key: cord-0875210-aejo3ryd
authors: Schein, C. H.; McClellan, S. F.; Levine, C. B.; Negi, S. S.; Braun, W.; Dreskin, S.; Anaya, E. S.; Schmidt, J.
title: Synthetic protein antigens for COVID-19 diagnostics
date: 2021-03-01
journal: nan
DOI: 10.1101/2021.02.23.21251934
sha: 3373d3df9e678b5ba087da078b77d76f59b5ad07
doc_id: 875210
cord_uid: aejo3ryd

There is an urgent need for inexpensive, rapid and specific antigen-based assays to test for infection with SARS-CoV-2 and distinguish variants arising as the COVID-19 pandemic spreads. 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 N501Y, can be quickly synthesized in a 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.

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 showing three circulating human variants (red arrows, yellow side chains) at L452R (recent California), E484K 4 and N501Y 15 .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted March 1, 2021. ; https://doi.org/10.1101/2021.02.23.21251934 doi: medRxiv preprint spectrum of JS7 (Fig. S2) suggests it forms the flexible 83 structure and β-strands that characterize the experimental 84 structure. 85 We compared JS7 to four recombinant proteins obtained Convalescent sera from 9 hospitalized patients with COVID-19 disease of varying severity, from 98 "mild" to critical, were used for dotspots, ELISAs and an assay for inhibition of association of the RBD 99 with its cellular receptor, ACE2. As shown in Table 1S , these patients had co-morbidities that have 100 been found, in other studies, to be associated with hospitalization due to COVID-19. Of the 9, 5 were 101 known to be diabetic, and 6 had elevated glucose levels at time of diagnosis. Four of the 6 patients who 102 had severe or critical disease were obese (BMI>30). Notably, the one patient who succumbed did not 103 have these risk factors except for age >70. As Fig. 3 shows, sera from patients with severe or critical 104 disease had antibodies that interfered with the binding of the SARS-CoV-2 RBD (protein 4) to an ACE2 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Table 1 . Antibody binding to recombinant S protein and JS7 from sera of 3 COVID 19 patients and a control serum. Methods and data for all 9 patients and controls are described in Supplementary material. Recombinant proteins (1-4) or synthetic proteins (5,6) (see Fig. 2 ) were bound to nitrocellulose for dotspots 9 and reacted with sera diluted 1:100. ELISA, for binding to recombinant SAR-CoV-2 RBD (Protein 4) or JS7 (6) is the highest dilution factor of serum where significant OD450 is measured; maximum dilution in the assay was 3.3 x 10 5 . ACE2 inhibition is the degree to which each serum inhibited the binding of an ACE2 fragment to SARS-CoV-2 RBD (protein 4). A negative number indicates inhibition, where protein 4 inhibition (of binding to itself) = -0.82. (average of triplicates is shown). (protein 4, Fig. 2) and an ACE2 receptor fragment correlate with disease severity ((decreasing OD650 indicates increasing inhibition of the interaction).

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 4 or 6, Fig. 2, respectively) in 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 in competitive ELISA (maximum serum dilution to see binding). See supplementary material for peptide sequences, patient (Table S1 ) and control sera from spring 2019 (Table S2) . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 1, 2021. ; https://doi.org/10.1101/2021.02.23.21251934 doi: medRxiv preprint patients with only mild COVID-19 disease; this was confirmed by ELISA (Figs. 4,5) . This binding 153 could be due to previous infections with other coronaviruses 37 , or simply reflect germline antibody 154 recognition. In support of the latter, others have found that neutralizing monoclonal antibodies against 155 the SARS-CoV-2 S protein, isolated from many different donors, were from public clonotypes, bound 156 autoantigens and contained relatively few somatic mutations from germline 38 .

The greatest advantage of using JS7 for diagnostic purposes is that its sequence can be rapidly SARS-CoV-1 antibody, CR3022, whose epitope is located in the conserved area just upstream of the 165 JS7 site, 28 was greatly reduced by the single amino acid change, P to A found in the SARS-CoV-2 166 protein. 29 Further, while all 9 COVID-19 patients in this study had antibodies that recognize the RBD of 167 SARS-CoV-2, one patient with mild illness did not recognize the whole S protein of SARS-CoV-1 168 (protein 2), despite the 80% overall sequence identity (Table 1, is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted March 1, 2021. ;

representing areas of maximum variation in the RBD that can be used to rapidly determine specific variants and support planning clinical treatments. Reagents and linkers needed for assays can be 176 integrated into variants of JS7 during synthesis, while controlling its sequence, stereochemistry and 177 disulfide patterns. The JS7 protein, further modified to display variants and to be serum stable, may also 178 have a future as a vaccine additive or booster. For example, recent methods have been developed to 179 generate one component-synthetic proteins with incorporated adjuvant 42 .

In conclusion, we show here that short synthetic proteins, which can be produced quickly in large 181 quantities, can be the basis of specific assays to detect antibodies in sera that that are produced in 182 response to infection with SARS-CoV-2. Antibodies in sera of 9 patients who had PCR confirmed, 183 COVID-19 of differing severity recognized the JS7 protein. The sera from 8 of these patients was taken 184 up to 2 months after diagnosis, while the 9 th patient died during hospitalization. JS7 and future variants 185 should prove to be another tool for eventually defeating the current outbreak. 

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV 222 antibody

Characterization of a novel coronavirus associated with severe acute respiratory 224 syndrome

SARS-CoV-2 RBD219-N1C1: A Yeast-Expressed SARS-CoV-2 Recombinant 226

Receptor-Binding Domain Candidate Vaccine Stimulates Virus Neutralizing Antibodies and T-227 cell Immunity in Mice. bioRxiv : the preprint server for biology

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal 229

Covid-19: New coronavirus variant is identified in UK

Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-24

D-graph clusters flaviviruses and beta-coronaviruses

We thank Wendy S. Baker for expert technical assistance, and Wen-Hsian