international patent publication WO 2009/143413 relates to immunogenic compositions containing fusion proteins, including proteins of the mtb complex Mycobacterium tuberculosis conjugated to a polysaccharide such as dextran, the claimed compositions giving an enhanced T-cell response.

The invention according to the application US 2010/0015171 A1 relates to an immunogenic composition comprising antigens of the Ag85 family and TB 10.4 Mycobacterium tuberculosis; various combinations of these antigens; the use of various adjuvants (DDA, polycationic peptides, oligonucleotides, IC31® adjuvant) for the introduction of antigens.

The invention RU 2520737 C1 relates to biochemistry and biotechnology and is a recombinant plasmid pESAT6-DBD, consisting of an artificial bacterial operon of a chimeric protein, including the promoter region of the early promoter of the bacteriophage T5, a gene of the chimeric protein, consisting of the sequence of the ESAT-6 protein antigen from Mycobacterium tuberosis fused to the sequence dextran binding domain (DBD) dextransurase Leuconostoc citreum KM20 and transcription terminator; a bacterial operon of beta-lactamase; and a bacterial replication initiation site of ColE1 type. The invention also includes a strain of Escherichia coli, a producer of the chimeric protein ESAT6-DBD, as well as a method for immobilizing, concentrating and purifying the obtained protein on dextran. In addition, the invention relates to the recombinant ESAT6-DBD protein itself and an immunogenic composition containing it, aimed at inducing immunity against tuberculosis infection. The invention allows to obtain a producer strain that provides a high level of production of stable immunogenic proteins that can be obtained, immobilized and purified in one stage, as well as to obtain effective immunogenic compositions against tuberculosis.

The invention RU 2539026 C1 relates to genetic engineering, biochemistry, biotechnology and immunology and is a recombinant plasmid pESAT6-CFP10-DBD, a recombinant strain of Escherichia coli M15 [pREP4, pESAT6-CFP10-DBD], a method for the preparation, immobilization, concentration and purification of a recombinant protein Dextran ESAT6-CFP10-DBD, 26.4 kDa recombinant ESAT6-CFP10-DBD protein, ESAT6-CFP10-DBD immunogenic composition immobilized on dextran, specifically activating T lymphocytes that synthesize IFN-gamma upon stimulation Mycobacterium antigens. EFFECT: invention allows to obtain a high level of protein expression, which effectively induces an immune response to mycobacterial antigen.

The closest analogue of the invention is a recombinant anti-tuberculosis vaccine (RU 2665817 C1) containing the first antigen, which is a purified recombinant protein Ag85A of M. tuberculosis Mycobacterium tuberculosis, the second antigen, which is a hybrid recombinant protein ESAT6-CFP10 of M. tuberculosis Mycobacterium tuberculosis, including purified tuberculosis M. tuberculosis ESAT-6 mycobacterial protein, fused to purified CFP10 mycobacterial protein, and an adjuvant that includes the first water-soluble high molecular weight polysaccharide dextran for immobilization of the Ag85A and ESAT6-CFP10 proteins of the indicated antigens, unmethylated CpG oligonucleotide with a phospho-receptor of the receptor group 3 and a second water-soluble high molecular weight dextran polysaccharide, different from the first water-soluble high molecular weight dextran polysaccharide, for immobilizing said CpG oligonucleotide.

The GamTBvak vaccine, which is in phase II of clinical trials, is positioned as a prophylactic drug, it is supposed to be used as a “booster vaccine” (“boost”) for use in BCG-vaccinated adults. Conducted preclinical trials and a clinical trial of phase I vaccine GamTBvak showed its safety and immunological potential [39].

Since the number of infected people with latent TB is high, one of the most urgent tasks is to develop a vaccine that can not only protect against active forms of the disease, but also control the reactivation of mycobacteria in already infected patients and, thus, maintain a dormant state in the host’s body the condition of the pathogen cells, and thus significantly limit the natural reservoir of tuberculosis, and in addition, help reduce treatment time when used together with antibiotic therapy.

Therefore, the object of the invention was the development of a subunit vaccine (LTPvac) containing recombinant MTB antigens expressed at different stages of infection.

In addition to the preventative model, it is being tested as a therapeutic vaccine. The vaccine includes three antigens expressed at an early stage of infection (Ag85a, CFP10, ESAT6), and one antigen expressed in the latent phase of the infection (Rv2660c).

This choice of proteins is due to the need for immunization of tuberculosis, which allows you to control all stages of the infectious process.

The main antigenic components of the vaccine are immunodominant recombinant proteins ESAT-6 (Rv3875, Early secreted antigenic target 6 kDa protein) and CFP10 (Rv3874, Culture filtrate protein 10). They are secreted antigens found in the filtrate of an early mycobacterial culture. Their inclusion is due to the strong immune response they provoke at all stages of infection, and confirmed immunogenicity when vaccinated in trials in animal models and humans.

The genes encoding these proteins are located in unique regions of the genomes of virulent strains of M. tuberculosis, and are not found in any of the eight used BCG vaccine sub-strains. They are included in the composition of the ESX-1 secretion system and, apparently, partially determine the virulence of mycobacteria, allowing the pathogen to bypass the host's immune response. Recombinant antigens entering the body have been shown to be able to induce IFN-γ synthesis in case of tuberculosis infection, activate the specific immune response of the organism upon contact with M. tuberculosis, and form protective immunity until it is in direct contact with tuberculosis pathogens - mycobacteria.

In addition, the vaccine includes the Ag85a antigen (Rv3804c), which plays an important role in the formation of anti-tuberculosis immunity. This protein is capable of stimulating the specific production of IFN-γ in tuberculosis infection; it contains epitopes for recognition by the T-cells of the CD4 + and CD8 + populations. In addition, Ag85a induces the production of specific antibodies, which are found both in the sera of patients with positive tuberculin samples and in the active form of TB. Thus, the antigen stimulates the formation of a balanced immune response, with the predominance of the Th1 type, which is necessary for the development of protective immunity.

Since the vaccine under development is aimed, inter alia, at preventing the reactivation of latent forms and the occurrence of secondary tuberculosis, the recombinant Rv2660c protein belonging to the DosR proteins of the M. tuberculosis regulon was included in the antigenic composition of LTPvac. It is known that the dormant state of mycobacteria is characterized by a low level of metabolic activity of cells, as well as increased expression of proteins related to the DosR regulon. It was the DosR genes that were identified as key regulators of the transition to the dormant state in vitro, and are necessary to maintain this state in vivo. It is assumed that the inclusion of such antigens in the composition of the developed vaccines can increase the effectiveness of the control of the reactivation of mycobacterial infection and reduce the bacterial load.

Thus, the vaccine preparation is formulated as follows.

The recombinant TB vaccine contains an effective amount of the recombinant protein ESAT6-CFP10-Ag85a-Rv2660c, which is a fused mycobacterial protein ESAT-6, CFP10, Ag85a, Rv2660c with a histidine tag of 8 residues and the adjuvant represented by the recombinant protein and an adjuvant immobilized on carrier particles from a lactic and glycolic acid copolymer of PLGA. The PLGA carrier is a copolymer of lactic and glycolic acids with a molecular weight of 66,000-108,000 Yes, with a ratio of monomer units of 75:25. In this case, the average particle size of PLGA with immobilized protein is in the range of 100-200 nm. The adjuvant in the claimed vaccine is a combination of a CPG oligonucleotide and a muramyl dipeptide, where the muramyl dipeptide is a D-L isomer, an N-acetylmuramyl-L-alanyl-D-isoglutamine, and a CPG oligonucleotide is a class A CpG-ODN.

The choice of this formulation is due to the fact that each component plays a role important for the formation of effective protective immunity.

The technical result from the use of the proposed recombinant anti-tuberculosis vaccine is that the recombinant antigen ESAT6-CFP10-Ag85a-Rv2660c in combination with the adjuvant represented by CPG-oligonucleotide and muramyl dipeptide has an increased immunogenic effect, primarily due to its increased mass of antigen sizes, which causes a more active absorption of such proteins by the recognizing cells of the immune system. The use of the ESAT6-CFP10-Ag85a-Rv2660c whole-protein fusion protein provides a uniform steady high level of cellular and humoral immune response. This provides a synergistic effect of the epitopes of the hybrid protein in comparison with the individual antigens that make up its composition. The content of the vaccine of three antigens expressed at an early stage of infection (Ag85a, CFP10, ESAT-6), and one antigen expressed in the latent phase of the infection (Rv2660c) allows you to control all stages of the infection process.

A brief description of the drawings.

FIG. 1 - Schematic representation of the location of key elements of the expression vector. Designations: lad, rop, ori, KanR, fi ori, T7, DsbA — service regions of the plasmid pET-42b (+); ESAT-CFP, AG85, 2660 s - coding regions of the target antigens ESAT-6, CFP10, Ag85a, Rv2660c, respectively; 8His is an oligohistidine tag.

FIG. 2 - Growth curves of E. coli strain Clear coli BL21 (DE3) on various nutrient media.

FIG. 3 - Analysis of protein expression in E. coli cells (electrophoresis in 12% PAAG).

(1) ESAT6-CFP10-Ag85a-2660c (8His) (before induction) (2) ESAT6-CFP10-Ag85a-2660 s (8His) (after induction) (3) ESAT6-CFP10-Ag85a-2660c (8His) (lysate supernatant) (4) ESAT6-CFP10-Ag85a-2660c (8His) (insoluble precipitate) (5) Molecular weight marker (14.4, 21.5, 31.45, 66.2, 97.4 kDa).

FIG. 4 - Chromatogram of a recombinant hybrid protein ESAT6-CFP10-Ag85a-Rv2660c on a column with a sorbent Ni Sepharose High Performance.

FIG. 5 - Electrophoretic separation in PAGE by Laemmli of protein fractions obtained in the process of isolation and purification of the recombinant fusion protein ESAT6-CFP10-Ag85a-Rv2660c. (1) supernatant proteins after biomass lysis, not bound to the Ni-NTA sorbent; (2-12) protein ESAT6-CFP10-Ag85a-Rv2660c (68.2 kDa), eluted with a gradient of an imidazole solution of 10-500 mM, pH 8.8; (13) Molecular weight marker (14.4, 21.5, 31.45.66.2, 97.4 kDa).

FIG. 6 - Electrophoretic separation in PAGE on Laemmli combined preparation of a hybrid protein after dialysis. (1) ESAT6-CFP10-Ag85a-Rv2660c protein (68.2 kDa), after dialysis against 20 mM Tris pH 8.8, 5 mM β-mercaptoethanol; (2) Molecular weight marker (14.4,21.5, 31, 45.66.2, 97.4 kDa).

FIG. 7 - Study of the proliferation of CD4 +, CD8 + T-lymphocyte populations in mice with various vaccine formulations. The proportion of proliferating effector (CD4 +) and cytotoxic (CD8 +) from all T-lymphocytes in response to the introduction of the components of the vaccine antigen separately and the hybrid protein is shown. The data are presented in the form of cumulative (summing) diagrams.

FIG. 8 - Production of INF-γ by splenocytes of mice upon stimulation with vaccine antigens. Designations: AB — results of an IGRA test when stimulated by individual components of a hybrid protein-antigen; D — results of an IGRA test when stimulated by a hybrid protein. Shows the average values for the groups.

FIG. 9 - The cumulative effect of the production of INF-γ by splenocytes upon stimulation with all vaccine antigens.

FIG. 10 - Serum levels of test animals of class G antibodies to peptides that are part of a specific antigen. A humoral immune response to each of the peptides (A-G) is shown. Data are presented as all values against the background of the mean +/- standard error of the mean (mean +/- SEM).

FIG. 11 - Serum levels of test animals of class G antibodies to the LTPvak vaccine fusion protein. Data are presented as all values against the background of the mean +/- standard error of the mean (mean +/- SEM).

FIG. 12 - Results of seeding of target organs (spleen - upper panel, lung - middle panel, dynamics of animal weight change - lower panel) for the presence of M. tuberculosis in them.

FIG. 13 - The results of seeding of target organs (spleen, lung) on the presence of M. tuberculosis in them for 15 weeks in animals of the studied groups. The data are presented in the form of a span chart indicating the median, lower and upper quartiles.

DETAILED DESCRIPTION OF THE INVENTION

A subunit polyantigenic genetic engineering vaccine has been developed for the prophylaxis and adjunctive treatment of tuberculosis (including the latent form) with the working name "LTBvak", the components of which are recombinant mycobacterial proteins expressed both in the early (active) Ag85a, ESAT-6, CFP10, and at the latent (Rv2660c) stages of infection, combined by covalent bonds into a common (“fusion”) recombinant protein ESAT6-CFP10-Ag85a-Rv2660c; carrier (poly (lactic-co-glycolic acid), PLGA); and a complex molecular adjuvant represented by CpG oligonucleotides (CpGODNs), which are ligands of various types of receptors of the innate immunity system (toll-like receptors-TLR9), as well as muramyl peptide (MDP), which is a non-specific ligand of NOD2 receptors.

The process of obtaining recombinant secretory mycobacterial proteins intended for use as specific stimulants of the immune response when used as a component of a subunit tuberculosis vaccine consists of the following steps:

- creation of a genetic vector for the expression of mycobacterial proteins in a fused (fusion) form;

- obtaining a culture producer of a soluble recombinant mycobacterial protein based on E. coli cells of Clear coli strain BL21 (DE3);

- scaling of the producer culture and obtaining cell biomass in the fermenter;

- obtaining a substance of recombinant secretory mycobacterial antigens in the form of a fusion protein of the composition ESAT6-CFP10-Ag85a-Rv2660c.

EXAMPLE 1

Preparation of Recombinant Ag85a-ESAT6-CFP10-Rv2660c Fusion Protein

1.1. Creating a genetic vector for the expression of genes encoding mycobacterial proteins

The generally recognized problem of recombinant protein production technologies is to obtain a sufficiently significant amount of a soluble and functionally complete product. Despite the fact that Escherichia coli remains the most common producer of recombinant antigens, this producer is characterized by the absence of helper proteins common in other organisms - intracellular chaperones. The consequence of this is often the incorrect folding of the polypeptide chain with the formation of insoluble aggregates (inclusion bodies), or soluble forms lacking functional activity.

The insolubility and lack of functional activity of the recombinant product may also be caused by inconsistencies in the use of E. coli codons and the natural producer.

It is known that cloning of the fbpA, esxA, esxB, rv2660 genes as part of E. coli expression vectors leads to the production of large amounts of protein antigens Ag85a, ESAT6-CFP10 and Rv2660c as part of inclusion bodies.

Genes encoding mycobacterial antigens were synthesized by CJSC Evrogen (Russia). To obtain soluble native forms of antigens, the genes encoding them were cloned into the vector pET-42b (+) (Novagen of Merck Millipore, USA). An expression vector was created where the target mycobacterial antigens connected by spacers and the 8-His-tag, necessary for affinity chromatographic purification of the resulting protein, were located in one reading frame (Fig. 1).

The primary sequences of the resulting genetic constructs and the polypeptide sequences encoded by them are shown in Appendix A.

1.2. Preparation of culture-producers of soluble recombinant mycobacterial proteins based on E. coli cells of Clear coli strain BL21 (DE3).

Recombinant mycobacterial antigens were produced in a specific suspension prokaryotic culture of E. coli strain clear coli BL21 (DE3). This cell culture allows you to build biomass with low pyrogenic activity. This effect is explained by genomic divisions (ΔgutQ ΔkdsD ΔlpxL ΔlpxM ΔpagP ΔlpxP ΔeptA) and one compensatory mutation (msbA148), which block the synthesis of native lipopolysaccharide molecules, which are the main pyrogenicity factor.

The producer of the recombinant mycobacterial hybrid antigen was obtained by chemical transformation using the generated genetic construct.

After obtaining colonies of producers of the recombinant mycobacterial antigen on solid nutrient media, the suitability of the process conditions for cultivation in Erlenmeyer conical flasks with temperature control and stirring was studied.

The three most popular modifications of LB nutrient medium were tested: LB-Miller, LB-Lennox, LB-Luria, during the experiment it was established (Fig. 2.) that the optimal medium for producer growth would be Miller LB nutrient medium.

When choosing the ratio of the volumes of liquid culture to the volume of the flask, two factors were taken into account - on the one hand, a decrease in the volume of the culture leads to an improvement in its aeration, on the other hand, it reduces the biomass yield in grams. During the experiment (table 2) it was found that the optimal ratio of the volume of liquid culture to the volume of the flask 1 to 5 at a stirring speed of 250 rpm

When studying the dependence of the growth of the culture of E. Coli strain Clear coli BL21 (DE3) on ambient temperature, it was found that cultivation at temperatures below 29 ° C and above 39 ° C significantly worsened the dynamics of bacterial growth.

Taking into account the above, the following were considered optimal parameters for the cultivation of producer cells:

- nutrient medium LB-Miller;

- the ratio of the volume of liquid culture to the volume of the flask 1 to 5;

- cultivation temperature 37 ° C;

- the stirring speed during incubation is 250 rpm.

The next stage of the study was the selection of conditions for the induction of expression of genes encoding recombinant antigens. It was necessary to establish the current concentration of the inductor, the time of its addition, the total duration of induction. The results of the selection of conditions for the induction of genes of recombinant mycobacterial antigens are presented in table 3.

It was also shown during experiments that when cultivating the culture of producers at a temperature of 37 ° C and above, a small part of the produced recombinant proteins appeared in the insoluble fraction of the cell lysate, while lowering the temperature to 30 ° C resulted in the appearance of all recombinant proteins in the soluble fraction.

In addition, it turned out that recombinant proteins reach their maximum native concentration in cells as a result of exposure to an inducer from 4 hours to 6 hours, after six hours of induction, recombinant proteins began to accumulate in the form of insoluble aggregates.

As a result of experiments, it was shown that the largest production of recombinant products in cells is achieved under the following conditions:

- the addition of IPTG must be done at optical density

- culture 0.5-0.75 p.u. (measurement at a wavelength of 600 nm);

- final concentration of IPTG in the medium of 0.4 mm

- the temperature of the culture medium after adding the inductor 30 ° C

- the cultivation time after adding the inductor should not exceed 6 hours.

Selected conditions for the growth and induction of producer cultures allowed us to achieve the production of intein chimeras of antigens in quantities of about 10 mg / g of cell biomass; receive biomass of cells 2 g / l of the medium; at the same time, the produced chimeras were soluble and did not fall into the composition of the inclusion bodies.

1.3. Concentration of producer cells, their lysis and protein fractionation by solubility

The development of a method for isolation and purification of the ESAT6-CFP10-Ag85A-Rv2660c hybrid protein from induced cells of producer strains included the following stages: deposition of cells from the culture medium by centrifugation, lysis and destruction of cells by sonication and in a laboratory cell separator, separation by differential centrifugation of proteins is readily soluble fractions and insoluble proteins, purification of recombinant proteins using affinity metal-chelate chromatography, sterilizing filtration. The developed technique allows purification of recombinant proteins from the biomass of producer strains with the possibility of subsequent scaling of this process to industrial volumes.

The following is a detailed stepwise description of all stages of purification of a recombinant protein.

The cells were concentrated by collecting them by centrifugation followed by resuspension in lysis buffer. It was experimentally established that centrifugation of a cell suspension at 6000 g for 20 min at a temperature of 4 ° C optimally concentrates the original cellular material, while maintaining the ability of the sediment to resuspend.

After resuspension of the cells, it is necessary to completely destroy and achieve complete release of recombinant proteins in the conditions of industrial production volumes. The method should be not only effective, but also technological.

The method of cell destruction, proposed in the laboratory version of the purification of the recombinant fusion protein ESAT6-CFP10-Ag85A-Rv2660c, consists in treating the cells with a lytic buffer with lysozyme that destroys the cell wall of E. coli and ultrasound. Lysing buffer made it possible to maintain a stable slightly alkaline environment, since when the cells are destroyed, their contents enter the solution, which reduces the pH. For lysis of the producers, a lysis buffer of the following composition was used: 20 mM Tris pH 8.8, 0.1% Triton X-100, 5 mM EDTA, 200 μM PMSF (phenylmethyl sulfonyl fluoride). Depending on the amount of biomass, based on the ratio of 1 g of cells in 20 ml of lysis buffer.

Nucleic acids of cells released during lysis make the suspension more viscous, therefore, the samples were treated with ultrasound on an ultrasonic disintegrator in the following mode: 5 s. scoring - 5 s., pause for 30 minutes at an amplitude of 40%. This method of lysis can be used in the future when scaling the process and industrial purification of proteins.

The cell suspension in lysis buffer was passed through a laboratory cell separator at a pressure of 1500 Psi, after which the lysate was centrifuged for 20 min at 15000 g at a temperature of (9 ± 1) ° С. The obtained precipitate after centrifugation was washed three times with lysis buffer, each time the precipitate of inclusion bodies was broken by ultrasound and precipitated by centrifugation for 20 min at 15000 g at a temperature of (9 ± 1) ° С. The resulting precipitate was dissolved in a solution of the following composition: 8 M Urea, 20 mm Tris pH 8.8, 5 mm β-mercaptoethanol, 0.1% Triton X-100, 10 mm Imidazole and passed under pressure through a filter nozzle with a PVDF filter and pores with a diameter 0.22 μm for sterilization of the solution (Fig. 2).

1.4. Metal Chelate Affinity Chromatography

The 8His sequence (affinity for divalent metals) was included in the amino acid sequence of the ESAT6-CFP10-Ag85A-Rv2660c fusion protein polypeptide; therefore, metal chelate affinity chromatography (MXAX) may be used to purify the fusion protein.

In metal chelate affinity chromatography, the property of protein domains with specific amino acid sequences to bind to metal ions such as Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ^{2+ is used} . Specific binding is due to the presence of free electron-donating groups on the surface of the protein. Under metal chelate chromatography (neutral pH and high salt concentrations), the imidazole group of histidine (pK ~ 6.7), the thiol group of cysteine (pK ~ 8.5), and the indole group of tryptophan (pK ~ 9.41). Under certain conditions, C-terminal amino acids (pK ~ 7.7), as well as residues of aspartic and glutamic acids (pK ~ 3.9), can take part in such interactions. According to the strength of the complexes formed, these amino acids are located in the series: His, Cys> Asp, Glu >> other amino acids. The presence of histidine residues on the surface of a protein molecule is a necessary and sufficient condition for its sorption on a chelating sorbent: sorption requires the presence of at least two closely spaced histidine residues, which can be brought together in both the primary and tertiary structure of the protein.

Sorbents for MXAX are charged with divalent metal ions, usually Ni ²⁺ . Divalent metal ions specifically bind to proteins containing polyhistidine tag (usually 6His tag), both soluble and initially insoluble, but dissolved under denaturing conditions. Successful purification using metal chelate chromatography gives a high yield of active protein; it is possible to refolde a protein immobilized on an affinity sorbent. However, due to the fact that many proteins have histidine and / or cysteine residues in their natural sequence, they can bind to a metal chelate sorbent in the same way as a protein with His tag. In such cases, it is often necessary to select the conditions for protein sorption, washing and elution in order to elute the target protein as clean as possible. In particular, an increase in the concentration of imidazole during sorption of the target proteins can reduce the amount of adsorbed impurities. Therefore, when isolating a recombinant fusion protein, the imidazole-HCl buffer system with an imidazole concentration of 20 mM, pH 8.0, was used. Protein binding on a metal-chelate sorbent usually occurs in the pH range 5.5–8.5 (depending on the sorbent), but it is stronger at alkaline pH, which determines our choice of pH. NaCl to a concentration of 0.15-0.5 M is added to the buffer solutions for MXAX in order to increase the ionic strength of the protein solution and minimize non-specific ionic interactions with the affinity matrix. The presence of detergents and denaturing agents usually does not affect protein sorption.

The following chelating groups are widely used to hold the metal ion on the surface of the affinity sorbent: iminodiacetate (IDA from the English iminodiacetate), nitriloacetic acid (NTA from the English. Nitrilotriaceticacid), carboxymethylaspartate (CM-Asp from the English carboxymethyl-ethylenediamine), TED from English Tris (carboxymethyl) ethylendiamin). These groups form coordination bonds with ions of divalent metals of various numbers and quality. Ions have different sizes, and the possibility of forming bonds for each individual chelating group depends on this. So, IDA forms two bonds with a divalent metal ion, NTA - 3, CM-Asp - 4, TED - 5. In our work, we used Ni-containing sorbent WorkBeads 40 Ni with a chelating iminodiacetate group (IDA) for metal-chelate chromatography.

There are several ways to elute a protein from a metal-chelate sorbent:

- imidazole, histidine or other substances that have an affinity for chelated metal ions.

- lowering the pH (linearly or in steps). Most proteins elute in the pH range from 6 to 4.

- chelating agents such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA). These substances bind metal ions and thereby cause protein elution. In this case, protein separation by affinity for divalent metal ions will not occur.

The first elution method is the most specific and therefore preferred over others.

To purify the hybrid protein ESAT6-CFP10-Ag85A-Rv2660c, a chromatographic column was prepared at the rate of 1 ml of a chromatographic sorbent Ni Sepharose High Performance (GE, USA) for every 20 ml of cell lysate, the sorbent was packed in cylindrical columns with a diameter of 1.5-5 cm, volume 35-100 ml under a pressure of 30 psi, after which it is balanced by 15 volumes of a Buffer solution of the composition: 8 M Urea, 20 mm Tris pH 8.8, 5 mm β-mercaptoethanol, 0.2 mm PMSF, 0.1% Triton X-100, per speeds of 1-4 ml / min.

The precipitate solution in the urea buffer solution obtained at the stage of producer cell lysis was applied to a pre-balanced column with Ni-Sepharose High Performance (GE, USA) at a speed of 1 ml / min.

The column was washed with 10-15 column volumes with Buffer solution of the composition: 8 M Urea, 20 mM Tris pH 8.8, 5 mM β-mercaptoethanol, 0.2 mM PMSF, 0.1% Triton X-100, at a rate of 2-4 ml / min with a pressure limit of 15 psi, while controlling the optical density (OD) of 220 nm, 260 nm, 280 nm, 320 nm, column washing was completed with decreasing OD at the above wavelengths to the baseline. At the stage of column loading, the recombinant fusion protein was bound to the sorbent (the stationary phase of the column), and unbound impurities were washed out with the mobile phase. The presence of impurities was detected by the first peak in the chromatogram (Fig. 3).

After washing the column from non-specifically bound impurities, the chromatography is transferred to non-denaturing conditions: a gradient is launched into the Buffer solution of the composition: 20 mM Tris pH 8.8, 5 mM β-mercaptoethanol, 0.1% Triton X-100, 10 mM Imidazole, the gradient is 15 column volumes at a flow rate of 1 ml / min.

The elution is carried out with a Buffer solution of the composition: 20 mM Tris pH 8.8, 5 mM β-mercaptoethanol, 0.1% Triton X-100, 500 mM Imidazole, at a flow rate of 1 ml / min, the eluate was collected and subsequent electrophoretic analysis of the protein composition of the eluate fractions was carried out (Fig. 4).

Fractions containing the hybrid protein of recombinant mycobacterial antigens were combined and dialyzed three times against a 500-fold excess of a solution of 20 mM Tris pH 8.8, 5 mM β-mercaptoethanol in three series of 4-12 hours to remove imidazole and urea (Fig. 5).

The yield of ESAT6-CFP10-Ag85A-Rv2660c protein during the purification process according to the developed technology amounted to 1.5 mg from 1 g of producer cell biomass.

1.5. Sterilizing filtration During sterilization by filtration, any particles (including microorganisms) are removed whose size exceeds the threshold filter transmission. As a result, the final product does not contain viable pathogenic or other microorganisms. Filtration was carried out by forcing through a membrane filter made of polyethersulfone with a pore diameter of 0.22 μm (disposable sterile system for sterilizing vacuum filtration from Corning, USA) in a sterile box. By measuring the optical density of protein solutions at 280 nm, quantitative filtering losses were evaluated. It amounted to 10-20%.

The preparation obtained after dialysis was filtered through a PVDF filter with a pore diameter of 0.22 μm, the concentration of the protein component and the homogeneity of the preparation were evaluated, the endotoxin content (LAL test) was studied, and it was poured into hermetically sealed polypropylene tubes.

EXAMPLE 2

Packaging antigens and molecular adjuvants in nanosized particles of poly (lactic-co-glycolic) acid (PLGA)

One of the most commonly used vaccine carriers is the biodegradable PLGA polymer, which consists of copolymerized residues of lactic and glycolic acids. PLGA is widely used in the creation of materials for implants, prostheses, and surgical instruments. When poly (lactide-co-glycolide) particles enter the body, hydrolysis of the copolymer of lactic and glycolic acids occurs with the formation of lactic and glycolic acid molecules that are biologically compatible and involved in the metabolism, which are ultimately eliminated from the body through the Krebs cycle. In addition to the chemical structure of the polymer, when creating a specific vaccine, it should be borne in mind that the interaction of nanoparticles with the immune system is affected by their physical parameters: size, surface charge, hydrophobicity / hydrophilicity. As a result, by changing the material of the particles and modifying their surface, the necessary effect on the immune response can be achieved. It has been shown that microparticles with an encapsulated hepatitis B virus surface antigen (HBsAg) with a diameter of 2-8 μm sit on the surface of macrophages and cause a long and strong production of antibodies. In addition, they stimulate the production of interleukin-4, a cytokine characteristic of the humoral Th2 immune response, and the synthesis of MHC II molecules. On the other hand, nanoparticles 100-600 nm in size are efficiently absorbed by macrophages and lead to less antibody production, while at the same time stimulating the synthesis of MHC I and the INF-γ characteristic of the cellular Th1 response.

In this regard, to formulate the GLF of the LTB vaccine, it was PLGA that was chosen as the carrier. The experiments on the final development of the technological process for producing PLGA microparticles with vaccine components for the prevention of tuberculosis packed into them included the immobilization of antigenic components of the drug in PLGA microparticles in three different mixing variants. The particle size of PLGA, the concentration of antigen incorporated into nanosized particles were checked, and the numbers of agonists of pattern recognition receptors in nanosized particles were measured.

Features of the developed technological processes are described below.

Antigens and agonists were dissolved in phosphate buffered saline to final concentrations:

белковой фармацевтической субстанции антигена 3,2 мг/мл;

3.2 mg / ml protein pharmaceutical antigen substance;

олигонуклеотид CpG 2 мг/мл;

CpG oligonucleotide 2 mg / ml;

МДП 2,5 мг/мл (водная фаза -W1)

MDP 2.5 mg / ml (aqueous phase -W1)

After that, a 2% solution of PLGA (75:25) in ethyl acetate (oil phase — O) was prepared and a 10% solution of PVA in mQ water (aqueous phase — W2) was prepared. To prepare the primary emulsion (W1 / O), 50 μl (I formulation), 500 μl (II formulation), or 1000 μl (III formulation) of antigen and agonist solution were added to the PLGA solution. Then emulsified using an ultrasonic unit (Branson Sonifer) for 30 seconds. To obtain a double emulsion (W1 / O / W2), a solution of 10% polyvinyl alcohol was added to the primary emulsion. Then re-voiced using an ultrasonic setup (Branson Sonifer) for 60 seconds. The resulting double emulsion was transferred into sterile glasses, covered with foil and placed on a magnetic stirrer overnight at room temperature, with constant stirring.

To precipitate the particles, the resulting formulation was centrifuged at 2000 g, 30 min, 4 ° C. To get rid of PVA, the supernatant was washed 5 times with water, each time the particles were precipitated by centrifugation at 10,000 rpm, 4 ° C, for 60 minutes.

Particle Size Measurement of Poly (Lactic-Co-Glycolic) Acid

Particle size was determined using a Zetasizer Nano ZS nanosizer (Malvern). For the Zetasizer Nano ZS PLGA, particles were diluted to a concentration of 100 μg / ml in milliQ water. Then transfer 1 ml of particles to a cuvette for measurement. The cuvette was placed in the device. Measurement and calculation of the results was performed using the software Zetasizer Software (table 4).

The average particle size of PLGA with encapsulated antigens is 126 nm and is within acceptable values (100 nm - 200 nm) necessary for the delivery of antigens into phagocytes.

Measurement of the amount of antigen incorporated into nanoscale particles

The antigen concentration per 1 mg of nanoscale particles of PLGA (table 5) was determined as highly sensitive and highly specific

immunodiagnostic method of heterogeneous solid-phase

enzyme immunoassay (GF XIII, OFS.1.7.2.0033.15). To construct a calibration curve, standard solutions of the ESAT6-CFP10-Ag85A-Rv2660c fusion protein were prepared in concentrations from 0.19 μg / ml to 50 μg / ml. An enzyme immunoassay was carried out to determine the content of the hybrid protein ESAT6-CFP10-Ag85A-Rv2660cB of a series of dilutions of a standard sample. All measurements were carried out in three technical replicates. The optical density is measured using a tablet spectrophotometer in two-wave mode: the main filter is 450 nm, the reference filter is in the range of 620-655 nm.

Measurement of the number of agonists of pattern recognition receptors incorporated into nanoscale particles

The concentration of pattern recognition receptor agonists (PRPs) internalized into nanosized particles was determined using the HEK293-NOD2 and HEK293-TLR9 reporter cell lines that express the individual PRPs NOD2 and TLR9 on their surface, respectively. These cell lines carry the gene NF-kB / AP-1 - dependent alkaline phosphatase (reporter gene). To quantify the incorporated agonists, a calibration curve was constructed using the corresponding agonists (MDP or CpG, respectively) at the initial concentration of 1 mg / ml to 1 μg / ml, and dilutions of the test samples (after lysis in 0.1 N NaOH) from 1 mg / ml to 100 μg / ml, in two steps. Samples and controls were added in 10 μl, to the wells of a 96-well plate with a 24-hour cell culture of HEK293-NOD2 or HEK293-hTLR4-CD14. The day after the introduction of the samples, the level of the enzymatic activity of the alkaline phosphatase reporter gene was measured by the colorimetric method, determining the rate of cleavage of the colorless substrate of disodium nitrophenyl phosphate into yellow-colored para-nitrophenol. For this, a freshly prepared solution of the substrate in SEAP buffer (0.5 M NaHCO3, 0.5 mM MgCl2, pH 9.8) was dispensed with 150 μl into each well of a new 96 well plate. Then, 50 μl of culture medium were taken from the wells of the plate in which the cells with the test samples of pattern-recognizing receptors were incubated, and transferred to the wells of the tablet with SEAP measurement buffer. Immediately after transferring the medium from all samples, the optical density of the solution was measured at a wavelength of 405 nm (OD1). Next, the tablet was incubated at + 37 ° C in a thermostat, for 10-60 minutes (time depends on the color of the solution). After that, optical density (OD2) was measured. The level of activity of alkaline phosphatase was calculated by the formula where EA is the unit of activity of alkaline phosphatase, OD0 is the optical density of the first measurement, OD1 is the optical density of the last measurement, T0 is the time of the first measurement, T1 is the time of the last measurement.

The result of the amount of molecular adjuvant included in the composition of the PLGA particles is presented in table 6.

EXAMPLE 3

The study of the specific pharmacological activity of the in vivo vaccine LTPvak

Female and male mice (F1 BALB / c x C57BL / 6 hybrids) pre-immunized with live BCG vaccine at a dose of 5 × 10 ⁶ CFU / animal (on day 0 of the study), in different doses (5.0 and 0.5 μg / dose according to the content of vaccine mycobacterial antigen) was administered the test drug - vaccine LTPvak. The first immunization was carried out subcutaneously in the third week, the injection volume was 0.2 ml / animal (while maintaining the amount of the administered LTPvak drug of 5 μg and 0.5 μg), the second immunization was intranasal in the fifth week of the study, the volume of administration was 0.02 ml / animal (similarly with the preservation of the number of LTPvak 5 μg and 0.5 μg). The comparison drug was Adjuvant, which is part of the vaccine, administered in equivalent volumes and doses. Saline solution (0.9% NaCl solution) and BCG vaccine preparation were used as control preparations.

The following formulations were used to study the specific pharmacological activity in vivo and the mechanism of action of the LTPvak vaccine:

In all experiments, with the exception of experiments to study the specific pharmacological activity of the drug, all tested drugs and comparison drugs were prepared immediately before administration by dissolving in water or saline based on 0.5 ml of solvent per dose of administration. In this case, the preparations LTPvak (5 μg), LTPvak (0.5 μg), Adjuvant were dissolved in saline, and the Antigen preparation was dissolved in water.

In experiments to study the specific pharmacological activity of the drug, LTPvak (5 μg) and LTPvak (0.5 μg) were dissolved in saline in volumes depending on the method of immunization of animals: 0.2 ml or 0.02 ml, while maintaining the amount of drug administered .

EXAMPLE 4

Evaluation of activation of cellular immunity by the proliferation level of T-lymphocytes of immunized mice in response to stimulation with a specific vaccine antigen.

We studied the contents of general populations of immunocompetent lymphocytes and their main subpopulations upon stimulation with the mycobacterial antigens that make up the LTPvak vaccine.

The study of proliferating effector (CD4 +) and cytotoxic (CD8 +) T cells in mice that received different doses of the vaccine showed no significant differences when compared with control groups (groups 1-3) (Fig. 7, table 7).

In the study of a separate CD4 + population, the effect was least pronounced in group 5 (LTPvak, a hybrid protein content of 0.5 μg / dose), where the proportion of proliferating CD4 + lymphocytes was 0.16% of the total T-lymphocyte population, while for the rest the studied groups, this value was 0.50-0.76%. Moreover, the main contribution to the proliferative activity of populations in all groups was made by stimulation with a full fledged hybrid antigen, and there was practically no influence of the individual antigens ESAT-6, CFP10 and Rv2660c (Fig. 7, upper part, table 7).

For a population of cytotoxic CD8 + T-lymphocytes, the effect of stimulation with vaccine antigens was more pronounced (but also statistically unreliable in comparisons with control groups). The proliferative activity of CD8 + T cells was observed in response to stimulation by both the hybrid protein and all its components in the study in all the studied groups. This effect is especially pronounced for the studied groups 4-5 (immunized with LTPvac at doses corresponding to 5 and 0.5 μg of protein, respectively), where the proportion of the CD8 + subpopulation reached 1.5% of all T-lymphocytes (Fig. 7, lower part, table 7).

Thus, based on the results obtained by the method of assessing lymphoproliferation, it can be concluded that the administration of the LTPvak drug at a dose equivalent to 5 μg of the vaccine protein causes the maximum immunogenic effect.

EXAMPLE 5

Evaluation of activation of cellular immunity by the level of production of INF-y by lymphocytes (splenocytes) of immunized mice in response to stimulation with a specific vaccine antigen.

10 weeks after the start of the study (5 weeks after the second injection of the LTP vaccine necessary for the formation of immunity), 5 animals in each group were euthanized, followed by sampling of biomaterial (blood, splenocytes, etc.) to determine immunogenicity. The production of INF-y by lymphocytes (splenocytes) of immunized mice was evaluated in response to stimulation with the ESAT6-CFP10-Ag85a-Rv2660c vaccine hybrid antigen, which are part of the vaccine (and its individual components) in the IGRA test. To do this, in the obtained supernatants by ELISA, a study of the level of INF-γ was carried out using a commercial IFN gamma Mouse ELISA Kit (eBioscience, USA).

To compare the results in the study groups according to the values of indicators, non-parametric statistics methods were used, since the volumes of the groups fall under the definition of small samples. To assess the significance of the differences, one-way ANOVA and the Kruskell-Wallace criterion with Dunn correction were used for multiple comparisons of several and multiple comparisons with the control group (groups 1-3, who received saline, BCG or adjuvant, respectively, table 8) . Differences were considered significant at p <0.05.

The obtained quantitative data are presented as an average indicating +/- standard deviation. In FIG. 8, 9, the significance level of the differences is presented in the form of symbols: “*” - p <0.05; "***" - p <0.001 of different colors. In green, significant differences are shown, in red, the absence of significant differences between groups.

Activation by a non-specific mitogen, phytohemagglutinin, showed that all experimental groups retain their mitogenetic potential and respond to exposure by active release of INF-γ into the supernatant. It was shown that the hybrid antigen, and the individual components included in its composition, are able to activate the production of cytokine and contribute to the formation of cellular immunity during immunization with LTPvac (Fig. 8). It is seen that all groups of immunized animals show a significant increase in the secretion of INF-γ. However, depending on the groups, the results of stimulation of interferon production varied.

A significant increase in the production of INF-γ by splenocytes in response to stimulation with antigens relative to the control groups is shown for almost all of the studied antigens that are part of the tested vaccine preparations, regardless of the dose received (Fig. 8, 9, table 8). An exception is stimulation with the Rv2660c antigen in group 4.

When splenocytes of mice of the studied groups were stimulated with a hybrid protein, a significant level of INF-γ production was demonstrated with complete absence of such in two control groups (groups 1 and 2) and the absence or significantly reduced level of production in the adjuvant group (group 3) (Fig. 8, D ) At the same time, it is obvious that the epitopes of the CFP10 protein appear to make the largest contribution to the immune response (Fig. 8, B), while the ESAT6 and Rv2660c antigens to a lesser extent influence the formation of the immune cellular response during vaccination (Fig. 8, A, B). For group 4 (LTPvak 5 μg / dose, CpG + MDP), the ESAT6 antigen also contributes to the formation of the cellular immune response. The highest concentration of INF-γ was observed in group 5 (LTPvak 0.5 μg / dose, CpG + MDP).

Moreover, if we evaluate the total level of response to stimulation with vaccine antigens of splenocytes, there are no statistically significant differences between the study groups 4 and 5, who received LTPvak in different dosages (Fig. 9, table 8). It is worth noting that groups 4 and 5 show significant differences in the activation of INF-γ secretion compared with the adjuvant (in the groups receiving the drug, the average values of INF-γ secretion are 17–25 times higher than in the “Adjuvant” group, not to mention the other two control groups in which the specific secretion of INF-γ was zero (Fig. 9, table 8.) This suggests that the antigens in the vaccine make a statistically significant contribution to the immunogenicity of the vaccine composition.

Since the profile of the cellular response during stimulation with vaccine antigens (both a hybrid protein and the individual antigens included in it) is very close in the two studied groups (groups 4 and 5), both formulations can be used in the further development of the vaccine.

Taken together, the data show that, when LTPvac is immunized, a cellular response is formed, including through the formation of specific subpopulations of CD4 + and CD8 + that can produce a significant level of INF-γ, the main marker of protective immunity against tuberculosis, established to date.

EXAMPLE 6

Evaluation of the activation of the humoral immune response by increasing the titer of class G antibodies specific for the components of the developed LTB vaccine

When studying the control groups, it was shown that nonspecific antibody reactions in response to the introduction of solutions and adjuvants included in the vaccine are absent (table 9, 10). As seen in FIG. 10, in the sera of the control groups there is no increase in titer specific for the hybrid antigen or its individual antibody components.

The study of the dynamics of the accumulation in the blood serum of animal antibodies of class G to peptides that are part of the antigenic hybrid protein showed that three of the four peptides with a high degree of confidence induce the production of antibodies - CFP10, Ag85A, Rv2660c (Fig. 10, table 9, 10) . The ESAT6 peptide had no effect on the humoral immune response — no significant increase in antibody level relative to control groups was detected for any of the animal immunization options (Fig. 10A).

The remaining components of the hybrid protein CFP10, Ag85A and Rv2660c according to the results of the study can be recognized as equally and positively affecting the production of a humoral immune response (p <0.01). This conclusion is confirmed by significantly high IgG levels in all three groups of immunized animals with some assumption regarding Rv2660c in group 4 (5 μg / ml CpG + MDP) (Fig. 10G). However, on the whole, a certain heterogeneity of the results is characteristic of the serodiagnosis of tuberculosis, when, for example, for CFP10, the IgG values in the blood serum of three out of 10 mice do not differ from the values in the control groups, while in the rest they are located in a rather wide range of MFI (Fig . 10B).

For the hybrid protein ESAT6-CFP10-Ag85A-Rv2660, the presence of a reliable humoral response was shown in all studied groups (Groups 4-5) compared with the control groups (Groups 1-3) (Fig. 11). The average MFI for the studied LTPvak groups of 5 μg and 0.5 μg was about 16,000 and 17,500 units, respectively, which is more than 10 times higher than the similar values in all control groups (table 9, 10). It is worth noting that, for individual antigens, the humoral response was generally less pronounced compared to the hybrid protein. Moreover, the heterogeneity of the level of the humoral response described above for the peptide components of the hybrid protein is much less pronounced in this case - one case per fifteen test animals. For group 5 (LTPvak 0.5 μg / ml, CpG + MDP), the values can be described as pronounced and compactly located. Such differences in the heterogeneity of responses can be associated with a partial synergistic effect of the epitopes of the hybrid protein in comparison with the individual antigens that make up its composition. In this case, these results are in favor of using not individual antigens as part of the vaccine, but a whole-fused hybrid protein that can provide a uniform, stable high level of the immune response of the humoral link.

EXAMPLE 7

Evaluation of vaccine protective parameters

Aerosol spraying of M. tuberculosis was performed at 10 weeks of the experiment in an amount of 50-100 CFU.

Since the protective effect of the vaccine is determined by the suppression of the multiplication of M. tuberculosis in target organs, the second and third animal euthanasia was performed at 11 and 15 weeks (6 animals in each group). Target organs (spleen and lungs) were selected from animals for bacteriological culture and assessment of the presence of M. tuberculosis in them. Seeding results for 11 weeks were performed to control infection of animals.

It was shown that the concentration of bacteria in the lungs at 11 weeks in all groups is the same, does not differ significantly and ranges from 65 to 135 CFU / ml.

Inoculation of spleen homogenates did not show the presence of bacteria, with the exception of the control group OKO (Fig. 12). Thus, it can be argued that the infection of animals occurred in all the studied groups, while its effectiveness was identical.

In the seeding of animal organs after the third euthanasia (week 15), bacteria were found both in the lungs and in the spleen, and there were significant differences in the concentrations of bacteria in the experimental groups: the minimum seeding of both target organs was observed in groups 4 and 5 (LTPvak 5 μg / dose, CpG + MDP and LTPvak 0.5 μg / ml, CpG + MDP) averaged 729 and 15 CFU / ml for the lung and 1902.7, and 5958.3 CFU / ml for the spleen, respectively (Fig. 13). Group 3 (Adjuvant LTBvak) did not show significant differences from control group 1, which indicates the effectiveness of the formulation of the vaccine containing the hybrid protein.

Thus, according to the results of the studies, it was shown that when immunized with the LTP vaccine, there is a significant decrease in the contamination of target organs with infection of animals with M. tuberculosis. Observation of animals, histological and bacteriological studies allowed us to evaluate the effect of the vaccine as protective. It was shown that the maximum effect of the vaccine appears at a protein concentration of 5 μg / dose (similar to immunogenic effects).

Conclusions The study of specific pharmacological activity in vivo According to the results of a preclinical study, it was shown that the LTPvak vaccine has a pronounced specific pharmacological activity in the form of immunogenic and protective effects on the body of experimental animals with a double administration with an interval of 8 weeks. In this case, a pronounced effect is achieved in a dosage equivalent to 5 μg of vaccine protein per dose.

When assessing the cellular immune response in the IGRA test (stimulation of splenocytes of experimental mouse mice with a hybrid protein), a significant level of INF-γ production was demonstrated in groups receiving LTPvac vaccine at a dose of 5 and 0.5 μg, in the absence of such in two control groups, received BCG or saline, and a significantly reduced level of production in the adjuvant group. Vaccine-treated groups showed significant differences in INF-γ secretion compared with all three control groups (in the drug-treated groups, the average INF-γ secretion values in response to stimulation with the full vaccine protein ESAT6-CFP10-Ag85A-Rv2660c average secretion values amounted to 120.24 and 225.6 pg / ml INF-γ for vaccine doses of 5 and 0.5 μg, which is 17-25 times higher than in the Adjuvant group; in the other two control groups receiving saline or BCG, INF-γ secretion was zero). At the same time, there was no significant difference in the indicators between the groups receiving the test drug LTPvak in different doses.

Assessment of cellular immunity by the method of determining lymphocyte proliferation of lymphocytes after stimulation with vaccine antigens did not reveal a significant difference between the groups receiving LTPvak and control drugs. In this case, the highest lymphoproliferation rates were observed in the group receiving LTPvak at a dose of 5 μg.

Evaluation of the humoral (antibody response) showed that the ESAT6-CFP10-Ag85A-Rv2660 hybrid vaccine protein reliably forms a humoral response in the groups receiving LTPvac in both dosages compared to the control groups. The average obtained value of antibody titers for the studied LTPvak groups of 5 μg and 0.5 μg was about 16000 and 17500 conventional units, respectively, which is more than 10 times higher than the same values in all control groups.

A significant decrease in the bacterial load on the target organs of animals of the studied groups (LTPvak at a dose of 5 μg and 0.5 μg) relative to control groups 1 and 3 (saline and adjuvant vaccines) was shown, which indicates the formation of protective immunity in mice after immunization.

The efficacy of the selected formulation of the recombinant subunit vaccine against tuberculosis LTPvak was confirmed, as well as the administration schedule for the vaccine preparation. It was shown that, at the chosen immunization regimen, the formulation of the LTP vaccine with a hybrid protein content of 5 μg / dose and the adjuvant CpG + MDP incorporated in PGA is effective from the point of view of activation as a cellular (basic, from the point of view of formation of protective immunity to tuberculosis, according to modern ideas), and humoral immunity in the studied animals.

The data obtained indicate the ability of the studied medicinal product, a vaccine of the subunit recombinant LTPB, to form a specific protective immunity of the cell type (Th1) to pathogenic microorganisms of M. tuberculosis. The use of highly immunogenic antigens (recombinant proteins of mycobacteria characteristic of the active phase of the disease - ESAT6, CFP10, Ag85A, as well as the latent phase of the disease - Rv2660c), incorporated into a biodegradable non-toxic polymer from poly (lactic-co-glycolic) acid (PLGA), together with a highly effective complex molecular adjuvant (represented by ligands of the TLR9-CpG ODN and NOD2 innate immunity system receptors - Muramyldipeptide, MDP), which stimulates the polarization of the immune response, leads to stimulation of proliferation of CD4 + and CD8 + T cells, an increase in IFN-γ synthesis, which indicates activation of IFN-γ cellular immunity and determines the body's resistance to M. tuberculosis.

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Note: The average fraction of proliferating effector (CD4 +) and cytotoxic (CD8 +) from all T-lymphocytes in response to the introduction of the components of the vaccine antigen separately and the hybrid protein is shown. The standard deviation is not given due to the unreliability of differences between groups.

Claims (5)

1. Recombinant tuberculosis vaccine containing an effective amount of a recombinant protein ESAT6-CFP10-Ag85a-Rv2660c, which is a fused mycobacterial protein ESAT-6, CFP10, Ag85a, Rv2660c with a histidine tag of 8 residues, and adjuvant where the recombinant protein and adjuvant are immobilized on carrier particles from a PLGA lactic and glycolic acid copolymer. 2. The recombinant anti-tuberculosis vaccine according to claim 1, characterized in that the PLGA carrier is a copolymer of lactic and glycolic acids with a molecular weight of 66,000-108,000 Yes, with a ratio of monomer units of 75:25. 3. The recombinant anti-tuberculosis vaccine according to claim 1, characterized in that the average particle size of the PLGA with the immobilized protein is in the range of 100-200 nm. 4. The recombinant anti-tuberculosis vaccine according to claim 1, characterized in that the adjuvant muramyl dipeptide is a D-L isomer, N-acetylmuramyl-L-alanyl-D-isoglutamine. 5. The recombinant anti-TB vaccine according to claim 1, characterized in that the CPG oligonucleotide is represented by a class A CpG-ODN.

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