Abstract
A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155–71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155–171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.
Highlights
In the frame of the ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reliable diagnostic and prognostic tools are constantly needed for the appropriate management of patients, to guide the development and implementation of surveillance and prevention measures, and to assess the efficacy of therapeutic interventions and vaccines
While molecular and antigenic testing of respiratory samples are used for the diagnosis of acute SARS-CoV-2 infection, serology testing allows the identification of individuals with recent or past exposure to SARS-CoV-2
Preliminary data on IgG response in sera of Covid-19 patients were generated using high-density peptide array made of 4883 different peptides, 15 amino acids long, 13 amino acids overlap (PEPperCHIP® SARS-CoV-2 Proteome Microarray) displaying sequences of ORF1ab polyprotein, surface glycoprotein (S), ORF3a protein, envelope protein (E), membrane glycoprotein (M), ORF6 protein, ORF7a protein, ORF8 protein, nucleocapsid phosphoprotein (N) and ORF10
Summary
In the frame of the ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reliable diagnostic and prognostic tools are constantly needed for the appropriate management of patients, to guide the development and implementation of surveillance and prevention measures, and to assess the efficacy of therapeutic interventions and vaccines. While molecular and antigenic testing of respiratory samples are used for the diagnosis of acute SARS-CoV-2 infection, serology testing allows the identification of individuals with recent or past exposure to SARS-CoV-2. Serological tests can be combined with molecular testing to improve the sensitivity and specificity of diagnosis [1] and represent key instruments for epidemiological surveillance to guide public health interventions [2]. Data from representative and reliable seroprevalence studies will greatly support decision-making practices in terms of physical distancing and other restrictions. The use of seroprevalence data to inform policymaking strictly depends on the accuracy and reliability of tests. Currently available serological assays are biased by false-positive and false-negative results as pointed out by sub-optimal sensitivity and specificity performance (https://www.fda.gov/medical-devices/emergency-situationsmedical-devices/eua-authorized-serology-test-performance)
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