Cells and viruses
All the virus strains used in this work were propagated and titrated in Vero cells (ATCC CCL-81) using standard plaque assays. Vero cells were grown in complete Eagle’s Minimal Essential Medium (ATCC 30-2003) supplemented with 10 % FBS. The CHIKV-033 isolate, was kindly donated by Dra. Rosalia Lira from Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, IMSS, México. The VEEV strain TC-83, derived from ATCC VR-1249TM, was directly amplified in Vero cells from the VEEV vaccine (EQUIVAC TC-83, Productora Nacional de Productos Veterinarios, Mexico). The ZIKV isolate (Yucatán, México), the DENV-1 strain Puerto Rico/PR159-S1/1969 and the DENV-2 strain YUC17438 were kindly donated by Dr. Ma Isabel Salazar Sánchez from the Escuela Nacional de Ciencias Biológicas, National Polytechnic Institute, México.
Virus assays
The alphaviruses (CHIKV-033 and VEEV strain TC-83) and flaviviruses (ZIKV, DENV-1 and DENV-2) were routinely propagated in our laboratory. Briefly, Vero cells were grown in 75 cm2 flasks until a confluence of 80% was reached. Afterwards, the growth media was removed, and the cells were infected with alphaviruses at a MOI of 0.001, or Flaviviruses at a MOI of 0.1 using 1 mL of viral inoculum per 75 cm2 bottle. After an adsorption period of 2 h, the viral inoculum was removed and maintenance media (EMEM supplemented with 3% FBS) was added. The infection was left to proceed for 3 days for the alphaviruses, and 4 days for the Flavivirus ZIKA and 7 days for DENV-1 and DENV-2, afterwards the infected cultures were frozen and thawed once, and the viral lysates were supplemented with 20% FBS, clarified and kept at −80 ºC until further use.
CHIKV purification
Before virus purification, CHIKV-033 lysates were inactivated by three cycles of UV light for periods of 30 min. The inactivation of final preparations was verified using standard plaque assays in accordance with Warter and co-workers [21]. CHIKV-033 particles were precipitated from the inactivated viral lysates with cold PEG-NaCl at least 12 h. The mixture was centrifugated at 3000xg for 30 min at 4 ºC. The precipitated CHIKV particles were resuspended in TNE buffer (50 mM Tris–HCl, 100 mM NaCl, 0.1 mM EDTA, pH 7.4) at 4 °C. The CHIKV stock from PEG precipitation (25 mL) was then layered over 10 mL of a 60−30 % (w/v) sucrose step gradient prepared in ultracentrifuge tubes (Beckman Coulter, Cat No. 349622) on ice. Ultracentrifugation was carried out at 121,000×g for 3 h at 4 °C using a Beckman SW-28 rotor. After centrifugation, the CHIKV particles were observed as a white band above the 60% (w/v) sucrose layer. The particles were collected using a Pasteur pipette and diluted in TNE buffer. To pellet down the virus, 10 mL of sample with TNE buffer was layered over 2 mL of 30% (w/v) sucrose to a 12 mL ultracentrifuge. A second ultracentrifugation step was carried out at 281,000×g for 1 h at 4 °C using a Beckman SW41Ti rotor. The supernatant was removed, and the pellet was resuspended with 200 µL of TNE buffer.
Transmission electron microscopy (TEM)
Negative staining TEM was used to analyze the shape and size of the purified viral particles. Chikungunya particles (10 µL) were fixed for 1 min on copper grids coated with Formvar-carbon (Electron Microscopy Sciences, Cat No. CF200-Cu) and stained with 2.5% (v/v) uranyl acetate for 15 s. After air dry, samples were observed in a Zeiss Libra 120 transmission electron microscope at 80 kV. The images of the CHIKV were displayed using ImageJ software (National Institutes of Health, Bethesda, MD).
Control antibodies
Three control antibodies were used in this work: two human antibodies (4J21 and 4N12) reported by [13] and another antibody (5F10) reported by [21]. 4J21 and 4N12 were cloned as human IgG1/kappa molecules from the V regions disclosed in the patent WO 2016/168,417 and Protein-A purified in house—See IgG conversion below. 5F10 was purchased at Novus Biologicals (Clone E26D9.02, Cat No. DDX9 100P-100).
Direct binding ELISA
Nunc MaxiSorp™ flat-bottom plates (Thermo Scientific; Cat No. 44-2404-21) were coated with purified CHIKV particles at 3 µg/ml in PBS overnight at 4 °C. After blocking with 3% skim milk prepared in PBS (MPBS) for 1 h, IPTG-induced cultures, purified scFvs or IgGs in MPBS were added to the wells in 3-fold serially dilutions and incubated for 1 h at 37 °C. Bound samples were detected with recombinant Protein-A conjugated to HRP (1:8000, Invitrogen, Thermo Fisher Scientific; Cat No. 101123). The assay was revealed with TMB Substrate Reagent Set (BD OptEIA, BD Biosciences; Cat No. 555214). The reaction was stopped by the addition of 1 M phosphoric acid. The absorbance was read at 450/570 nm using an automated BioTek´s 2 microplate reader.
ALTHEA Gold Libraries™ and panning using inactivated CHIKV particles
ALTHEA Gold Libraries™ consist of two semisynthetic libraries built with synthetic human germline genes combined with natural human HCDR3/JH (H3J) fragments obtained from peripheral blood mononuclear cells (PBMCs) of a large pool of 180 donors. One of the libraries, called SL1, was built with a VL scaffold assembled with the human IGKV3-20*01 germline gene combined with the human IGJV4*01 joining region. The other library, called SL2, was made with a VL scaffold built with the human IGKV4-01*01 germline gene, also combined with the IGJV4*01 germline gene. Both libraries have a universal VH scaffold, which was partially built with the human IGHV3-23*01 germline gene. SL1 has a short LCDR1, whereas SL2 has a long one. By changing the length of LCDR1 from short to long, antibodies alter the preference to bind protein or peptide targets, respectively [26, 27]. Therefore, by using the library with the proper VL scaffold, antibodies against protein or peptide targets can be selected. When used in combination, SL1 and SL2 would potentially produce antibodies that bind diverse epitopes on a given target.
Selection of anti-CHIKV antibodies was performed in solid phase. Nunc MaxiSorp™ flat-bottom plates (Thermo Scientific; Cat No. 44-2404-21) were coated with purified CHIKV-033 at a concentration of 5 µg/mL in PBS. Aliquots of 1011–1012 cfu from ALTHEA Gold Libraries™, covering 10 to 100 times the initial library diversity, were used in the first round of panning. Subsequent rounds were performed with the output of the previous round at 1012 cfu.
Screening for positive and unique anti-CHIKV scFvs
Soluble scFvs were induced with IPTG (1 mM) and tested in plates with CHIKV or BSA as negative control. As reporter reagent, Protein-A/HRP (Thermo Fisher; Cat No. 101,123) was used. PCR of the positive clones were submitted to Sanger sequencing and unique clones were expressed as soluble scFvs, purified using Protein-A and re-tested for binding to CHIKV and BSA in direct ELISA assays.
IgG conversion
The scFvs identified in the ELISA-based screening were converted to human IgG1 via PCR of the V regions and cloning in TGEX expression vectors (Antibody Design Labs, Inc). The V regions of the sequences identified by NGS were PCR amplified with a reverse primer matching the HCDR3 sequences and a universal forward primer hybridizing in the leader peptide. The IgGs were expressed in HEK 293 cells (ATCC CRL-3216) by co-transfection of plasmid DNA containing the heavy and light chains. After a 4-day incubation, the supernatants containing the IgGs were harvested, filtrated and purified using Protein A MabSelect SuRe column (5 mL, GE Healthcare). The IgGs were captured in PBS (20 mM, 150 mM NaCl, pH 7.4) and eluted with 20 mM citrate buffer pH 3.5. The monomeric content of the purified IgGs was estimated by UPLC BEH200 150 mm Size Exclusion Chromatography (SEC) columns (Waters). The integrity of the IgG was evaluated by SDS-PAGE and Mass Spectrometry (Intact Mass).
Specificity assay
The cross-reactivity experiment was performed in MaxiSorp 96-well plates coated with 100 µL of purified particles from alphavirus genus: CHIKV (3 µg/mL) and VEEV (1 µg/mL) in PBS, pH 7.4, for 16 h at 4 °C. Additionally, inactivated flaviviruses lysates from ZIKV, DENV-1 and − 2 (30 µg/mL) were used. The plates were then incubated with MPBS for 1 h at RT, washed with 0.1% Tween-20 in PBS (PBS-T), and then further incubated with the anti-CHIKV antibodies (10 µg/mL) for 1 h at 37 °C, followed by further incubation with HRP-conjugated Protein A (1:8000, Invitrogen, Thermo Fisher Scientific) for 1 h at 37 °C. The positive controls: VEEV, hyperimmune serum 1:10 (RIH Research); DENV-1, monoclonal antibody D2-1F1-3 1:10 (Biotem, Le Rivier d’Apprieu, France); DENV-2 and ZIKV, mouse Anti-flavivirus Envelope Protein Antibody (4G2, 1:10; Native Antigen Company, UK) were included. All measurements were made at least three times. Statistical analysis was performed by one-way ANOVA followed by post hoc Tukeys’s multiple comparisons test.
Western blot
CHIKV (2 µg/mL) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (0.2 μm; Biorad, CA, USA) at 25 V and 1.3 A. After blocking with 3% skim milk in PBS-T, the membranes were incubated with the anti-CHIKV antibodies (10 µg/mL) overnight at room temperature. Afterwards, the membranes were washed three times with PBS-T and further incubated with goat anti-human IgG Fc-HRP (1:5,000, Abcam, US) for 1 h. The immunoreactive bands were detected using ECL Western Blotting Substrate (Pierce, Thermo Scientific) and visualized in a Chemidoc™ MP System.
Detection of CHIKV by a sandwich ELISA
Nunc MaxiSorp™ flat-bottom plates were coated with 100 µL of 10 µg/mL of the C7 antibody in carbonate buffer pH 9.4 overnight at 4 °C and blocked with 3% MPBS for 60 min at 37 °C. Serial dilutions of the CHIKV purified and UV-inactivated extract, starting at a concentration of 40 µg/mL, were incubated for 1 h at 37 °C. After washing the non-bond CHIKV, biotinylated 4N12 antibody (0.1 µg/mL) was added and incubated at 37 °C for 1 h. The assay was revealed with Streptavidin conjugated to HRP (1:1,000; Invitrogen Cat. No. S911) and TMB Substrate Reagent Set (BD OptEIA, BD Biosciences; Cat No. 555214). The reaction was stopped with 1 M phosphoric acid, and the absorbance was read at 450/570 nm by using automated BioTek´s 2 microplate reader.
Number of virions in the purified CHIKV preparation
To estimate the number of CHIKV particles per mL in the UV-inactivated extract, we first determined the mass of E2 protein in our preparation as follows. Three volumes: 10, 5 and 2 µL of the CHIKV stock at a total protein concentration of 691 µg/mL 2D-Quant Kit (Sigma Aldrich) were loaded into an SDS-PAGE gel and ran side-by-side with a standard of known mass (0.5 µg of BSA). The density of the E2 bands was quantified by comparison with the density of the BSA standard. The concentration of E2 was estimated in 36 µg/mL, which is 5.15% of the CHIKV UV-inactivated stock. The number of CHIKV particles per mL (CHIKV/mL) in 40 µg/mL of our CHIKV preparation was calculated in 1.2 × 1010 CHIKV/mL as follows: # of molecules of CHIKV/mL = {[(40 µg/mL × 0.0515)/ 240)] / 46,000 g/mol} × 6.022 × 1023 number of particles/mol x 10− 6; where 40 µg/mL is the concentration of the CHIKV UV-inactivated in the starting dilution (see above); 0.0515 is the fraction of E2 protein; 46,000 g/mol is the molecular weight of E2; 240 is number of E2 copies per CHIKV particle [28]; 6.022 × 1023 number of particles/mol is Avogadro’s number; and 10− 6 is a factor to convert µmol into mol.
Statistical analysis
Data are shown as the average ± standard error of the mean (SEM). To determine the affinity constants by direct binding ELISA, the results were adjusted following the 4-Parameter Logistic (4PL) curve model and each experiment was carried out at least three times with independent samples. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons tests for the cross-reactivity experiments. Differences were considered statistically significant with a p value < 0.05. Statistical analyses were performed in Prism 9.2.0 (Graphpad Software, LLC).