Plasmids
Plasmid pcDNA3A-Hsp65 contained T7 RNA Polymerase promoter and cDNA encoding the gene for M. Leprae Hsp65 that has been previously described [20]. Plasmid DNA was purified as described in Endo-Free QIAGEN plasmid purification kit (QIAGEN AG, Basel, Switzerland). Spectrophotometry analysis revealed 260/280 nm ratios of 1.80 or more. The purity of the DNA was confirmed in a 1% agarose gel. Plasmid concentration was determined by spectrophotometry at the wave lengths 260 and 280 nm using Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, USA).
mRNA preparation
Plasmids containing the full length cDNA of Hsp65 gene (pcDNA3A-Hsp65) were linearized with APA I enzyme and purified at a final concentration of 1 μg/μL by standard protocols. Plasmid pTRI-Xef, used as a control template containing 1.85-kbp elongation factor 1-α gene from Xenopus laevis (EF-1α), was already linearized by Ambion (Austin, Texas, USA). RNA replicons were in vitro transcribed using linearized replicon plasmids and mMessage mMachine Ultra Kit (Ambion), according to the manufacturer's recommendations. After the RNA synthesis was complete, the in vitro transcription reactions were treated with RNase-free DNase (Ambion) at 37°C for 15 min to degrade the DNA templates. The next step was the synthesis of poly-A tail, and then the RNA was purified by acidic phenol/chloroform extraction followed by mRNA isopropanol precipitation. mRNAs were quantified by absorbance at 260 and 280 nm using Nanodrop (Thermo), and the proportion of full-length transcripts was checked by formaldehyde denaturing agarose gel electrophoresis. All formulations were tested for endotoxin levels with QCL-1000 Limulus amebocyte lysate. The levels found were under 0.01 EU/mL.
mRNA structure analysis
Plasmid pcDNA3A-Hsp65 template (data not shown) was sequenced to assure that the sequences required to the perfect translation process were present. Since the plasmid had all these sequences, in silico mRNA structure analysis was performed using mFOLD default parameters.
Cell assay
Transfections was made in HEK 293T cells with mRNA-Hsp65 were made using Transmessenger Transfection Reagent (Qiagen), according to the manufacturer's instructions. Briefly, 10 μg of mRNA-Hsp65 produced in vitro was mixed with Transmessenger Reagent for 1 hour and then applied to a 70% confluent culture of HEK 293T cells maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Gaithersburg, USA) without serum. After 30 minutes, 2, 4, and 8 hours, total RNA was extracted using Trizol (Invitrogen, Carlsbad, USA), according to the manufacturer's recommendation.
RT-PCR
Total cellular RNA (10 μg/mL) was reverse transcribed using oligo(dT) primers and Superscript reverse transcriptase (Invitrogen), according to the manufacturer's recommendations. The contaminating plasmid DNA was removed by treatment with DNAse I, amplification-grade (Invitrogen). The sequences of PCR primers for the amplification of Hsp cDNA were 5'-ACC AAC GAT GGC GTG TCC AT-3' (sense) and 5'-TAG AAG GCA CAG TCG AGG-3' (antisense), resulting in a 400-bp PCR product. Primers for β-actin were used as a control for the quantity of RNA used. β-actin specific primers amplified a 450-bp product and were composed of the following sequences: 5'-GTG GGC CGC TCT AGG CAC CAA-3' (sense) and 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3' (antisense). All primers were purchased from Invitrogen. The cDNA (2 μg) was submitted to an initial denaturation step (95°C, 5 minutes), followed by 35 cycles of denaturation (94°C, 30 seconds), annealing (60°C, 45 seconds), and extension (72°C, 1.5 minute), and a final extension step (72°C, 3 min). PCR amplification products were analyzed by agarose gel 1% electrophoresis and stained with ethidium bromide. In order to avoid cross-contamination, all procedures including the PCR, were performed in separate laminar flow hoods.
Western Blot
Total protein content from transfected HEK 293T cells was extracted 30 minutes, 2, 4, and 8 hours after the mRNA transfection using RIPA buffer. The Hsp65 polyclonal antibody and the Hsp65 recombinant protein used in this blot were kindly provided by Dr. Célio Lopes Silva. The total protein extract was separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with blocker solution [(3% BSA (w/v) in PBS containing 0.05% Tween-20 (v/v) (PBS-T)] for 2 hours at 37°C, followed by a incubation with mouse polyclonal anti-Hsp65 IgG for 18 hours at 4°C. After washing with PBS-T, the membranes were incubated with goat anti-mouse IgG peroxidase conjugate (Invitrogen). Reactions were revealed by DAB Substrate Kit for peroxidase (Vector, London, UK).
mRNA uptake experiments
mRNA labeling
mRNA encoding full-length Hsp65 gene was labeled with Alexa Fluor 488 by Universal Linkage System (ULS(tm)) using the ULYSIS nucleic acid labeling kit (Molecular Probe Inc, Eugene, USA) with some modifications. Briefly, mRNA (10 μg) was incubated in labeling buffer (25 μL) and denatured at 95°C for 5 min and cooled on ice. The ULS labeling reagent stock solution (2 μL) was added to the tube and the reaction incubated at 80°C for 15 min. The labeled mRNA was purified by ethanol precipitation, followed by suspension in Ringer Lactate solution (RL).
Mice and labeled mRNA immunization
BALB/c mice, 6 to 8 weeks old, were obtained from the Animal Facilities of the Medical School of Ribeirão Preto, University of São Paulo, and were maintained under standard laboratory conditions. The Alexa 488 labeled mRNA (10 μg per mouse in 100 μL volume) formulated in RL was given by intranasal route with 50 μL in each nostril. Control mice were immunized with the same dose of unlabeled mRNA. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of the University.
Preparation of lung cells
Lungs were washed with sterile PBS and each was placed in a Petri dish containing incomplete RPMI-1640 medium (Sigma). Lungs were fragmented and transferred to a conical tube containing digestion solution, prepared with Liberase Blendzyme 2 (Roche, Indianapolis, IN) diluted (0·5 μg/ml) in incomplete RPMI-1640. Samples were incubated at 37° under agitation for 30 min After incubation, the cells were dispersed by using a 10-ml syringe and pelleted by centrifugation for 10 min at 400 g . Cells were then washed with complete RPMI-1640, passed through a Nytex screen (Sigma) and resuspended in complete RPMI-1640. Total cell counts were determined in a Neubauer chamber.
FACS analysis
Single-cell suspensions (1 × 106) isolated from lungs of immunized mice obtained 30 minutes, 2, 4 and 8 hours after the injection of labeled mRNA were suspended in RPMI 1640 medium (Life Technologies, Grand Island, NY). After lysis of red blood cells (RBCs) with ACK buffer, cells were pre-incubated with anti-CD16/32 (FcBlock - 2.4G2) monoclonal antibodies (mAb) to block FcγR, and then incubated with the mAb anti-CD19+ (PE), anti-CD11b+ (PerCP) and anti-CD11c+ (APC) for 30 min at 4°C (all antibodies were purchased from Becton Dickinson, San Diego, USA). Mice immunized with unlabeled mRNA were used as a control. Analytical flow cytometry was carried out using a FACSCANTO II (Becton Dickinson, San Jose, USA) and the data were processed using the FACSDIVA software (Becton Dickinson). A biparametric gate was drawn around the mononuclear populations in the forward (FSC) and side (SSC) scatter dot plot. The gated populations were then selected according to CD19+, CD11b+, or CD11c+ staining. Alexa488 positive population was considered after background analysis from appropriate isotype controls. All antibodies were purchased from BD (BD PharMingen, San Diego, USA). The gating strategy was shown on Additional file 5.
Mice vaccination and challenge with MTB
Animals
Female 6-week-old BALB/c mice were obtained from the Animal Facilities of the Medical School of Ribeirão Preto, University of São Paulo. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of the University. Infected animals were kept in biohazard facility of the Level 3 Biosafety Laboratory and housed in cages within a laminar flow safety enclosure under standard conditions.
Immunization procedures
Immunization was performed by the following treatments, using five animals per group. For BCG immunization, one dose of Moreau strain was given by subcutaneous injection of 105 live bacteria in 100 μL of saline. For mRNA vaccination the mRNA-Hsp65 (10 or 5 μg per mouse in 100 μL volume) was formulated in RL and given by intranasal route with 50 μL drop in each nostril. EF-1α mRNA was given by the same process and formulations. For cytokine evaluation mice were immunized with one dose of mRNA-Hsp65 (10 μg per mouse in 100 μL volume) was formulated in RL and given by intranasal route with 50 μL drop in each nostril, as control we immunize mice only with Ringer solution.
Experimental infection with M. tuberculosis
The H37Rv strain of M. tuberculosis (American Type Culture Collection, Rockville, MD) was grown in 7H9 Middlebrook broth (Difco Laboratories, Detroit, USA) for seven days. The culture was harvested through centrifugation and the cell pellet was resuspended in sterile phosphate buffered saline (PBS) and vigorously agitated. The homogeneous suspension was filtered through 2 μm filters (Millipore, Bedford, MA). Viability of the M. tuberculosis suspension was pre-tested with fluorescent diacetate (Sigma, Saint Louis, MO) and ethidium bromide at least 80% viable. Thirty days after mRNA immunization all mice groups were challenged with M. tuberculosis. Intranasal challenge was made by introducing 1×105 viable CFU of M. tuberculosis H37Rv in 100 μL of PBS by 50 μL drop in each nostril. Control mice received PBS. Mice from all groups were euthanized on day 30 after infection and their lungs were aseptically removed. Lungs from each mouse were used for histopathology analyses and quantification of the bacterial loads.
Determination of M. tuberculosisCFU in lungs
The number of live bacteria was determined by extracting the right lobes of the lung, washed with sterile PBS, followed by plating 10-fold serial dilutions of homogenized tissue on Middlebrook 7H11 agar media (Difco) [supplemented with 0.2% (v/v) glycerol and 10% (v/v) bovine fetal serum], and counting colonies after 28 days at 37°C. The colony-forming units (CFU) are expressed as log10 of CFU/g lung.
Histology
30 days post-infection, the left lobe of each mouse lung was removed and fixed in 10% formalin. Paraffin blocks were prepared and then sectioned for light microscopy. Sections (5 μm each) were stained with hematoxylin & eosin (HE). Slides were evaluated using a Leitz Model Aristoplan microscope (Germany) connected to a Leica Model DFC280 color camera (Heerbrugg, Germany) linked to a PC computer.
Evaluation of cytokine production
Two weeks after mRNA-Hsp65 immunization the animals were sacrificed and splenic cells were collected and adjusted to 5 × 106 cells/ml in RPMI 1640 medium, supplemented with 5% FCS, 20 mM glutamine and 40 IU/l of gentamicin. The cells were cultured in 48-well flat-bottomed culture plates (Nunc, Life Tech. Inc., Maryland, MA, US) in the presence of 20 μg/ml of Concanavalin A (ConA) or Hsp65 recombinant protein. Cytokine levels in culture supernatants were evaluated 48 hours later by ELISA. Cytokines were measured following manufacturer instructions (PharMingen). Purified monoclonal antibodies anti-IFN-γ (R4-6A2) and anti-TNF-alpha (G281-2626) were used at 1 μg/ml as capture antibodies and the following biotinylated antibodies were used for detection: anti-IFN-γ (XMG1.2) and anti-TNF-alpha (MP6-XT3) at 0,5 μg/ml.
In vitro TLR 7 assay
Cell Culture, plasmids and transfection. HEK293T cells were grown in DMEM with 10% fetal bovine serum, supplemented with penicillin and streptomycin. All transfections were performed in 24-well plates. One day before transfection, cells were plated at 3 × 105 cells/well. Cells were transfected with pcDNA3.1 (1 μg/well) as empty vector or the TLRs plasmids (1 μg/well), All TLR vectors was kindly provided by Dr. Aristóbolo Mendes da Silva. The next day cells were left untreated or treated with hsp65 mRNA (2, 5 or 10 μg/ml) or agonists specific for each TLR for 7 h before harvest. The following TLRs agonists were used: poly-IC 100 μg/ml (TLR3), LPS 1 μg/ml (O55:B55) (TLR4) and R-848 1 μg/ml (TLR7 and 8). Nitrite, the end product of NO metabolism, was measured from 50 μl of cell culture supernatants by using Griess reagent, as described elsewhere [44]
Statistical analysis
All values are expressed as mean±SEM. Data were investigated by analysis of variance (anova) using graphpad instat software. When the values indicated the presence of a significant difference, a Tukey-Kramer multiple comparisons test was used. Values of P < 0·05 were considered significant.
