Cell lines and viruses
Vero cells were obtained from the ATCC and cultured in DMEM supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin and streptomycin at 5% CO2, 37℃. Chikungunya/human/China/GD134/2010 (GenBank Accession: HQ846359) was isolated from serum of an infected female patient at Guangdong Provincial Center for Disease Control and Prevention. Zika/human/China/GD01/2016 (GenBank Accession: KU740184) was isolated from Guangdong inbound passengers. All experiments involving the CHIKV and ZIKA authentic virus were conducted in Biosafety Level 3 and 2 laboratories, respectively. Virus virions were inactived using β-propiolactone (Sigma-Aldrich) when its concentration was 1:2000 at 4℃ for 24 hours. Complete inactivation of virus was confirmed by the lack of replication in a Vero infection experiment. Supernatants containing virus particles concentrated using PEG-it (SBI Biosciences) overnight at 4 °C and resuspended in PBS for further use.
Nanobodies screening from naïve phage display library
A VHH phage display library (AlpaLife) constructed in the vector pADL-10b and containing ~2×109cfu was inoculated into 2×YT supplemented with 100μg/mL ampicillin (2×YTA) and infected with M13KO7 helper phage to obtain a library of VHH-presenting phages. Biopannning of phages displaying VHHs specific for the Chikungunya/SL-CK1 E2 (Sino Biological) were performed as described previously [24-25]. After the three rounds of panning, 384 individual clones were picked to inoculate 2×YTA and were grown overnight at 37 °C, supernatant of clonal phage were detected by phage-ELISA with HRP-conjugated goat anti-M13 IgG antibody (Sino Biological). OD450 values ≥0.5 and P (positive OD450)/N (negative OD450) greater than 3 P/N ratios was determined as positive clones. Positive candidates were sequenced (Sangon Biotech) and aligned with complementary determining regions (CDRs) amino acid sequence. Five percent BSA was used as a negative control for each round.
Expression and purification of VHHs in Escherichia coli (E.coli)
For monovalent nanobodies, sequences were synthesized (Generay Biotech), and subcloned into pET-SUMO with a tandem N-terminal His-tag, SUMO-tag, and plasmids were subsequently transformed into E. coli BL21(DE3) cells. All protein expression overnight at 18 °C was induced at OD600 of 0.6 by addition of 0.5 mM IPTG. The His-tagged protein was purified by Talon Metal Affinity Resin (Clontech) according to the manufacturer. The eluates were concentrated using a 1.5 mL Microsep advance centrifugal devices (PALL) with a molecular weight cut-off of 3 kDa. Purity quality was analyzed by Coomassie-stained SDS-PAGE.
Expression and purification of Fc conjugated multivalent nanobodies in HEK293 cells
Bivalent and trivalent VHHs were fused to the Fc region of human IgG1 and cloned into the pcDNA 3.4. Multivalent nanobody units were connected through (GGGGS)×3 flexible linkers. The Fc-fusion constructs were expressed in HEK 293 cells at 8% CO2 37°C for 1 week. Nanobodies in the supernatant were purified using protein A. Purity of all samples was analyzed by SEC-HPLC.
Indirect ELISA to quantitate initial binding
Microtiter plates (Corning) were coated with 2µg/mL Chikungunya E2, 10µg/mL purified virus virions, or the control protein SUMO in carbonate buffer (CBS, pH 9.6) overnight at 4 °C, and blocked with 3% BSA(Sigma-Aldrich) PBS pH 7.4 at 37 ℃. Serial 10-fold dilutions of SUMO-tagged VHHs in 3% BSA were incubated with the immobilized antigen, followed by incubation with HRP-conjugated goat anti-SMT3 (1:2000, CUSABIO). After wash, 100 μL of TMB substrate (TIANGEN) was added the wells and reactions were stopped with 100μL of 1M HCl. Absorbance was measured at 450 nm on a EpochTM microplate reader (BioTek Instruments Inc., Winooski, VT, USA). EDIII protein of ZIKA was used as the negative control.
Nanobodies were validated by western blotting
Purified Chikungunya E2 protein (20μg) and purified Chikungunya virion (50μg) were loaded onto 12% SDS-PAGE and electro-transferred to a PVDF membrane (Millipore). Following blocking of membranes with 5% BSA in 0.05% TBST and incubated overnight with 1:500 dilution of nanobodies. Membranes were probed to a SMT3-HRP conjugated antibody (1:1000, CUSABIO) and revelation with SuperSignal West Dura ECL reagent (Thermo Fisher Scientific). Chemiluminescence images were generated with FluorChem E scanner system (ProteinSimpleSan, Jose, CA, USA).
Localized Surface Plasmon Resonance (LSPR) assay
LSPR measurements were performed using a OpenSPRTM instrument (Nicoyalife) to determine the affinity of monovalent nanobodies to E2 protein. The COOH chip (Nicoyalife, Canada) was loaded onto the OpenSPRTM instrument following the standard OpenSPRTM procedure. Run with PBS (pH 7.4) at maximum flow rate (150 µL/min) to reach signal baseline. Sample 200 µL of isopropanol and run for 10 s to evacuate air. After baseline is reached, the PBS buffer is rinsed through the sample loop and evacuated with air. Slow down the flow rate of PBS (pH7.4) to 20 µL/min, and then load 200 µL of EDC/NHS (1:1) solution to activate the COOH sensor chip. Dilute 200 µL of ligand E2 protein (0.4 mg/mL) for 4 min and rinse the sample loop with PBS (pH7.4). Sample with 200 µL blocking solution, the sample loop is rinsed with PBS and evacuated with air. Baseline was observed for 5 min to ensure stability. Selected nanobodies were diluted into a series of different concentrations and sampled at 20 µL/min. Both nanobodies and ligand binding times were 240 s and natural dissociation was 360 s. Kinetic parameters for the binding reactions were calculated using Trace Drawer software (Ridgeview Instruments AB), One to One analytical model.
ELISA-based Mxra8-Fc binding assay
A fragment of cDNA encoding the Mouse-Mxra8 extracellular domain (residues 23-336, GenBank accession number NM_024263.4) or Human-Mxra8 extracellular domain (residues 24-337, GenBank accession number NM_032348.3) was appended with a TEV enzyme site and a human IgG1 Fc at the C-terminus as well as the IL-2 signal peptide at the N-terminus in pcDNA 3.4 expression vectors and transiently transfected into HEK293 cells followed by media collection and purification using protein A sepharose. Mxra8-Fc binding assay was adapted from previously described . MaxiSorp ELISA plates (Corning) were coated with 2µg/mL anti-mouse CHKV E2 monoclonal antibodies in CBS (pH 9.6) overnight at 4 °C. Washed four times with PBS and blocked with 4% BSA(Sigma-Aldrich) for 1h at room temperature (RT). CHIKV virions (1µg/ml) were diluted and added for 1 h at RT. After washing, MoNb-2E8 and MoNb-3C5 or Mouse Mxra8-Fc fusion protein (all at 10 µg/ml) were incubated for 30min. Plates were washed and Human Mxra8-Fc (10-fold serial dilutions) were added to the plates and incubated for 1h at RT. Plates were washed again and incubated with secondary Rabbit Anti-Human IgG-Fc (1:5000, Bioss). After washing, the plates were developed with TMB substrate (TIANGEN) and 2N H2SO4. Absorbance was measured at 450 nm.
Indirect immunofluorescence assay
An indirect immunofluorescence assay was developed on CHIKV-infected Vero cells, as previously described . Briefly, infected-cells (MOI, 1) were cultured for 72h and then fixed in 4% paraformaldehyde (Biosharp) for 20min. PFA-fixed cells were permeabilized and blocked with 1% Triton X-100(MP)/1% BSA(Sigma-Aldrich)/PBS for 1h at room temperature. The cells were incubated with nanobodies (10μg/mL) at 37°C for 30 min, followed by Alexa Fluor 488 Anti-6×His tag antibody (Abcam) or Cy3-labeled Goat Anti-Rabbit IgG (H+L) (Beyotime) for 30 min at 37°C. After washing with PBS, cells were observed under a fluorescence microscope (Mshot). Anti-CHIKV rabbit serum was obtained from a rabbit immunized with β-propiolactone CHIKV GD134 virion and used as a positive control. Anti-CHIKV E1 mouse IgG antibody (R&D Systems) was used as a control antibody.
Flow cytometry assay
The binding of nanobodies to virus on cell surface was assessed by flow cytometry assay. Monolayers of Vero cells were infected with CHIKV and ZIKA at an MOI of 0.1 and harvested at 48h post infection. Cells were then fixed with 4% paraformaldehyde, and permeabilized with permeabilization buffer (0.05% Triton-X in PBS). 10μg/ml nanobodies were added into cells and incubated for 60 min on ice. Mouse anti-E2 monoclonal antibody was used as a positive control. After washing twice with PBS, cells were stained with 1:1000 diluted goat anti-human H&L (FITC) or FITC Rabbit polyclonal to 6×His tag (Abcam) for 45 min and analyzed using flow cytometry (Beckman CytoFlex, Brea, CA, USA).
Human sera samples were detected in a sandwich ELISA
HRP-coupled bivalent VHHs performed using HRP Conjugation Kit (Abcam) following manufacturer instructions. 2 μg/mL of BiNb-2E8 in CBS (pH9.6) was added per well of a coated 96-Well Plate at 4℃ overninght. After washing with PBST and blocking with 5% BSA in PBST for 1 h, 100 μL of E2 protein diluted in a 2-fold dilution series (starting dilution 5 in 10240) or human sera samples (dilution 1:10) were added and incubated at room temperature for 1h. Wells were washed 3×5 min, and 100 μL of HRP labeled BiNb-3C5 (1 μg/mL) was added to each. After 1 h of incubation, plates were washed 5 times with PBST. TMB was added to the wells for colorimetric development and the absorbance was read at 450 nm. Human Mxra8-hFc served as the control. Samples were considered seropositive if OD450 values higher than the mean obtained for the negative samples plus 3 standard deviations. The CHIKV positive sera samples were sourced from Center for Disease Control and Prevention of Southern Theater Command. Twenty CHIKV negative sera from healthy subjects were used for the calculation and validation of the cut-off value and excluded the sample matrix.
Epitope-binding using peptide-based ELISA
A pool of 10-mer peptides with 5 amino acid overlap spanning the Chikungunya/SL-CK1 E2 glycoprotein were generated by chemistry to a purity of 90% (GL Biochem). All peptides were provided as lyophilised power, reconstituted in DMSO to a concentration of 1mg/mL, and stored at −80 °C. Each peptide was coated at 1ug/mL in CBS (pH 9.6) overnight at 4 °C and then blocked with 5% BSA(Sigma-Aldrich) in PBST. An irrelevant SARS-CoV-2 peptide was used as the negative control. Binding of the coated peptide was characterized by incubation with 2µg/mL nanobodies. HRP Mouse monoclonal to 6× His tag (Abcam) used at 1:5000 dilution in blocking buffer, and further absorbance measurement of the enzymatic reaction in TMB substrate (TIANGEN), were used to detect the bound epitopes.
Prediction of the conformation of nanobodies complex with CHIKV E2 protein
The homology model of protein structure was built on the DeepMind algorithm AlphaFold system (https://deepmind.com/). The complex model of Nb-2E8 or Nb-3C5 to CHIKV E2 protein were genetated by pyDOCK (https://life.bsc.es/pid/pydockweb). All structural data of the docking models were visualized using PyMOL software (https://pymol.org/2/).
All experiments were performed at least two times in duplicate, and representative data or pooled data from repeat experiments were recorded. Statistical analyses were carried out using GraphPad Prism Software (San Diego, CA, USA). Data were presented as the mean ± SD. Students t-test was used for two groups comparison. Two-sided P-values<0.05 were considered statistically significant.