RU2262351C1 - Vaccine composition for prophylaxis and treatment of tuberculosis infection and genetic constructions for preparing acting components of this composition - Google Patents

Abstract

FIELD: medicine, immunology, phthisiology.

SUBSTANCE: invention proposes a vaccine composition used in prophylaxis and treatment of tuberculosis infection. The composition comprises a mixture of fused recombinant protein ESAT-HSP70 from Mycobacterium tuberculosis, protein HSP65 from M. tuberculosis and an inert vehicle. Also, the proposed composition can comprise timogen additionally. Also, invention proposes recombinant plasmid DNA pE HSP70 and pQE HSP65 encoding corresponding proteins. The proposed vaccine composition enhances effectiveness in treatment and prophylaxis of tuberculosis infection. Genetic constructions provide carrying out the stable expression of indicated proteins and to enhance purity the end preparing proteins. Invention can be used in treatment and prophylaxis of tuberculosis.

EFFECT: valuable medicinal properties of composition.

5 cl, 6 dwg, 6 ex

Description

The invention relates to the field of medicine, immunology, can be used for the treatment and prophylactic vaccination of tuberculosis infection.

Studies conducted by the World Health Organization (WHO) have shown that the problem of tuberculosis (TB) is still extremely acute, as evidenced by the level of infection of the world's population of more than 1 billion people. About 8 million people get tuberculosis each year. At the same time, 44% suffer from a pulmonary form of this disease. About 2 million people die annually, which accounts for 23% of deaths worldwide and exceeds 50% of mortality in African countries (Dye C., Scheele S., Dolin P., Pathania V. and Ravinglione M.C., 1999, JAMA, 282, 7, 677-686).

The problem of tuberculosis on a global scale is becoming even more acute due to several factors, including the emergence of multiresistant strains of M. tuberculosis. An analysis of drug resistance in 35 countries showed the spread of acquired resistance to four first-level anti-TB drugs: isoniazid, rifampin, ethambutol and streptomycin; with a percentage distribution from 5.3% (New Zealand) to 100% (Ivanovo Oblast, Russia) and an average level of 36% (Pablos-Mender A., Ravinglione M.C. et al., 1998, N. Eng.J. Med. , 338.23.13641-1649).

The acquired immunodeficiency syndrome (AIDS) epidemic has further complicated the TB situation. According to data published by WHO in 1997, 8% of TB cases were infected with HIV. In HIV-infected patients with TB infection, immunodeficiency is associated with increased dissemination of mycobacteria and the fulminant course of the disease. The combination of TB and HIV has a very poor prognosis with an average survival of two months (Ormerod L.P., Shaw R.J. and Mitchell D.M., 1994, Thorax, 49, 1, 1085-1089).

To control the spread of TB and significantly reduce the risk of the spread of this disease, it is necessary to create an effective vaccine and reagents for specific diagnosis. The only tuberculosis vaccine currently available is the bacillus Calmette Guerin-BCG, BCG, a live, attenuated strain of the virulent bovine tuberculosis bacillus Mycobacterium bovis. Based on the promising results of tests on animal models and tests on more than three hundred children from 1921 to 1924. BCG vaccine has been introduced into healthcare practice around the world to prevent the spread of TB. Since then, several billion people have been vaccinated with BCG. Although BCG is the most common vaccine in the world and is directed against the leading cause of death among infectious diseases, it is also the most controversial vaccine. Estimates of the protective activity of BCG against pulmonary TB vary from 0 to 80% (Fine P.E., 1995, Lancet, 346 (8986), 1339-1345). Moreover, the use of BCG vaccine poses two additional problems:

a) BCG induces a delayed-type hypersensitivity reaction (RGT), which cannot be distinguished from infection with M. tuberculosis, and therefore compromises the use of tuberculin test in skin tests (PPD) for diagnosis or for epidemiological purposes,

b) BCG, as a living vaccine, is contraindicated for HIV-infected patients for fear of causing the disease itself (Lowrie D.V., Tascon RE and Silva C.L., 1995, Int.Arch. Allergy Immunol. 108 (4), 309 -312).

According to the Center for Infectious Disease Control (Atlanta, USA), degeneration of M. bovis strains used for the commercial production of BCG is observed. The loss of the protective properties of BCG, according to experts, is due to long-term laboratory passages of the strains without proper control of quality characteristics.

Monitoring the spread of tuberculosis infection is not possible without a reliable TB vaccine, devoid of the above disadvantages.

The completion of studies to determine the nucleotide sequence of the M. tuberculosis genome has opened up new possibilities for the identification of M. tuberculosis antigens, which may be candidates for the production of improved vaccines with universal efficacy.

The development of biotechnology, in particular the use of DNA cloning and expression technologies, greatly facilitated the identification of several major M. tuberculosis antigens.

As a result of screening of libraries of recombinant mycobacteria using monoclonal and polyclonal antibodies, several mycobacterial antigens were identified and characterized (Young R.A., Mehra V., Sweetser D., Buchanan T. et al., 1985, Nature, 316 96027), 450-452; Young R.A., Bloom B.R. et al., 1985, Proc. Natl. Acad. Sci, USA 82 (9), 2583-2587; Young D.B., Kent L. and Young R.A., 1987, Infect immunity, 55 (6), 1421-1425).

Recently, most attention has been paid to M. tuberculosis proteins (antigens) secreted from infected macrophages as promising vaccine components.

It has been established that immunity to TB includes several types of effector cells. The activation of macrophages by cytokines, such as γ-interferon, leads to an effective reduction in intracellular mycobacterial reproduction. However, complete protection against TB requires the activation of T lymphocytes, such as CD8 + or CD4 + T cells.

Antigenic stimulation of T cells requires the representation of the main histocompatibility complex (GCC) by the molecules.

It is believed that proteins secreted by mycobacteria from infected macrophages are readily available for proteolytic processing and subsequent presentation in the form of a peptide fragment associated with receptors of the major histocompatibility complex (HCH).

Among these secreted proteins, such as the antigenic 85 complex of proteins (85A, 85B, 85C) (Wiker et al., 1992, Microbiol.Rev., 56, 648), a 6 kDa protein called ESAT6 (Andesson, 1994, Infect. Immunity, 62, 2536), 38 kDa lipoprotein with Dhos homology, 65 kDa heat shock protein GroEL (Siva et al., 1994, Immunology, 82, 244), 10 kDa protein CFP-10 ( Mustafa AS, 2001, Corrent Pharmaceutical Biotechnology, 2.157) and several others.

Based on such proteins or their peptides, a number of diagnostic systems and vaccines were created, among which the most interesting are those based on the secretory proteins ESAT6 or CFP-10, which are absent in M. bovis and most non-pathogenic mycobacteria.

Thus, Patent WO00262248 describes a diagnostic system based on a mixture of ESAT6 peptides.

Applicant's application No. 2003123149 describes a diagnostic system based on the hybrid protein ESAT6-CFP-10.

The closest technical solution to the present invention is a vaccine composition for the prevention and treatment of tuberculosis infection, containing an inert carrier and / or diluent and antigenic component in an effective amount, which is used as a hybrid recombinant protein ESAT6-85B (published patent application US No. 2002216867) .

A protein can be obtained by genetic engineering - expression of the corresponding genes (nucleotide sequences up to 75% corresponding to these genes) in a plasmid vector. Despite the fact that the protein contains two secreted M. tuberculosis proteins, the vaccine's protective ability was not greater than that of BCG. The low efficiency of the ESAT6-85B hybrid protein vaccine, in our opinion, in the insufficient repertoire of antigenic determinants presented in the hybrid protein necessary for effective protection against M. tuberculosis, as well as in the low efficiency of the presentation of the protein in the context of MHC for recognition of antigenic determinants by cells the immune system.

An inventive task is to expand the arsenal of vaccines for the prevention and treatment of tuberculosis infection, as well as increasing their effectiveness by increasing the protective ability of the antigenic composition.

The problem is solved in that a vaccine composition for the prevention and treatment of tuberculosis infection is proposed, containing an inert carrier and / or diluent and antigenic component in an effective amount and characterized in that as the latter it contains a mixture of the hybrid recombinant protein ESAT6-HSP70 M.tuberculosis and heat shock protein HSP65 M. tuberculosis.

The composition may additionally contain thymogen (glutamyl tryptophan), a pharmacopeia drug, which is a nonspecific peptide immunomodulator of thymic origin. Advantageously, the ratio of ESAT6 to HSP70: HSP65 proteins in the patented composition is in the range of 1:10 to 10: 1, preferably 1: 1.

The HSP70 and HSP65 proteins of M. tuberculosis belong to the family of heat shock proteins (HSPs), which are found in any tissue and in any organism, and the HSP70 and HSP65 classes are found in mammalian, bacterial, and mycobacterial cells, such as M.leproe, M .tuberculosis, M.vaccoe, M. bovis et al.

It has been found that heat shock proteins are also recognized by T cells and, therefore, can induce protective immunity (Mustafa AS, 1999, Ind. J. Lepr, 71 (1), 75-86; Oftung F., Borka E. and Mustafa AS, 1999, Fems Immunol. Med. Microbiol, 20 (4), 319-325; Pramod srivastava, Interaction of heat shock protein with peptides and antigen presenting cells, Annu Rev Immunol, 2002, 20, 395-425), c. including specific antiviral (antibacterial) immunity, for which special “constructs” are created to immobilize these specific antigens with “heat shock” proteins. For example, see PCT Application 97/068916 Barriocs et al., Eur. J. Immunol, 22 1365-1372, 1994; K. Suzue et al. Journal of Immunology, 19, 159-164, 1999).

Moreover, in some cases, it is possible to significantly enhance the immunogenicity of viral (or bacterial) proteins, that is, to provide an adjuvant effect (see positive decision on the application of the Russian Federation of the applicant No. 2002128131/14).

Thus, when developing a new vaccine antigen (hybrid protein ESAT6-HSP70 M. tuberculosis), the applicant proceeded from certain laws, knowledge and experience, including his own, but to predict the properties and characteristics (especially in quantitative terms) of a new substance from a previous level of knowledge does not seem possible. They are established by the Applicant for the first time empirically. The same applies to the synergistic effect (synergistic protective properties) of the vaccine composition as a whole.

The specified set of features of the invention has not been previously described, thus, the proposed solution meets the criteria of "inventive step" and "novelty."

Appropriate genetic constructs have been developed to produce the ESAT6-HSP70 and HSP65 proteins.

A recombinant plasmid DNA (pE HSP70) encoding the synthesis of M. tuberculosis ESAT6-HSP70 fusion protein is proposed, which is characterized in that it is a pQE30 vector having a size of 3461 nucleotide base pairs, with a lac promoter, an ampicillin resistance gene and a sequence of 18 nucleotides encoding 6 histidine residues into which the ESAT6 gene gene is operatively inserted, having 288 nucleotides (between the restriction enzyme sites of BglII and BamHI) and the heat shock protein gene HSP70 M. tuberculosis, having 1878 nucleotides (between the restriction enzyme sites of BamH I and HindIII).

Thus, a recombinant plasmid DNA (pQE HSP65) encoding the synthesis of M. tuberculosis HSP65 protein, which is characterized by the fact that it is a pQE30 vector having 3461 bp, with a lac promoter, ampicillin resistance gene, is also proposed. and a sequence of 18 nucleotides encoding 6 histidine residues into which the heat shock protein gene HSP65 M. tuberculosis, which has 1623 nucleotides (between the restriction enzyme sites KpHI and HindIII), is operatively inserted.

The proposed genetic constructs allow stably expressing these proteins, provide technological methods for their isolation and purification, high purity of the target proteins.

The invention is illustrated using graphic materials:

Figure 1 contains the scheme for obtaining the plasmid PQE30-ESAT-dnak (pE HSP70) to obtain a hybrid protein ESAT6-HSP70 M. tuberculosis,

Figure 2 contains data on the molecular weight of the hybrid protein ESAT6-HSP70 M. tuberculosis by electrophoresis,

Figure 3 contains data on the purity of the secreted fusion protein ESAT6-HSP70 M. tuberculosis electrophoregram on Lemmli in 12% SDS page,

Figure 4 contains the scheme for obtaining the plasmid PQE30-groEL2 (pQE HSP65) to obtain the protein HSP65,

Figure 5 contains data on the molecular weight of the hybrid protein HSP65 M. tuberculosis by electrophoresis,

6 illustrates the protective activity of HSP65, a hybrid protein of ESAT6-HSP70 M. tuberculosis, the proposed composition and a composition containing additional thymogen in comparison with the protective activity of the BCG vaccine in an in vivo model experiment.

The invention is illustrated by the following examples.

Example I.

Obtaining a producer of a hybrid protein containing a hybrid protein ESAT6-DnaK (Hsp70) M. tuberculosis.

1. PCR amplification of the esat-6 gene of M. tuberculosis.

The esat-6 gene was amplified on M. tuberculosis H37Rv DNA using ES-F and ES-R primers

The initiating codon is in the same reading frame with the 6HIS tag sequence; there is no terminating codon.

2. Cloning of the esat-6 gene sequence in a recombinant vector producing DnaK.

The previously obtained pQE30-dnaK-Y vector was used (see. Scheme), providing expression of the DnaK. Protein fused to the 6HIS sequence at the N-terminus. The PCR fragment of the esat-6 gene was digested with restriction enzymes BamHI and BglII and cloned at the BamHI site of plasmid pQE30-dnaK-Y. Recombinants having the insert in the correct orientation were identified by restriction analysis. The patent design scheme is shown in Figure 1. It should be noted that the recombinant plasmid pQE30-esat-6-dnaK provides the production of a hybrid protein 6HIS-esat6-DnaK.

Scheme for obtaining plasmids pQE30-esat-dnaK (pE HSP70) (see Figure 1).

3. Esat6-DnaK Products

Synthesis of the Esat6-DnaK fusion protein (HSP70) was induced using IPTG in the following manner.

The night culture DLT1270 / pQE30-esat-dnaK grown in LB broth was diluted 1: 100 and grown in a LB broth on a shaker to OD ₆₀₀ = 0.5. Then, 0.1 tM IPTG was added and cultivation was continued for 3 hours.

Protein production was monitored by SDS-PAGE. Observed protein synthesis of the expected molecular weight, the yield was 50-70% (see Figure 2).

Example II

Protocol for isolating the recombinant protein ESAT6-HSP70 from the biomass of the E. coli producer strain from inclusion bodies by refolding.

1. Buffer solutions.

A. 0.05 M Tris-HCl buffer, 0.4 M NaCl, 0.1% tween-20, 2 mM PMSF, pH 8.0.

B. 0.05 M Tris-HCl buffer, 0.4 M NaCl, pH 8.0.

C. 0.1 M Tris-HCl buffer, 6 M guanidine chloride, pH 8.0.

D. 0.05 M Tris-HCl buffer, 6 M urea, 0.4 M NaCl, pH 8.0.

E. 0.01 M Tris-HCl buffer, 0.15 M NaCl, pH 8.0.

2. Obtaining Taurus inclusion.

500 μl of biomass is suspended in 5 ml of buffer A, sonicated at 0 ° C (1 min, 5 × 5 s, 22 kHz, 225 W). The homogenate is centrifuged for 10 min at 13,000 rpm (Jouan BR4 centrifuge) and at + 4 ° C. The supernatant is discarded, the precipitate is washed 2 times with buffer A and 3 times with buffer B, each time suspending the precipitate with ultrasound and centrifuging under the above conditions. The precipitate was stored at -18 ° C.

3. Obtaining a protein extract.

Inclusion bodies were dissolved by ultrasound in 5 ml of buffer C, the solution was centrifuged for 20 min at 13000 rpm (Jouan centrifuge) and at + 4 ° C, the precipitate was discarded.

4. Refolding.

The supernatant from p. D is diluted with stirring in 50 ml of cold (+ 4 ° С) buffer D and after mixing it is dialyzed (3 × 3 L, 18-8-18 h) against pre-cooled (+ 4 ° С) buffer E. Dialysate centrifuged for 1 h (+ 4 ° C, 5000 rpm, Jouan BR4 centrifuge), the precipitate was discarded, the supernatant was filtered through a membrane filter (capron) with a pore size of 0.22 μm. In the filtrate, the protein content is determined by the ICA method, aliquoted in penicillin vials of 2 ml, frozen at -70 ° C and freeze-dried.

Typical electrophoregram of HSP70-ESAT6 protein in 12% Laemmly PAGE (see Figure 3).

Example III

Obtaining producer GroEL2 (HSP65) M. tuberculosis.

1. Cloning in pQE30.

The groEL2 M tuberculosis gene was amplified from the genomic DNA of strain H37RV using primers:

(The KpnI site used for cloning is underlined; the ATG codon of the groEL2 gene is shown in bold.)

(The HindIII site used for cloning is underlined; the sequence complementary to the TGA termination codon is shown in bold.)

The resulting 1.65 kb PCR fragment was digested with KpnI + HindIII and cloned between the KpnI and HindIII sites of pQE30. The resulting recombinant plasmid pQE30-groEL2 was maintained in the E. coli strain DLT1270 containing a chromosomally integrated copy of the lacI repressor.

Design diagram pQE30-groEL2 (pQE HSP65) (see Fig. 4).

2. GroEL2 products (HSP65).

Synthesis of GroEL2 was induced using IPTG in the following manner.

The night culture DLT1270 / pQE30-groEL2 grown in LB broth was diluted 1: 100 and grown in a LB broth on a shaker to OD ₆₀₀ = 0.5. Then, 0.1 mM IPTG (as small as possible) was added and cultivation continued for 2 hours.

Protein production was monitored by SDS-PAGE (see Figure 5). The protein synthesis of the expected molecular weight was observed.

Example IV

A vaccine composition comprising a mixture of the hybrid protein ESAT6-HSP70 and HSP65 from M. tuberculosis.

The mixture is a white powder, soluble in water, physiological saline and phosphate-buffered saline Dilbecco.

The composition is stored under aseptic conditions in sealed glass capsules in the absence of acid, in dry form or dissolved in these solvents.

The composition with a protein ratio of 1: 1 is used for immunotherapy according to example 6 in a wide range of doses from 0.01 to 10 μg.

The composition at a ratio of protein, ESAT6-HSP70 M.tuberculosis and protein HSP65 M.tuberculosis 98: 1 is used for immunization at a dose of 0.1 μg, and at a ratio of these proteins of 3:97 at a dose of 10 μg according to example 6.

Example V.

A vaccine composition containing a mixture of the hybrid protein ESAT6-HSP70, HSP65 from M. tuberculosis and thymogen.

The composition is the composition of example 4 and thymogen in an amount of 10 μg.

Example VI

Study of the protective effect of the vaccine composition on an in vivo model of tuberculosis infection.

Mice were immunized subcutaneously three times with the appropriate vaccine preparation without adjuvant at a two-week interval. 10 weeks after the first vaccination, the mice were infected with M. tuberculosis, for which they were infected intravenously by the introduction of M. tuberculosis H37RV bacteria suspended in a PBS solution in a volume of 0.2 ml and 5 × 10 ⁴ microbial bodies. 6 weeks after infection, the mice were sacrificed, the lungs and spleen were examined for M. tuberculosis. For this, the organs were homogenized in a sterile buffer and serial dilutions were plated on Middlebruck agar plates.

The numbers are presented on the graph as log ₁₀ CFU (see Figure 6). One dose of BCG (5 × 10 ⁴ ) was administered subcutaneously once.

Thymogen was administered in an amount of 10 μg per animal in all experiments. The value of CFU _(log10) at a concentration of 0.1 μg at a protein ratio of (ESAT6-HSP70: HSP65) 98: 2 is 4.9, and at a concentration of 10 μg at a ratio of 3:97 it is 4.6.

Claims (5)

1. A vaccine composition for the prevention and treatment of tuberculosis infection, containing an inert carrier and / or diluent and antigenic component in an effective amount, characterized in that as the latter it contains a mixture of hybrid recombinant protein ESAT6-HSP70 M.tuberculosis and HSP65 protein M.tuberculosis . 2. The vaccine composition according to claim 1, characterized in that it further comprises thymogen. 3. The vaccine composition according to claim 1, characterized in that the ratio of M. tuberculosis ESAT6-HSP70 protein and M. tuberculosis HSP65 protein is 1: 1. 4. Recombinant plasmid DNA (pE HSP70) encoding the synthesis of the M. tuberculosis ESAT6-HSP70 fusion protein, which is characterized in that it is a pQE30 vector having a size of 3461 nucleotide base pairs, with a lac promoter, an ampicillin resistance gene and a sequence of 18 nucleotides encoding 6 histidine residues in which the ESAT6 protein gene has 288 nucleotides (between the Bgl II and BamHI restriction enzyme sites) and the heat shock protein gene HSP70 M. tuberculosis having 1878 nucleotides (between the sites for restriction enzymes BamHI and HindIII). 5. Recombinant plasmid DNA (pQE HSP65) encoding the synthesis of M. tuberculosis HSP65 protein, which is characterized by the fact that it is a pQE30 vector having a size of 3461 bp, with a lac promoter, an ampicillin resistance gene and a sequence of 18 nucleotides encoding 6 histidine residues into which the heat shock protein gene HSP65 M. tuberculosis, which has 1623 nucleotides (between the restriction enzyme sites KpHI and HindIII), is operatively inserted.

Images