The study subjects were 30 patients (22 men and 8 women, median age 54; range: 25–69 years) diagnosed clinically with COVID-19 for the first time after visiting the National Center for Global Health and Medicine (NCGM), Tokyo, Japan, between June 2020 and March 2021. Twenty five of the 30 (83.3%) patients developed co-existing severe pneumonia, and of these, 5 required positive pressure ventilation, and 3 of the latter also required treatment with extracorporeal membrane oxygenation. Plasma test samples were obtained between 33 and 316 days (median 96 days) after the onset of clinical features of COVID-19 infection. The Human Ethics Committee of the NCGM approved the study (#NCGM-G-003472–02) and each patient provided a written informed consent. The study also conformed to the principles of the Declaration of Helsinki.
Measurement of anti-SARS-CoV-2 antibody titers
Samples from each participant were analyzed for the levels of two anti-SARS-CoV-2 antibodies (anti-S-IgG and anti-N-IgG) using the chemiluminescence enzyme immunoassay (CLEIA) platforms (HISCL) purchased from Sysmex Co. (Kobe, Japan) as reported previously14.
Cells, viruses, antibodies and isolation of IgG fractions from COVID‑19‑convalescent patients
Vero-E6TMPRSS2 cells23 and HeLahACE2-TMPRSS2 cells24 were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). Each cell type was maintained in D-MEM supplemented with 10% FCS, 100 μg/ml of penicillin, 100 μg/ml of streptomycin, and 1 mg/mL of G418. PANGO lineage B, wild type (WT) Wuhan strain [SARS-CoV-2 NCGM-05-2N (SARS-CoV-205-2N)] and B.1.1.529 (omicron) variants [hCoV-19/Japan/IC-2279/2021 SARS-CoV-2 NCGM-929-1N (SARS-CoV-2929-1N)] were isolated from nasopharyngeal swabs of a patient with COVID-19, who was admitted to the NCGM7,13,16,17. Seven clinically isolated SARS-CoV-2 mutant strains were used in the present study: two B.1.1.7 (alpha) variants [hCoV-19/Japan/QHN001/2020 (SARS-CoV-2QHN001, GISAID accession ID; EPI_ISL_804007) and hCoV-19/Japan/QK002/2020 (SARS-CoV-2QK002, GISAID Accession ID; EPI_ISL_768526)] and a B.1.351 (beta) variant [hCoV-19/Japan/TY8-612-P0/2021 (SARS-CoV-2TY8-612, GISAID accession ID; EPI_ISL_1123289)] and a P.1 (gamma) variant [hCoV-19/Japan/TY7-501/2021 (SARS-CoV-2TY7-501, GISAID Accession ID; EPI_ISL_833366)] were obtained from the National Institute of Infectious Diseases, Tokyo. The B.1.617.2 (delta) variant [hCoV-19/Japan/TKYK01734/2021 (SARS-CoV-21734, GISAID Accession ID; EPI_ISL_2080609)], B.1.617.1 (kappa) variant [TKYTK5356_2021 (SARS-CoV-25356, DDBJ Accession ID; LC633761)] and R.1 variant [hCoV-19/Japan/TKY76107/2021 (SARS-CoV-276107, GISAID Accession ID; EPI_ISL_1041946)] were provided by Tokyo Metropolitan Institute of Public Health, Tokyo. Each variant was confirmed to contain each variant of concern-specific amino acid substitutions before the assays conducted in the present study (vide infra). The mAb1414, mAb2414 and mAb40591, anti-SARS-CoV-2 monoclonal antibodies, were purchased from Active Motif (Carlsbad, CA) and Sino Biological (Beijing, China), respectively. The pAbA19215, anti-SARS-CoV-2 polyclonal antibody was purchased from ABclonal (Woburn, MA). Plasma or serum samples were collected from patients, and IgG fractions were purified using a spin column-based antibody purification kit (Cosmo Bio, Tokyo) according to the instructions provided by the manufacturer. Briefly, serum or plasma was collected, heat-inactivated for 30 min at 56 °C, and spin columns were centrifuged at 3500 rpm for 5 min. The IgG fractions in the supernatants were eluted and collected.
The neutralizing activities of IgG fractions from COVID‑19‑convalescent plasma were determined by quantifying the IgG antibody suppression of the cytopathic effect (CPE) of each SARS-CoV-2 strain in VeroE6TMPRSS2 cells and HeLahACE2-TMPRSS2 cells, using the procedures described previously7,13,16,17. Briefly, each of the purified IgG fraction was two-fold serially diluted in the culture medium. The diluted IgG fractions were incubated with 100 50% tissue culture infectious dose (TCID50) of the viruses at 37 °C for 20 min (final IgG dilution range: 6.3–100 µg/ml), after which the IgG-virus mixtures were inoculated into VeroE6TMPRSS2 cells and/or HeLahACE2-TMPRSS2 cells (1.0 × 104/well) in 96-well plates. The SARS-CoV-2 strains used in this assay were as follows: wild type strain, SARS-CoV-205-2N (PANGO lineage B), two alpha variants (SARS-CoV-2QHN001 and SARS-CoV-2QK002), beta variant SARS-CoV-2TY8-612, gamma variant SARS-CoV-2TY7-501, delta variant SARS-CoV-21734, kappa variant SARS-CoV-25356, omicron variant SARS-CoV-2929-1N and R.1 variant SARS-CoV-276107. After 3-day culture of the cells, the level of cytopathic effect (CPE) observed in SARS-CoV-2-exposed cells was determined using the WST-8 assay, employing Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). The IgG antibody dilution that yielded 50% inhibition of CPE was defined as the 50% Inhibition Concentration (IC50). Each of the purified IgG fractions was tested in duplicate.
Data are expressed as mean ± standard deviation (SD). Differences between groups were analyzed for statistical significance using Kruskal–Wallis test. When the latter test was significant, post-hoc Dunn’s multiple comparisons test was applied. Correlations between two assays were analyzed for statistical significance using nonparametric Spearman test. A p value < 0.05 denoted the presence of statistically significant difference. All statistical analyses were performed using the GraphPad Prism software version 8 (GraphPad Software, San Diego, CA).
Institutional review board statement
The Ethics Committee at the NCGM approved the present study (#NCGM-G-003472-02). Each patient provided written informed consent. The study also conformed to the Declaration of Helsinki principles.