key: cord-0984401-clefny7s
authors: Klumpp-Thomas, Carleen; Kalish, Heather; Hicks, Jennifer; Mehalko, Jennifer; Drew, Matthew; Memoli, Matthew J; Hall, Matthew D; Esposito, Dominic; Sadtler, Kaitlyn
title: D614G Spike Variant Does Not Alter IgG, IgM, or IgA Spike Seroassay Performance
date: 2020-12-01
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa743
sha: 8558513840671ce208e23fcf3d1eeafc7effcd3e
doc_id: 984401
cord_uid: clefny7s

Emergence of a new spike protein variant (D614G) with increased infectivity has prompted many to analyze its role in the SARS-CoV-2 pandemic. There is concern regarding whether an individual exposed to one variant of a virus will have cross-reactive memory to the second. Accordingly, we analyzed the serologic reactivity of both variants, and found that antibodies from 88 donors from a high-incidence population reacted toward both the original spike and the D614 spike variant. These data suggest patients who are exposed to either variant have cross-responsive humoral immunity. This represents an important finding both for SARS-CoV-2 disease biology and for therapeutics.

recognize multiple SARS-CoV-2 proteins including nucleocapsid (N), envelope (E), and spike (S) proteins. Antibodies are highly specific for presented epitopes, and mutations in viruses can lead to reductions in immune responses based on acquired immunity to prior viral exposure [1] , or even during the course of a single infection (so-called viral escape). It was recently demonstrated by Korber et al. through variant tracking that the original SARS-CoV-2 genome spike protein sequence has been supplanted by a changed amino acid at position 614, from D614 to G614 [2] . Furthermore, evidence suggests that the emergent (and now dominant) G614 virus is more infectious, but a great deal remains to be elucidated including a patient's potential to be infected by both variants at once [3] .

The SARS-CoV-2 spike protein is an outward facing homotrimer presented on the surface of the nucleocapsid that mediates binding to the host cell's angiotensin-converting enzyme 2 (ACE2). To test for the presence of antibodies against SARS-CoV-2, we and others have developed enzymelinked immunosorbent assay (ELISA) based seroassays. Our protocol utilizes two types of recombinant primary antigens--full spike ectodomain protein (SARS-CoV-2 S2P) and RBD domain protein (RBD)--as the primary antigens in separate assays for IgG, IgA, and IgM levels [4] . The spike 614 position is not within the RBD domain (although, it is represented in full spike ectodomain constructs), and reported seroassays (including ours) are based on the originally observed aspartic acid at position 614 (D614) [5] .

The consequence of utilizing D614 spike domain in seroassays is that it could impact the specificity of these assays, given it is likely that most infections in the US are now occurring with the G614 variant of SARS-CoV-2. Knowledge of cross-reactivity is essential to interpreting serosurveys and A c c e p t e d M a n u s c r i p t 4 clinical antibody tests [6] . As serosurveys are well underway, we sought urgently to clarify whether serum from recovering/convalescent donors was cross-reactive to both forms of spike protein. Here, we generated a G614 full spike ectodomain construct and incorporated this protein as antigen in an ELISA assay. This spike G614-based assay was compared to the original D614-based assay, and a set of 88 positive samples from a hard-hit (high-incidence) community were applied to both assays.

ELISAs were performed as previously published [4] . Briefly, plates are coated with 1 ug/ml full spike ectodomain trimer (D614G) in 1x PBS overnight at 4 o C. Plates are washed three times with PBST (1x PBS + 0.05% Tween 20), then blocked in 5% non-fat dry milk in PBST for 2 hours at room temp.

Plates are washed again three times with PBST, then samples are added at a 1:400 dilution of serum into blocking buffer and incubated for 1 hour at room temperature. Plates are washed 3 times as previously described, then incubated with anti-IgG, IgM, or IgA cross-adsorbed HRP-linked secondary antibody (ThermoFisher, 1:4000 in blocking buffer) for 1 hour at room temperature. Plates are washed again as described, then 100 ul of TMB substrate (1 Step Ultra TMB Substrate, ThermoFisher) is added for 10 minutes before stopping the reaction with 100ul of stop solution (ThermoFisher). Plates are read on a BioTek Epoch2 plate reader at 450nm and 650nm. Resulting 650 reading is subtracted from the 450 reading prior to data analysis using GraphPad Prism. D614G data were compared to previously published WT spike data [4] . Assay limits of detection were described utilizing monoclonal recombinant antibodies against the D614 spike protein as described previously signed written informed consent prior to enrollment. All other samples were collected under an IRB exemption since these were fully deidentified samples. Eighty-eight (88) convalescent donors were fully de-identified and collected from a high incidence community in New York and New Jersey between April and May, 2020. Sequencing of virus was not completed to evaluate which variant these donors were infected with due to sample availability and scope of study.

To evaluate the ability of antibodies developed during SARS-CoV-2 infection to react against both D614 and G614 variants of the spike protein, we measured serologic reactivity via an enzyme linked immunosorbent assay detecting IgG, IgM and IgA binding to full spike ectodomain trimers. Soluble spike trimers were produced that contained a protein sequence identical to the original S-2P spike variant [7] except for the addition of the D614G mutation. DNA constructs were generated by synthesis (ATUM, Inc.) with gene optimization for expression in human cells and were subcloned into a high-yielding mammalian expression vector driven by a strong CMV51 promoter. Proteins were expressed in Expi293 cells and purified as previously published [5] . In these vectors, the yields of both D614 and G614 spike proteins were similar (approximately 8-10 mg/L). Both proteins purified similarly, and no difference in protein behavior was observed in analytical size exclusion chromatography, demonstrating that both proteins equivalently formed the expected trimeric structures. Using the same ELISA conditions described previously, we tested 88 samples from a high-incidence community and 100 pre-pandemic negative controls (archived pre-2019 sourced from pre-existing NIH study NCT01386424) and compared them to our previously published data for the original D614 spike. We found that sera from donors who tested positive for anti-spike antibodies using D614 spike also tested positive using G614 spike for IgG, IgM and IgA antibodies (Fig. 1a) .

These data positively correlated for all antibody sub-classes, and all donors who tested positive for D614 antibodies also displayed strong reactivity to G614 spike (Fig. 1b) . To further evaluate this A c c e p t e d M a n u s c r i p t 6 correlation, we normalized the IgG values within the linear range of the detector (OD < 3) with the mean of the archival negative controls (Fig. 1c) . After running a linear regression and correlation analysis, we found a strong correlation (Pearson R = 0.9735, p < 0.0001) and good fit (R 2 = 0.9476) between both variants. Furthermore, the slope of the regression line was 1.091 suggesting a 1:1 signal intensity ratio.

A number of seroassays have been published that utilize various spike constructs, and ELISA assays do not provide detection coverage for all possible SARS-CoV-2 antigens, but rather utilize a single protein construct to ascertain seropositivity. Our data show that use of the full spike protein construct should not impact seroassay performance or "miss" seropositive samples. The fact that D614 and G614 both elicited seropositivity is perhaps expected, given that the human immune response is polyclonal [8, 9] . While there may be antibodies produced that recognize spike protein epitopes specific for D614 or G614, these would be among the many antibodies recognizing the SARS-CoV-2 spike protein used in our seroassays [10] . We conclude that human antibody response to SARS-CoV-2 can be detected using D614 or G614 spike protein in ELISA assays. 

Viral escape mechanisms--escapology taught by viruses

Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus

Making sense of mutation: what D614G means for the COVID-19 pandemic remains unclear

Standardization of enzyme-linked immunosorbent assays for serosurveys of the SARS-CoV-2 pandemic using clinical and athome blood sampling

et al Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays

Serologic cross-reactivity of SARS-CoV-2 with endemic and seasonal

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Immune Response to Viruses: Antibody-Mediated Immunity. Encyclopedia of Virology

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Antibody responses to SARS-CoV-2 in patients with COVID-19

The authors would like to thank members of the FNLCR Protein Expression Laboratory (William Gillette, Simon Messing, and Vanessa Wall) for support in DNA production and protein purification.This research was supported in part by the Intramural Research Program of the NIH, including the National Institute of Biomedical Imaging and Bioengineering, the National Institute of Allergy and Infectious Disease, and the National Center for Advancing Translational Sciences. This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract number HHSN261200800001E. Disclaimer: The NIH, its officers, and employees do not recommend or endorse any company, product, or service.

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The authors declare no conflict of interest.A c c e p t e d M a n u s c r i p t