Synthetic proteins for COVID-19 diagnostics

Abstract

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for vaccine efficacy and detect infection with SARS-CoV-2 and its variants. We have identified a small, synthetic protein (JS7), representing a region of maximum variability within the receptor binding domain (RBD), which binds antibodies in sera from nine patients with PCR-verified COVID-19 of varying severity. Antibodies binding to either JS7 or the SARS-CoV-2 recombinant RBD, as well as those that disrupt binding between a fragment of the ACE2 receptor and the RBD, are proportional to disease severity and clinical outcome. Binding to JS7 was inhibited by linear peptides from the RBD interface with ACE2. Variants of JS7, such as E484K or N501Y, can be quickly synthesized in pure form in large quantities by automated methods. JS7 and related synthetic antigens can provide a basis for specific diagnostics for SARS-CoV-2 infections.

Keywords: ACE2 interaction; COVID-19 variants; Neutralizing antibodies; Peptide vaccines; Receptor binding domain; S protein epitopes; Structure based design.

Fig. 1

The JS7 synthetic protein (S443-Y505,red ribbon structure) represents the most variable region of SARS-CoV-2 S protein (relative to SARS-CoV-1 from 2003) which mediates its interaction with ACE2. Top) A cryo-EM structure (PDB entry 7KMB) of the complex shows how the JS7 segment (red) of the RBD (blue) lies at the intersection with the ACE2 cell receptor (gray). Sidechains are shown for the N- (S443) and C- (Y505) terminal residues of JS7 and the 4 Y residues forming hydrogen bonds across the interface. Bottom) Alignment comparing the area of the S proteins of SARS-CoV -2 covered by JS10 (green and yellow highlighted area) and JS7 (green highlighted) with the SARS 2003 sequence. Three peptides from the interface between the S protein and ACE2 are underlined in the JS7 sequence. Three circulating human variants in the JS7 sequence, L452R (recent California), E484K [5] and N501Y [13] are indicated by red arrows, yellow side chains in the structure above.

Fig. 2

Structural characterization of JS7. The CD spectrum of JS7 suggests an antiparallel beta sheet can form, where one is expected from the cryo-EM structure of the SARS-CoV-2 S protein with ACE2 (PDB entry 7KMB). A detailed view of the JS7 protein region in that complex is shown on the right.

Fig. 3

Recombinant and synthetic proteins from SARS-CoV-1 and -2 used for dotspots and ELISA assays (je 1.25 μg) on a 17 % Tris/tricine PAGE developed with Coomassie blue gel stain. Proteins: 1 &2: SARS-CoV-2 RBD-SD1 (SSM-1175) [1] and JSP-657 SARS-Cov-1 528 [2]; 3&4: SARS RBD 219-N1 [3] and SARS CoV-2 RBD [4]; 5 & 6: synthetic antigens JS10 and JS7 (10,130D and 7158D respectively) from the ACE2 interacting area of SARS-CoV-2 S protein. The exact sequences of proteins 1-4 are included in the supplementary material; the sequences of JS10 and JS7 are shown in Fig. 1.

Fig. 4

Serum antibodies inhibit the interaction between the RBD and ACE2. Level of antibodies in patient sera that block the interaction between the RBD (protein 4, Fig. 3) and an ACE2 receptor fragment correlates with disease severity (decreasing OD₆₅₀ indicates increasing inhibition of the interaction).

Fig. 5

Antibody binding to the RBD of SARS-CoV-2 or JS7 (protein 4 or 6, respectively inFig. 3) using sera of a mild, a moderate, 2 severe and 2 critical cases of COVID-19 is inhibited by 3 peptides from the RBD/ACE2 interface (underlined in Fig. 1) in competitive ELISA. The maximum serum dilution to see binding is shown, whereby the maximum serum dilution in the assay was 3.3 × 10⁵. See Supplementary Material for details of sera from patients (Table S1) and controls from spring 2019 (Table S2).

Fig. 6

Competitive ELISA: Binding to JS7 by antibodies in convalescent sera of a mild, a moderate, a severe and two critical cases of COVID-19 is inhibited by Peptide 1: SKVGGNYNYLYRLF from the RBD/ACE2 interface. (maximum serum dilution to see binding is shown, maximum dilution in the assay is 1.1 × 10⁵).