Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2’) known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.

Peptide N' to C' PNA C' to N'

Synthesis of the PNA-Peptide conjugates
All reagents and solvents for the organic synthesis were purchased from commercial sources and were used without further purification. Automated solid phase synthesis was carried out on an Intavis AG Multipep RS instrument. MALDI-TOF Mass spectra were measured using a Bruker Daltonics Autoflex spectrometer operated in positive mode.
HPLC purification was performed with an Agilent Technologies 1260 infinity HPLC using a ZORBAX 300SB-C18 column (9.4 x 250 mm).
After 20 minutes the mixture was filtered, the resin was washed with DMF, and a new premixed reaction solution was added to the resin for another 20 minutes. Finally, the resin was washed with 2 x DMF, 2 x CH2Cl2 and 2 x DMF.

Procedure 2: Fmoc deprotection
A solution of 20% piperidine in DMF was added to the resin and allowed to react for 5 minutes. The mixture was filtered, the resin was washed with DMF and the sequence was repeated a second time for another 5 minutes. Finally, the resin was washed with 2 x DMF and 2 xCH2Cl2 and 2 x DMF.

Procedure 3: Capping
The resin was treated with a capping mixture (0.92 mL of acetic anhydride and 1.3 mL of 2,6 lutidine in 18 mL of DMF; 10 mL of solution/g of resin) for 5 minutes. After flushing the solution, the resin was washed with 2 x DMF, 2 x CH2Cl2 and 2 x DMF.     After incubation, the slide was washed for 5minutes with PBS-t and 30 seconds with water. Finally, the slide was dried by centrifugation for 1 minute at 1000g and ready for next step.

-Incubation of the secondary antibody
1.0 μL of Goat Anti-Human IgG H&L Cy3 (ab97170 from Abcam) was diluted with 450 μL of PBS-t and 50 μL of BSA 5% and centrifuged for 1 minute at 12krpm. This mixture, 82 μL, was added into the microarray and incubated for 30 minutes at room temperature.
After incubation, the slide was washed for 5 minutes with PBS-t and 30 seconds with de ionized water. The slide was finally dried by centrifugation for 3 minutes at 1000g and scanned on the Cy3 channel with GenePix 4100A Microarray Scanner.

-Data Analysis
Heat The band corresponding to the spike protein (MW= 141'108 Da, omitting glycosylation, lane 2) is progressively converted to two new bands (expected in S1 MW=79'307 Da and S2 MW=61'819 Da, omitting glycosylation) over time (lane 3-5). The difference in fluorescence intensity of the S1 and S2 bands might correspond to different quantity of dye conjugation since the S1 domain is more exposed.