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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-337676

ABSTRACT

Protein-biomolecule interactions play pivotal roles in almost all biological processes, the identification of the interacting protein is essential. By combining a substrate-based proximity labelling activity from the pupylation pathway of Mycobacterium tuberculosis , and the streptavidin (SA)-biotin system, we developed S pecific P upylation as IDE ntity R eporter (SPIDER) for identifying protein-biomolecular interactions. As a proof of principle, SPIDER was successfully applied for global identification of interacting proteins, including substrates for enzyme (CobB), the readers of m 6 A, the protein interactome of mRNA, and the target proteins of drug (lenalidomide). In addition, by SPIDER, we identified SARS-CoV-2 Omicron variant specific receptors on cell membrane and performed in-depth analysis for one candidate, Protein-g. These potential receptors could explain the differences between the Omicron variant and the Prototype strain, and further serve as target for combating the Omicron variant. Overall, we provide a robust technology which is applicable for a wide-range of protein-biomolecular interaction studies.

2.
STAR Protoc ; 3(2): 101238, 2022 06 17.
Article in English | MEDLINE | ID: covidwho-1704569

ABSTRACT

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).


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Escherichia coli , Humans , Proteome
4.
EBioMedicine ; 75: 103773, 2022 01.
Article in English | MEDLINE | ID: covidwho-1587928
5.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-296391

ABSTRACT

mRNA vaccine was approved clinically in 2020. Future development includes delivering mRNA to dendritic cells (DCs) specifically to improve effectiveness and avoid off-target cytotoxicity. Here, we developed virus-like particles (VLPs) as a DC tropic mRNA vaccine vector and showed the prophylactic effects in both SARS-CoV-2 and HSV-1 infection models. The VLP mRNA vaccine elicited strong cytotoxic T cell immunity and durable antibody response with the spike-specific antibodies that lasted for more than 9 months. Importantly, we were able to target mRNA to DCs by pseudotyping VLP with engineered Sindbis virus glycoprotein and found the DC-targeting mRNA vaccine significantly enhanced the titer of antigen-specific IgG, protecting the hACE-2 mice from SARS-CoV-2 infection. Additionally, we showed DC-targeted mRNA vaccine also protected mice from HSV-1 infection when co-delivering the gB and gD mRNA. Thus, the VLP may serve as an in situ DC vaccine and accelerate the further development of mRNA vaccines.

6.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-293476

ABSTRACT

mRNA vaccine was approved clinically in 2020. Future development includes delivering mRNA to dendritic cells (DCs) specifically to improve effectiveness and avoid off-target cytotoxicity. Here, we developed virus-like particles (VLPs) as a DC tropic mRNA vaccine vector and showed the prophylactic effects in both SARS-CoV-2 and HSV-1 infection models. The VLP mRNA vaccine elicited strong cytotoxic T cell immunity and durable antibody response with the spike-specific antibodies that lasted for more than 9 months. Importantly, we were able to target mRNA to DCs by pseudotyping VLP with engineered Sindbis virus glycoprotein and found the DC-targeting mRNA vaccine significantly enhanced the titer of antigen-specific IgG, protecting the hACE-2 mice from SARS-CoV-2 infection. Additionally, we showed DC-targeted mRNA vaccine also protected mice from HSV-1 infection when co-delivering the gB and gD mRNA. Thus, the VLP may serve as an in situ DC vaccine and accelerate the further development of mRNA vaccines.

7.
J Adv Res ; 36: 133-145, 2022 02.
Article in English | MEDLINE | ID: covidwho-1536633

ABSTRACT

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.


Subject(s)
COVID-19 , Antibodies, Viral , Humans , Immunoglobulin G , SARS-CoV-2 , Severity of Illness Index
8.
Genomics Proteomics Bioinformatics ; 2021 Nov 05.
Article in English | MEDLINE | ID: covidwho-1499887

ABSTRACT

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.

9.
J Proteome Res ; 20(12): 5241-5263, 2021 12 03.
Article in English | MEDLINE | ID: covidwho-1483082

ABSTRACT

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.


Subject(s)
COVID-19 , Proteome , Databases, Protein , Humans , Mass Spectrometry , Proteome/genetics , Proteomics , SARS-CoV-2
10.
Curr Med Sci ; 41(6): 1065-1074, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1449989

ABSTRACT

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.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Epitopes, B-Lymphocyte/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , COVID-19/immunology , COVID-19/virology , Epitope Mapping , Humans , Models, Molecular , Mutation , Pandemics , Receptors, Virus/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
13.
Cell Discov ; 7(1): 67, 2021 Aug 17.
Article in English | MEDLINE | ID: covidwho-1360193

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.

14.
STAR Protoc ; 2(3): 100707, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1307268

ABSTRACT

Host humoral immunological response plays an essential role in protection against pathogens. Identification of B-cell epitopes on antigens is required for accurate diagnosis and vaccine development. To map SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) spike linear epitopes, we developed a protocol of profiling sera from patients with COVID-19 (coronavirus disease 2019) via a peptide microarray designed according to spike protein. The protocol is also applicable for other antigens or sample types. This protocol is rapid, high throughput, and the cost is acceptable while it needs specialized microarray facilities. For complete details on the use and execution of this protocol, please refer to Li et al. (2020, 2021a, 2021b).


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/blood , COVID-19/immunology , Epitopes, B-Lymphocyte/immunology , Peptide Fragments/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/blood , COVID-19/virology , Humans , Peptide Fragments/blood , Spike Glycoprotein, Coronavirus/blood
15.
Cell Rep ; 36(2): 109391, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1303454

ABSTRACT

The immunogenicity of the SARS-CoV-2 proteome is largely unknown, especially for non-structural proteins and accessory proteins. In this study, we collect 2,360 COVID-19 sera and 601 control sera. We analyze these sera on a protein microarray with 20 proteins of SARS-CoV-2, building an antibody response landscape for immunoglobulin (Ig)G and IgM. Non-structural proteins and accessory proteins NSP1, NSP7, NSP8, RdRp, ORF3b, and ORF9b elicit prevalent IgG responses. The IgG patterns and dynamics of non-structural/accessory proteins are different from those of the S and N proteins. The IgG responses against these six proteins are associated with disease severity and clinical outcome, and they decline sharply about 20 days after symptom onset. In non-survivors, a sharp decrease of IgG antibodies against S1 and N proteins before death is observed. The global antibody responses to non-structural/accessory proteins revealed here may facilitate a deeper understanding of SARS-CoV-2 immunology.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Viral Nonstructural Proteins/immunology , Viral Regulatory and Accessory Proteins/immunology , Adult , Aged , Antibodies, Viral/immunology , Antibody Formation , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Male , Middle Aged , Protein Array Analysis
16.
Acta Biochim Biophys Sin (Shanghai) ; 53(9): 1134-1141, 2021 Aug 31.
Article in English | MEDLINE | ID: covidwho-1280062

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2-host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.


Subject(s)
COVID-19/drug therapy , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/chemistry , Drug Delivery Systems , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Binding , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Vacuolar Proton-Translocating ATPases/chemistry , Vacuolar Proton-Translocating ATPases/metabolism , Viral Nonstructural Proteins/metabolism
17.
Allergy ; 76(2): 551-561, 2021 02.
Article in English | MEDLINE | ID: covidwho-1140085

ABSTRACT

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.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Carrier State/immunology , SARS-CoV-2/immunology , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/blood , COVID-19/diagnosis , COVID-19 Testing/methods , Carrier State/blood , Carrier State/diagnosis , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Male , Middle Aged
18.
Cell Rep ; 34(13): 108915, 2021 03 30.
Article in English | MEDLINE | ID: covidwho-1128919

ABSTRACT

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.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , COVID-19/epidemiology , COVID-19/genetics , China/epidemiology , Disease Models, Animal , Epitope Mapping/methods , Epitopes/immunology , Female , Humans , Immunoglobulin G/immunology , Male , Mice , Mice, Inbred BALB C , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
20.
Mol Cell Proteomics ; 20: 100059, 2021.
Article in English | MEDLINE | ID: covidwho-1087559

ABSTRACT

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.


Subject(s)
COVID-19/immunology , Epitope Mapping/methods , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes/metabolism , Escherichia coli Proteins/immunology , High-Throughput Nucleotide Sequencing , Humans , Immune Sera/blood , Immune Sera/immunology , Peptide Library
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