Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 24
Filter
2.
Cell Chem Biol ; 29(2): 215-225.e5, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1664751

ABSTRACT

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.


Subject(s)
Anticoagulants/pharmacology , Aptamers, Nucleotide/pharmacology , Blood Coagulation/drug effects , Factor V/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Amino Acid Sequence , Anticoagulants/chemistry , Anticoagulants/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Pairing , Binding Sites , COVID-19/blood , COVID-19/drug therapy , Cell Membrane/chemistry , Cell Membrane/metabolism , Factor V/chemistry , Factor V/genetics , Factor V/metabolism , Factor Va/chemistry , Factor Va/genetics , Factor Va/metabolism , Heparin, Low-Molecular-Weight/chemistry , Heparin, Low-Molecular-Weight/metabolism , Humans , Immune Sera/chemistry , Immune Sera/metabolism , Models, Molecular , Nucleic Acid Conformation , Protamines , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , SELEX Aptamer Technique , Substrate Specificity
3.
Signal Transduct Target Ther ; 7(1): 18, 2022 01 19.
Article in English | MEDLINE | ID: covidwho-1639142

ABSTRACT

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants' spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011-2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10-100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19 Vaccines/metabolism , COVID-19/prevention & control , Immunogenicity, Vaccine , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Substitution , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Binding Sites , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/chemistry , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Gene Expression , Humans , Immune Sera/chemistry , Influenza A Virus, H3N2 Subtype/chemistry , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H3N2 Subtype/immunology , Influenza Vaccines/administration & dosage , Influenza Vaccines/chemistry , Influenza Vaccines/metabolism , Influenza, Human/immunology , Influenza, Human/prevention & control , Influenza, Human/virology , Models, Molecular , Mutation , Neutralization Tests , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
4.
Cell Host Microbe ; 29(12): 1738-1743.e4, 2021 12 08.
Article in English | MEDLINE | ID: covidwho-1574127

ABSTRACT

Different SARS-CoV-2 vaccines are approved in various countries, but few direct comparisons of the antibody responses they stimulate have been reported. We collected plasma specimens in July 2021 from 196 Mongolian participants fully vaccinated with one of four COVID-19 vaccines: Pfizer/BioNTech, AstraZeneca, Sputnik V, and Sinopharm. Functional antibody testing with a panel of nine SARS-CoV-2 viral variant receptor binding domain (RBD) proteins revealed marked differences in vaccine responses, with low antibody levels and RBD-ACE2 blocking activity stimulated by the Sinopharm and Sputnik V vaccines in comparison to the AstraZeneca or Pfizer/BioNTech vaccines. The Alpha variant caused 97% of infections in Mongolia in June and early July 2021. Individuals who recover from SARS-CoV-2 infection after vaccination achieve high antibody titers in most cases. These data suggest that public health interventions such as vaccine boosting, potentially with more potent vaccine types, may be needed to control COVID-19 in Mongolia and worldwide.


Subject(s)
Antibodies, Viral/blood , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Mass Vaccination , SARS-CoV-2/drug effects , Adult , Aged , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Viral/biosynthesis , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , Female , Gene Expression , Humans , Immune Sera/chemistry , Immunogenicity, Vaccine , Male , Middle Aged , Mongolia/epidemiology , Retrospective Studies , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
6.
Virus Res ; 305: 198555, 2021 11.
Article in English | MEDLINE | ID: covidwho-1412516

ABSTRACT

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. ß-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly resulting in insufficient inactivation, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.


Subject(s)
Antiviral Agents/pharmacology , COVID-19 Vaccines/chemistry , Propiolactone/pharmacology , SARS-CoV-2/drug effects , Virion/drug effects , Virus Inactivation/drug effects , Animals , Antigens, Viral/chemistry , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Flocculation/drug effects , Humans , Immune Sera/chemistry , RNA, Viral/chemistry , RNA, Viral/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Vaccines, Inactivated , Vero Cells , Virion/chemistry , Virion/immunology
7.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Article in English | MEDLINE | ID: covidwho-1366850

ABSTRACT

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.


Subject(s)
Amino Acid Substitution , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/pharmacology , Binding Sites , COVID-19/genetics , COVID-19/virology , Chlorocebus aethiops , Convalescence , Gene Expression , Humans , Immune Evasion , Immune Sera/chemistry , Models, Molecular , Mutation , Neutralization Tests , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
8.
Biochim Biophys Acta Gen Subj ; 1865(11): 129974, 2021 11.
Article in English | MEDLINE | ID: covidwho-1330651

ABSTRACT

Background Since December 2019, the newly emerged SARS-CoV-2 virus continues to infect humans and many people died from severe Covid-19 during the last 2 years worldwide. Different approaches are being used for treatment of this infection and its consequences, but limited results have been achieved and new therapeutics are still needed. One of the most interesting biotherapeutics in this era are Nanobodies which have shown very promising results in recent researches. Scope of review Here, we have reviewed the potentials of Nanobodies in Covid-19 treatment. We have also discussed the properties of these biotherapeutics that make them very suitable for pulmonary drug delivery, which seems to be very important route of administration in this disease. Major conclusion Nanobodies with their special biological and biophysical characteristics and their resistance against harsh manufacturing condition, can be considered as promising, targeted biotherapeutics which can be administered by pulmonary delivery pharmaceutical systems against Covid-19. General significance Covid-19 has become a global problem during the last two years and with emerging mutant strains, prophylactic and therapeutic approaches are still highly needed. Nanobodies with their specific properties can be considered as valuable and promising candidates in Covid-19 therapy.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/therapy , Immunologic Factors/therapeutic use , SARS-CoV-2/drug effects , Single-Domain Antibodies/therapeutic use , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Neutralizing/isolation & purification , Antiviral Agents/isolation & purification , Antiviral Agents/metabolism , COVID-19/immunology , COVID-19/virology , Camelus , Drug Delivery Systems , Humans , Immune Sera/chemistry , Immunologic Factors/biosynthesis , Immunologic Factors/isolation & purification , Lung/drug effects , Lung/immunology , Lung/virology , Molecular Targeted Therapy/methods , Peptide Library , Protein Binding/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Single-Domain Antibodies/biosynthesis , Single-Domain Antibodies/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
9.
PLoS One ; 16(7): e0255096, 2021.
Article in English | MEDLINE | ID: covidwho-1325440

ABSTRACT

The COVID-19 pandemic raises the need for diverse diagnostic approaches to rapidly detect different stages of viral infection. The flexible and quantitative nature of single-molecule imaging technology renders it optimal for development of new diagnostic tools. Here we present a proof-of-concept for a single-molecule based, enzyme-free assay for detection of SARS-CoV-2. The unified platform we developed allows direct detection of the viral genetic material from patients' samples, as well as their immune response consisting of IgG and IgM antibodies. Thus, it establishes a platform for diagnostics of COVID-19, which could also be adjusted to diagnose additional pathogens.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/immunology , Single Molecule Imaging/methods , Viral Proteins/genetics , Antibodies, Viral/blood , Base Sequence , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Nucleic Acid Testing/standards , COVID-19 Serological Testing/standards , Enzyme-Linked Immunosorbent Assay , Humans , Immune Sera/chemistry , Immunoglobulin G/blood , Immunoglobulin M/blood , Nasopharynx/virology , Polyproteins/blood , Polyproteins/genetics , RNA, Viral/blood , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Single Molecule Imaging/instrumentation , Viral Proteins/blood
10.
PLoS One ; 16(7): e0253551, 2021.
Article in English | MEDLINE | ID: covidwho-1325433

ABSTRACT

BACKGROUND: The novel coronavirus SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.5 million people, but no cure exists. Although passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable therapeutic option, the selection of optimal units for therapy in a timely fashion remains a barrier. STUDY DESIGN AND METHODS: Since virus neutralization is a necessary characteristic of plasma that can benefit recipients, the neutralizing titers of plasma samples were measured using a retroviral-pseudotype assay. Binding antibody titers to the spike (S) protein were also determined by a clinically available serological assay (Ortho-Vitros total IG), and an in-house ELISA. The results of these assays were compared to a measurement of antibodies directed to the receptor binding domain (RBD) of the SARS-CoV2 S protein (Promega Lumit Dx). RESULTS: All measures of antibodies were highly variable, but correlated, to different degrees, with each other. However, the anti-RBD antibodies correlated with viral neutralizing titers to a greater extent than the other antibody assays. DISCUSSION: Our observations support the use of an anti-RBD assay such as the Lumit Dx assay, as an optimal predictor of the neutralization capability of CCP.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/therapy , Immunoglobulin G/blood , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Blood Donors , COVID-19/diagnosis , COVID-19/immunology , COVID-19/virology , Enzyme-Linked Immunosorbent Assay , Gene Expression , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immune Sera/chemistry , Immunization, Passive/methods , Neutralization Tests , Predictive Value of Tests , Protein Binding , Protein Domains , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/immunology
11.
J Biol Chem ; 296: 100536, 2021.
Article in English | MEDLINE | ID: covidwho-1126904

ABSTRACT

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from humans to animals has been reported for many domesticated species, including farmed minks. The identification of novel spike gene mutations appearing in minks has raised major concerns about potential immune evasion and challenges for the global vaccine strategy. One genetic variant, known as "cluster five," arose among farmed minks in Denmark and resulted in a complete shutdown of the world's largest mink production. However, the functional properties of this new variant are not established. Here we present functional data on the cluster-five variant, which contains a mutation resulting in a Y453F residue change in the receptor-binding domain (RBD) of the spike protein. Using an ELISA-based angiotensin-converting enzyme-2/RBD inhibition assay, we show that the Y453F variant does not decrease established humoral immunity from previously infected individuals or affect the neutralizing antibody response in a vaccine mouse model based on the original Wuhan strain RBD or spike as antigens. However, biolayer interferometry analysis demonstrates that it binds the human angiotensin-converting enzyme-2 receptor with a 4-fold higher affinity than the original strain, suggesting an enhanced transmission capacity and a possible challenge for viral control. These results also indicate that the rise in the frequency of the cluster-five variant in mink farms might be a result of the fitness advantage conferred by the receptor adaptation rather than evading immune responses.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/epidemiology , COVID-19/transmission , Mink/virology , Pandemics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/chemistry , Antibodies, Viral/metabolism , COVID-19/immunology , Convalescence , Denmark/epidemiology , Gene Expression , HEK293 Cells , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immune Sera/chemistry , Immunity, Innate , Models, Molecular , Mutation , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/genetics , Recombinant Proteins/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Virus Internalization
12.
Virology ; 557: 15-22, 2021 05.
Article in English | MEDLINE | ID: covidwho-1071993

ABSTRACT

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of ß-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , SARS-CoV-2/immunology , Amino Acid Sequence , COVID-19/blood , COVID-19/immunology , COVID-19 Serological Testing/methods , Case-Control Studies , Cloning, Molecular , Coronavirus Nucleocapsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay/standards , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Immune Sera/chemistry , Immunoglobulin M/blood , Phosphoproteins/genetics , Phosphoproteins/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity
13.
Commun Biol ; 4(1): 129, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1054066

ABSTRACT

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Adult , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , Convalescence , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Female , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Immune Sera/chemistry , Immunity, Humoral , Lentivirus/genetics , Lentivirus/immunology , Male , Middle Aged , Neutralization Tests , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/metabolism , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Survival Analysis
14.
Bioessays ; 43(3): e2000257, 2021 03.
Article in English | MEDLINE | ID: covidwho-995863

ABSTRACT

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Immunoassay , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Microscopy/methods , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/virology , COVID-19 Testing/methods , Fluorescent Antibody Technique , High-Throughput Screening Assays , Humans , Image Processing, Computer-Assisted/statistics & numerical data , Immune Sera/chemistry , Machine Learning , Sensitivity and Specificity
15.
J Clin Virol ; 134: 104690, 2021 01.
Article in English | MEDLINE | ID: covidwho-926280

ABSTRACT

BACKGROUND: These last months, dozens of SARS-CoV-2 serological tests have become available with varying performances. A major effort was completed to compare 17 serological tests available in April 2020 in Switzerland. METHODS: In a preliminary phase, we compared 17 IgG, IgM, IgA and pan Ig serological tests including ELISA, LFA, CLIA and ECLIA on a panel of 182 sera, comprising 113 sera from hospitalized patients with a positive RT-PCR, and 69 sampled before 1st November 2019, expected to give a positive and negative results, respectively. In a second phase, the five best performing and most available tests were further evaluated on a total of 582 sera (178 and 404 expected positive and negative, respectively), allowing the assessment of 20 possible cross-reactions with other viruses. RESULTS: In the preliminary phase, among eight IgG/pan-Ig ELISA or CLIA/ECLIA tests, five had a sensitivity and specificity above 90 % and 98 % respectively, and on six IgM/IgA tests, only one was acceptable. Only one LFA test on three showed good performances for both IgG and IgM. For all the tests IgM and IgG aroused concomitantly. In the second phase, no test showed particular cross-reaction. We observed an important heterogeneity in the development of the antibody response. CONCLUSIONS: The majority of the evaluated tests exhibited high performances of IgG/pan-Ig sensitivity and specificity to detect the serological response of moderately to critically ill hospitalized patients. The IgM and IgA tests showed mostly insufficient performances with no added value for the early diagnostic on the cohort tested in this study.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/blood , COVID-19/diagnosis , Immunoglobulin G/blood , Immunosorbent Techniques/statistics & numerical data , SARS-CoV-2/immunology , COVID-19/pathology , COVID-19/virology , COVID-19 Testing/methods , Cross Reactions , Humans , Immune Sera/chemistry , Immunoglobulin A/blood , Immunoglobulin M/blood , Immunosorbent Techniques/classification , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/pathogenicity , Sensitivity and Specificity , Severity of Illness Index , Switzerland
16.
J Gen Virol ; 102(1)2021 01.
Article in English | MEDLINE | ID: covidwho-873186

ABSTRACT

Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.


Subject(s)
Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , COVID-19/diagnosis , Henipavirus Infections/diagnosis , High-Throughput Screening Assays , Respiratory Syncytial Virus Infections/diagnosis , Viral Fusion Proteins/antagonists & inhibitors , Animals , Antibodies, Neutralizing/isolation & purification , Antibodies, Neutralizing/metabolism , Antibodies, Viral/isolation & purification , Antibodies, Viral/metabolism , COVID-19/immunology , COVID-19/virology , Cell Fusion , Convalescence , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Henipavirus Infections/immunology , Henipavirus Infections/virology , Humans , Immune Sera/chemistry , Luciferases/genetics , Luciferases/metabolism , Models, Molecular , Nipah Virus/immunology , Nipah Virus/pathogenicity , Protein Conformation , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/immunology , Respiratory Syncytial Virus, Human/pathogenicity , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Swine , Viral Fusion Protein Inhibitors/chemistry , Viral Fusion Protein Inhibitors/metabolism , Viral Fusion Protein Inhibitors/pharmacology , Viral Fusion Proteins/genetics , Viral Fusion Proteins/immunology
17.
Cell Mol Immunol ; 17(10): 1098-1100, 2020 10.
Article in English | MEDLINE | ID: covidwho-772968
18.
Cell Mol Immunol ; 17(10): 1095-1097, 2020 10.
Article in English | MEDLINE | ID: covidwho-748174
19.
Infection ; 49(1): 75-82, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-725312

ABSTRACT

OBJECTIVE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti-SARS-CoV-2 antibody levels. METHODS: In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA- and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. RESULTS: The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. CONCLUSIONS: With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/immunology , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/standards , Neutralization Tests/standards , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Neutralizing/blood , Antigens, Viral/chemistry , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Cross-Sectional Studies , Humans , Immune Sera/chemistry , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Protein Domains , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/chemistry
20.
Clin Microbiol Infect ; 26(10): 1386-1394, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-628848

ABSTRACT

OBJECTIVES: To validate the diagnostic accuracy of a Euroimmun SARS-CoV-2 IgG and IgA immunoassay for COVID-19. METHODS: In this unmatched (1:2) case-control validation study, we used sera of 181 laboratory-confirmed SARS-CoV-2 cases and 326 controls collected before SARS-CoV-2 emergence. Diagnostic accuracy of the immunoassay was assessed against a whole spike protein-based recombinant immunofluorescence assay (rIFA) by receiver operating characteristic (ROC) analyses. Discrepant cases between ELISA and rIFA were further tested by pseudo-neutralization assay. RESULTS: COVID-19 patients were more likely to be male and older than controls, and 50.3% were hospitalized. ROC curve analyses indicated that IgG and IgA had high diagnostic accuracies with AUCs of 0.990 (95% Confidence Interval [95%CI]: 0.983-0.996) and 0.978 (95%CI: 0.967-0.989), respectively. IgG assays outperformed IgA assays (p=0.01). Taking an assessed 15% inter-assay imprecision into account, an optimized IgG ratio cut-off > 2.5 displayed a 100% specificity (95%CI: 99-100) and a 100% positive predictive value (95%CI: 96-100). A 0.8 cut-off displayed a 94% sensitivity (95%CI: 88-97) and a 97% negative predictive value (95%CI: 95-99). Substituting the upper threshold for the manufacturer's, improved assay performance, leaving 8.9% of IgG ratios indeterminate between 0.8-2.5. CONCLUSIONS: The Euroimmun assay displays a nearly optimal diagnostic accuracy using IgG against SARS-CoV-2 in patient samples, with no obvious gains from IgA serology. The optimized cut-offs are fit for rule-in and rule-out purposes, allowing determination of whether individuals in our study population have been exposed to SARS-CoV-2 or not. IgG serology should however not be considered as a surrogate of protection at this stage.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus/immunology , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Immunoassay/standards , Immunoglobulin A/blood , Immunoglobulin G/blood , Pneumonia, Viral/diagnosis , Adult , Area Under Curve , COVID-19 , COVID-19 Testing , Case-Control Studies , Child , Coronavirus Infections/immunology , Coronavirus Infections/physiopathology , Coronavirus Infections/virology , Female , Humans , Immune Sera/chemistry , Male , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/physiopathology , Pneumonia, Viral/virology , ROC Curve , SARS-CoV-2 , Sensitivity and Specificity , Severity of Illness Index
SELECTION OF CITATIONS
SEARCH DETAIL