Study subject, blood sample collection and processing
The HIV-1 seropositive patient (# 254) enrolled for the construction of the anti-V3 scFv phage library was recruited after obtaining written informed consent from the Department of Medicine, AIIMS, New Delhi. The study was approved by the institute ethics committee. Whole blood was collected in EDTA vaccutainers. The plasma was separated from whole blood, aliquoted and stored at −70°C until tested. The peripheral blood mononuclear cells (PBMCs) were separated by phycoll hypaque centrifugation and processed immediately for EBV transformation.
The plasma sample of the patient # 254 was previously tested and found to exhibit good neutralization potential against a diverse panel of viruses (Table
1). It was also tested for the presence of binding anti-V3 antibodies and the data is shown in Figure
Screening of the patient plasma (#254) for cross reactive neutralizing antibodies
The neutralization efficiency of the plasma #254was tested against a standard panel of pseudoviruses of clades A, B and C obtained from the NIH AIDS Research and Reference Reagent Program, by TZM-bl assay
. The standard panel of pseudoviruses has been categorized from tier 1 to tier 3, based on the decreasing order of susceptibility to neutralization by the known monoclonal antibodies
. The neutralization assay was carried out in 96-well tissue culture plates. Briefly, 50 μl of the heat inactivated plasma/purified scFv, at different dilutions in duplicate, was added to 200 TCID 50 of the virus and incubated for 1 h. A cell control well containing culture media only and a virus control well containing both the culture media and the virus were tested in parallel. The rest of the procedure is the same as described for calculating TCID 50. The scFv (HepB), an scFv against the Hepatitis B surface antigen
 and 1418, a human antibody to parvovirus B19 protein, were used as negative controls. For all dilutions of test plasma, the percent neutralization was calculated based on the relative luminescence units (RLU) in the presence of plasma divided by the virus control. The cell control value was subtracted from the plasma RLU value as the background cutoff. The 50% neutralization titer (ID50 titer) was determined for the plasma sample #254 against each virus by plotting percentage neutralization against the dilution of the plasma tested. A non-linear regression straight line was drawn by the method of least squares, and the reciprocal ID50 titers were extrapolated. The experiments were performed in duplicate and repeated at least twice and the mean ID50 titers were calculated.
Quantitation of the levels of anti-V3 Abs by Peptide ELISA
The anti-V3 Antibody content in the HIV-1 seropositive plasma sample #254 and in the supernatants of the EBV transformed PBMCs in culture at different stages was determined using V3 peptide ELISA. Thirty five mer peptides of V3C (CTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHC) and V3B (CTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHC) were synthesised (Sigma Aldrich, USA), based on the consensus V3 sequences. The V3 peptides (1 μg/ml) were coated onto 96 well Nunc-Immuno plates (Nunc: Cat# 439454) using antigen coating buffer (150 mM Na2CO3, 350 mM NaHCO3, 30 mM NaN3, pH 9.6) at 4°C overnight. Plates were washed using phosphate buffered saline with 0.1% Tween-20 (0.1% PBST) thrice using a plate washer. Plates were then blocked with 100 μl of 15% fetal calf serum and incubated at 37°C for 1.5 h. Following blocking and washing, heat inactivated plasma (100 μl, dilution range=300-100000) or 100 μl of supernatants from EBV transformed PBMC cultures was added to each well and incubated for 1hour at 37°C. After 3 washings with PBST (0.1%), the bound V3 specific antibodies were detected by addition of 100 μl of alkaline-phosphatase conjugated anti-human IgG Fc (1:2000 in PBST). Immune complexes were revealed with AP-Substrate in DAE buffer and the colorimetric reaction was stopped by the addition of 6N NaOH. The optical density was read at 405 nm. ID50 titers were calculated for the plasma sample against each of the peptide by plotting the absorbance at 405 nm against the dilutions of the plasma sample tested. A non-linear regression straight line was drawn by the method of Least squares and the ID50 titers were extrapolated.
Epstein –Barr Virus (EBV) induced transformation of PBMCs
B95 cell line comprises of EBV transformed human lymphoblastoid cells which secrete EBV in the supernatant. EBV transforms human B cells
. We obtained the B95 cell line from American Type Cell Culture (Cat. No. VR-1492). B95 cells were grown in T-75 tissue culture flasks, at 100,000 cells/ml at 37°C, 5% CO2, in complete RPMI media. After 10 days, the viral supernatant was centrifuged at 300 × g and filtered with 0.45 μm filters. Peripheral blood mononuclear cells (PBMC) (100,000 cells/well) from the patient #254 were EBV transformed by mixing with 100 μl of the viral supernatant in a 96-well plate and cultured with a polyclonal B cell activator, CpG (2 μg/ml), which enhanced EBV infection and B cell transformation
 and CsA (0.5 μg/ml). The plate was incubated at 37°C in a 5% CO2 incubator overnight. Next day, cells were fed with 100 μl of complete medium containing CsA and CpG. Cultures were fed twice per week and half of the culture supernatant was replaced with fresh complete media, 200 μl/well (no CsA and CpG).
Screening of B-lymphocytes producing anti-V3 antibodies
After two weeks of culturing the B cells in the 96 well plate, we screened for the presence of V3 antibodies by V3 peptide binding ELISA as described above. The positive B cell clones were further expanded to 24 well plates and screened in the 3rd and 4th week; V3 positive clones were transferred to a six well plate, pooled and further expanded to the T-25 flask (Table
Construction of Human anti-V3 scFv phage library
Total RNA from the V3 specific antibody producing B cells (from flask stage) was isolated by Trizol reagent (Sigma, USA) and then reverse transcribed to cDNA, using the reverse aid MMuLV reverse transcriptase (Fermentas,USA). A total of 200 ng of RNA was reverse transcribed in a reaction volume of 50 μl containing, 10ng of random hexamer, 20μM oligo-dT, 1.5 μl of RNase inhibitor (40U/μl), all dissolved in 1X RT buffer. The RNA was heated to 65°C for 5 min and then immediately chilled on ice for at least five minutes. Following the addition of the reaction mixture, the tube was incubated at 42°C for 60 min, at 70°C for 5 min and quickly chilled on ice and stored in −20°C. Heavy chain variable region genes were amplified using a total of 24 combinations (6 forward primers and 4 reverse primers representing all human immunoglobulin subfamilies) and for light chain kappa, a total of 30 combinations were used (6 forward primers and 5 reverse primers) as described previously
. A total of 54 independent reactions were performed to generate the variable regions of heavy and light chains. The heavy chain 5′ primers included a SfiI site and the light chain 3′primer included a NotI site. Light chain 5′ primer included part of the linker region (Gly4Ser)3 and this was compatible with the heavy chain 3′ primer. Each variable heavy region was amplified using Hot start Taq DNA polymerase (Fermentas) in a PCR reaction of 50 μl containing 2.5 μl cDNA, primers 1 μl (10 pmole each) both forward and reverse. PCR reaction was performed for 34 cycles (94°C for 3 min initial denaturation, 94°C for 1 min, annealing at 63°C for 1 min, extension at 72°C for 2 min) using eppendorf Master Cycler. Light chain variable region were amplified with the similar protocol except the annealing temperature used was 57°C. Each variable region gene was purified from the agarose gel using gel extraction kit (Qiagen, Germany). An equimolar mixture of pooled heavy and light chain DNA was used in the second round assembly PCR. The assembly PCR reaction was cycled 20 times (94°C for 1 min, 94°C for 45 sec, 62°C for 50 sec, 72°C for 2 min) the assembly reaction was performed using Pfu DNA polymerase and without primers. Full length scFvs were amplified using a pull through PCR reaction using Taq DNA polymerase and the following primers PTfw 5′ CCT TTC TAT GCG GCC CAG CCG GCC ATG GCC 3′ and PTrv 5′ CAG TCA TTC TCG ACT TGC GGC CGC ACG 3′ (94°C for 1 min, annealing at 62°C for 1min, extension at 72°C for 1 min and final extension at 72°C for 5 min). The scFvs were agarose gel purified using gel extraction kit (Qiagen, Germany).
Cloning of anti-V3 scFv into pCANTAB -5E vector
The scFv DNA fragments and pCANTAB-5E vector were digested with NotI /SfiI (New England Biolabs, USA) respectively. The digested scFv DNA fragments and pCANTAB-5E vector were gel purified using gel extraction kit (Qiagen, Germany). The scFv DNA was ligated into vector at a 3:1 molar ratio using T4 DNA ligase (New England Biolabs, USA). The ligated DNA was transformed into chemically competent cells of E.coli TG1 and placed on ice for 1h followed by heat shock treatment for 90 seconds and chilled on ice for 5 min. Next, 800 μl of 2XYT media was added and incubated in a rotating shaker at 200 rpm for 40 min. The transformed cells were plated on to 2XYT medium agar plates containing ampicillin (50 mg/ml) and 2% glucose, and incubated overnight at 37°C. The following day, colonies were scraped into 1ml of 2XYT medium with 20% glycerol and stored at −70°C. Sequence of the assembled scFv was confirmed by an automated ABI prism sequencer using gene specific primers.
Panning of the scFv phage library
The phage were rescued by infection with helper phage (M13-KO7), followed by precipitation with PEG/NaCl, resuspension in PBS and titration for the determination of phage concentration. The phage were then subjected to a single round of enrichment by bio-panning.
The panning procedure was carried out in Immuno 96 microwell plates. Plates were coated with 100 μl of V3 peptide 1 μg/ml in 0.1 M NaHCO3 (pH 8.6) overnight at 4°C. A phage library of 1012 phage was incubated for 1h in milk coated wells to remove the non-specific binders. After one hour, unbound phage were transferred to the peptide coated wells for 30 min at RT. The unbound phage was eliminated by washing 10–15 times with PBS containing 0.1% Tween 20. The bound phage was eluted with 0.2 M glycine pH 2.2 for 10 min at RT. The eluted phage were neutralized with 1M Tris HCl pH 9.2 and immediately infected onto TG1 (OD 0 .4 to .5) for 30 min at 37°C and for 30 min with shaking at 37°C. Cells were spun down and plated on 2XTY agar containing ampicillin (50 mg/ml) and 2% glucose. Individual colonies were picked and grown in 96 well sterile culture plates (Corning) and a glycerol stock was made and stored at −70°C.
Phage Rescue (24 well plate)
Individual colonies were grown in 1ml 2XYT broth containing ampicillin and 2% glucose overnight with shaking at 37°C at 160 rpm. A small inoculum was transferred to 1ml 2XYT broth containing ampicillin (50 mg/ml) and 2% glucose at 37°C with shaking at 200 rpm till the OD reached 0.4 to 0.5. Helper phage were added and the plate was incubated at 37°C without shaking and then for 30 min with shaking at 180 rpm at 37°C. The cells were spun down at 1500 × g, the supernatant was discarded and pellet was washed with 2XYT broth. The pellet was then resuspended in 2XYT broth containing ampicillin (50 mg/ml) and kanamycin (100 mg/ml) (no glucose) with shaking at160 rpm at 30°C for 16–18 h. It was then centrifuged at 6000 × g and the supernatant was collected and stored at 4°C and an aliquot was tested in the phage ELISA.
The ELISA plates were coated with 100 μl of V3 peptide (1 μg/ml) in 0.1 M NaHCO3 (pH 8.6) and incubated overnight at 4°C. The plates were washed once with 1X PBS and blocked with 4% non-fat milk (Titan Biotech, India) for 2 h at 37°C. The plates were then washed three times with 1X PBS. Phage supernatant (100 μl) was added to each well and incubated for 1h at RT. Phage supernatant was discarded and the plates were washed four times with PBST(0.1%). 100 μl of anti M13 antibody (diluted 1:2000) was added (Sigma) and incubated for 1h at RT. The plates were washed four times with PBST (0.1%). 100 μl of anti rabbit HRP (Jackson) diluted 1:3000 were added and incubated at RT for 1h. The plates were then washed four times with PBST (0.1%). 100 μl of TMB substrate was added, and the reaction was stopped by adding 8N H2SO4. Absorbance was measured at 450nm.
PEG precipitation of phage (pure phage preparation)
Individual bacterial colonies were grown in 5ml 2XYT medium containing ampicillin (50 mg/ml) and 2% glucose with shaking at 200 rpm at 37°C till the O.D reached 0.4 to 0.5. Next, 1 μl of KO7 helper phage (1018) was added and incubated at 37°C for 30 min without shaking, followed by 30 min shaking at 200 rpm. The cells were centrifuged at 2500 × g for 10 min, supernatant was discarded and the pellet was washed again with 2XYT broth. The pellet was resuspended in 50ml 2XYT broth containing Ampicillin (50 mg/ml) and Kanamycin (100 mg/ml) (no glucose) at 30°C with shaking at 160 rpm for 12–16 h. It was then centrifuged at 10000 × g for 20 min at 4°C. The supernatant was transferred to glass bottles and Â¼ volume of PEG/NaCl was added and kept on ice for 4 to 6 h followed by centrifugation at 20000 × g for 20 min at 4°C. The supernatant was discarded and the pellet was dissolved in sterile and autoclaved 1X PBS and stored at 4°C. The transformation unit (TU) was calculated.
DNA sequencing and sequence analysis
Twenty nine scFvs clones were randomly selected from the unselected library and sequenced by Macrogen (South Korea). The sequences were analysed using immunoglobulin BLAST
 and V BASE software
DNA fingerprinting of antibody fragments
The diversity of the scFv repertoire was analysed by comparing the restriction digestion pattern of scFvs. Ten clones were randomly selected from the primary phage library and the plasmid was isolated. The scFv sequences were amplified using primers PTfw 5′ CCT TTC TAT GCG GCC CAG CCG GCC ATG GCC 3′ and PTrv 5′ CAG TCA TTC TCG ACT TGC GGC CGC ACG 3′ (94°C for 1 min, annealing at 62°C for 1min, extension at 72°C for 1 min and final extension at 72°C for 5 min). The amplified PCR products were digested with a frequent cutter restriction enzyme BstN1 (NEB) and analysed on 2% agarose gel.
Soluble scFv expression
Clones showing positivity/binding in phage ELISA were selected for soluble scFv expression. These were than transformed into HB2151 using calcium chloride mediated transformation for soluble scFv expression. The HB2151 cells carrying pCANTA-5E plasmid were grown in 10 ml 2XTY medium overnight at 37°C with shaking at 200 rpm. The next day, 1/100th volume of the overnight culture was inoculated in 1 litre of 2XTY medium and grown at 37°C with shaking at 240 rpm till the OD reached 0.6 and then the culture was induced by 1 mM IPTG for 6 to 8 h at 24°C
. Cells were harvested and different fractions were prepared. scFvs were purified from the periplasmic fraction. Briefly, the cells were harvested by centrifugation at 4000 × g for 15 min at 4°C. The supernatant was discarded and pellet was resuspended in 30 mM Tris-Cl. 20% sucrose, pH 8.0 at 80 ml/ gram wet weight. The cells were placed on ice for 20 min and 500 mM EDTA was added to a final concentration of 1 mM EDTA. The cells were spun down at 8000 × g for 15 min at 4°C. The pellet was resuspended in 5 mM MgSO4 and the cells were placed on ice for 10 min with slowly stirring, pelleted down at 8000 × g for 15 min at 4°C and the supernatant collected for purification.
Purification of E-tagged scFv
Purification of E-tagged scFv was carried out using the Recombinant Phage Antibody System (RPAS) Purification Module (Amersham Biosciences) as per the manufacturer’s instructions. The periplasmic extract was filtered through a 0.45 μm filter to remove any remaining cell debris. The Anti-E Tag column was regenerated by washing the column with 15ml of elution buffer (0.1 M glycine, pH 3.0) and was then equilibrated with 25 ml binding buffer (0.02 M phosphate buffer, 0.005% NaN3, pH 7.0). The E-tagged scFv in the periplasmic extract was then allowed to bind to the column by passing the extract through the column. The unbound excess E. coli proteins were removed from the column by washing it with 25 ml binding Buffer. The flow rate at each step was maintained at 5 ml/min through the column. Finally, the bound scFv was eluted by 15ml elution buffer. Several fractions of the eluted scFv (900 μl each) were collected in tubes containing 100 μl neutralization buffer (1 M Tris, 0.05% NaN3, pH 8.2). The amount of protein in each fraction was estimated using BCA method and the fractions containing considerable amount of scFv were pooled together and concentrated.
Soluble ELISA was performed, as described in the phage ELISA. Hundred microliter of soluble scFv periplasmic extract/purified scFv was added and incubated at room temperature for 1h. ELISA plates were washed three times with 0.1% PBST and incubated with 1:1000 dilution of primary antibody in 2% MPBS. The plates were washed three times with 0.1% PBST and incubated with 1:2000 diluted anti rabbit HRP conjugated secondary antibody in 2% MPBS. The ELISA plates were washed again as described above. 100 μl of TMP substrate was added and incubated at RT till the colour developed. Reaction was stopped by adding 8NH2SO4. Absorbance was read at 450 nm.
SDS-PAGE and Western blot
SDS-PAGE was done as described
. Proteins were separated on a 12% running gel and 5% stacking gel and visualized by Coomassie Brilliant Blue (CBB) staining. For Western blotting, gel was blotted onto nitrocellulose membrane using electroblotting, (100 V for 1h) and probed with primary antibody. Anti-rabbit HRP was used as secondary antibody and colour was developed with DAB as the substrate.