Linear epitopes of SARS-CoV-2 spike protein elicit neutralizing antibodies in COVID-19 patients

SARS-CoV-2 outbreak is a world-wide pandemic. The Spike protein plays central role in cell entry of the virus, and triggers significant immuno-response. Our understanding of the immune-response against S protein is still very limited. Herein, we constructed a peptide microarray and analyzed 55 convalescent sera, three areas with rich linear epitopes were identified. Potent neutralizing antibodies enriched from sera by 3 peptides, which do not belong to RBD were revealed.

during the period from 2020-1-25 to 2020-3-8 with variable stay time (Supplementary  1 . Briefly, the key points of the discharge criteria are: 1) Body temperature is back to normal for more than three days; 2) Respiratory symptoms improve obviously; 3) Pulmonary imaging shows obvious absorption of inflammation; 4) Nuclei acid tests negative twice consecutively on respiratory tract samples such as sputum and nasopharyngeal swabs (sampling interval being at least 24 hours). From 2020-2-28, standard criteria of discharge were modified by adding one item that nuclei acid tests should be negative on anal swab sample. Sera of the control group from Lung cancer patients and healthy donors were collected from Ruijin Hospital, Shanghai, China. All the sera were inactivated at 56 °C for 30 min and stored at -80℃ until use.

Microarray-based serum analysis
A 7-chamber rubber gasket was mounted onto each slide to create individual chambers for the 7 identical subarrays. The microarray was used for serum profiling as described previously with minor modifications 2 . Briefly, the arrays stored at -80°C were warmed to room temperature and then incubated in blocking buffer (3% BSA in 1×PBS buffer with 0.1% Tween 20) for 3 h. A total of 400 μL of diluted sera or antibodies was incubated with each subarray for 2 h. The sera were diluted at 1:200 for most samples and for competition experiment, free peptides were added at a concentration of 0.25 mg/mL. For the enriched antibodies, 0.1-0.5 μg antibodies were included in 400 μL incubation buffer . The arrays were washed with 1×PBST and bound antibodies were   detected by incubating with Cy3-conjugated goat anti-human IgG and Alexa Fluor 647conjugated donkey anti-human IgM (Jackson ImmunoResearch, PA, USA), which were diluted for 1: 1,000 in 1×PBST. The incubation was carried out at room temperature for 1 h. The microarrays were then washed with 1×PBST and dried by centrifugation at room temperature and scanned by LuxScan 10K-A (CapitalBio Corporation, Beijing, China) with the parameters set as 95% laser power/ PMT 550 and 95% laser power/ PMT 480 for IgM and IgG, respectively. The fluorescent intensity was extracted by GenePix Pro 6.0 software (Molecular Devices, CA, USA).

Purification of epitope-specific antibodies
Depends on the availability，200-500 μL serum from COVID-19 convalescent patient was two-fold diluted in 1×PBS and then pre-incubated with streptavidin beads to eliminate non-specific binding. For each epitope, 100 µg peptides conjugated with biotin-BSA were coated to 100 µL streptavidin magnetic beads (Invitrogen, MA, USA) in 1×PBS buffer at room temperature for 1 h. The protein-coated streptavidin beads were washed 4 times in 1×PBS containing 0.1% BSA, and incubated with the precleaned serum in 1×PBS at 4°C for 4 h. The streptavidin beads were then washed 3 times in 1×PBS containing 0.1% BSA, and eluted with 0.2 M glycine, 1 mM EGTA, pH 2.2. Finally, the antibodies were neutralized with 1M Tris-HCl, pH8.0. The concentration of the purified antibody was monitored by silver staining.

Pseudotyped Virus Neutralization
The neutralization assay was performed as described 3

Data analysis and software
Signal Intensity was defined as the median of the foreground subtracted by the median of background for each spot and then averaged the triplicate spots for each peptide or protein. IgG and IgM data were analyzed separately. Pearson correlation coefficient between two proteins or indicators and the corresponding p value was calculated by SPSS software under the default parameters. Cluster analysis was performed by pheatmap package in R 4 . To calculate the response frequency of each epitope specific antibody, mean signal + 3*SD of the control sera were used to set the threshold. The epitope map was generated by ImmunomeBrowser issued by IEDB (Epitope Prediction and Analysis Tools) 5 . Visualization of the structural details were processed by Pymol