Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Protein-biomolecule interactions play pivotal roles in almost all biological processes. For a biomolecule of interest, the identification of the interacting protein(s) is essential. For this need, although many assays are available, highly robust and reliable methods are always desired. By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin (SA)-biotin system, we developed the Specific Pupylation as IDEntity Reporter (SPIDER) method for identifying protein-biomolecule interactions. Using SPIDER, we validated the interactions between the known binding proteins of protein, DNA, RNA, and small molecule. We successfully applied SPIDER to construct the global protein interactome for m6A and mRNA, identified a variety of uncharacterized m6A binding proteins, and validated SRSF7 as a potential m6A reader. We globally identified the binding proteins for lenalidomide and CobB. Moreover, we identified SARS-CoV-2-specific receptors on the cell membrane. Overall, SPIDER is powerful and highly accessible for the study of protein-biomolecule interactions.

Rapid and Accurate Detection of SARS-CoV-2 Using an iPad-Controlled, High-Throughput, Portable, and Multiplex Hive-Chip Platform (HiCube).

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the most effective measures to control the coronavirus disease 2019 (COVID-19) pandemic. However, there is still lack of an ideal detection platform capable of high sample throughput, portability, and multiplicity. Herein, by combining Hive-Chip (capillary microarray) and reverse transcriptional loop-mediated isothermal amplification (RT-LAMP), we developed an iPad-controlled, high-throughput (48 samples at one run), portable (smaller than a backpack), multiplex (monitoring 8 gene fragments in one reaction), and real-time detection platform for SARS-CoV-2 detection. This platform is composed of a portable Hive-Chip device (HiCube; 32.7 × 29.7 × 20 cm, 5 kg), custom-designed software, and optimized Hive-Chips. RT-LAMP primers targeting seven SARS-CoV-2 genes (S, E, M, N, ORF1ab, ORF3a, and ORF7a) and one positive control (human RNase P) were designed and prefixed in the Hive-Chip. On-chip RT-LAMP showed that the limit of detection (LOD) of SARS-CoV-2 synthetic RNAs is 1 copy/µL, and there is no cross-reaction among different target genes. The platform was validated by 100 clinical samples of SARS-CoV-2, and the results were highly consistent with those of the traditional real-time PCR assay. In addition, on-chip detection of 6 other respiratory pathogens showed no cross-reactivity. Overall, our platform has great potential for fast, accurate, and on-site detection of SARS-CoV-2.

Longitudinal neutralization activities on authentic Omicron variant provided by three doses of BBIBP-CorV vaccination during one year.

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.

Nucleic acid testing of SARS-CoV-2: A review of current methods, challenges, and prospects.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has brought a huge threat to public health and the global economy. Rapid identification and isolation of SARS-CoV-2-infected individuals are regarded as one of the most effective measures to control the pandemic. Because of its high sensitivity and specificity, nucleic acid testing has become the major method of SARS-CoV-2 detection. A deep understanding of different diagnosis methods for COVID-19 could help researchers make an optimal choice in detecting COVID-19 at different symptom stages. In this review, we summarize and evaluate the latest developments in current nucleic acid detection methods for SARS-CoV-2. In particular, we discuss biosensors and CRISPR-based diagnostic systems and their characteristics and challenges. Furthermore, the emerging COVID-19 variants and their impact on SARS-CoV-2 diagnosis are systematically introduced and discussed. Considering the disease dynamics, we also recommend optional diagnostic tests for different symptom stages. From sample preparation to results readout, we conclude by pointing out the pain points and future directions of COVID-19 detection.

Characterization and epitope mapping of monoclonal antibodies against PEDV N protein.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets. The nucleocapsid (N) protein of PEDV is a highly conserved protein with strong immunogenicity and palys an important role in PEDV diagnosis. However, epitopes on the PEDV N protein have not yet been well characterized. Here, 32 monoclonal antibodies (mAbs) against the PEDV N protein were produced and identified. Six new epitopes were first identified by using a high-throughput epitope mapping method named AbMap. Sequence analysis revealed that among the six epitopes five epitopes were highly conserved among different PEDV strains. We also confirmed that the mAbs derived from the six epitopes of PEDV N protein, have no cross-reactivity with transmissible gastro enteritis virus or porcine delta coronavirus. These mAbs and their defined epitopes will help to understand the N protein structure and immunological characteristics, and to develop a rapid, accurate PEDV diagnosis method.

Age- and Severity-Associated Humoral Immunity Response in COVID-19 Patients: A Cohort Study from Wuhan, China.

Age has been found to be the single most significant factor in COVID-19 severity and outcome. However, the age-related severity factors of COVID-19 have not been definitively established. In this study, we detected SARS-CoV-2-specific antibody responses and infectious disease-related blood indicators in 2360 sera from 783 COVID-19 patients, with an age range of 1-92 years. In addition, we recorded the individual information and clinical symptoms of the patients. We found that the IgG responses for S1, N, and ORF3a and the IgM for NSP7 were associated with severe COVID-19 at different ages. The IgM responses for the S-protein peptides S1-113 (aa 673-684) and S2-97 (aa 1262-1273) were associated with severe COVID-19 in patients aged <60. Furthermore, we found that the IgM for S1-113 and NSP7 may play a protective role in patients aged <60 and >80, respectively. Regarding clinical parameters, we analyzed the diagnostic ability of five clinical parameters for severe COVID-19 in six age groups and identified three-target panel, glucose, IL-6, myoglobin, IL-6, and NT proBNP as the appropriate diagnostic markers for severe COVID-19 in patients aged <41, 41-50, 51-60, 61-70, 71-80, and >80, respectively. The age-associated severity factors revealed here will facilitate our understanding of COVID-19 immunity and diagnosis, and eventually provide meaningful information for combating the pandemic.

Ultrasensitive monitoring of SARS-CoV-2-specific antibody responses based on a digital approach reveals one week of IgG seroconversion.

COVID-19 is still unfolding, while many people have been vaccinated. In comparison to nucleic acid testing (NAT), antibody-based immunoassays are faster and more convenient. However, its application has been hampered by its lower sensitivity and the existing fact that by traditional immunoassays, the measurable seroconversion time of pathogen-specific antibodies, such as IgM or IgG, lags far behind that of nucleic acids. Herein, by combining the single molecule array platform (Simoa), RBD, and a previously identified SARS-CoV-2 S2 protein derivatized 12-aa peptide (S2-78), we developed and optimized an ultrasensitive assay (UIM-COVID-19 assay). Sera collected from three sources were tested, i.e., convalescents, inactivated virus vaccine-immunized donors and wild-type authentic SARS-CoV-2-infected rhesus monkeys. The sensitivities of UIM-COVID-19 assays are 100-10,000 times higher than those of conventional flow cytometry, which is a relatively sensitive detection method at present. For the established UIM-COVID-19 assay using RBD as a probe, the IgG and IgM seroconversion times after vaccination were 7.5 and 8.6 days vs. 21.4 and 24 days for the flow cytometry assay, respectively. In addition, using S2-78 as a probe, the UIM-COVID-19 assay could differentiate COVID-19 patients (convalescents) from healthy people and patients with other diseases, with AUCs ranging from 0.85-0.95. In summary, the UIM-COVID-19 we developed here is a promising ultrasensitive biodetection strategy that has the potential to be applied for both immunological studies and diagnostics.

Evaluation of Spike Protein Epitopes by Assessing the Dynamics of Humoral Immune Responses in Moderate COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike protein (S) of SARS-CoV-2 is a major target for diagnosis and vaccine development because of its essential role in viral infection and host immunity. Currently, time-dependent responses of humoral immune system against various S protein epitopes are poorly understood. In this study, enzyme-linked immunosorbent assay (ELISA), peptide microarray, and antibody binding epitope mapping (AbMap) techniques were used to systematically analyze the dynamic changes of humoral immune responses against the S protein in a small cohort of moderate COVID-19 patients who were hospitalized for approximately two months after symptom onset. Recombinant truncated S proteins, target S peptides, and random peptides were used as antigens in the analyses. The assays demonstrated the dynamic IgM- and IgG recognition and reactivity against various S protein epitopes with patient-dependent patterns. Comprehensive analysis of epitope distribution along the spike gene sequence and spatial structure of the homotrimer S protein demonstrated that most IgM- and IgG-reactive peptides were clustered into similar genomic regions and were located at accessible domains. Seven S peptides were generally recognized by IgG antibodies derived from serum samples of all COVID-19 patients. The dynamic immune recognition signals from these seven S peptides were comparable to those of the entire S protein or truncated S1 protein. This suggested that the humoral immune system recognized few conserved S protein epitopes in most COVID-19 patients during the entire duration of humoral immune response after symptom onset. Furthermore, in this cohort, individual patients demonstrated stable immune recognition to certain S protein epitopes throughout their hospitalization period. Therefore, the dynamic characteristics of humoral immune responses to S protein have provided valuable information for accurate diagnosis and immunotherapy of COVID-19 patients.

SARS-CoV-2 proteome microarray for COVID-19 patient sera profiling.

The immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome is largely unknown. Here we describe a protocol for analyzing sera samples with SARS-CoV-2 proteome microarray. The proteins were expressed by either E. coli expression system or eukaryotic cell expression systems and obtained by affinity purification. The protocol includes microarray fabricating and sera profiling, which will be used to build an antibody response landscape for IgG and IgM. The protocol may help to facilitate a deeper understanding of immunity related to SARS-CoV-2. For complete details on the use and execution of this protocol, please refer to Li et al. (2021c).

Anti-SARS-CoV-2 IgG responses are powerful predicting signatures for the outcome of COVID-19 patients.

Introduction: The COVID-19 global pandemic is far from ending. There is an urgent need to identify applicable biomarkers for early predicting the outcome of COVID-19. Growing evidences have revealed that SARS-CoV-2 specific antibodies evolved with disease progression and severity in COIVD-19 patients. Objectives: We assumed that antibodies may serve as biomarkers for predicting the clinical outcome of hospitalized COVID-19 patients on admission. Methods: By taking advantage of a newly developed SARS-CoV-2 proteome microarray, we surveyed IgG responses against 20 proteins of SARS-CoV-2 in 1034 hospitalized COVID-19 patients on admission and followed till 66 days. The microarray results were further correlated with clinical information, laboratory test results and patient outcomes. Cox proportional hazards model was used to explore the association between SARS-CoV-2 specific antibodies and COVID-19 mortality. Results: Nonsurvivors (n = 955) induced higher levels of IgG responses against most of non-structural proteins than survivors (n = 79) on admission. In particular, the magnitude of IgG antibodies against 8 non-structural proteins (NSP1, NSP4, NSP7, NSP8, NSP9, NSP10, RdRp, and NSP14) and 2 accessory proteins (ORF3b and ORF9b) possessed significant predictive power for patient death, even after further adjustments for demographics, comorbidities, and common laboratory biomarkers for disease severity (all with p trend < 0.05). Additionally, IgG responses to all of these 10 non-structural/accessory proteins were also associated with the severity of disease, and differential kinetics and serum positive rate of these IgG responses were confirmed in COVID-19 patients of varying severities within 20 days after symptoms onset. The area under curves (AUCs) for these IgG responses, determined by computational cross-validations, were between 0.62 and 0.71. Conclusions: Our findings might have important implications for improving clinical management of COVID-19 patients.

COVID-ONE-hi: The One-stop Database for COVID-19-specific Humoral Immunity and Clinical Parameters.

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (named COVID-ONE-hi). COVID-ONE-hi is based on the data that contain the IgG/IgM responses to 24 full-length/truncated proteins corresponding to 20 of 28 known SARS-CoV-2 proteins and 199 spike protein peptides against 2360 serum samples collected from 783 COVID-19 patients. In addition, 96 clinical parameters for the 2360 serum samples and basic information for the 783 patients are integrated into the database. Furthermore, COVID-ONE-hi provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the "START" button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-hi is freely available at www.COVID-ONE.cn.

Advances and Utility of the Human Plasma Proteome.

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.

Epitope Analysis of Anti-SARS-CoV-2 Neutralizing Antibodies.

Coronavirus disease 2019 is threatening thousands of millions of people around the world. In the absence of specific and highly effective medicines, the treatment of infected persons is still very challenging. As therapeutics, neutralizing antibodies (NAbs) have great potential. Many NAbs have been reported, and most target various regions on the receptor-binding domain of the spike (S) protein, or the N-terminal domain. Several NAbs and NAb cocktails have been authorized for emergency use, and more are in clinical trials or are under development. In this review, considering the angle of binding epitopes on the S protein, we summarize the functions and the underlying mechanisms of a set of well-recognized NAbs and provide guidance for vaccine design and the combinatorial use of these antibodies. In addition, we review the NAbs and NAb cocktails that have been approved for emergency use and discuss the effectiveness of these NAbs for combating severe acute respiratory syndrome coronavirus 2 mutants.

Systematic profiling of SARS-CoV-2-specific IgG responses elicited by an inactivated virus vaccine identifies peptides and proteins for predicting vaccination efficacy.

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.