WO2012088577A2 - Protéine recombinante de mycobacterium sp., test immunodiagnostique et vaccin contre la tuberculose - Google Patents

Protéine recombinante de mycobacterium sp., test immunodiagnostique et vaccin contre la tuberculose Download PDF

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WO2012088577A2
WO2012088577A2 PCT/BR2011/000517 BR2011000517W WO2012088577A2 WO 2012088577 A2 WO2012088577 A2 WO 2012088577A2 BR 2011000517 W BR2011000517 W BR 2011000517W WO 2012088577 A2 WO2012088577 A2 WO 2012088577A2
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tuberculosis
protein
immunodiagnostic test
group
test kit
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PCT/BR2011/000517
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Portuguese (pt)
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WO2012088577A3 (fr
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Sérgio costa OLIVEIRA
Lucas de Lima NOGUEIRA
André BAFICA
Benildo Sousa CAVADA
Manoel BARRAL-NETO
Henrique Couto TEIXEIRA
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Universidade Federal De Minas Gerais - Ufmg
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Publication of WO2012088577A3 publication Critical patent/WO2012088577A3/fr

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    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis

Definitions

  • the present invention relates to the description of a diagnostic test and a vaccine for tuberculosis comprising the use of a novel secreted protein (sMTL-13) with lectin characteristics isolated from Mycobacterium tuberculosis.
  • sMTL-13 novel secreted protein
  • EP 2226635 "IMMUNODETECTION ASSAY FOR MYCOBACTERIUM TUBERCULOSIS COMPLEX", which describes a method for detecting M. tuberculosis secreted protein MPT64 by immunoassay using specific antibody capable of recognizing epitopes of such protein;
  • the present invention demonstrates the identification of a new secreted 13 kDa lectin not yet described in the prior art (secreted M. tuberculosis Lectin, sMTL-13; Rv1419), belonging to the beta-trefoil type-ricin family, present only M. tuberculosis complex species, being absent in non-tuberculous mycobacterial species (MNT) such as M. avium, M. kansasii and M. leprae.
  • MNT non-tuberculous mycobacterial species
  • the sMTL-13 lectin was able to agglutinate rabbit erythrocytes in a dose-dependent manner, suggesting that this protein has functional lectin domains.
  • sMTL-13 can be used as a biomarker in the treatment of tuberculosis in immunodiagnostic testing and as a vaccine.
  • FIG. 1 In silico analysis and molecular cloning of the Mycobacterium tuberculosis gene Rv1419.
  • A Rv1419p is composed of a 33 amino acid signal peptide (gray bar), with the cleavage site between Ala33 and Asp34 ( * ). The ricin-like lectin domain is found from Asp45 to Asp 154 (table). Putative sugar binding sites are present in the mature sequence as indicated by circles. The presence of a predicted QW pattern is underlined.
  • B Isolation of the Rv1419 gene. Lines: 1, standard molecular weight; 2, expected PCR product.
  • FIG. 1 Predicted Rv1419 protein is detected in the Mycobacterium tuberculosis H37Rv CFP fraction.
  • A Western Blot analysis in which the M. tuberculosis CFP fraction was resolved on SDS-PAGE, transferred to nitrocellulose membrane and probed with monoclonal supernatant produced against Rv1419p.
  • Mtb Mycobacterium tuberculosis; Atypical mycobacteria (clinical isolates of M. avium, M. fortuitum and M. kansasii).
  • B subcellular preparations of Mtb H37Rv. As a control the membrane was again analyzed with the 19-kD lipoprotein monoclonal antibody (IT-19, 1: 1000 dilution). The data shown are representative of two independent experiments. In situ marking of sMTL-13 in patients with active tuberculosis.
  • Figure 3 Adaptive immune response in patients with active tuberculosis.
  • PBMC Peripheral blood mononuclear cells
  • B Serum from tuberculosis patients was evaluated for sMTL-13 specific IgG by ELISA.
  • C based on anti-ESAT-6 and anti-sMTL-13 antibody levels (active disease versus endemic area controls), a ROC curve was used to compare the accuracy of the indicated variables (variable 1: ESAT-6; variable 2 : sMTL-13) during the active phase of tuberculosis. For each indicator, sensitivity is plotted against specificity and accuracy is measured by the area over the curve (AUC).
  • the recombinant protein Rv1419p from Mycobacterium sp. was produced and from it the lectin sMTL-13 was obtained.
  • the ability of sMTL-13 to agglutinate erythrocytes in a dose-dependent manner was then demonstrated, suggesting that this protein has functional lectin domains.
  • serum from patients with active tuberculosis showed high levels of IgG antibodies against sMTL-13, and this response decreases after successful anti-tuberculosis treatment.
  • the immunodiagnostic test may be selected from the group comprising ELISA, Western blot, dot blot, immunodiffusion and immunochromatography. In the following examples the ELISA test was used.
  • Example 1 Obtaining Mycobacterial Fractions.
  • Subcellular fractions of the standard M. tuberculosis H37Rv laboratory strain used in this invention as total cell lysate (WCL), culture protein filtrate (CFP), membrane (MEM), and cell wall (CW) fractions were obtained by culturing the strain. late log phase (day 14) in GAS (glycerol-alanine-salts) media as described elsewhere (Sonnenberg MG and Belisle, JT. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry Infect Immun 1997; 65: 4515-24; Lee BY Hefta SA and Brennan P.
  • a database of non-redundant lectins was generated to search lectinic domains in the Araidopsis thaliana genome using this organism as a model (Moreno FB, Facó F, Ceccatto VM, Sampaio AH, Costa ABS, Freitas JLT, Nogueira LL, Lima ME, Lima JL, Cavada BS Matching carbohydrate-binding domains in Arabidopsis thaliana genome: development of a lectin database Online J Bioinform 2003; 4: 96-105.). To assess the presence of such domains in an important human pathogen, Mycobacterium tuberculosis, this database was adapted and a unique hypothetical lectin encoded by the Rv1419 gene was identified.
  • Figure 1 shows the bioinformatic characterization of the Rv1419 gene (SEQ IDN # 3).
  • Its open reading module (ORF) contains 474 nucleotides and the amino acid sequence (aa) encodes a hypothetical 157 residue protein containing signal peptide and an estimated molecular mass of 16.8 kDa.
  • the primary amino acid sequence of the protein encoded by Rv1419 contains a 33 residue signal peptide, with a type I cleavage signal between Ala33 and Asp34.
  • Analysis of mature protein (without signal peptide) showed molecular mass of 13.6 kDa.
  • Precursor protein positions 31-32 contain the Ala-x-Ala sequence, a standard sequence commonly found preceding the cleavage site (Tuteja R.
  • Rv1419p contains a carbohydrate-binding B-chain ricin domain and belongs to the beta-trefoil-like ricin family of proteins, consisting of three homologous subdomains as well as the presence of a QW (Hazes B.
  • the (QxW) 3 domain a) flexible lectin scaffold (Protein Sci 1996; 5: 1490-501).
  • B-chain ricin domains have been shown to bind cell surface glycolipids and glycoproteins that contain beta-1,4-linked galactose and mannose molecular moieties [Hartley MR, Lord JM.
  • Rv1419p a recombinant protein (SEQ ID NO: 1) was produced.
  • the sequence encoding the complete Rv1419, including the signal peptide was amplified by PCR using M. tuberculosis H37v DNA as a template.
  • a 496 bp DNA fragment was obtained ( Figure 1 B), purified and then cloned into the pMOSBIue vector.
  • the sequencing procedure was employed to confirm cloning and amplification experiments generated an unchanged Rv1419 sequence.
  • the product was named pMOSBIue-Mtb and digested by restriction enzymes XhoI and Ndel to subclon the pET-15b expression vector. The fragment was then inserted in-frame with the start codon present at the Ndel cleavage site to allow total production of Rv1419p. Heterologous expression was then performed using E. coli strain BL21 (DE3).
  • Figure 1C shows a typical SDS-PAGE experiment of the recombinant Rv1419 (rRv1419) obtained, showing a single band of ⁇ 17 kDa molecular weight.
  • This recombinant protein (SEQ ID NO: 1) was produced as inclusion bodies and purified under denaturing conditions by affinity chromatography. Refolding of the denatured protein was performed using pH 7.4 renaturation PBS buffer, resulting in the complete soluble protein used thereafter.
  • Example 4 Rv1419p is expressed in the fraction of the M. tuberculosis culture protein filtrate, but not in the non-tuberculous mycobacteria (NTM).
  • FIG. 2A shows a single band of -13 kDa mm in M. tuberculosis H37Rv CFP, but not in CFP fractions obtained from the M. avium, M. kansasii and M. fortuitum MNT species.
  • M. tuberculosis H37Rv cell wall or membrane preparations showed discrete bands when incubated with the same monoclonal antibody ( Figure 2B).
  • tuberculosis CFP preparations revealed that sMTL-13 is at least as abundant as 19 kDa lipoprotein, a well-known component of CFP (Gasteiger, E., Gattiker, A., Hoogland, C, Ivanyi, I., Appel, RD and Bairoch, A., ExPASy: The proteomics server for in-depth protein knowledge and analysis (Nucleic Acids Res. 2003. 31: 3784-3788). As previously shown by Rosenkrands et al., High levels of the M.
  • tuberculosis H37Rv 19 kDa lipoprotein band were observed in fractions incubated with the 19 kDa anti-lipoprotein monoclonal antibody (Figure 2B) (Rosenkrands I, King A, Weldingh K, Moniatte M, Moertz E and Andersen P. Towards the proteome of Mycobacterium tuberculosis (Electrophoresis 2000; 21: 3740-56). Together, these data indicate that Rv1419p is a secreted lectin present at reasonable levels in M. tuberculosis CFP. We therefore name this protein 'Secreted M. tuberculosis Lectin' (sMTL) -13.
  • Open reading modules recorded as hypothetical proteins of unknown or putative function were filtered from the entire M. tuberculosis genome using a Peari script (Moreno FB, Phaco F, Ceccatto VM, Sampaio AH, Costa ABS, Freitas JLT, Nogueira LL, Lima ME, Lima JL, Cavada BS Matching carbohydrate-binding domains in Arabidopsis thaliana genome: development of a lectin database Online J Bioinform 2003; 4: 96-105.).
  • the deduced amino acid sequence of the entire Rv1419 ORF was subjected to primary structure analysis using Proteomis Server Expert Protein Analysis System (ExPASy), Gattiger E, Hoogland C, Ivanyi I, Appel RD and Bairoch A.
  • ExPASy The proteomics server for in-depth protein knowledge and analysis (Nucleic Acids Res 2003; 31: 3784-8).
  • the SignalP 3.0 server was used to identify potential Sec-type signal peptides and cleavage sites based on various Neural Network methods and Hidden Markov models (Nielsen H, Engelbrecht J, Brunak S and von Heijne G.
  • the sMTL-13 protein containing the signal peptide was expressed as a histidine-tagged protein (His) in an E. coli strain (DE3).
  • the Rv1419 gene was subcloned into the pET15b expression vector (Novagen, Madison, USA) encoding six histidine residues located in the N-terminal region, allowing expression of a fusion protein.
  • an Rv1419 PCR fragment representing the entire open reading module was generated with specific primers designed to produce Ndel and Xhol enzyme restriction sites in the resulting PCR product using M.
  • tuberculosis H37Rv DNA as a template: Ndel for sense (5'-GGAATTCCATATGGGTGAATTACGGTTGG-3 ') and Xhol for antisense (5'-CCGCTCGAGTCATTACGGCACGCTATCCC-3').
  • the parameters for the PCR reaction were as follows: initial denaturation for 4 min at 94 ° C, denaturation for 1 min at 94 ° C, annealing for 1 min at 56 ° C and extension for 1 min at 72 ° C (36 cycles). Then the sequence was confirmed by DNA sequencing.
  • E E.
  • coli BL21 (DE3) was grown at 37 ° C for an A 60 (nm) of 0.6 and expression was performed in the presence of 1 mM isopropylthiogalactoside (IPTG). After further induction for 4 h, cells were harvested by centrifugation and resuspended in 50 ml 10 mM Na 2 HP0 4, 10 mM NaH 2 P0 4 , 0.5 mM NaCl and 10 mM imidazole. Cells were sonicated lyses 3 times at 30% amplitude and centrifuged at 5,400xG, 4 ° C for 20 min.
  • IPTG isopropylthiogalactoside
  • Recombinant sMTL-13p was recovered as inclusion bodies and resuspended in 50 ml of 10 mM Na 2 HP0 4, 10 mM NaH 2 P0 4 , 0.5 mM NaCl, 10 mM imidazole and 8 M urea.
  • the recombinant protein was purified by nickel column affinity chromatography (GE Healthcare, Sao Paulo) under denaturing conditions following the methodology described in the manufacturer's protocol. Then the denaturation buffer was removed by dialysis and exchanged for renaturation PBS buffer before in vivo use in experiments.
  • Example 7- Generation of monoclonal antibody against sMTL-13p (clone 276.B7).
  • mice Three Balb / c mice were immunized with subcutaneous injection of 100 ⁇ g of recombinant sMTL-13p diluted in incomplete Freund's adjuvant and then three intraperitoneal injections of 100 ⁇ g of lectin were administered at weekly intervals. Splenocytes from mice showing the highest ELISA detector serum titers were fused to NS1 myeloma cells (ATCC number TIB-108) at a 7: 1 ratio using 50% polyethylene glycol as fusogen.
  • NS1 myeloma cells ATCC number TIB-108
  • Hybridoma supernatants were screened by ELISA, where purified recombinant sMTL-13 was used as a capture antigen Out of 900 initially screened clones, 12 positive clones were selected based on the production of high lectin antibody titers. The most stable hybridoma was selected and subcloned by dilution. The murine immunoglobulin class and subclass produced was determined by ELISA to be IgGlkappa using SBA Clonotyping. System / HRP (Southern Biotech, Birmingham, USA) following manufacturer's instructions.
  • Example 8 sMTL-13p-induced haemagglutination assay.
  • sMTL-13p a classic hemagglutination experiment was performed [38-40]. Briefly, different doses of recombinant sMTL-13p (2.5-10 pg / ml, 100 ⁇ l) were incubated with 100 ⁇ l to 2% of a rabbit erythrocyte suspension. Samples were then incubated for 30 min at 37 ° C and left at room temperature for a further 16 h. Lectinic activity was assessed by optical microscopy (Olympus, 10X objective) and images were acquired with digital camera (Nikkon, Japan).
  • rRv1419p induces agglutination of erythrocytes at doses of 2.5 ⁇ g / ml or 10 ⁇ g / ml ( Figure 1 D).
  • pretreatment with trypsin or papain two enzymes known to expose hidden carbohydrate binding sites on the cell surface (Denis M, Palatty PD, Bai NR, Suriya SJ. Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii Eur J Biochem 2003; 270 (21): 4348-55.), increase hemagglutination by Rv1419p (data not shown).
  • rRv1419p has at least two carbohydrate-binding functional domains, a prerequisite for promoting hemagglutination.
  • Maxsorb plates (Nunc, Roskilde, Denmark) were sensitized overnight with recombinant sMTL-13p in carbonate buffer at 4 ° C. Plates were washed with PBS / 0.05% Tween-20 and blocked with PBS / 0.1% Tween-20 and 0.05% BSA. Serum from patients and controls was diluted 1: 20 in PBS / 0.05% Tween-20 and incubated overnight at 4 ° C. Then the plates were washed and incubated with HRP-conjugated anti-human IgG (Sigma, St. Louis, MO, USA) at a dilution of 1: 1,000. Color development was performed with the addition of ABTS ® Peroxidase substrate. The reaction was stopped with 10% SDS and A * 05 was measured in an ELISA reader.
  • Anti-sMTL-13 IgG antibodies are increased during active tuberculosis.
  • a feature of mycobacterial infection is the generation of an intense immune response against secreted antigens.
  • Several antigens secreted by M. tuberculosis are believed to be virulence factors and influence the development of tuberculosis.
  • FIG. 3B newly diagnosed tuberculosis patients who are either treatment-naive or up to 15 days of chemotherapy (0-15 days) had high levels of total anti-sMTL-13 IgG antibodies.

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Abstract

La présente invention concerne la description d'un test diagnostique et d'un vaccin contre la tuberculose, caractérisés en ce qu'ils impliquent l'utilisation d'une nouvelle protéine sécrétée (sMTL-13), à caractéristiques de lectine, isolée de Mycobacterium tuberculosis.
PCT/BR2011/000517 2010-12-29 2011-12-29 Protéine recombinante de mycobacterium sp., test immunodiagnostique et vaccin contre la tuberculose WO2012088577A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066143A1 (fr) * 1999-05-04 2000-11-09 The Public Health Research Institute Of The City Of New York, Inc. Proteines secretees par mycobacterium tuberculosis et utilisations comme vaccins et reactifs de diagnostic
EP2226635A1 (fr) * 2007-12-28 2010-09-08 BL Co., Ltd Test d'immunodétection du complexe mycobacterium tuberculosis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000066143A1 (fr) * 1999-05-04 2000-11-09 The Public Health Research Institute Of The City Of New York, Inc. Proteines secretees par mycobacterium tuberculosis et utilisations comme vaccins et reactifs de diagnostic
EP2226635A1 (fr) * 2007-12-28 2010-09-08 BL Co., Ltd Test d'immunodétection du complexe mycobacterium tuberculosis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NOGUEIRA L ET AL.: 'Mycobacterium tuberculosis Rv1419 encodes a secreted 13 kDa lectin with immunological reactivity during human tuberculosis' EUR. J. IMMUNOL. vol. 40, March 2010, ISSN 1521-4141 pages 744 - 753 *

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