key: cord-0687651-p9h9mgr6
authors: Chen, Yuxin; Shen, Han; Huang, Rui; Tong, Xin; Wu, Chao
title: Serum neutralising activity against SARS-CoV-2 variants elicited by CoronaVac
date: 2021-05-27
journal: Lancet Infect Dis
DOI: 10.1016/s1473-3099(21)00287-5
sha: 295b8aa6ff47a273d117289c04851693273d6f80
doc_id: 687651
cord_uid: p9h9mgr6

nan

assigned a value of 10 for geometric mean calculation and was considered as seronegative. Statistical analyses were performed using two-tailed Kruskal-Wallis test after the adjusting the false discovery rate, and statistical difference of each pseudovirus compared to wild-type strain was shown with the following notation: ****p<0.0001. (B) Fold decrease in neutralization for each pseudovirus relative to wild-type virus, which was calculated by dividing pNT50 titer of the SARS-CoV-2 variant by that of wild-type from the same vaccinee sample. Box plots indicated the median and interquartile range (IQR) of decreased fold. The average value of the fold decrease was shown at the top of each bar.

(C) Serum anti-RBD IgG antibody titers against RBD wild-type versus RBD E484 antigen measured by ELISA assay. Wilcoxon signed-ranked test was performed for statistical analysis of matched pairs, and statistical difference is shown with the following notation: ****p<0.0001.

A total of 93 healthy healthcare professional was enrolled in a prospective study (NCT04729374) in a tertiary hospital in Nanjing, China, between Jan 26 and Feb 25, 2020.

Participants received two doses of CoronaVac which was administered 14-28 days apart.

The serum samples were collected before the first dose and at day 14 after two doses. This protocol was approved by Nanjing Drum Tower Hospital Ethics Committee (2021-034-01). All participants provided written informed consent before any study procedures were undertaken.

Pseudovirus neutralization assay was performed as previously described 1 

An in-house enzyme-linked immunosorbent assay (ELISA) was performed as previously described 2 . Briefly, 96-well plates were coated with 500 ng/mL of recombinant RBD wildtype or RBD E484K protein in coating buffer (pH 9.6) at 4˚C overnight, respectively. After washing, blocking buffer with 5% non-fat milk in PBS was added and incubated for 1 hour.

After washing 5 times, the plates were incubated with 100 μl serial diluted serum samples, followed by incubation with anti-human IgG conjugated with HRP (Abcam, ab6759).

Following washing, the plates were subsequently incubated with 3,3',5,5'-Tetramethylbenzidine (TMB, sigma) substrate for 1 hour and the reaction stopped with 1M H2SO4. The preliminary cut-off value for each ELISA assay was determined as the mean of the negative control serum OD values plus 2 standard deviation (SD) from 45 archived healthy individuals before COVID-19 pandemic. Optical density (OD) value at 450nm was measured by Multiskan TM FC microplate photometer (Thermo Fisher Scientific). Reciprocal RBD-specific IgG titer was calculated by the highest dilution which gives an OD value higher than cut off value of the negative control group at the same dilution.

Multiple group comparisons for serum neutralization titers against a panel of SARS-CoV-2 pseudoviruses were applied using Kruskal-wallis test with false discovery rate (FDR) method, and multiple testing correction was performed for each comparison using Benjamini-Hochberg (BH) procedure at a 5% FDR threshold. For comparison of serum IgG titer specific to RBD wild-type versus RBD E484K, Wilcoxon signed-ranked test was performed for matched pairs. Data were analyzed using GraphPad Prism (version 9.0.1, La Jolla, California, USA). The statistical tests were performed in a two-sided manner, and p value <0.05 was considered as statistically significant.

Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

A comprehensive, longitudinal analysis of humoral responses specific to four recombinant antigens of SARS-CoV-2 in severe and nonsevere COVID-19 patients