Design, synthesis and cloning of cDNA coding for antigenic proteins
Synthetic cDNAs coding for TB10.4 and Ag85B antigenic proteins were designed in silico by back translation of the amino acid sequences reported in the database (TB10.4: GenBank Accession no. CAA17363.1; Ag85B: UniProtKB/Swiss-Prot Accession no. P0C5B9.1). A third cDNA, in which the nucleotide sequence coding for TB10.4 is upstream to the sequence coding for Ag85B (full cDNA) was also designed. All synthetic cDNAs were produced by Eurofins Medigenomix GmbH (Ebersberg, Germany), after optimization of the nucleotidic sequences for expression in E. coli (see Additional file 2: Figure S1). The three synthetic genes were cloned into pET32b using Bam HI and Eco RI restriction sites. By this cloning strategy, the genes were fused with thioredoxin (Trx) encoding gene and tagged by an His6-tag. Two additional constructs of the full cDNA were also produced. To prepare His-full 1 form, the full cDNA was extracted from pET32b plasmid using Kpn I and Eco RI restriction sites and cloned into pColdI plasmid (TaKaRa Bio Inc., Otsu, Japan). For the production of His-full 2 form, site-directed mutagenesis was carried out on the full cDNA using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA), to insert a Nde I restriction site at the 5′ of the full cDNA, including the triplet coding for the initial methionine. The following mutagenesis primers were used: 5′GCCATGAGCTCGGATCAT ATGAGCCAGATCATG3′; 5′CATGATCTGGCTCATAT GATCCGAGCTCATGGC3′ (mutated bases are in italics). The presence of the desired mutation was confirmed by automated DNA sequencing. The mutated cDNA was then cloned into pColdI plasmid using Nde I and Eco RI restriction sites. The gene sequences are reported in Figure 1.
Strain and growth conditions
For protein expression, all plasmids were transferred to the E. coli hosts BL21(DE3) and BL21(DE3)pLysS. The following media were used: M9/glucose (6.78 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl, 0.1 mM CaCl2, 2 mM MgSO4, 10 g/L glucose); Luria-Bertani (LB, 10 g/L bacto-tryptone, 5 g/L yeast extract, 5 g/L NaCl); Terrific Broth (TB, 12 g/L bacto-tryptone, 24 g/L yeast extract, 8 mL/L glycerol, 2.2 g/L KH2PO4, 9.4 g/L K2HPO4); Terrific Broth/NaCl (TB, 12 g/L bacto-tryptone, 24 g/L yeast extract, 8 mL/L glycerol, 2.2 g/L KH2PO4, 9.4 g/L K2HPO4, 5 g/L NaCl); Super Broth (SB, 32 g/L bacto-tryptone, 20 g/L yeast extract, 5 g/L NaCl); Super Broth/NaCl (SB/NaCl, 32 g/L bacto-tryptone, 20 g/L yeast extract, 10 g/L NaCl) [15, 20]. Starter cultures were prepared from a single colony of E. coli carrying the recombinant plasmid, employing the same medium used for protein expression containing ampicillin (100 μg/mL, final concentration) and, in the case of BL21(DE3)pLysS strain, also chloramphenicol (34 μg/mL, final concentration). Cultures were grown overnight under vigorous shaking at 37°C. For chaperone co-expression, BL21(DE3) E. coli cells were co-transformed with pKJE7 (coding for AraB, DnaK, DnaJ and GrpE) or with pG-Tf2 (coding for GroES, GroEL and TiG) plasmids (Chaperone Plasmid Set, TaKaRa Bio Inc., Otsu, Japan), adding chloramphenicol (30 μg/mL, final concentration) to the growth medium. Expression of the chaperone proteins was induced by adding 1 mg/mL L-arabinose (for pKJE7) or with 2 ng/mL tetracyclin (for pG-Tf2), according to the manufacturer’s instructions. Protein production trials were carried out in 500 mL or 2 L baffled Erlenmeyer flasks containing 80 mL or 500 mL of each medium inoculated with the starter cultures (initial OD600nm = 0.1). Cells were grown at 37°C with shaking (150 rpm) until protein expression was induced by adding IPTG: cells were then grown at 15 or 18°C until harvesting. Collected cells were washed with STE buffer and stored at -20°C.
3 L bioreactor cultivations
SB for E. coli BL21(DE3) cells containing the pET32b-Trx-Ag85B plasmid, LB for those transformed with pET32b-Trx-TB10.4 plasmid and SB/NaCl for those transformed with pColdI-His-full 2 plasmid were used as production media in 2 L working volume P-100 Applikon 3 L glass reactor (height 25 cm, diameter 13 cm) equipped with a AD1030 Biocontroller and AD1032 motor [20, 21]. Cultivations in bioreactor were conducted in batch-mode at 37°C, 500 rpm stirring (corresponding to 1.17 m/s of tip speed) and 2 L/min aeration rate. Foam production was controlled by the addition of Hodag antifoam through an antifoam sensor. Starter cultures were grown overnight in SB, LB or SB/NaCl medium and diluted up to an initial OD600nm of 0.1. For pET32b-Trx-Ag85B and pET32b-Trx-TB10.4 recombinant cells, after 2–4 hours of growth (corresponding to an OD600nm of 2 for the previous and of 0.8 for the latter), 0.1 mM IPTG was added and the temperature was decreased to 18°C. pColdI-His-full 2 recombinant cells grown for 4–6 hours (corresponding to an OD600nm of 2) were added of 0.1 mM IPTG and 25 g/L NaCl and the temperature was decreased to 15°C. After 16 hours, cells were harvested by centrifugation, washed with STE buffer and stored at -20°C.
Crude extract preparation and purification of fusion proteins
Cell paste was re-suspended in phosphate buffer saline (PBS) (8.1 mM Na2HPO4, 1.76 mM KH2PO4, 0.137 M NaCl, 2.68 mM KCl), pH 7.0, 1 mM phenylmethanesulphonylfluride (PMSF), 10 μg/mL deoxyribonuclease. Cells were disrupted by sonication (5 cycles of 30 s each, in ice, with 30-s interval). The insoluble fraction was removed by centrifugation at 39000 × g for 1 hour at 4°C. The crude extract (added of 0.9 M NaCl) was loaded on a HiTrap Chelating column (GE Healthcare, Piscataway, NJ, USA), pre-loaded with Ni2+ and equilibrated in PBS, 0.9 M NaCl, pH 7.0. Elution of the fusion protein was performed increasing the amount of PBS buffer containing 0.5 M imidazole, pH 7.0 . The pure proteins were then equilibrated in 20 mM TrisHCl, 50 mM NaCl, pH 7.4 by size-exclusion chromatography on a PD10 desalting column or by extensive dialysis.
Enterokinase cleavage of the fusion proteins
Enterokinases (EK, Recombinant Enterokinase, and Tag · off High Activity Recombinant Enterokinase) and Recombinant Enterokinase Capture Agarose were purchased from Novagen. One EK unit is defined as the amount of enzyme that cleaves 50 μg of a control protein (supplied by the producer) in 16 hours at room temperature and in 20 mM TrisHCl, 50 mM NaCl, 2 mM CaCl2, pH 7.4.
SDS-PAGE electrophoresis and Western blot analysis
Proteins from total cell extracts or from both soluble and insoluble cell fractions were separated by SDS-PAGE: gels were stained for proteins with Coomassie Blue R-250. For total cell extracts and for insoluble fractions after cell disruption, cell pellets were directly resuspended in an appropriate volume of Laemmli sample buffer. For Western blot analysis, upon separation by SDS-PAGE, proteins were transferred electrophoretically onto a nitrocellulose membrane. The fusion proteins were detected using anti-His-tag mouse monoclonal antibody (His-probe, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-TB10.4 polyclonal antibodies (Antibodies-online GmbH, Aachen, Germany), or rabbit anti-Ag85B polyclonal antibody (DIATHEVA, Fano, Italy), in combination with the appropriate secondary antibody: goat anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA), donkey anti-mouse and donkey anti-rabbit (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) IgG HRP-conjugated antibody. The immunorecognition was detected by a chemioluminescence method (ECL Plus Western Blotting Detection System, GE Healthcare, Piscataway, NJ, USA). The amount of protein was estimated by evaluating the intensity of the signal using the public domain, Java-based image processing program ImageJ (National Institutes of Health, http://rsb.info.nih.gov/ij/). His-tagged D-amino acid oxidase  was used as standard.
Extinction coefficients of native proteins were determined using a Jasco V-560 spectrophotometer by complete denaturation of appropriate dilutions of protein samples in 6 M urea assuming as extinction coefficients of the fully denatured proteins those calculated by ExPASy Bioinformatic Resource Portal (http://www.expasy.org/) using ProtParam tool .
Circular dicroism (CD) spectra were recorded on a J-815 Jasco spectropolarimeter : cell path-lenth was 1 cm for measurements above 250 nm (0.4 mg protein/mL) and 0.1 cm for measurements in the 190–250 nm range (0.1 mg protein/mL). Secondary structure fractions were calculated from deconvolution of the CD spectra with the program K2D2 (http://www.ogic.ca/projects/k2d2/) .
Temperature ramp experiments were carried out by using the same instrumentation equipped with a software-driven Peltier-based temperature controller (temperature gradient: 0.5°C/min). All spectral experiments were carried out in 20 mM MOPS, 0.4 M NaCl, pH 7.0 or in 10 mM ammonium acetate, pH 7.0 at 15°C.
Mass spectrometry analyses
Intact MS experiments were in general carried out on a LTQ-MS (Thermo Electron, San Jose, CA, USA) with an ESI source. The protein samples were prepared in 10 mM ammonium bicarbonate (pH 7.4), 0.1% acetonitrile/trifluroacetic acid (50/50) to a final concentration of 0.3 mg/mL. Full scan intact MS experiments were carried out under the following instrumental conditions: positive ion mode, mass range 700–2000 m/z, source voltage 4.5 kV, capillary voltage 35 V, sheat gas 15, auxiliary gas 2, capillary temperature 220°C, tube lens voltage 140 V. Five spectra were acquired for each data and each spectrum was the composite of two averaged scans. Data processing was performed using Bioworks Browser (Thermo Electron, revision 3.1). Theoretical average masses were calculated by Ion Source Bioinformatic Resource Portal (http://www.ionsource.com/) using Peptide Mass Calculator tool. Identification of truncated forms of the proteins was carried out by MS-non specific database search program Protein Prospector v 5.10.4 (http://prospector.ucsf.edu).
The His-full2 protein was dissolved in acetonitrile and analyzed using a MALDI-TOF/TOF Ultraflex III spectrometer (Bruker Daltonics, Bremen, Germany) in linear positive mode. A solution of sinapinic acid (SA) 20 mg/mL solved in a 70/30 acetonitrile/trifluoroacetic acid (TFA) 0.1% solution, was used as matrix. The spectra were acquired in a mass range of 6000–52000 m/z and processed using Flex Analysis software 3.0 (Bruker Daltonics).
Immunological studies: study population
The study population included 19 subjects. Twelve patients with newly diagnosed, untreated active pulmonary TB and 7 healthy individuals without any history of TB exposure (hereafter indicated as TB unexposed controls). Study subjects were recruited at the Department of Infectious Diseases of the Fondazione IRCCS-Policlinico San Matteo of Pavia (Italy), after informed consent. In all cases, the diagnosis of active TB was confirmed by M. tuberculosis culture isolation.
Peripheral venous blood was obtained for serum samples from all the participants to the study to perform ELISA assay. Additionally, peripheral blood was collected into tubes containing heparin for ELISPOT assay. Peripheral blood mononuclear cells (PBMC) were isolated by standard density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). Isolated PBMC were cryopreserved in freezing medium: 10% v/v DMSO (Sigma-Aldrich, St. Louis, MO, USA), 25% v/v human albumin (Grifolds Biologicals, Los Angeles, CA, USA) and 65% v/v RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Euroclone, Milan, Italy) and kept in liquid nitrogen until ELISPOT analyses.
ELISA assay was performed with the method of  with minor modifications. Microplates 96-wells high-binding capacity flat-bottom (Greiner-bio-one GmbH, Frickenhausen Germany) were coated with different concentrations (400, 200, 100 ng/well) of Ag85B, TB10.4, His-full 2 in 50 mM bicarbonate buffer (pH 9.6) and incubated at room temperature overnight. Plates were washed three times with 0.05% (v/v) Tween-20 in phosphate buffered saline (PBS-T, Sigma-Aldrich, St. Louis, MO, USA) and incubated with 200 μL/well of blocking buffer (PBS with 1% BSA) at room temperature for at least 1 hour and then washed three times with PBS-T. Serum samples were diluted with dilution buffer (PBS-T with 1% BSA) at scalar dilution of 1:50 to 1:800. One-hundred microliter of the diluted sera was added to each well and incubated for 1 hour at 37°C. Plates were then washed four times with PBS-T. Anti-human specific IgG antibody labeled with horseradish peroxidase (MP Biomedicals, LLC Santa Ana, CA, USA) was added to each well and incubated at 37°C for 1 hour. Plates were again washed four times with PBS-T and 100 μL of o-phenyl-diamine/H2O2 substrate (Sigma-Aldrich, St. Louis, MO, USA) was added for 30 minutes. The reaction was stopped by adding 50 μL of 0.5 M H2SO4. The absorbance at 492 nm was measured with an ELISA plate spectrophotometer (Titertek Plus MS212 ICN, Eschwege, Germany).
Evaluation of peptide specific single T-cell IFN-γ release
The enumeration of peptide specific T-cell producing IFN-γ was performed at the single cell level by using ELISPOT assay, as previously described, with minor modifications .
In detail, PBMC were thawed, washed and re-suspended in the culture medium: RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated fetal calf serum (Euroclone, Milan, Italy). Cells were kept overnight at 37°C in a humidified 5% CO2 atmosphere. Cells were then transferred to a 24-well flat-bottom plate (5 × 105 cells/mL per well), stimulated with 1 μg/mL of each protein (one protein per well) or culture medium only or phytohemagglutinin (PHA) (5 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), and cultured at 37°C in a humidified 5% CO2 atmosphere for 10 days. On days 3 and 7, half of the supernatant from each well was removed and replaced with wet culture medium supplemented with 20 IU/mL recombinant human IL-2 (Peprotech, London, UK). After 10 days, cells from each well were harvested, washed three times with culture medium and re-suspended at a concentration of 4 × 105 cells/mL before their use in ELISPOT assay. Un-stimulated PBMC were included as negative controls.
In ELISPOT assay, multiScreen-IP 96-well plates (Merck Millipore, Darmstadt, Germany) were coated with IFN-γ capture antibody and incubated overnight at 4°C. Plates were then washed 5 times with PBS and blocked with culture medium for 2 hours at room temperature. Antigen-stimulated cells for 10 days were added in duplicate (4 × 104 cells/well) and re-stimulated with each protein used for stimulation during the 10-day period. Cells stimulated with PHA during the 10-day period were re-stimulated with Staphylococcus aureus Enterotoxin B (SEB) superantigen (2 μg/mL; Sigma-Aldrich, St. Louis, MO, USA). After incubation at 37°C in a humidified 5% CO2 atmosphere for 24 hours, plates were washed five times with PBS-T (Sigma-Aldrich, St. Louis, MO, USA). Biotinylated detection antibody for IFN-γ was added and incubated overnight at 4°C. After five washes with PBS-T, streptavidin-alkaline phosphatase conjugate was added and plates were incubated at 37°C, 5% CO2 atmosphere for 1 hour. Plates were then washed, 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) was added and incubated at room temperature for 20 minutes. Wells were then washed several times under running water and air-dried. Spot-forming cells (SFCs) were counted in duplicate wells using an automated ELISPOT reader system (Autoimmun Diagnostika GmbH, Strassberg, Germany). The mean value of the SFCs was calculated, and the value of the control subtracted to the SFCs of the stimuli: values were referred to million PBMC.
Data are expressed using mean and standard deviation of the mean or median and percentiles as appropriate. Comparison between groups have been made using Mann–Whitney and χ2 tests. A p value below 0.05 was considered significant. All tests were performed using the GraphPad Prism 4.0 (Graphpad software, San Diego, CA, USA) software package.