Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray

Assis, Rafael Ramiro de; Jain, Aarti; Nakajima, Rie; Jasinskas, Algis; Felgner, Jiin; Obiero, Joshua M.; Adenaiye, Oluwasanmi; Tai, Sheldon; Hong, Felix T.; Norris, Philip J.; Stone, Mars; Simmons, Graham; Bagri, Anil; Schreiber, Martin A.; Buser, Andreas; Holbro, Andreas; Battegay, Manuel; Milton, Donald K.; Davies, Huw; Corash, Laurence; Busch, Michael P.; Felgner, Philip L.; Khan, Saahir

doi:10.1101/2020.04.15.043364

Cited by 76 publications

(93 citation statements)

References 17 publications

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“…Furthermore, it should be appreciated that the current platform is programmed to detect the capture of IgG antibodies; thus, the resultant signal (or lack thereof) for a particular antigen could be the consequence of prevalences between different isotypes (IgA, IgD, IgE, IgG, IgM), which are known to evolve during the course of infection and subsequent resolution. Although currently focused on three antigens, this platform can be readily expanded to study differential antibody responses to different SARS-CoV-2 antigens and subdomains of those antigens amongst individuals, which are actively being investigated by others [52][53][54][55]. We are currently working to include not only other antigens from SARS-CoV-2 (E protein, M protein, etc.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Herrera

Morano

Celikgil

et al. 2020

Preprint

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Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F™ and ExpiCHO-S™ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

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Section: Discussionmentioning

confidence: 99%

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Herrera

Morano

Celikgil

et al. 2020

Preprint

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“…Second, we applied a robust SARS-CoV-2 Antigen Microarray technology which has superior sensitivity and specificity relative to what were currently available FDA-approved tests used by others. 15 Third, we recruited a sufficiently large sample of adults to calculate seroprevalence by race/ethnicity, age, and gender, which may uncover important differences across these groups. Fourth, we recruit subjects by administering a questionnaire without initially disclosing an offer for an antibody test.…”

Section: Introductionmentioning

confidence: 99%

Estimated Seroprevalence of SARS-CoV-2 Antibodies Among Adults in Orange County, California

Bruckner

Parker

Bartell

et al. 2020

Preprint

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Background: Clinic-based estimates of SARS-CoV-2 may considerably underestimate the total number of infections. Access to testing in the US has been heterogeneous and symptoms vary widely in infected persons. Public health surveillance efforts and metrics are therefore hampered by underreporting. We set out to provide a minimally biased estimate of SARS-CoV-2 seroprevalence among adults for a large and diverse county (Orange County, CA, population 3.2 million). Methods: We implemented a surveillance study that minimizes response bias by recruiting adults to answer a survey without knowledge of later being offered SARS-CoV-2 test. Several methodologies were used to retrieve a population-representative sample. Participants (n=2,979) visited one of 11 drive-thru test sites from July 10th to August 16th, 2020 (or received an in-home visit) to provide a finger pin-prick sample. We applied a robust SARS-CoV-2 Antigen Microarray technology, which has superior measurement validity relative to FDA-approved tests. Findings: Participants include a broad age, gender, racial/ethnic, and income representation. Adjusted seroprevalence of SARS-CoV-2 infection was 11.5% (95%CI: 10.5% to 12.4%). Formal bias analyses produced similar results. Prevalence was elevated among Hispanics (vs. other non-Hispanic: prevalence ratio [PR]= 1.47, 95% CI: 1.22 to 1.78) and household income <$50,000 (vs. >$100,000: PR= 1.42, 95% CI: 1.14 to 1.79). Interpretation: Results from a diverse population using a highly specific and sensitive microarray indicate a SARS-CoV-2 seroprevalence of ~12 percent. This population-based seroprevalence is seven-fold greater than that using official County statistics. In this region, SARS-CoV-2 also disproportionately affects Hispanic and low-income adults.

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“…Moreover, infection with SARS-CoV-2 has recently been shown to increase IgG seroreactivity to HCoVs 21 , supporting the existence of shared epitopes. Numerous serology assays that are being developed have also detected SARS-CoV-2reactive antibodies in a considerable proportion of SARS-CoV-2-uninfected individuals, which has often been considered as unspecific binding, rather than HCoV-elicited specific antibodies [9][10][11][12][13][14][15][16][17][18][19][20]22 . Based on our data, we would argue that this is the result of cross-reactivity between HCoVs.…”

mentioning

confidence: 99%

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans

Ng¹,

Faulkner²,

Cornish³

et al. 2020

Preprint

126

170

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Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections 1 . Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 , is a recent zoonotic infection that has quickly reached pandemic spread 2,3 . Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 Spike (S) glycoprotein, we demonstrate the presence of pre-existing immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies, exclusively of the IgG class, were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals with recent HCoV infection and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 Sreactive IgG antibodies, as well as concomitant IgM and IgA antibodies throughout the observation period of 6 weeks since symptoms onset. HCoV patient sera also variably reacted with SARS-CoV-2 S and nucleocapsid (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, HCoV patient sera exhibited specific neutralising activity against SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for serology, seroprevalence and vaccine studies, as well as for our understanding of susceptibility to and natural course of SARS-CoV-2 infection. ResultsImmune cross-reactivity among seasonally spreading human coronaviruses (HCoVs) has long been hypothesised to provide cross-protection, albeit transient, against infection with distinct HCoV types 1,4,5 . To determine the degree of cross-reactivity between HCoVs and the recently introduced zoonotic coronavirus SARS-CoV-2, we developed a sensitive flow cytometry-based assay for detection of SARS-CoV-2-binding antibodies. Sera from COVID-19 patients at University College London Hospitals (UCLH) (Table S1), contained high levels of IgG, IgM and IgA antibodies recognising the wild-type Spike (S) glycoprotein of SARS-CoV-2 expressed on the surface of HEK293T cells, whereas control sera did not (Fig. 1a). Notably, sera from a proportion patients with confirmed HCoV infection collected before or during the early spread of SARS-CoV-2 in the UK (Table S1), also contained SARS-CoV-2 S-specific antibodies (Fig. 1a). However, the latter sera contained only lower levels of S-specific IgG and no IgM or IgA antibodies, which clearly distinguished them from COVID-19 patient sera (Fig. 1a). The SARS-CoV-2 S protein is proteolytically processed into the S1 and S2 subunits that mediate target cell attachment and entry, respectively 6,7 . S2 exhibits a higher degree of homology among coronaviruses than S1 (Extended data Fig. 1...

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Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray

Cited by 76 publications

References 17 publications

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

Estimated Seroprevalence of SARS-CoV-2 Antibodies Among Adults in Orange County, California

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans

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