COVID-19 is caused by SARS-CoV-2. 1, 2 By July 25, 2020, globally, 15,672,841 diagnosed cases and 638,352 deaths were reported (https://coronavirus. jhu. edu/map. html). 3 High titers of Spike protein (S protein)-specific antibodies are found in the blood of COVID-19 patients, especially IgG for both SARS-CoV 4 and SARS-CoV-2. 5, 6 Because of the central role that S protein plays in the entry of the virus into the host cell, S1 and, more specifically, the RBD (receptorbinding domain) is the most targeted region for the development of COVID-19 therapeutic antibodies 7, 8 and vaccines. 9 It is known that in addition to the RBD, other areas/epitopes of S protein may also elicit neutralizing antibodies. 10 However, antibody responses to full-length S protein have not been investigated at epitope resolution, and the capability of linear epitopes to elicit neutralizing antibodies has still not been explored. To precisely decipher the B-cell linear epitopes of the S protein, we constructed a peptide microarray. A total of 211 peptides (Supplementary Table 1) were synthesized and conjugated to BSA (Supplementary Fig. 1a-c). The conjugates along with control proteins were prepared in triplicate at three dilutions. High reproducibility among triplicate spots or repeated arrays for serum profiling was achieved (Supplementary Fig. 1d, e). Peptides with variable concentrations may enable dynamic detection of antibody responses and indicate that antibodies against different epitopes may have different kinetic characteristics (Supplementary Figs. 1f and 2a). Moreover, an inhibitory assay using free peptides verified the specificity of the signals generated against the peptides (Supplementary Fig. 2b). Fifty-five sera from convalescent COVID-19 patients and 18 control sera (Supplementary Table 2) were screened on the peptide microarray for both IgG and IgM responses (Fig. 1 a and Supplementary Fig. 3). For IgG, COVID-19 patients were completely separated from controls, and distinct and specific signals were shown for some peptides. In contrast, the assay results were not distinct enough for IgM responses. We then focused on IgG for further analysis. Epitope maps of S protein were generated based on the response frequency (Fig. 1 b). Primarily, there are three hot epitope areas across S protein. The first is on the CTD (C terminal domain) that immediately follows the RBD, ie, from S1-93 to S1-113. Interestingly, the identified epitopes, S1-93, 97, 100/101, 105/106, 111, and 113, are located predominantly in flexible loops (Fig. 1 c). In addition, the signals of some epitopes had moderate correlations with others (Fig. 1 d), and most of these epitopes were positively correlated with S1 (Supplementary Fig. 4c-f). The second hot area is from S2-14 to S2-23, including the FP (fusion peptide, aa 788-806) region and the S2′ cleavage site (R815)(Fig. 1 e). In contrast to those for the first hot region, the antibody responses against epitopes of this region had poor correlations with each other (Fig. 1 f), possibly due to the capability of this region to generate continuous but competitive epitopes. Moreover, part of this area is shielded by other parts in trimeric S (Fig. 1 e), suggesting that this part would be easily accessed by the immune system after the departure of S2 from S1. The third hot area is S2-78 or aa 1148-1159, connecting HR1 (heptad repeat 1) and HR2 (heptad repeat 2) on the S2 subunit. IgG antibodies against this epitope can be detected in~ 90% of COVID-19 patients, indicating that it is an extremely dominant epitope. In addition to these three areas, S2-34 (aa 884-895) and S2-96/97 (aa 1256-1273) also elicited antibodies in some patients.

The RBD can elicit a high titer of …