WO2016201825A1 - Antigènes de mycobacterium tuberculosis et leurs applications - Google Patents

Antigènes de mycobacterium tuberculosis et leurs applications Download PDF

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WO2016201825A1
WO2016201825A1 PCT/CN2015/091068 CN2015091068W WO2016201825A1 WO 2016201825 A1 WO2016201825 A1 WO 2016201825A1 CN 2015091068 W CN2015091068 W CN 2015091068W WO 2016201825 A1 WO2016201825 A1 WO 2016201825A1
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tuberculosis
nucleic acid
seq
immunogenic
rv0792c
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Chinese (zh)
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刘军
张鹭
郭沛
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复旦大学
成都永安制药有限公司
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Priority to CN201580079653.6A priority Critical patent/CN107531764A/zh
Publication of WO2016201825A1 publication Critical patent/WO2016201825A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of novel tuberculosis vaccines.
  • it relates to corresponding immunogenic components, vaccines or therapeutic ingredients for the prevention or treatment of pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae, Infection caused by Mycobacterium ulcerans.
  • the immunogenic component, vaccine or therapeutic component consists of the M. tuberculosis antigen Rv0976c, Rv1255c, Rv3160c and/or Rv0792c, or a corresponding encoding nucleic acid thereof.
  • Tuberculosis has been the most common infectious disease caused by a single cause worldwide. It is estimated that one third of the world’s population is infected with M. tuberculosis, and in 2013 alone 1.5 million people died of tuberculosis. On a global scale, effective control of tuberculosis still faces many difficulties and challenges, including the lack of rapid and accurate diagnostic techniques, the lack of effective anti-tuberculosis vaccines, and treatments for months. Co-infection of tuberculosis and HIV, as well as the spread of multiple drug resistance (MDR-TB) and extensive drug-resistant TB (XDR-TB), further complicate the prevention and control of tuberculosis.
  • MDR-TB multiple drug resistance
  • XDR-TB extensive drug-resistant TB
  • BCG Bacille Calmette-Guérin
  • M. tuberculosis vaccine is the only attenuated M. tuberculosis vaccine and is the only approved anti-tuberculosis vaccine to date.
  • BCG has two major drawbacks: one is that the protective effect on adult tuberculosis is very limited; the other is that it may cause disseminated BCG disease in people with low immunity.
  • Clinical studies have shown that BCG has more than 80% protective effect on severe tuberculosis in children including miliary tuberculosis and tuberculous meningitis. However, the protective effect on adult tuberculosis is limited, and the protective effect of clinical research is uneven (0-80%). ).
  • BCG BCG is not an ideal vaccine and has limited protection time for the human body.
  • the immunoprotective effect of BCG on newborn priming can last up to 10-20 years, so there is almost no protective effect on adult tuberculosis.
  • tuberculosis vaccines are used as an adjuvant for chemotherapy.
  • This “priming-boosting” strategy includes: after initial immunization with BCG or recombinant BCG, boosting vaccination with infants and children who have not been exposed to tuberculosis, supplemented with subunit vaccines (protein/peptide or DNA); or The unit vaccine is boosted alone; or the subunit vaccine is used as an adjuvant for chemotherapy.
  • the primary development strategy for subunit vaccines has been to find protein antigens that induce strong IFN- ⁇ release to construct subunit vaccines (protein or DNA).
  • the method used is to screen for protein antigens that induce IFN- ⁇ release by biochemical fractionation of a mixture of tuberculosis proteins, especially bacterial supernatants.
  • Esx family proteins EsxA, B, G, H, G, N
  • antigen 85 complex Ag85A, B, C
  • PE/PPE family proteins such as: PPE18, PPE14
  • H1 Ag85B-ESAT-6
  • H4 Ag85B-TB10.4
  • M72 PPE18-Rv0125
  • DNA subunit vaccines such as MVA85A (adenovirus expressing antigen 85A, Ag85A) and AERAS-402 (vaccinia virus expression) have been developed using replication-deficient viruses such as adenovirus or vaccinia virus as vectors.
  • MVA85A enhances the protective efficiency of BCG (Verreck FA, et al. (2009) MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques.
  • PLoS ONE 4(4): e5264 AERS-402 which has entered clinical phase IIa trials, also potentiates T cell responses after BCG priming (Magalhaes I, et al. (2008) rBCG induces strong antigen-specific T cell responses in rhesus Macaques in a prime-boost setting with an adenovirus 35 tuberculosis vaccine vector.
  • MVA85A has now completed the clinical Phase IIb trial, which is the first subunit vaccine to complete the clinical validation of the protective effect, but the results are disappointing.
  • MVA85A does not significantly improve BCG protection, anti-tuberculosis, or anti-tuberculosis infection (Tameris MD, et al. (2013) Safety and efficacy of MVA85A, a new tuberculosis vaccine , in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 381 (9871): 1021-1028).
  • the results of MVA85A suggest that it is necessary to continue to search for new and more effective subunit vaccine candidate antigens (Geluk A, van Meijgaarden KE, Joosten SA, Commandeur S, & Ottenhoff TH (2014) Innovative Strategies to Identify M.
  • the present invention relates to an immunogenic composition, vaccine or therapeutic composition consisting of M. tuberculosis antigens Rv0976c, Rv1255c, Rv3160c and/or Rv0792c, or a coding nucleic acid corresponding thereto.
  • the invention also encompasses short/long overlapping or non-overlapping peptides produced by synthetic or recombinant methods.
  • the present invention relates to the use of the Rv0976c, Rv1255c, Rv3160c and/or Rv0792c proteins, or the coding genes corresponding to these proteins, for the diagnosis, treatment and/or prevention of M. tuberculosis infection.
  • Rv0976c, Rv1255c, Rv3610c and Rv0792c are highly immunogenic in animal models and have a protective effect against Mycobacterium tuberculosis infection. Therefore, the antigens Rv0976c, Rv1255c, Rv3610c and Rv0792c are very promising candidate antigens for the construction of anti-tuberculosis vaccines.
  • the present invention discloses an immunogenic composition, a vaccine or therapeutic composition comprising one or more of the following polypeptides:
  • the invention also discloses an immunogenic composition, a vaccine or therapeutic composition consisting of any one or more of the following nucleic acid molecules:
  • the above nucleic acid is a DNA fragment.
  • immunogenic compositions, vaccines or therapeutic compositions of the invention may have the following applications:
  • the polypeptide vaccine of the present invention is applied to humans or other mammals or animals to enhance the body against pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium. Bovis), Mycobacterium africanum, Mycobacterium leprae or ulcer Resistance to tuberculosis infection caused by Mycobacterium ulcerans.
  • the immunogenicity of a polypeptide or immunogenic fragment may be enhanced by fusion with an adjuvant, or may be enhanced by the addition of other mycobacterial polypeptides, or other organisms such as bacteria, viruses, mammalian polypeptides.
  • the added polypeptide is also included in the composition composition and is bound to the polypeptide or immunogenic fragment in a cross-linked or non-cross-linked form.
  • the nucleic acid of the present invention is randomly inserted into a vector, such as an adenovirus or vaccinia virus vector, directly used in human or other mammals or animals as a DNA vaccine, and expresses antigen in vivo, resulting in pathogenic branching of the body.
  • Bacteria such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, or Mycobacterium ulcerans Resistance to tuberculosis infection.
  • the polypeptides and nucleic acids of the invention may constitute a therapeutic composition for use in humans or other mammals or animals to prevent and/or treat M. tuberculosis infection.
  • the present invention provides a vaccine for human or other mammalian or animal immunization against pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium Bovis infection, tuberculosis caused by Mycobacterium africanum, Mycobacterium leprae or Mycobacterium ulcerans.
  • pathogenic mycobacteria such as Mycobacterium tuberculosis, Mycobacterium Bovis infection, tuberculosis caused by Mycobacterium africanum, Mycobacterium leprae or Mycobacterium ulcerans.
  • a microorganism such as a free plasmid or into a microbial genome
  • compositions, polypeptide, and nucleic acid of the present invention are used in an in vitro and in vivo assay for detecting an antibody response or a cellular immune response against Mycobacterium tuberculosis, and are suitable for diagnosis of infection or monitoring of disease progression.
  • a polypeptide may be used as an in vivo diagnostic reagent during a skin test.
  • Polypeptides can also be used for in vitro testing in ELISA or T-spot assays of blood samples from tuberculosis patients.
  • the nucleic acid or polypeptide may be used in non-human animals to extract antibodies against M. tuberculosis, which antibodies can be used to detect target antigens by in vivo or in vitro experiments.
  • the invention provides an immunogenic component comprising one or more polypeptides or immunological fragments thereof selected from the group consisting of an immunogenic component (ie, the invention provides one selected from the group consisting of Application of one or more polypeptides or immunological fragments thereof as an immunogenic component):
  • the immunological fragment is, for example, a T cell epitope.
  • the invention provides an immunogenic component comprising a nucleic acid molecule encoding the above polypeptide or an immunological fragment thereof.
  • the immunogenic component of the present invention comprises one or more nucleic acid molecules selected from the group consisting of the following as an immunogenic component (ie, the present invention provides one or more nucleic acid molecules selected from the group consisting of: Application of the original ingredients):
  • nucleic acid molecule consisting of the nucleotide sequence shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8 or a complement thereof;
  • nucleic acid molecule consisting of a nucleotide sequence encoding the same amino acid sequence as the nucleic acid molecule of (a) or a complement thereof;
  • nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule as defined in (a) or (b) and which has a length of at least 10 nucleotides.
  • the present invention provides an immunogenic composition comprising the aforementioned one or more polypeptides or immunological fragments thereof, and/or one or more of the aforementioned nucleic acid molecules as an immunogenic component . That is, the present invention also provides the use of the immunogenic component described in the preparation of an immunogenic composition.
  • the immunogenic composition of the invention may further comprise an adjuvant.
  • the immunogenicity of a polypeptide or immunogenic fragment may be enhanced by fusion with an adjuvant, or by the addition of other mycobacterial polypeptides, or other organisms such as bacteria, viruses, mammalian polypeptides. strengthen.
  • the added polypeptide may also be included in the compositions of the invention to bind to the polypeptide or immunogenic fragment in a cross-linked or non-cross-linked form.
  • the present invention provides an expression vector or a non-pathogenic microorganism in which at least one copy of a DNA fragment comprising the aforementioned nucleic acid molecule of the present invention is integrated (for example, placed in a free plasmid or integrated into a microbial genome) And the DNA fragment can be expressed in the form of a polypeptide in a microorganism.
  • the expression vector or non-pathogenic microorganism is vaccinia, adenovirus, BCG or transformed cells.
  • the expression vector or non-pathogenic microorganism is used to express antibodies against mycobacteria in humans or other animals.
  • the present invention provides the immunological component, the immunogenic composition, or the expression vector or the non-pathogenic microorganism in the preparation of a medicament for anti-tuberculous mycobacterial infection.
  • the anti-tuberculous mycobacterial infection refers to enhancing the body caused by pathogenic mycobacteria, such as: Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae or Mycobacterium ulcerans Resistance to tuberculosis infection.
  • the present invention also provides a medicament for infection against Mycobacterium tuberculosis, comprising the immune component of the present invention, the immunogenic composition, or the expression vector or non-pathogenic microorganism;
  • the medicament may be a vaccine.
  • the drug against M. tuberculosis infection can be applied to humans or other animals, preferably mammals, to prevent and/or treat M. tuberculosis infection.
  • the invention provides a method of immunizing against tuberculosis in a human or other animal, preferably a mammal, comprising administering to the individual (by means of intradermal, subcutaneous transdermal, muscle, or mucosal delivery)
  • the immune component, the immunogenic composition, or the expression vector or non-pathogenic microorganism of the invention are administered to the individual (by means of intradermal, subcutaneous transdermal, muscle, or mucosal delivery.
  • the invention also provides the use of the immunological component or the immunogenic composition described in the preparation of a formulation for detecting and/or diagnosing a Mycobacterium tuberculosis infection.
  • Detection of M. tuberculosis infection includes detection of an antibody response or cellular immune response against M. tuberculosis. It can be used for in vitro and/or in vivo detection for the detection of antibodies or cell-mediated immune responses against M.tb in body fluids for disease diagnosis, infection diagnosis, or monitoring disease progression.
  • Nucleic acids and polypeptides can be used to produce anti-M.tb antibodies in non-human animals.
  • the antibody can be used to detect a target antigen in vivo and/or in vitro.
  • the immunogenic components (polypeptides and nucleic acids) of the invention can be used in diagnostic methods to screen for antibodies reactive with the antigen associated with different populations (eg, active, latent infection, healthy humans). serum.
  • the experiments of the present invention show that the levels of antibodies specifically recognizing Rv0976c, Rv1255c, and Rv3160c antigens are significantly higher than those of active TB and latent infected people, and the levels of antibodies specifically binding to Rv0792c in the serum of latent infected populations are significant. Higher than active tuberculosis and healthy controls.
  • the immunogenic components of the invention can also be used in a variety of in vitro cytokine release assays to determine the level of secretion of cytokines induced by the polypeptides of the invention, including IFN-[gamma], TNF and IL-2. At least one of them.
  • the invention provides a diagnostic kit for a tuberculosis sample comprising an immunological component of the invention, or an immunogenic composition of the invention.
  • the Rv0976c, Rv1255c, Rv3160c and Rv0792c of the present invention can be used for differential diagnosis between active tuberculosis, latent tuberculosis infection and healthy individuals.
  • the levels of antibodies specifically recognizing Rv0976c, Rv1255c, and Rv3160c in the serum of active tuberculosis and latent infection were significantly higher than those in healthy people.
  • the level of antibody specifically binding to Rv0792c in the serum of latent infected population was significantly higher than that of active tuberculosis and healthy control group. .
  • the invention also provides the use of a protein (including polypeptide), nucleic acid or composition of the invention in the treatment or prevention of tuberculosis.
  • the invention also provides the use of the following proteins or nucleic acids in the preparation of a tuberculosis vaccine or in the preparation of a medicament for the diagnosis and/or detection of tuberculosis:
  • amino acid sequences of the above proteins Rv0976c, Rv1255c, Rv3160c, and Rv0792c are shown in SEQ ID Nos. 1 to 4, respectively.
  • nucleic acid sequences encoding the proteins Rv0976c, Rv1255c, Rv3160c, Rv0792c are shown in SEQ ID Nos. 5-8, respectively.
  • the invention provides a method of treating or preventing tuberculosis comprising administering a protein, nucleic acid or vaccine of the invention to a human or other animal.
  • the administration is by intradermal, subcutaneous transdermal, muscle or mucosal delivery.
  • the protein or polypeptide fragment thereof provided by the invention and the nucleic acid encoding the same have high immunogenicity against Mycobacterium tuberculosis, can provide effective protection to the organism, can be used as a vaccine candidate or prepared for diagnosis and/or detection of tuberculosis. Reagents.
  • FIG. 1 Expression and purification of Rv0976c protein; wherein each lane represents: 1, molecular weight marker; 2. whole cells induced by IPTG; 3. flow through Ni 2+ affinity chromatography column; 4, 50 mM imidazole washing buffer 5, 100 mM imidazole washing buffer; 6, 150 mM imidazole elution buffer; 7, 300 mM imidazole elution buffer; 8, 300 mM imidazole elution buffer; 9, 500 mM imidazole elution buffer. Fragments eluted with 300 mM imidazole elution buffer (lanes 7 and 8) were collected as purified Rv0976c protein.
  • FIG. 1 Protein-induced T cell response of the invention: production of Th1 cytokines (TNF, IL-2, IL-12) as determined by ELISA. The data was homogenized to Ag85A, and Ag85A was included in parallel in each experiment.
  • Th1 cytokines TNF, IL-2, IL-12
  • FIG. 1 Protein-induced B cell response of the invention; wherein A is Rv0792c, B is Rv1255c, C is Rv0976c, and D is Rv3160c.
  • FIG. 1 Experimental results of protective efficacy of Rv0976c; BALB/c mice were immunized with DNA vaccine and infected with M. tuberculosis H37Rv. Five weeks after the infection, the mice were sacrificed and the number of M. tuberculosis in the lungs and spleen was counted. A is the result of counting the amount of Mycobacterium tuberculosis in the mouse lung, and B is the result of counting the amount of Mycobacterium tuberculosis in the mouse spleen. Among them, pVAX is a M.
  • pVAX: Ag85A is a M. tuberculosis count result of a mouse inoculated with plasmid DNA expressing Ag85A
  • pVAX: PPE18 is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing PPE18
  • pVAX: Rv0976c is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing Rv0976c.
  • FIG. 7 Experimental results of protective efficacy of Rv1255c, Rv3160c and Rv0792c; BALB/c mice were immunized with DNA vaccine and infected with M. tuberculosis H37Rv. After 9 weeks of infection, the mice were sacrificed and the number of M. tuberculosis in the lungs and spleen was counted. A is the spleen count result, and B is the lung count result. Among them, sham and pcDNA are M. tuberculosis count results of mice inoculated with PBS or empty plasmid vector (negative control), and pcDNA: Ag85A is the result of M.
  • pcDNA :HspX is the result of M. tuberculosis in mice inoculated with plasmid DNA expressing HspX
  • pcDNA: Rv1255c is the result of M. tuberculosis inoculation of a plasmid DNA expressing Rv1255c
  • pcDNA: Rv3160c is a plasmid inoculated with Rv3160c.
  • pcDNA: Rv0792c is a result of counting M. tuberculosis in mice inoculated with plasmid DNA expressing Rv0792c.
  • FIG. 8 Serological test results for active tuberculosis (TB), latent tuberculosis (LTBI), and healthy controls (HC) with selected proteins; A is Rv0792c, B is Rv0976c, C is Rv1255c, and D is Rv3160c .
  • the International Tuberculosis Vaccine Research field has conducted animal protection experiments on more than 200 antigens over the past 20 years and found that only a few antigens have potential as subunit anti-tuberculosis vaccines, including the well-known antigens Ag85A, PPE18, HspX, Esat- 6. CFP-10, etc. These antigens have been patented by relevant researchers and constructed corresponding subunit vaccines for clinical trials, such as MVA85A above.
  • the inventors of the present invention conducted an animal experiment on about 130 tuberculosis proteins that have not been used to construct a subunit anti-tuberculosis vaccine, and found that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins of the M. tuberculosis H37Rv strain are highly immunogenic. It can produce a significant protective effect against Mycobacterium tuberculosis infection.
  • Rv0976c is a conserved putative protein with unknown biological functions in Mycobacterium tuberculosis, including Mycobacterium tuberculosis CDC1551, Mycobacterium bovis (including BCG), Mycobacterium tuberculosis, and tuberculosis Mycobacteria and Mycobacterium marinum have homologous genes among a plurality of mycobacterial species.
  • Rv1255c, Rv3160c and Rv0792c are both transcriptional regulatory proteins and are highly conserved within Mycobacterium. The amino acid sequences of Rv0976c, Rv1255c, Rv3160c and Rv0792c are shown in SEQ ID No. 1 to SEQ ID No.
  • the present invention cloned the Rv0976c, Rv1255c, Rv3160c and Rv0792c open reading frames into the pET28a plasmid, and expressed and purified using the E. coli BL21 strain using a standardized procedure (Fig. 1 and Fig. 2). 10 ⁇ g of the purified protein was mixed with Freund's incomplete adjuvant, and C57BL/6 mice were immunized every other week for 3 times to evaluate the immunogenicity of these antigens. After 8 weeks of the first immunization, the mice were sacrificed to isolate spleen cells.
  • the spleen cells were stimulated by Rv3160c and Rv0792c antigens at a concentration of 5 ⁇ g/ml and 10 ⁇ g/ml, respectively, and PBS was used as the antigen-free negative control.
  • the purified Ag85A antigen was used as a positive control, and the spleen cells were cultured under stimulation.
  • the cell culture supernatant was collected 3 days later, and the release levels of Th1 type cytokines (IFN- ⁇ , TNF- ⁇ , and IL-2) were measured by ELISA.
  • the present invention cloned the DNA sequence encoding these antigens (Table 2) into the mammalian expression vector pVAX1 (Rv0976c) or pcDNA3.1 containing the CMV promoter. (Rv1255c, Rv3160c and Rv0792c) (Fig. 5).
  • the pVAX1 and pcDNA3.1 vectors were purchased from Invitrogen.
  • the coding sequences of Ag85A were also cloned into these two vectors as experimental controls. Mice were immunized with these recombinant DNAs, followed by M.
  • tuberculosis H37Rv infection challenge experiments The specific experiment was as follows: BALB/c mice were immunized once a week with 100 ⁇ g of DNA, and immunized 3 times. After 8 weeks of the first immunization, mice were infected with M. tuberculosis (6 ⁇ 10 5 CFU/mouse) via the tail vein or aerosol (dose: 100-300 CFU/lung/mouse), and sacrificed after 5 or 9 weeks of challenge. The mice were counted for the number of M. tuberculosis in the lungs and spleen. The constructed Ag85A DNA was run in parallel as a control. In the same batch of experiments, the number of M.
  • tuberculosis in the lung and spleen of mice inoculated with Rv0976c DNA vaccine after 5 weeks of infection with M. tuberculosis was significantly lower than that of the negative control (pVAX) or the mice inoculated with Ag85A DNA vaccine.
  • the number of Mycobacterium species was similar to that of M. tuberculosis in mice inoculated with the PPE18 DNA vaccine (Fig. 6).
  • PPE18 is also a positive control, and fusion protein vaccine M72 (PPE18-Rv0125) with it as an important component has entered clinical trials (Andersen P& Kaufmann SH (2014) Novel vaccination strategies against tuberculosis. Cold Spring Harb Perspect Med 4 (6)).
  • HspX is a latent-associated antigen of M. tuberculosis and was also used to construct a multi-level subunit vaccine, which was used as a positive control in this experiment (Mir FA, Kaufmann SH, & Eddine AN (2009) A multicistronic DNA vaccine induces significant protection against Tuberculosis in mice and offers flexibility in the expressed antigen repertoire.
  • Rv0976c, Rv1255c, Rv3160c and Rv0792c can react with specific antibodies in human serum, and then distinguish between healthy people and different disease populations, the present invention will purify Rv0976c, Rv1255c, Rv3160c and Rv0792c.
  • the protein was subjected to an ELISA reaction with serum from different populations (active tuberculosis group, tuberculosis latent infection group, healthy control group). The results showed that Rv0976c, Rv1255c, Rv3160c and Rv0792c differed from those of different disease patterns in the serum.
  • serum derived from active tuberculosis and latent infections specifically recognized Rv0976c, Rv1255c, and Rv3160c antigens.
  • Antibody levels were significantly higher than those in healthy populations; the level of antibodies specifically binding to Rv0792c in the serum of latently infected populations was significantly higher than in active tuberculosis and healthy controls (Figure 8).
  • the present invention relates to the use of the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins as vaccines.
  • the protein, polypeptide or peptide of the present invention includes a native protein, polypeptide or peptide, or a homologue functionally identical to a native protein, polypeptide or peptide. These homologues include at least 60%, 70%, 80% or more (more preferably), and most preferably 90% or more (e.g., 95%, 96%, 97%, 98%) of the native protein, polypeptide or peptide contained in Table 1. Or 99%) a protein, polypeptide or peptide of amino acid sequence homology.
  • homologues include substitution, addition and deletion of one or more (eg, 1-50, 1-20, 1-10, 1-5) amino acid residues based on the amino acid sequence of the native protein, polypeptide or peptide in Table 1. a protein, polypeptide or peptide.
  • Such homologs include, inter alia, proteins, polypeptides or peptides containing conservative amino acid substitutions.
  • Nucleic acid sequence in the present invention means a nucleotide sequence encoding such a polypeptide. Further within the framework of the invention are overlapping or non-overlapping peptides which are either long or short and which have at least 70% amino acid sequence identity to any of the polypeptides of the invention.
  • the present invention provides the use of the above polypeptide or nucleic acid as a preparation of an immunogenic component (composition), vaccine or therapeutic composition, which can be combined with BCG as a prophylactic vaccine as an antipathogenic branching Bacteria, such as Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium ulcerans. Enhanced vaccine or therapeutic vaccine.
  • the immunogenic component, vaccine or therapeutic composition of the present invention can be used as a prophylactic vaccine for a population not infected with pathogenic mycobacteria or a population inoculated with BCG, and can also be used as a therapeutic vaccine. A population infected with pathogenic mycobacteria.
  • the invention provides a class of susceptible expression vectors, such as vaccinia, adenovirus or BCG comprising a nucleic acid fragment of the invention, and a class of transformed cells comprising at least one such vector.
  • susceptible expression vectors such as vaccinia, adenovirus or BCG comprising a nucleic acid fragment of the invention
  • the present invention also relates to a method for diagnosing tuberculosis caused by pathogenic mycobacteria in an animal body including a human.
  • Intradermal injection of the polypeptide of the present invention produces a detectable positive skin reaction at the injection site of a tuberculosis individual, and no skin reaction is detected at the injection site of the individual without tuberculosis.
  • a monocyte-containing blood sample eg, T lymphocytes
  • the positive reaction may be T cell proliferation or extracellular. Release such as IFN- ⁇ -like cytokines.
  • a type of serum sample is mixed with a polypeptide sample of the invention, which may be in an individual who has been or is being infected. It was observed that the antibody in the serum sample binds to the polypeptide of the present invention.
  • a monoclonal or polyclonal antibody which specifically reacts with a polypeptide of the present invention in an immunoassay, or a specific binding fragment of the above antibody, is also within the scope of the present invention.
  • a sample of a bodily fluid or a potentially infected organ from an animal including a human is mixed with the above antibody, and the binding of the sample and the antibody can be detected in the infected individual.
  • Nucleic acid probes encoding the polypeptides of the invention can also be used in a variety of diagnostic assays to detect the presence of pathogens in a particular sample.
  • Such a tuberculosis diagnostic method can comprise at least a portion of a nucleic acid sequence, using a PCR or mature hybridization technique to hybridize an animal sample, including a human, to a nucleic acid fragment (or full length fragment) to detect the presence of a nucleic acid sequence in the sample.
  • nucleotide molecules in the DNA sequences provided herein may be modified.
  • the invention includes nucleotide modifications of sequences (or fragments thereof) that can be expressed directly in bacterial or mammalian cells. Modifications include substitutions, insertions or deletions of nucleotides, changes in the relative position of nucleotides or sequential changes.
  • Nucleic acid molecules of the invention include sequences encoding substitutions, additions or deletions that result in non-conservative amino acids in the sequence of Table 1.
  • Nucleic acid molecules (DNA and RNA) comprising corresponding functions that alter the non-conservative amino acid sequence in the amino acids of Table 1, which encode amino acid sequences with non-conservative amino acid substitutions (especially substitutions, additions, or deletions of chemical approximations, but also The amino acid sequence identical or similar to Table 1 is retained. These DNA or RNA may encode fragments or variants of the amino acid sequence shown in Table 1.
  • Fragments can be used as immunogens and immunogenic compositions. Such fragments or variants can be identified by the methods described below.
  • the nucleic acid molecule (or a fragment thereof) referred to in the present invention has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least at least the nucleic acid molecule sequence set forth in the application.
  • a consistency of 97%, at least 98%, or, preferably, 99% or 99.5% or greater, can be expressed in bacterial or mammalian cell lines.
  • Consistency refers to the similarity of two nucleotide sequences, the highest value that can be matched after sequence alignment. Consistency is calculated according to existing mature methods. For example, if a stretch of nucleotide sequence (referred to as sequence A) is 90% identical to the reference fragment of SEQ ID No. 5, then in addition to the reference nucleotide sequence of every 100 SEQ ID No. 5, the sequence Except for 10 point mutations (eg, substitutions with other nucleotides), the remaining sequences are identical.
  • Sequence identity is preferentially set such that the sequence has at least 70%, at least 80%, at least 90%, of SEQ ID No. 5 or its complement provided in the invention, At least 95%, at least 96%, at least 97%, at least 98%, or, preferably, 99% or 99.5% identity.
  • Sequence consistency first uses the GCG program in bioinformatics Perform calculations (Wisconsin University). Other programs can also be used for consistent calculations, such as the Clustal W program (better for default parameters; Thompson, JD et al., Nucleic Acid Res.
  • BLAST P Mycobacterium tuberculosis BLASTN (http:tigrblast.tigr.org/) from the Genomics Institute, Mycobacterium bovis, at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/Microbes/), Tuberculosis of Mycobacterium bovis BCG (Pastuer), Mycobacterium marinum, Mycobacterium leprae, Mycobacterium tuberculosis BLASTN, Pasterculist (http://genolist.pasteur.fr/TubercuList/) BLASTN study by the Pasteur BLASTN study, Leproma (http://genolist.pasteur.fr/Leproma/), University of Minnesota (http://www.cbc.umn.edu/ResearchProjects/Ptb/ And http://www.cbc.umn.edu/ResearchProjects/AGAC/M
  • tuberculosis BLASTN NCBI USA-(http://www.ncbi.nlm. Various BLAST studies by nih.gov/BLAST/), and GenomeNet (Bioinformatics Center - Institute of Chemistry) (http://blast.genome.ad.jp/) Research various BLAST.
  • nucleic acid sequences given in Table 2 are not only the only sequences encoding the polypeptides in Table 1.
  • the invention encompasses nucleic acid molecules having the same basic genetic information as the nucleic acid molecules of Table 2.
  • Nucleic acid molecules which produce one or more nucleotide changes (including RNA) as compared to the nucleic acid sequences in the application, but which produce the same polypeptide product as in Table 1, are also within the scope of the present invention.
  • nucleotide functional equivalents encoding the above polypeptides which can be detected by conventional DNA-DNA or DNA-RNA hybridization techniques are also within the scope of the present invention.
  • the DNA of the present invention has sufficient sequence identity with the nucleic acid molecule provided in the application, and hybridization can be achieved under strict hybridization conditions (low salt washing solution: about 0.2% SSC, reaction temperature of 50-65 ° C). Common hybridization techniques).
  • the invention also encompasses nucleic acid molecules that are capable of hybridizing to one or more of the sequences in Table 2 or its complements. This nucleic acid molecule achieves hybridization under highly stringent conditions (Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). It is preferred to use a low salt wash (about 0.2% SSC) at a reaction temperature of about 50-65 °C.
  • the preparation of vaccines is well known. Typically, injectable liquid solutions or suspensions; solid forms which are readily dissolved or suspended in the liquid prior to injection, may be prepared by emulsifying, proteolipidization.
  • the immunogenic composition is often mixed with a pharmaceutically acceptable excipient that is compatible with the active composition. Suitable excipients include: water, physiological saline, dextrose, glycerol, ethanol or mixtures thereof.
  • the vaccine may contain other minor excipients, such as infiltrants or emulsifiers, pH buffers, and/or adjuvants that enhance the effectiveness of the vaccine, if desired.
  • the active ingredients of the adjuvant may include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine ( CGP 19835A, known as MTP-PE) and RIBI.
  • thr-MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • CGP 11637 known as nor-MDP
  • CGP 19835A known as MTP-PE
  • RIBI RIBI
  • RIBI is extracted three kinds of bacterial components: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a mixture of 2% squalene / Tween 80 TM manufactured by emulsion An adjuvant.
  • the efficiency of the adjuvant is assessed by the level of antibody directly induced by the immunogenic polypeptide containing the immunogenic sequence of M. tuberculosis, immunized with a mixture of different adjuvants.
  • the vaccine is vaccinated either subcutaneously or intramuscularly, as well as suppositories or oral formulations.
  • conventional binders or carriers include, for example, polyethylene glycol, triglycerides, and such suppositories are mixtures containing from 0.5% to 10% (1% to 2% by weight) of the active ingredient.
  • Oral formulations include excipients that are normally used, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These ingredients are in the form of solutions, suspensions, tablets, pills, capsules, sustained release agents or powders with from 10% to 95% (25% to 70% by weight) of the active ingredient.
  • Appropriate vaccine immunization doses are administered depending on the purpose of the prophylactic and/or therapeutic vaccine.
  • the vaccine can be administered in a single dose regimen, or preferably in a multiple dose regimen.
  • a multi-dose regimen is one in which the initial vaccination procedure can be used in 1-10 separate doses, followed by administration of other doses at subsequent intervals required to maintain and or boost the immune response, for example, at 1-4 months. Two doses, if needed, are given a subsequent dose a few months later.
  • the dosage regimen is at least partially determined by the individual and depends on the judgment of the practitioner.
  • the vaccine may be used in conjunction with other immunoglobulin-like immunomodulators, and a sub-part of the invention is also a multivalent vaccine formulation consisting of a mixture of the vaccine and other vaccines, particularly BCG or recombinant BCG.
  • the therapeutic component (pharmaceutical composition) of the present invention is used for the treatment or prevention of a mammal against Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium leprae or Mycobacterium ulcerans, and is also used for Treatment of degenerative diseases, abnormal physiological conditions, such as malignant tumors.
  • compositions can be administered to humans or other animals in the form of tablets, sprays, tracheal perfusions or intravenous injections.
  • FIG. 15 The open reading frame of Rv0976c by using the upstream PCR primer 5'-TAGGATCCGTGCGTATCGGAAACTGCTCG-3' (SEQ ID No. 15) and the downstream PCR primer 5'-TAGAAGCTTTCACAAC AGGGTCTCCGG GATCT-3' (SEQ ID No. 16) was amplified.
  • the PCR amplification primers for Rv1255c, Rv3160c and Rv0792c are: 5'-CATGGATCCATGGCGGGTACCGACTGGCTG-3' (upstream, Rv1255c, SEQ ID No. 17), 5'-CTGAAGCTTTCACTCGGGTCCAGGGTGAC-3' (downstream, Rv1255c, SEQ ID No. 18) ; 5'-CGTGGATCCATGCCGAGGCAGGCCGGCCGCTG-3' (upstream, Rv3160c, SEQ ID No. 19), 5'-GCAAAGCTTCTAGAGCCCGCGGTCGGGGGGTGCG-3' (downstream, Rv3160c, SEQ ID No.
  • PCR reaction using M. tuberculosis H37Rv (ATCC93009) genomic DNA as a template (50 ⁇ l system) containing template DNA (10 ng), upstream and downstream primers 0.5 ⁇ M each, 0.2 mM dNTPs, 1 ⁇ reaction buffer, 1.25 units of PrimeSTAR HS DNA polymerase (Clontech). Cycling conditions: denaturation at 95 ° C for 5 min; denaturation at 30 cycles (98 ° C, 10 sec), annealing (65 ° C, 20 sec), extension (72 ° C, 2 min); last 72 ° C extension for 5 min, 4 ° C cooling.
  • the PCR amplification product was subjected to agarose gel electrophoresis and purified using a gel purification kit (Qiagen).
  • the product was purified by digestion with BamHI and HindIII at 37 ° C for 3 h, and the digested target fragment was purified using a gel purification kit (Qiagen).
  • the pET28a plasmid (Novagen) was recovered by restriction enzyme digestion under the same conditions.
  • the ligation reaction system total 10 ⁇ l included: 2 ⁇ l of PCR fragment, 2 ⁇ l of pET28a fragment, 1 ⁇ l of 10 ⁇ T4 ligase buffer, and 1 ⁇ l of DNA T4 ligase (NEB).
  • the reaction was stopped by ligation at room temperature for 3 h and incubation at 65 ° C for 20 min.
  • the plasmid-gene fragment ligation products pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were transformed into E. coli DH5 ⁇ , respectively.
  • the ligation reaction mixture was mixed with E. coli DH5 ⁇ competent cells, plated on LB plates containing kanamycin (50 ⁇ g/ml), cultured at 37 ° C overnight, and randomly selected for inoculation into LB liquid medium.
  • Recombinant plasmids pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were extracted from E. coli DH5 ⁇ cells using Qiagen Miniprep kit, and the insertion sequence was verified by DNA sequencing.
  • the recombinant plasmids pET28a-Rv0976c, pET28a-Rv1255c, pET28a-Rv3160c and pET28a-Rv0792c were separately transformed into E. coli BL21 and plated on LB plates containing kanamycin (50 ⁇ g/ml), 37 Incubate overnight at °C, randomly select the monoclonal inoculation into LB liquid medium, and expand to 1L. After incubation at 26 ° C for 3 h, 1 mM IPTG was added. The culture was induced overnight at 26 °C. The cells were collected by centrifugation at 12,000 rpm for 10 min at 4 ° C and resuspended in BugBuster (Novagen) protein extract.
  • mice 50 ⁇ l of the purified protein of Example 1 (10 ⁇ g) and 50 ⁇ l of Freund's incomplete adjuvant (Sigma) were thoroughly mixed, and subcutaneously injected with C57BL/6 mice (Shanghai Slack, SPF grade, 6-8 weeks female) (each group) 4)). Immunization was performed once every other week for 3 times to evaluate the immunogenicity of these antigens. After 8 weeks of the first immunization, the mice were sacrificed and the spleens were isolated.
  • lymphocytes After obtaining spleen lymphocytes, 1 ⁇ 10 6 lymphocytes were added to 24-well cell culture plates and stimulated with Rv0976c, Rv1255c, Rv3160c and Rv0792c antigens respectively, and the stimulation concentrations were 5 ⁇ g/ml and 10 ⁇ g/ml, respectively; The antigen served as an antigen-free negative control. Incubate for 60 hours at 37 ° C, 5% CO 2 , 100% saturated humidity.
  • the cell culture supernatant was collected by centrifugation, and the levels of Th1 type cytokines IFN- ⁇ , TNF- ⁇ , and IL-2 were measured, and the OptEIATM pre-coated ELISA kit (BD Biosciences) was used, and the procedure recommended by the kit was followed.
  • Purified Ag85A was used as a reference, and the expression results of different antigenic cytokines were homogenized according to the results of Ag85A. The results showed that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins were highly immunogenic and capable of inducing Th1 type cytokine secretion levels close to or stronger than Ag85A (Fig. 3).
  • mice sera were collected and antigen-specific antibody levels were measured by ELISA.
  • the serum was serially diluted (1:400 to 1:51200) and added to a 96-well plate pre-coated with Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins.
  • ELISA assays for antibody subtypes were performed using HRP-labeled anti-mouse IgG1 (Youinwei, sc2969) or IgG2c (Youinwei, ab97255) secondary antibody.
  • the results showed that the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins induced high levels of antigen-specific antibodies in the immunized mice, showing strong B cell reactivity (Fig. 4).
  • Candidate gene clones enter mammalian expression vector
  • the scheme for cloning the gene encoding Rv0976c, Rv1255c, Rv3160c and Rv0792c into the mammalian expression vector pVAX1 or pcDNA3.1 is shown in FIG.
  • the primers for the Rv0792c clone into pVAX1 were: 5'-TAGAATTCGCCACCATGGGCATGCGTATCGGAAACTG-3' (upstream, SEQ ID No. 29) and 5'-TAACTGCAGCTAGTGATGGTGATGGTGATGCAACAGGGTCTCCG-3' (downstream, SEQ ID No. 30).
  • the system and reaction conditions of the PCR were the same as in Example 1.
  • the PCR product was digested with EcoRI and PstI, and ligated into the same
  • the digested pVAX1 plasmid (Invitrogen, Cat. No. V260-20) produced pVAX1-Rv0976c.
  • Plasmids pVAX-Ag85A and pVAX-PPE18 were constructed in the same manner.
  • Rv1255c, Rv3160c and Rv0792c were cloned into pcDNA3.1, and the corresponding PCR primers were: 5'-ATATACTTAAGGCCGCCACCATGGCGGGTACCGACTGGCTGTC-3' (upstream, Rv1255c, SEQ ID No. 31), 5'-AATATTCTAGATCAATGGTGATGGTGATGATGCTCGGGTCCAGGGTGACCGGC-3' (downstream, Rv1255c, SEQ ID No. 32); 5'-ATATACTTAAGGCCGCCACCATGCCGAGGCAGGCCGGCCG-3' (upstream, Rv3160c, SEQ ID No.
  • the PCR product was digested with AflII and XbaI, and ligated into the same digested pcDNA3.1 plasmid (Invitrogen, Cat. No. V790-20) to produce pcDNA-Rv1255c, pcDNA-Rv3160c and pcDNA-Rv0792c.
  • the plasmids pcDNA-Ag85A and pcDNA-HspX were constructed in the same manner.
  • mice Human Fukang, SPF grade, 6-8 weeks female, 6 rats per group
  • mice were immunized every other week with 100 ⁇ g/mouse of pVAX-Rv0796c, pVAX-Ag85A or pVAX-PPE18.
  • M. tuberculosis H37Rv strain purchased from the China Microbial Culture Collection, ATCC93009, Shanghai Pulmonary Hospital, cultured and cultured
  • mice After 5 weeks of infection, the mice were sacrificed, and the organ homogenate was serially diluted, and then coated with 7H11 agar plates (adding OADC, namely Middlebrook OADC, mixed nutrient additive, 10% ratio), and counting the number of lung and spleen bacteria. .
  • the results showed that the protective effect of Rv0976c against M. tuberculosis was stronger than that of Ag85A, which was close to that of PPE18: the number of M. tuberculosis in the lung and spleen of mice infected with Rv0976c DNA vaccine was significantly lower than that after 5 weeks of infection with M. tuberculosis.
  • the number of M. tuberculosis in the control (pVAX) or mice inoculated with the Ag85A DNA vaccine was similar to the number of M. tuberculosis in the mice inoculated with the PPE18 DNA vaccine (Fig. 6).
  • mice were immunized every week with 100 ⁇ g/mouse of pcDNA-Rv1255c, pcDNA-Rv3160c, pcDNA-Rv0792c, pcDNA-Ag85A or pcDNA-HspX (Hua Fukang, SPF grade, 6-8 weeks female, each group) 6), a total of 3 immunizations.
  • M. tuberculosis H37Rv strain 100 CFU/lung was infected as an aerosol.
  • mice After 9 weeks of infection, the mice were sacrificed, and the organ homogenate was serially diluted, and then coated on a 7H11 agar plate (adding OADC, ibid.), and the number of lung and spleen organs was counted.
  • Example 4 Serological detection of active tuberculosis, latent tuberculosis, and healthy controls with selected antigens
  • the present invention purified the Rv0976c, Rv1255c, Rv3160c and Rv0792c proteins of Example 1 from different populations.
  • the serum was subjected to an ELISA reaction.
  • the experiment included three groups of population samples: active tuberculosis group (20 patients), tuberculosis latent infection group (25 samples), and healthy control group (24 individuals).
  • the active tuberculosis group included 11 sputum-positive patients and 9 patients with clinical signs of X-ray examination.
  • Tuberculosis latent sensation refers to those who have long-term, continuous close contact with TB patients (2-8 years, mean 4.3 years) but do not show the clinical symptoms of active tuberculosis.
  • a healthy individual is a population that does not have a history of close contact with TB patients, no history of tuberculosis or clinical TB symptoms. All individuals were from the same geographical area and had a history of BCG vaccination.
  • Each purified antigen (Rv0976c, Rv1255c, Rv3160c, and Rv0792c) was added to a 96-well plate at 0.25 ⁇ g/well; the serum from the experimental group of active tuberculosis, latent tuberculosis, and healthy controls was 1 : 100 dilution, added to the antigen-coated wells, antigen-specific antibody levels were detected by standard ELISA methods, and the results are shown in FIG.
  • Rv0976c, Rv1255c, Rv3160c and Rv0792c differed from those of different disease patterns in the serum.
  • serum derived from active tuberculosis and latent infections specifically recognized Rv0976c, Rv1255c, and Rv3160c antigens.
  • Antibody levels were significantly higher than in healthy populations (B, C, D in Figure 8).
  • the level of antibodies that specifically bind Rv0792c in the serum of latently infected populations was significantly higher than that of active tuberculosis and healthy controls (Figure 8 A).

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Abstract

L'invention concerne une composition induisant l'immunogénicité, comprenant des antigènes de mycobacterium tuberculosis Rv0976c, Rv1255c, Rv3160c et/ou Rv0792c et des gènes de codage de celle-ci, des vaccins ou une composition de traitement.
PCT/CN2015/091068 2015-06-15 2015-09-29 Antigènes de mycobacterium tuberculosis et leurs applications WO2016201825A1 (fr)

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