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Microbiol Spectr ; 9(2): e0141621, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1495015


The rapid worldwide spread of SARS-CoV-2 has accelerated research and development for controlling the COVID-19 pandemic. A multi-coronavirus protein microarray was created containing full-length proteins, overlapping protein fragments of various lengths, and peptide libraries from SARS-CoV-2 and four other human coronaviruses. Sera from confirmed COVID-19 patients as well as unexposed individuals were applied to multicoronavirus arrays to identify specific antibody reactivity. High-level IgG, IgM, and IgA reactivity to structural proteins S, M, and N of SARS-CoV-2, as well as accessory proteins such as ORF3a and ORF7a, were observed that were specific to COVID-19 patients. Antibody reactivity against overlapping 100-, 50-, and 30-amino acid fragments of SARS-CoV-2 proteins was used to identify antigenic regions. Numerous proteins of SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and the endemic human coronaviruses HCoV-NL63 and HCoV-OC43 were also more reactive with IgG, IgM, and IgA in COVID-19 patient sera than in unexposed control sera, providing further evidence of immunologic cross-reactivity between these viruses. Whereas unexposed individuals had minimal reactivity against SARS-CoV-2 proteins that poorly correlated with reactivity against HCoV-NL63 and HCoV-OC43 S2 and N proteins, COVID-19 patient sera had higher correlation between SARS-CoV-2 and HCoV responses, suggesting that de novo antibodies against SARS-CoV-2 cross-react with HCoV epitopes. Array responses were compared with validated spike protein-specific IgG enzyme-linked immunosorbent assays (ELISAs), showing agreement between orthologous methods. SARS-CoV-2 microneutralization titers were low in the COVID-19 patient sera but correlated with array responses against S and N proteins. The multi-coronavirus protein microarray is a useful tool for mapping antibody reactivity in COVID-19 patients. IMPORTANCE With novel mutant SARS-CoV-2 variants of concern on the rise, knowledge of immune specificities against SARS-CoV-2 proteins is increasingly important for understanding the impact of structural changes in antibody-reactive protein epitopes on naturally acquired and vaccine-induced immunity, as well as broader topics of cross-reactivity and viral evolution. A multi-coronavirus protein microarray used to map the binding of COVID-19 patient antibodies to SARS-CoV-2 proteins and protein fragments as well as to the proteins of four other coronaviruses that infect humans has shown specific regions of SARS-CoV-2 proteins that are highly reactive with patient antibodies and revealed cross-reactivity of these antibodies with other human coronaviruses. These data and the multi-coronavirus protein microarray tool will help guide further studies of the antibody response to COVID-19 and to vaccination against this worldwide pandemic.

Antibodies, Viral/immunology , Coronavirus NL63, Human/immunology , Coronavirus OC43, Human/immunology , Epitopes/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , SARS-CoV-2/immunology , Antibodies, Viral/blood , Binding Sites, Antibody/immunology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Cross Reactions/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Phosphoproteins/immunology , Protein Array Analysis , Spike Glycoprotein, Coronavirus/immunology , Viral Proteins/immunology , Viroporin Proteins/immunology
Biosens Bioelectron ; 195: 113649, 2022 Jan 01.
Article in English | MEDLINE | ID: covidwho-1415226


Considering the worldwide health crisis associated with highly contagious severe respiratory disease of COVID-19 outbreak, the development of multiplexed, simple and rapid diagnostic platforms to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in high demand. Here, a nucleic acid amplification-free electrochemical biosensor based on four-way junction (4-WJ) hybridization is presented for the detection of SARS-CoV-2. To form a 4-WJ structure, a Universal DNA-Hairpin (UDH) probe is hybridized with two adaptor strands and a SARS-CoV-2 RNA target. One of the adaptor strands is functionalized with a redox mediator that can be detected using an electrochemical biosensor. The biosensor could simultaneously detect 5.0 and 6.8 ag/µL of S and Orf1ab genes, respectively, within 1 h. The biosensor was evaluated with 21 clinical samples (16 positive and 5 negative). The results revealed a satisfactory agreement with qRT-PCR. In conclusion, this biosensor has the potential to be used as an on-site, real-time diagnostic test for COVID-19.

Biosensing Techniques , COVID-19 , Diagnostic Tests, Routine , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity