We still know very little about how the human immune system responds to SARS-CoV-2. Here we construct a SARS-CoV-2 proteome microarray containing 18 out of the 28 predicted proteins and apply it to ...the characterization of the IgG and IgM antibodies responses in the sera from 29 convalescent patients. We find that all these patients had IgG and IgM antibodies that specifically bind SARS-CoV-2 proteins, particularly the N protein and S1 protein. Besides these proteins, significant antibody responses to ORF9b and NSP5 are also identified. We show that the S1 specific IgG signal positively correlates with age and the level of lactate dehydrogenase (LDH) and negatively correlates with lymphocyte percentage. Overall, this study presents a systemic view of the SARS-CoV-2 specific IgG and IgM responses and provides insights to aid the development of effective diagnostic, therapeutic and vaccination strategies.
The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral ...RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.
Background
The missing asymptomatic COVID‐19 infections have been overlooked because of the imperfect sensitivity of the nucleic acid testing (NAT). Globally understanding the humoral immunity in ...asymptomatic carriers will provide scientific knowledge for developing serological tests, improving early identification, and implementing more rational control strategies against the pandemic.
Measure
Utilizing both NAT and commercial kits for serum IgM and IgG antibodies, we extensively screened 11 766 epidemiologically suspected individuals on enrollment and 63 asymptomatic individuals were detected and recruited. Sixty‐three healthy individuals and 51 mild patients without any preexisting conditions were set as controls. Serum IgM and IgG profiles were further probed using a SARS‐CoV‐2 proteome microarray, and neutralizing antibody was detected by a pseudotyped virus neutralization assay system. The dynamics of antibodies were analyzed with exposure time or symptoms onset.
Results
A combination test of NAT and serological testing for IgM antibody discovered 55.5% of the total of 63 asymptomatic infections, which significantly raises the detection sensitivity when compared with the NAT alone (19%). Serum proteome microarray analysis demonstrated that asymptomatics mainly produced IgM and IgG antibodies against S1 and N proteins out of 20 proteins of SARS‐CoV‐2. Different from strong and persistent N‐specific antibodies, S1‐specific IgM responses, which evolved in asymptomatic individuals as early as the seventh day after exposure, peaked on days from 17 days to 25 days, and then disappeared in two months, might be used as an early diagnostic biomarker. 11.8% (6/51) mild patients and 38.1% (24/63) asymptomatic individuals did not produce neutralizing antibody. In particular, neutralizing antibody in asymptomatics gradually vanished in two months.
Conclusion
Our findings might have important implications for the definition of asymptomatic COVID‐19 infections, diagnosis, serological survey, public health, and immunization strategies.
The combination of NAT and serological testing for IgM antibody significantly improves the detection sensitivity of asymptomatic COVID‐19 infections, compared with NAT alone. S1‐specific IgM antibody response with rapid emergence and disappearance might be helpful to assist NAT for early identification of infectious individuals. A majority of asymptomatics induce very low levels of neutralizing antibody that disappear in two months. Abbreviations: NAT, nucleic acid testing; FI, fluorescence intensity; NT50, half‐maximal neutralizing titer.
Abstract
One of the best ways to control COVID-19 is vaccination. Among the various SARS-CoV-2 vaccines, inactivated virus vaccines have been widely applied in China and many other countries. To ...understand the underlying protective mechanism of these vaccines, it is necessary to systematically analyze the humoral responses that are triggered. By utilizing a SARS-CoV-2 microarray with 21 proteins and 197 peptides that fully cover the spike protein, antibody response profiles of 59 serum samples collected from 32 volunteers immunized with the inactivated virus vaccine BBIBP-CorV were generated. For this set of samples, the microarray results correlated with the neutralization titers of the authentic virus, and two peptides (S1-5 and S2-22) were identified as potential biomarkers for assessing the effectiveness of vaccination. Moreover, by comparing immunized volunteers to convalescent and hospitalized COVID-19 patients, the N protein, NSP7, and S2-78 were identified as potential biomarkers for differentiating COVID-19 patients from individuals vaccinated with the inactivated SARS-CoV-2 vaccine. The comprehensive profile of humoral responses against the inactivated SARS-CoV-2 vaccine will facilitate a deeper understanding of the vaccine and provide potential biomarkers for inactivated virus vaccine-related applications.
To fully decipher the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein, it is essential to assess which part is highly immunogenic in a systematic way. ...We generate a linear epitope landscape of the Spike protein by analyzing the serum immunoglobulin G (IgG) response of 1,051 coronavirus disease 2019 (COVID-19) patients with a peptide microarray. We reveal two regions rich in linear epitopes, i.e., C-terminal domain (CTD) and a region close to the S2′ cleavage site and fusion peptide. Unexpectedly, we find that the receptor binding domain (RBD) lacks linear epitope. We reveal that the number of responsive peptides is highly variable among patients and correlates with disease severity. Some peptides are moderately associated with severity and clinical outcome. By immunizing mice, we obtain linear-epitope-specific antibodies; however, no significant neutralizing activity against the authentic virus is observed for these antibodies. This landscape will facilitate our understanding of SARS-CoV-2-specific humoral responses and might be useful for vaccine refinement.
Display omitted
•A linear epitope landscape of the SARS-CoV-2 Spike from 1,051 COVID-19 patients•Responsive epitopes are highly variable among patients and correlate with severity•The RBD lacks linear epitopes, but two other regions are rich in linear epitopes•Little neutralization activity is observed for the linear-epitope-elicited antibodies
Li et al. construct a B cell linear epitope landscape of SARS-CoV-2 Spike protein, based on a large cohort of COVID-19 patients. The epitope responses were related to disease severity and outcome but mainly elicit non-neutralizing antibodies.
As a ubiquitous bacterial secondary messenger, c‐di‐GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase ...that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c‐di‐GMP strongly binds to CobB. Further, protein deacetylation assays showed that c‐di‐GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl‐CoA. Interestingly, we also found that one of the key enzymes directly involved in c‐di‐GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c‐di‐GMP. Our work establishes a novel negative feedback loop linking c‐di‐GMP biogenesis and CobB‐mediated protein deacetylation.
Synopsis
The bacterial secondary messenger c‐di‐GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. This study reveals a negative feedback loop between c‐di‐GMP biogenesis and CobB‐dependent protein deacetylation.
An Escherichia coli proteome‐wide microarray identifies deacetylase CobB as a c‐di‐GMP‐binding protein.
c‐di‐GMP binding inhibits deacetylase activity of CobB.
CobB deacetylates and activates diguanylate cyclase DgcZ.
DgcZ deacetylation promotes c‐di‐GMP production.
A negative feedback loop between c‐di‐GMP production and the Escherichia coli Sir2‐family deacetylase CobB modulates biogenesis of acetyl‐CoA.
The majority of people in China have been immunized with the inactivated viral vaccine BBIBP‐CorV. The emergence of the Omicron variant raised the concerns about protection efficacy of the ...inactivated viral vaccine in China. However, longitudinal neutralization data describing protection efficacy against Omicron variant is still lacking. Here we present one‐year longitudinal neutralization data of BBIBP‐CorV on authentic Omicron, Delta, and wild‐type strains using 224 sera collected from 14 volunteers who have finished three doses BBIBP‐CorV. The sera were also subjected for monitoring the SARS‐CoV‐2 specific IgG, IgA, and IgM responses on protein and peptide microarrays. The neutralization titers showed different protection efficacies against the three strains. By incorporating IgG and IgA signals of proteins and Spike protein derived peptide on microarray, panels as potential surrogate biomarkers for rapid estimation of neutralization titers were established. These data support the necessity of the 3rd dose of BBIBP‐CorV vaccination. After further validation and assay development, the panels could be used for reliable, convenient and fast evaluation of the efficacy of vaccination.
Database Development for Antibody Arrays Xu, Zhaowei; Huang, Likun; Tao, Sheng-Ce
Methods in molecular biology (Clifton, N.J.),
2021, Letnik:
2237
Journal Article
Antibody arrays have been widely applied in both basic research and clinical studies. Data analysis, archiving, and sharing of resulting data are very important for exploring and expanding the power ...of antibody microarray studies. The protein microarray database (PMD) has provided standards tailored for the management of protein microarray data and constructed an automated pipeline for array data analysis. In this chapter, we will describe the framework design, platform construction, and analysis tool integration of the PMD.
A pipette-actuated capillary array comb (PAAC) system operated on a smartphone-based hand-held device has been successfully developed for the multiplex detection of bacteria in a "sample-to-answer" ...manner. The PAAC consists of eight open capillaries inserted into a cylindrical plastic base with a piece of chitosan-modified glass filter paper embedded in each capillary. During the sample preparation, a PAAC was mounted into a 1 mL pipette tip with an enlarged opening and was operated with a 1 mL pipette for liquid handling. The cell lysate was drawn and expelled through the capillaries three times to facilitate the DNA capture on the embedded filter discs. Following washes with water, the loop-mediated isothermal amplification (LAMP) reagents were aspirated into the capillaries, in which the primers were pre-fixed with chitosan. After that, the PAAC was loaded into the smartphone-based device for a one-hour amplification at 65 °C and end-point detection of calcein fluorescence in the capillaries. The DNA capture efficiency of a 1.1 mm-diameter filter disc was determined to be 97% of λ-DNA and the coefficient of variation among the eight capillaries in the PAAC was only 2.2%. The multiplex detection of genomic DNA extracted from Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus provided limits of detection of 200, 500, and 500 copies, respectively, without any cross-contamination and cross reactions. "Sample-to-answer" detection of E. coli samples was successfully completed in 85 minutes, demonstrating a sensitivity of 200 cfu per capillary. The multiplex "sample-to-answer" detection, the streamlined operation, and the compact device should facilitate a broad range of applications of our PAAC system in point-of-care testing.
PDF
