Isolation of phages expressing scFvs specific to TSST-1
To isolate TSST-1-specific phages, a fully human scFv phage library with total diversity of 2×1010 was enriched against the TSST-1 protein as described previously with some modifications [1, 31]. Briefly, a 96-well MaxiSorp plate (Nunc, Roskilde, Denmark) was coated with 100 μl of the TSST-1 protein (2 μg/ml in NaHCO3) (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. Following blocking the plate (200 μl; 15 mg/ml BSA in PBS containing 0.1% tween-20 [PBS-T]), pre-blocked phages (~ 1012 plaque-forming unit/ml) amplified from the scFv phage library were added to the wells, and incubation was done for 90 minutes at room temperature (RT). Following several times washing with PBS-T, bound phages were eluted (output) and amplified (input) for further biopanning rounds. This procedure was repeated for four rounds. Washing steps were increased from round one (10 times) to round four (25 times). The output/input ratio of each round was determined to evaluate enrichment efficiency.
The polyclonal-phage ELISA was carried out to determine which biopanning round contained the phages specific to TSST-1 [31]. In brief, a 96-well MaxiSorp plate was coated with 100 μl of the TSST-1 protein (2 μg/ml) or BSA (2 μg/ml) (Merck, Darmstadt, Germany) overnight at 4°C. The wells were blocked, followed by the incubation with output phages obtained from the first to the fourth round of biopanning (output1-output4) for 60 minutes at RT. After multiple washing steps with PBS-T, horseradish peroxidase (HRP)-conjugated mouse anti-M13 antibody (1:2000 dilution in blocking buffer) (Santa Cruz Biotechnology, INC.) was added to the wells, followed by the incubation for 60 minutes at RT. Next, the wells were washed several times with PBS-T, and the signals were generated by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) (Thermo Scientific, MA, US). The reactions were stopped with sulfuric acid (1 M) (Merck), and the absorbance at 450 nm was measured using a microplate reader (Epoch, BioTek, USA).
Based on the data obtained from the polyclonal-phage ELISA, output phages of the third and fourth rounds of biopanning (output3 and output4), showing the highest signal intensity compared to the control, were further assessed by the monoclonal-phage ELISA [31]. Briefly, E. coli TG1 bacteria were infected with output phages (output3 and output4) and cultured on lysogeny broth (LB) agar (Merck) plates with 150 µg/ml ampicillin (Sigma-Aldrich). After incubation overnight at 37°C, the colonies were picked up randomly, and phage amplification was done as described previously [1]. The binding ability of phages to the TSST-1 protein was investigated by ELISA, as mentioned above in the polyclonal-phage ELISA.
Expression
Five phage clones, MS457, MS460, MS465, MS473, and MS475, which showed the highest binding to the TSST-1 protein compared to the control in the monoclonal-phage ELISA, were selected for more evaluations. To produce soluble scFv antibodies, the non-suppressor E. coli strain, HB2151, was infected with the phages obtained from five phage clones and cultured on LB agar plates containing ampicillin, followed by the incubation overnight at 37°C [1, 32]. The single colonies were picked up and cultured in terrific broth containing 100 µg/ml ampicillin. After adding IPTG (0.1 mM) (Thermo Scientific) and incubating overnight at 24°C, the cultures were centrifuged, and the pellets were incubated with the lysis buffer for 60 minutes at RT [32, 33]. Next, the existing level of scFvs in the periplasmic fraction of E. coli HB2151 bacteria, carrying the phagemids (MS457, MS460, MS465, MS473, or MS475), was investigated by a 12% SDS-PAGE gel, followed by western blot analysis. After electrophoresis, the proteins were transferred from the SDS-PAGE gel (12%) onto polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Little Chalfont, UK). The blocked membrane was incubated with mouse anti-human scFv polyclonal antibody (1:200 dilution) for 60 minutes at RT [1]. After several washing steps with tris-buffered saline (TBS) with 0.05% tween-20 (TBS-T) and TBS, the membrane was incubated with goat anti-mouse IgG-HRP-conjugated antibody (1:2000 dilution) (Santa Cruz) for 60 minutes at RT, followed by several washing steps and addition of 3,3′-diaminobenzidine substrate (DAB) (Sigma-Aldrich) and hydrogen peroxide (H2O2) (Merck).
Sequencing
The phagemid DNA of clones MS457, MS460, MS465, MS473, and MS475, was purified using the High Pure Plasmid Isolation Kit (Roche, Mannheim, Germany), based on the manufacturer's recommendation. For sequencing, the forward primer, 5'- CTA TGA CCA TGA TTA CGA ATT TCT A -3', was used. The nucleotide sequences of five scFvs were appraised using the Gene Runner program (version 6.0). Furthermore, the amino acid sequences of V-regions of MS473 was analyzed by the IMGT/V-QUEST tool (http://www.imgt.org/IMGT_vquest/analysis) [1].
Assessment of the binding ability of the purified scFv to TSST-1
The soluble scFv, MS473, was purified using a Ni-NTA column (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The bound proteins were eluted with 200 mM imidazole (Merck). Next, all the eluted fractions were pooled and then placed in a dialysis bag (cut off 14 kDa, Sigma-Aldrich), according to the manufacturer's instructions. The concentration of purified and dialyzed scFv (MS473) was measured via the Bradford assay. Moreover, the purity of the scFv was analyzed by a 12% SDS-PAGE gel.
The binding ability of purified scFv to TSST-1 was determined by ELISA as described previously with some modifications [31]. Briefly, a 96-well MaxiSorp plate was coated with 100 μl of the TSST-1 protein (2 μg/ml) or BSA (2 μg/ml) (as the control). Next, the wells were blocked and then incubated with MS473 or SP220 (an scFv against staphylococcal α-hemolysin) (400 μg/ml) for 60 minutes at RT. After several washing steps, mouse anti-human scFv polyclonal antibody was added to the wells, and incubation was done for 60 minutes at RT. The wells were washed several times with PBS-T and PBS, and goat anti-mouse IgG-HRP-conjugated antibody was added to the wells, followed by incubation for 60 minutes at RT. Moreover, the TSST-1-coated wells incubated with mouse anti-staphylococcal TSST-1 mAb (1/1000 dilution) (Santa Cruz), followed by goat anti-mouse IgG-HRP-conjugated antibody were used as the positive control. After multiple washing steps, the TMB substrate solution was added to the wells, and the color reactions were stopped with sulfuric acid. A microplate reader determined the absorbance at 450 nm.
Affinity determination
The binding affinity of MS473 to TSST-1 was determined as described previously [19, 31, 34]. In brief, a 96-well MaxiSorp plate was coated with 100 μl of the TSST-1 protein (1 and 2 μg/ml). After blocking, the wells were incubated with serial dilutions of the MS473 scFv (0.02-500 μg/ml) for 60 minutes at RT. After several washing steps, mouse anti-human scFv polyclonal antibody was added to the wells, and incubation was done for 60 minutes at RT. Next, the wells were washed multiple times, followed by the incubation with goat anti-mouse IgG-HRP-conjugated antibody for 60 minutes at RT. After adding the TMB substrate solution, the color development was stopped with sulfuric acid. The absorbance was read at 450 nm. The Kaff of MS473 to the TSST-1 protein was measured using the following formula:
Ag / Ag' = n
Kaff= n – 1 / 2 (n [scFv'] – [scFv])
Where Ag and Ag' are the TSST-1 protein at concentrations of 2 and 1 μg/ml, respectively; the scFv and scFv' are the concentration of MS473 at OD-50 and OD-50' for the wells coated with the TSST-1 protein at concentrations of 2 and 1 μg/ml, respectively.
Specificity
The binding specificity of MS473 to TSST-1 was assayed by ELISA as described previously [31]. In brief, a 96-well MaxiSorp plate was coated with 100 μl of the TSST-1 protein (2 μg/ml), the adiponectin protein (2 μg/ml) (R&D Systems, Minnesota, US), the α-hemolysin protein (2 μg/ml) (Merck, Calbiochem, Germany), BSA (2 μg/ml), and skimmed milk powder (1 mg/ml). After blocking, the wells were individually incubated with MS473. The wells were washed several times, and mouse anti-human scFv polyclonal antibody was added to the wells, followed by goat anti-mouse IgG-HRP-conjugated antibody. Next, the TMB substrate solution was added. After stopping the reaction with sulfuric acid, the absorbance at 450 nm was read using a microplate reader.
Assessment of the inhibition ability of MS473 on the TSST-1-induced mitogenesis and cytokine release in human PBMCs
The neutralizing activity of MS473 against the superantigenic activity of TSST-1 was examined on human PBMCs isolated from the whole blood of two healthy donors (men, 50 and 45 years) as described previously with some modifications [8, 12, 13, 29, 35, 36]. To determine the effective proliferative dose of TSST-1, fresh human PBMCs (~ 106 cells/ml) in RPMI 1640 medium (Gibco; Grand Island, NY, USA) with L-glutamine (2 mM) (Gibco) supplemented with heat-inactivated fetal bovine serum (10%) (Gibco) were seeded in 96-well round-bottom tissue culture plates (JET BIOFIL, Guangzhou, China) and were incubated with the TSST-1 protein at concentrations of 1, 10, 25, 50, and 100 ng/ml for 24 hours at 37°C, 5% CO2. The proliferation of PBMCs and the formation of cell clumps induced by TSST-1 were investigated using an inverted microscope [23]. Next, the cells were incubated concurrently with the TSST-1 protein (50 ng/ml) and MS473 (80 μg/ml) for 24 hours at 37°C, 5% CO2. The cells incubated with PBS or the TSST-1 protein (50 ng/ml) and PBS were used as the controls. The inhibitory effect of MS473 on the proliferation of PBMCs stimulated with TSST-1 was assessed by an inverted microscope. Moreover, the culture supernatant of PBMCs treated simultaneously with the TSST-1 protein (50 ng/ml) and MS473 (80 μg/ml) were collected after 24 hours incubation at 37°C, 5% CO2, followed by the centrifugation at 1000 g for 5 min. The cells treated with PBS or the TSST-1 protein (50 ng/ml) and PBS served as the controls. The concentrations of human IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17A, IFN-γ, TNF-α, G-CSF, and TGF-β in the culture supernatant of PBMCs (stimulated and unstimulated) were calculated using the standard curve provided in a Multi-Analyte ELISArray Kit (Qiagen), according to the manufacturer's instructions. Experimental procedures with human blood were approved by the Ethics Committee of the Pasteur Institute of Iran and were done in accordance with the Helsinki Declaration. The participant provided written informed consent before enrollment.
Statistical analyses
The one-way analysis of variance (ANOVA) and Student’s t-test were carried out in GraphPad Prism version v.6.0.7. The P-value < 0.05 was considered significant.