Cell culture, virus isolation and propagation: Vero cells (ATCC CCL-81) were cultured in T25cm2 flasks containing DMEM media supplemented with 10% Fetal Calf Serum (FCS), 1% L-glutamine 200mM, 1% penicillin G (100U/ml), streptomycin (100ug/ml) at 37°C and 5% CO2 until 80-90 confluency was achieved.
Nasopharyngeal swab (NPS) in viral transport medium from a PCR-positive SARS-CoV-2 case from June 2020, during the first wave of COVID-19 in Pakistan, was used for virus isolation. The particular viral isolate has not been sequenced but our data from that time period identified the G clade strains to be predominant in Pakistan (https://www.biorxiv.org/content/10.1101/2020.08.04.234153v1). Fifty microliters of serum-free DMEM were pipetted into columns 2–12 of a 96-well tissue culture plate; subsequently, 100μL of clinical specimens were pipetted into column 1 and serially diluted 2-fold across the plate (columns 2–12; from 1 log to 11 logs). Cultured Vero cells (80-90% confluent) were trypsinized and resuspended in DMEM containing 10% FCS and antibiotics at a concentration of 1×106 cells/mL. A hundred microliter of cell suspension was directly added to the wells of the 96-well plate containing dilutions of the clinical specimen (NPS) and mixed gently by pipetting. Inoculated cultures were grown in a humidified incubator at 37°C with 5% CO2 for 4 days. The infected Vero cell line was observed daily for the presence of CPE using an inverted optical microscope (Olympus, Japan), and the virus was harvested when 80%‐90% of the cells manifested CPE. The end‐point titers were calculated according to the Reed & Muench method [14] based on eight replicates for titration. The culture medium was centrifuged at +4°C 1600rpm for 8 minutes, to remove the cell debris, and then aliquoted and stored at 80°C. The study was approved by Aga Khan University, Ethical Review Committee (ERC# 2020-5152-11688).
Spike and RBD ELISA
Recombinant Spike and RBD proteins were kindly provided by Dr. Paula Alves, IBET, NOVA University, Portugal. We used ELISA to quantify serum antibodies against both SARS-CoV-2 Spike and RBD proteins. For this, a 96-well ELISA plate was coated with 50µl of Spike or RBD protein at a concentration of 2 µg/ml in PBS [15, 16]. The coated plate was stored at 4°C overnight. Next day, the wells were blocked with 200 µl of PBS + 0.1% Tween (PBS-T) + 3% non-fat milk and the plate was incubated for 1 hour at room temperature. After incubation, wells were washed with washing buffer. Subsequently, the serum samples were diluted 1:100 in PBS-T + 1% non-fat milk powder, and 100 µl of serum was added to the wells and the plate was incubated for 2 hours at room temperature. After incubation, the wells were washed with washing buffer, and stained with secondary antibody. For secondary staining, goat anti-human IgG Fc (HRP) was diluted 1:40,000 in PBS-T + 1% non-fat milk, and 50 µl was added to each well; the plate was incubated for 1 hour at room temperature. Following incubation, the plate was washed with washing buffer. Finally, for color development, 100 µl of TMB substrate solution was added to each well and the plate was incubated for 10 minutes. The reaction was stopped by adding 50 µl of 0.5M sulfuric acid to each well. The optical density of each well was read at 450nm immediately after adding the stop solution. A cut-off OD of 0.5 was used for both Spike and RBD proteins to detect positive samples.
PCR-based microneutralization assay
For the assay development and optimization, we used 19 serum samples containing anti-RBD antibodies as determined by laboratory-based ELISA. The samples were collected after obtaining informed consent from the patients. Patient serum samples were heat‐inactivated for 30 minutes at 56°C. Three ten-fold serum dilutions (1:10, 1:100, and 1:1000) were prepared in media. Each serum dilution was mixed with an equal volume of viral solution containing 100 TCID50 of SARS‐CoV‐2. The serum‐virus mixture was incubated for 1 hour at 37°C in a humidified atmosphere with 5% CO2. After incubation, 100ul of the mixture at each dilution was added in duplicates to a 96-well cell culture plate containing a semi ‐confluent Vero cell monolayer. The plates were incubated for 24hrs at 37°C in a humidified atmosphere with 5% CO2 [17]. Cells without virus served as ‘cell line control’, while cells with the virus without serum served as ‘virus control’.
After incubation of 96-well plates for 24 hours, the supernatant was carefully removed, and cells were washed with DMEM media. After the final wash, RNA was extracted from the cells using the Qiagen Viral Extraction Kit (cat no:52906; Qiagen-Germany) following the manufacturer’s instructions. The RNA was also used to perform a rapid real-time PCR using Novel Coronavirus (2019-nCOV) Nucleic Acid Diagnostic Kit (PCR Fluorescence Probing) of Sansure Biotech (S3102E) (Changsha, China) on CFX96™ Real-Time PCR thermal cycler (Bio-Rad Laboratories, Inc.). For PCR, 30 µL PCR-Master mix (including 2019-nCoV-PCR Mix and 2019-nCoV-PCREnzyme Mix, containing primers, probes, dNTPs, MgCl2, Rnasin and PCR buffer for the 2019-nCoV-PCR Mix and RT enzyme and Taq enzyme for the 2019-nCoVPCR-Enzyme Mix) was added to a PCR reaction tube with 20 µL of the extracted RNA sample. The first two steps “reverse transcription” and “cDNA preparation” were performed at 50°C (30 min) and 95°C (1 min), each with a single cycle, respectively. For PCR amplification following conditions were used: 45 cycles, comprising of denaturation for 15-second at 95°C, and annealing for 30-second at 60°C, followed by cooling to 25°C for 10 seconds to finalize the process. The CFX96 in-built software was used for the calculation of the cycle threshold (Ct) values. For 2019-nCoV-PCR, a negative control was defined as Ct value >40, while positive control was defined as Ct value No≤ 35, as per kit’s instructions [18]. Alternative to the commercial kit, the qPCR was also performed using SARS-CoV-2 specific primers as described in Supplementary File 1.
The SARS-CoV-2 PCR Ct values obtained for each serum-virus well, and control wells containing cells alone and virus control, were averaged for each sample. The average Ct values obtained were used to measure the percent inhibition/neutralization using the formula [4]: 100 − ((N‐average Ct of ʻcell line control’ wells)/(average Ct of ʻvirus controlʼ wells‐average of ʻ cell line control’ wells)*100), where N is the average Ct for each well/sample.
Correlation between the RBD titers and neutralizing potential was determined using the Pearson correlation test using the GraphPad prism. P-value <0.05 was considered significant.