Samples and ethics approvals
Individuals vaccinated with two or three doses of the NVX-CoV2373 vaccine were sampled at 14 days after the second dose or 35 days after the third dose. The third NVX-CoV2373 dose was administered 6 months after the first dose. This trial is registered under the ClinicalTrials.gov number, NCT04533399 (registered 17/09/2020), and the protocol was approved by the South African Health Products Regulatory Authority and by the institutional review board at each trial centre as described in detail by Shinde and colleagues13. Health care workers vaccinated with two dose of AD26.COV2.S (5 × 1010 viral particles) as part of the Sisonke implementation trial were sampled at 2 months after vaccination. This trial is registered under the ClinicalTrials.gov number, NCT05148845, and the protocol was approved by the South African Health Products Regulatory Authority. These Sisonke individuals were recruited at the National Institute for Communicable Diseases (NICD), Johannesburg. Individuals vaccinated with two and three doses of the BNT162b22 vaccine were sampled at 2 months after the second dose or 1–3 months after the third dose and were recruited from Johannesburg. This study was given ethics approval by the University of the Witwatersrand Human Research Ethics Committee (Medical) M210465. All individuals provided written informed consent and all research was performed in accordance with the relevant guidelines/regulations and in accordance with the Declaration of Helsinki.
Lentiviral pseudovirus production and neutralization assay
The 293 T/ACE2. MF cells modified to overexpress human ACE2 were kindly provided by M. Farzan (Scripps Research). Cells were cultured in DMEM (Gibco BRL Life Technologies) containing 10% heat-inactivated fetal bovine serum (FBS) and 3 μg ml−1 puromycin at 37 °C, 5% CO2. Cell monolayers were disrupted at confluency by treatment with 0.25% trypsin in 1 mM EDTA (Gibco BRL Life Technologies). The SARS-CoV-2, Wuhan-1 spike, cloned into pCDNA3.1 was mutated using the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies) to include D614G (ancestral D164G) or L18F,D80A, D215G, Δ242-244, K417N, E484K, N501Y, D614G, A701V (Beta) or Δ69-70, T915I, Δ143-145, Δ211, L212I, ins 214 EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (Omicron BA.1) or T19I, L24S, Δ25–27, Δ69–70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K (Omicron BA.4/BA.5). Pseudoviruses were produced by co-transfection with a lentiviral backbone (HIV-1 pNL4.luc encoding the firefly luciferase gene) and either of the SARS-CoV-2 spike plasmids with PEIMAX (Polysciences). Culture supernatants were clarified of cells by a 0.45 μM filter and stored at − 80 °C. Plasma samples were heat-inactivated and clarified by centrifugation. Pseudovirus and serially diluted plasma/sera were incubated for 1 h at 37 °C, 5% CO2. Cells were added at 1 × 104 cells per well after 72 h of incubation at 37 °C, 5% CO2, luminescence was measured using PerkinElmer Life Sciences Model Victor X luminometer. Neutralization was measured as described by a reduction in luciferase gene expression after single-round infection of 293 T/ACE2.MF cells with spike-pseudotyped viruses. Titers were calculated as the reciprocal plasma dilution (ID50) or monoclonal antibody concentration (IC50) causing 50% reduction of relative light units. Equivalency was established through participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey Concordance Survey 1 run by EQAPOL and VQU, Duke Human Vaccine Institute. Cell-based neutralization assays using live virus or pseudovirus have demonstrated high concordance, with highly correlated 50% neutralization titers (Pearson r = 0.81–0.89).