Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma

Data reporting

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Ethical statement

Nasopharyngeal and oropharyngeal swab samples and plasma samples were obtained from 20 hospitalized adults with PCR-confirmed SARS-CoV-2 infection who were enrolled in a prospective cohort study approved by the Biomedical Research Ethics Committee (BREC) at the University of KwaZulu–Natal (reference BREC/00001275/2020). The 501Y.V2 variant was obtained from a residual nasopharyngeal and oropharyngeal sample used for routine SARS-CoV-2 diagnostic testing by the National Health Laboratory Service through our SARS-CoV-2 genomic surveillance programme (BREC approval reference BREC/00001510/2020).

Whole-genome sequencing, genome assembly and phylogenetic analysis

cDNA synthesis was performed on the extracted RNA using random primers followed by gene-specific multiplex PCR using the ARTIC V.3 protocol (https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bibtkann). In brief, extracted RNA was converted to cDNA using the Superscript IV First Strand synthesis system (Life Technologies) and random hexamer primers. SARS-CoV-2 whole-genome amplification was performed by multiplex PCR using primers designed using Primal Scheme (http://primal.zibraproject.org/) to generate 400-bp amplicons with an overlap of 70 bp that covers the 30 kb SARS-CoV-2 genome. PCR products were cleaned up using AmpureXP purification beads (Beckman Coulter) and quantified using the Qubit dsDNA High Sensitivity assay on the Qubit 4.0 instrument (Life Technologies). We then used the Illumina Nextera Flex DNA Library Prep kit according to the manufacturer’s protocol to prepare indexed paired-end libraries of genomic DNA. Sequencing libraries were normalized to 4 nM, pooled and denatured with 0.2 N sodium acetate. Then, a 12-pM sample library was spiked with 1% PhiX (a PhiX Control v.3 adaptor-ligated library was used as a control). We sequenced libraries on a 500-cycle v.2 MiSeq Reagent Kit on the Illumina MiSeq instrument (Illumina). We assembled paired-end fastq reads using Genome Detective 1.126 (https://www.genomedetective.com) and the Coronavirus Typing Tool. We polished the initial assembly obtained from Genome Detective by aligning mapped reads to the reference sequences and filtering out low-quality mutations using the bcftools 1.7-2 mpileup method. Mutations were confirmed visually with BAM files using Geneious software (Biomatters). All of the sequences were deposited in GISAID (https://www.gisaid.org/). We retrieved all SARS-CoV-2 genotypes from South Africa from the GISAID database as of 11 January 2021 (

n

= 2,704). We initially analysed genotypes from South Africa against the global reference dataset (

n

= 2,592) using a custom pipeline based on a local version of NextStrain. The pipeline contains several Python scripts that manage the analysis workflow. It performs alignment of genotypes in MAFFT, phylogenetic tree inference in IQ-Tree20, tree dating and ancestral state construction and annotation (https://github.com/nextstrain/ncov).

Cells

Vero E6 cells (ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete DMEM with 10% fetal bovine serum (Hylone) containing 1% each of HEPES, sodium pyruvate,

l

-glutamine and nonessential amino acids (Sigma-Aldrich). Vero E6 cells were passaged every 3–4 days. H1299 cells were propagated in complete RPMI with 10% fetal bovine serum containing 1% each of HEPES, sodium pyruvate,

l

-glutamine and nonessential amino acids. H1299 cells were passaged every second day. HEK-293 (ATCC CRL-1573) cells were propagated in complete DMEM with 10% fetal bovine serum containing 1% each of HEPES, sodium pyruvate,

l

-glutamine and nonessential amino acids. HEK-293 cells were passaged every second day.Cell lines have not been authenticated. The cell lines have been tested for mycoplasma contamination and are mycoplasma negative.

H1299-E3 cell line for first-passage SARS-CoV-2 expansion

The H1299-H2AZ clone with nuclear-labelled YFP was constructed to overexpress human ACE2 as follows. Vesicular stomatitis virus G protein (VSVG)-pseudotyped lentivirus containing the human ACE2 was generated by co-transfecting HEK-293T cells with the pHAGE2-EF1alnt-ACE2-WT plasmid along with the lentiviral helper plasmids HDM-VSVG, HDM-Hgpm2, HDM-tat1b and pRC-CMV-Rev1b using the TransIT-LT1 (Mirus) transfection reagent. Supernatant containing the lentivirus was collected 2 days after infection, filtered through a 0.45-μm filter (Corning) and used to spinfect H1299-H2AZ at 1,000 rcf for 2 h at room temperature in the presence of 5 μg ml

−1

polybrene (Sigma-Aldrich). ACE2-transduced H1299-H2AZ cells were then subcloned at single-cell density in 96-well plates (Eppendorf) in conditioned medium derived from confluent cells. After 3 weeks, wells were trypsinized (Sigma-Aldrich) and plated in two replicate plates. The first plate was used to determine infectivity and the second plate was used as stock. The first plate was screened for the fraction of mCherry-positive cells per cell clone after infection with SARS-CoV-2 mCherry-expressing spike-pseudotyped lentiviral vector 1610-pHAGE2/EF1a Int-mCherry3-W produced by transfecting the cells as described above. Screening was performed using a Metamorph-controlled (Molecular Devices) Nikon TiE motorized microscope (Nikon Corporation) with a 20×/0.75 NA phase objective, 561 laser line, and 607-nm emission filter (Semrock). Images were captured using an 888 EMCCD camera (Andor). Temperature (37 °C), humidity and CO

2

(5%) were controlled using an environmental chamber (OKO Labs). The clone with the highest fraction of mCherry expression was expanded from the stock plate and denoted H1299-E3. This clone was used in the expansion assays.

Virus expansion

All work with live virus was performed in Biosafety Level 3 containment using protocols for SARS-CoV-2 approved by the Africa Health Research Institute Biosafety Committee. For first-wave virus, a T25 flask (Corning) was seeded with Vero E6 cells at 2 × 10

5

cells per ml and incubated for 18–20 h. After one DPBS wash, the subconfluent cell monolayer was inoculated with 500 μl universal transport medium diluted 1:1 with growth medium and filtered through a 0.45-μm filter. Cells were incubated for 1 h. The flask was then filled with 7 ml of complete growth medium and checked daily for cytopathogenic effects. After infection for 4 days, supernatants of the infected culture were collected, centrifuged at 300 rcf for 3 min to remove cell debris and filtered using a 0.45-μm filter. Viral supernatant was aliquoted and stored at −80 °C. For 501Y.V2 variants, we used ACE2-expressing H1299-E3 cells for the initial isolation followed by passaging in Vero E6 cells. ACE2-expressing H1299-E3 cells were seeded at 1.5 × 10

5

cells per ml and incubated for 18–20 h. After one DPBS wash, the subconfluent cell monolayer was inoculated with 500 μl universal transport medium diluted 1:1 with growth medium and filtered through a 0.45-μm filter. Cells were incubated for 1 h. Wells were then filled with 3 ml complete growth medium. After 8 days of infection, cells were trypsinized, centrifuged at 300 rcf for 3 min and resuspended in 4 ml growth medium. Then, 1 ml was added to Vero E6 cells that had been seeded at 2 × 10

5

cells per ml 18–20 h earlier in a T25 flask (approximately 1:8 donor-to-target cell dilution ratio) for cell-to-cell infection. The coculture of ACE2-expressing H1299-E3 and Vero E6 cells was incubated for 1 h and the flask was then filled with 7 ml of complete growth medium and incubated for 6 days. The viral supernatant was aliquoted and stored at −80 °C or further passaged in Vero E6 cells as described above. Two isolates were expanded, 501Y.V2.HV001 and 501Y.V2.HVdF002. The second isolate showed fixation of mutations in the furin cleavage site during expansion in Vero E6 cells and was not used except for data presented in Extended Data Fig. 1.

Microneutralization using the focus-forming assay

For plasma from donors infected with first-wave virus variants, we first quantified IgG targeting the spike RBD by enzyme-linked immunosorbent assay (ELISA) using the monoclonal antibody CR3022 (used at fourfold serial dilutions from 1,000 ng ml

−1

to 0.244 ng ml

−1

) as a quantitative standard (

n

= 13 excluding participant 039-13-0103, for whom ELISA data were not available). The mean concentration was 23.7 ± 19.1 μg ml

−1

(range, 5.7–62.6 μg ml

−1

). In comparison, control samples from donors who were not infected with SARS-CoV-2 had a mean of 1.85 ± 0.645 μg ml

−1

. To quantify neutralization, Vero E6 cells were plated in an 96-well plate (Eppendorf or Corning) at 30,000 cells per well 1 day before infection. Notably, before infection approximately 5 ml sterile water was added between wells to prevent wells at the edge drying more rapidly, which we have observed to cause edge effects (lower number of foci). Plasma was separated from EDTA-anticoagulated blood by centrifugation at 500 rcf for 10 min and stored at −80 °C. Aliquots of plasma samples were heat-inactivated at 56 °C for 30 min and clarified by centrifugation at 10,000 rcf for 5 min, after which the clear middle layer was used for experiments. Inactivated plasma was stored in single-use aliquots to prevent freeze–thaw cycles. For experiments, plasma was serially diluted twofold from 1:100 to 1:1,600; this is the concentration that was used during the virus–plasma incubation step before addition to cells and during the adsorption step. As a positive control, the GenScript A02051 anti-spike monoclonal antibody was added at concentrations listed in the figures. Virus stocks were used at approximately 50 focus-forming units per microwell and added to diluted plasma; antibody–virus mixtures were incubated for 1 h at 37 °C, 5% CO

2

. Cells were infected with 100 μl of the virus–antibody mixtures for 1 h, to allow adsorption of virus. Subsequently, 100 μl of a 1× RPMI 1640 (Sigma-Aldrich, R6504), 1.5% carboxymethylcellulose (Sigma-Aldrich, C4888) overlay was added to the wells without removing the inoculum. Cells were fixed at 28 h after infection using 4% paraformaldehyde (Sigma-Aldrich) for 20 min. For staining of foci, a rabbit anti-spike monoclonal antibody (BS-R2B12, GenScript A02058) was used at 0.5 μg ml

−1

as the primary detection antibody. Antibody was resuspended in a permiabilization buffer containing 0.1% saponin (Sigma-Aldrich), 0.1% BSA (Sigma-Aldrich) and 0.05% Tween-20 (Sigma-Aldrich) in PBS. Plates were incubated with primary antibody overnight at 4 °C, then washed with wash buffer containing 0.05% Tween-20 in PBS. Secondary goat anti-rabbit horseradish peroxidase (Abcam ab205718) antibody was added at 1 μg ml

−1

and incubated for 2 h at room temperature with shaking. The TrueBlue peroxidase substrate (SeraCare 5510-0030) was then added at 50 μl per well and incubated for 20 min at room temperature. Plates were then dried for 2 h and imaged using a Metamorph-controlled Nikon TiE motorized microscope with a 2× objective. Automated image analysis was performed using a custom script in MATLAB v.2019b (Mathworks), in which focus detection was automated and did not involve user curation. Image segmentation steps were stretching the image from minimum to maximum intensity, local Laplacian filtering, image complementation, thresholding and binarization. Two plasma donors initially measured from the second infection wave in South Africa did not have detectable neutralization of either 501Y.V2 or the first-wave variant and were not included in the study.

Statistics and fitting

All statistics and fitting were performed using MATLAB v.2019b. Neutralization data were fit to

$${\rm{T}}{\rm{x}}=1/1+(D{/{\rm{I}}{\rm{D}}}_{50}),$$

where Tx is the number of foci normalized to the number of foci in the absence of plasma on the same plate at dilution

D

. To visualize the data, we used percentage neutralization, calculated as (1 − Tx) × 100%. Negative values (Tx > 1, enhancement) were presented as 0% neutralization. Data were fitted to a normal distribution using the function normplot in MATLAB v.2019b, which compared the distribution of the Tx data to the normal distribution (see https://www.mathworks.com/help/stats/normplot.html).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.