SARS-CoV-2–Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence

Abstract Background The COVID-19 pandemic necessitates better understanding of the kinetics of antibody production induced by infection with SARS-CoV-2. We aimed to develop a high-throughput multiplex assay to detect antibodies to SARS-CoV-2 to assess immunity to the virus in the general population. Methods Spike protein subunits S1 and receptor binding domain, and nucleoprotein were coupled to microspheres. Sera collected before emergence of SARS-CoV-2 (n = 224) and of non-SARS-CoV-2 influenza-like illness (n = 184), and laboratory-confirmed cases of SARS-CoV-2 infection (n = 115) with various severities of COVID-19 were tested for SARS-CoV-2–specific IgG concentrations. Results Our assay discriminated SARS-CoV-2–induced antibodies and those induced by other viruses. The assay specificity was 95.1%–99.0% with sensitivity 83.6%–95.7%. By merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. Hospitalized COVID-19 patients developed higher IgG concentrations and the rate of IgG production increased faster compared to nonhospitalized cases. Conclusions The bead-based serological assay for quantitation of SARS-CoV-2–specific antibodies proved to be robust and can be conducted in many laboratories. We demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen–specific IgG determination.

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SARS-CoV-2-specific antibody detection for sero-epidemiology: a multiplex analysis approach accounting for accurate seroprevalence

10.1101/2020.06.18.20133660 ◽

2020 ◽

Cited By ~ 1

Author(s):

Gerco den Hartog ◽

Rutger M. Schepp ◽

Marjan Kuijer ◽

Corine GeurtsvanKessel ◽

Josine van Beek ◽

...

Keyword(s):

Multiplex Assay ◽

Spike Protein ◽

Antibody Detection ◽

Test Results ◽

Detailed Understanding ◽

Protein Subunits ◽

Specific Antibodies ◽

Single Antigen ◽

Specific Igg ◽

Kinetics Of

ABSTRACTBackgroundThe COVID-19 pandemic demands detailed understanding of the kinetics of antibody production induced by infection with SARS-CoV-2. We aimed to develop a high throughput multiplex assay to detect antibodies to SARS-CoV-2 to assess immunity to the virus in the general population.MethodsSpike protein subunits S1 and RBD, and Nucleoprotein were coupled to distinct microspheres. Sera collected before the emergence of SARS-CoV-2 (N=224), and of non-SARS-CoV-2 influenza-like illness (N=184), and laboratory-confirmed cases of SARS-CoV-2 infection (N=115) with various severity of COVID-19 were tested for SARS-CoV-2-specific concentrations of IgG.ResultsOur assay discriminated SARS-CoV-2-induced antibodies and those induced by other viruses. The assay obtained a specificity between 95.1 and 99.0% with a sensitivity ranging from 83.6-95.7%. By merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. Hospitalized COVID-19 patients developed higher IgG concentrations and the rate of IgG production increased faster compared to non-hospitalized cases.ConclusionsThe bead-based serological assay for quantitation of SARS-CoV-2-specific antibodies proved to be robust and can be conducted in many laboratories. Finally, we demonstrated that testing of antibodies against different antigens increases sensitivity and specificity compared to single antigen-specific IgG determination.

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Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals

10.1101/2020.05.13.092619 ◽

2020 ◽

Cited By ~ 43

Author(s):

Davide F. Robbiani ◽

Christian Gaebler ◽

Frauke Muecksch ◽

Julio C. C. Lorenzi ◽

Zijun Wang ◽

...

Keyword(s):

Virus Entry ◽

Protein S ◽

Memory B Cells ◽

Antibody Responses ◽

Spike Protein ◽

Human Antibody ◽

Receptor Binding Domain ◽

Single Digit ◽

Specific Antibodies ◽

Specific Memory

AbstractDuring the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21–5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

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The Slower Antibody Response in Myelofibrosis Patients after Two Doses of mRNA SARS-CoV-2 Vaccine Calls for a Third Dose

Biomedicines ◽

10.3390/biomedicines9101480 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1480

Author(s):

Fabio Fiorino ◽

Anna Sicuranza ◽

Annalisa Ciabattini ◽

Adele Santoni ◽

Gabiria Pastore ◽

...

Keyword(s):

Healthy Subjects ◽

Vaccine Dose ◽

Antibody Responses ◽

Inhibition Activity ◽

Specific Antibodies ◽

Slow Kinetics ◽

Binding Inhibition ◽

Specific Igg ◽

The Impact ◽

Kinetics Of

Immunization with mRNA SARS-CoV-2 vaccines has been highly recommended and prioritized in fragile subjects, including patients with myelofibrosis (MF). Available data on the vaccine immune response developed by MF patients and the impact of ruxolitinib treatment are still too fragmented to support an informed decision on a third dose for this category of subjects. Here, we show that 76% of MF patients develop spike-specific IgG after the second mRNA SARS-CoV-2 vaccine dose, but the response has a slower kinetics compared to healthy subjects, suggesting a reduced capability of their immune system to promptly react to vaccination. A reduced ACE2/RBD binding inhibition activity of spike-specific antibodies was also observed, especially in ruxolitinib-treated patients. Our results, showing slow kinetics of antibody responses in MF patients following vaccination with mRNA SARS-CoV-2 vaccines, support the need for a third vaccine dose.

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The Dynamic Changes of Antibodies against SARS-CoV-2 during the Infection and Recovery of COVID-19

10.1101/2020.05.18.20105155 ◽

2020 ◽

Author(s):

Kening Li ◽

Min Wu ◽

Bin Huang ◽

Aifang Zhong ◽

Lu Li ◽

...

Keyword(s):

Immune Response ◽

Protein S ◽

Spike Protein ◽

Receptor Binding Domain ◽

Dynamic Changes ◽

Laboratory Findings ◽

The Poor ◽

Specific Igg ◽

Critical Patients ◽

Nucleoprotein N

Deciphering the dynamic changes of antibodies against SARS-CoV-2 is essential for understanding the immune response in COVID-19 patients. By comprehensively analyzing the laboratory findings of 1,850 patients, we describe the dynamic changes of the total antibody, spike protein (S)-, receptor-binding domain (RBD)-, and nucleoprotein (N)- specific IgM and IgG levels during SARS-CoV-2 infection and recovery. Our results indicate that the S-, RBD-, and N- specific IgG generation of severe/critical COVID-19 patients is one week later than mild/moderate cases, while the levels of these antibodies are 1.5-fold higher in severe/critical patients during hospitalization (P<0.01). The decrease of these IgG levels indicates the poor outcome of severe/critical patients. The RBD- and S-specific IgG levels are 2-fold higher in virus-free patients (P<0.05). Notably, we found that the patients who got re-infected had a low level of protective antibody on discharge. Therefore, our evidence proves that the dynamic changes of antibodies could provide an important reference for diagnosis, monitoring, and treatment, and shed new light on the precise management of COVID-19.

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RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00402-5 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Zezhong Liu ◽

Wei Xu ◽

Shuai Xia ◽

Chenjian Gu ◽

Xinling Wang ◽

...

Keyword(s):

Subunit Vaccine ◽

Vaccine Candidate ◽

High Titer ◽

Neutralizing Antibody ◽

Spike Protein ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Specific Antibodies ◽

Good Potential ◽

Neutralizing Epitopes

AbstractThe pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed serious threats to global health and economy, thus calling for the development of safe and effective vaccines. The receptor-binding domain (RBD) in the spike protein of SARS-CoV-2 is responsible for its binding to angiotensin-converting enzyme 2 (ACE2) receptor. It contains multiple dominant neutralizing epitopes and serves as an important antigen for the development of COVID-19 vaccines. Here, we showed that immunization of mice with a candidate subunit vaccine consisting of SARS-CoV-2 RBD and Fc fragment of human IgG, as an immunopotentiator, elicited high titer of RBD-specific antibodies with robust neutralizing activity against both pseudotyped and live SARS-CoV-2 infections. The mouse antisera could also effectively neutralize infection by pseudotyped SARS-CoV-2 with several natural mutations in RBD and the IgG extracted from the mouse antisera could also show neutralization against pseudotyped SARS-CoV and SARS-related coronavirus (SARSr-CoV). Vaccination of human ACE2 transgenic mice with RBD-Fc could effectively protect mice from the SARS-CoV-2 challenge. These results suggest that SARS-CoV-2 RBD-Fc has good potential to be further developed as an effective and broad-spectrum vaccine to prevent infection of the current SARS-CoV-2 and its mutants, as well as future emerging SARSr-CoVs and re-emerging SARS-CoV.

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Anti-Spike, anti-Nucleocapsid and neutralizing antibodies in SARS-CoV-2 inpatients and asymptomatic carriers

10.1101/2020.05.12.20098236 ◽

2020 ◽

Cited By ~ 5

Author(s):

Etienne Brochot ◽

Baptiste Demey ◽

Antoine Touzé ◽

Sandrine Belouzard ◽

Jean Dubuisson ◽

...

Keyword(s):

Symptom Onset ◽

Neutralizing Antibodies ◽

Asymptomatic Carrier ◽

Secondary Infection ◽

List Type ◽

Receptor Binding Domain ◽

Plasma Therapy ◽

Specific Antibodies ◽

Asymptomatic Carriers ◽

Kinetics Of

SummaryObjectiveThe objective of this study was to monitor the anti-SARS-CoV-2 antibody response in infected patients.MethodsIn order to assess the time of seroconversion, we used 151 samples from 30 COVID-19 inpatients and monitored the detection kinetics of anti-S1, anti-S2, anti-RBD and anti-N antibodies with in-house ELISAs. We also monitored the presence of neutralizing antibodies in these samples as well as 25 asymptomatic carrier samples using retroviral particles pseudotyped with the spike of the SARS-CoV-2.ResultsWe observed that specific antibodies were detectable in all inpatients two weeks post-symptom onset. The detection of the SARS-CoV-2 Nucleocapsid and RBD was more sensitive than the detection of the S1 or S2 subunits. Neutralizing antibodies reached a plateau two weeks post-symptom onset and then declined in the majority of inpatients. Furthermore, neutralizing antibodies were undetectable in 56% of asymptomatic carriers.ConclusionsOur results raise questions concerning the role played by neutralizing antibodies in COVID-19 cure and protection against secondary infection. They also suggest that induction of neutralizing antibodies is not the only strategy to adopt for the development of a vaccine. Finally, they imply that anti-SARS-CoV-2 neutralizing antibodies should be titrated to optimize convalescent plasma therapy.HighlightsSpecific antibodies are detectable in 100% COVID-19 inpatients two weeks post-symptom onset.The detection of the SARS-CoV-2 Nucleocapsid and Receptor Binding Domain is more sensitive than the detection of the S1 or S2 subunits.Neutralizing antibodies reach a plateau two weeks post-symptom onset and then decline in the majority of inpatients.Neutralizing antibodies are undetectable in the majority of asymptomatic carriers.

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The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

Science Immunology ◽

10.1126/sciimmunol.abc8413 ◽

2020 ◽

Vol 5 (48) ◽

pp. eabc8413 ◽

Cited By ~ 111

Author(s):

Lakshmanane Premkumar ◽

Bruno Segovia-Chumbez ◽

Ramesh Jadi ◽

David R. Martinez ◽

Rajendra Raut ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Clinical Symptoms ◽

Severe Disease ◽

Population Level ◽

Respiratory Illness ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Specific Antibodies

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

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Prospective Cohort Study of the Kinetics of Specific Antibodies to SARS-CoV-2 Infection and to Four SARS-CoV-2 Vaccines Available in Serbia, and Vaccine Effectiveness: A 3-Month Interim Report

Vaccines ◽

10.3390/vaccines9091031 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1031

Author(s):

Olivera Lijeskić ◽

Ivana Klun ◽

Marija Stamenov Djaković ◽

Nenad Gligorić ◽

Tijana Štajner ◽

...

Keyword(s):

Real Life ◽

Vaccine Dose ◽

Igg Antibodies ◽

Post Vaccination ◽

Specific Antibodies ◽

Real Life Data ◽

Specific Igg ◽

Antibody Levels ◽

Kinetics Of ◽

Specific Igg Antibodies

Real-life data on the performance of vaccines against SARS-CoV-2 are still limited. We here present the rates of detection and levels of antibodies specific for the SARS-CoV-2 spike protein RBD (receptor binding domain) elicited by four vaccines available in Serbia, including BNT-162b2 (BioNTech/Pfizer), BBIBP-CorV (Sinopharm), Gam-COVID-Vac (Gamaleya Research Institute) and ChAdOx1-S (AstraZeneca), compared with those after documented COVID-19, at 6 weeks and 3 months post first vaccine dose or post-infection. Six weeks post first vaccine dose, specific IgG antibodies were detected in 100% of individuals fully vaccinated with BNT-162b2 (n = 100) and Gam-COVID-Vac (n = 12) and in 81.7% of BBIBP-CorV recipients (n = 148), while one dose of ChAdOx1-S (n = 24) induced specific antibodies in 75%. Antibody levels elicited by BNT-162b2 were higher, while those elicited by BBIBP-CorV were lower, than after SARS-CoV-2 infection. By 3 months post-vaccination, antibody levels decreased but remained ≥20-fold above the cut-off in BNT-162b2 but not in BBIBP-CorV recipients, when an additional 30% were seronegative. For all vaccines, antibody levels were higher in individuals with past COVID-19 than in naïve individuals. A total of twelve new infections occurred within the first 3 months post-vaccination, eight after the first dose of BNT-162b2 and ChAdOx1-S (one each) and BBIBP-CorV (six), and four after full vaccination with BBIBP-CorV, but none required hospitalization.

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ACE2 peptide fragment interacts with several sites on the SARS-CoV-2 spike protein S1

10.21203/rs.3.rs-289393/v1 ◽

2021 ◽

Author(s):

Aleksei Kuznetsov ◽

Piret Arukuusk ◽

Heleri Heike Härk ◽

Erkki Juronen ◽

Ülo Langel ◽

...

Keyword(s):

Binding Sites ◽

Peptide Binding ◽

Inhibitory Effect ◽

Spike Protein ◽

Receptor Binding Domain ◽

Peptide Fragment ◽

Dissociation Kinetics ◽

Angiotensin Converting Enzyme 2 ◽

Significant Inhibitory Effect ◽

Kinetics Of

Abstract The influence of the peptide QAKTFLDKFNHEAEDLFYQ on the kinetics of the SARS-CoV-2 spike protein S1 binding to angiotensin-converting enzyme 2 (ACE2) was studied to model the interaction of the virus with its host cell. This peptide corresponds to the sequence 24–42 of the ACE2 α1 domain, which is the binding site for the S1 protein. The on-rate and off-rate of S1-ACE2 complex formation were measured in the presence of various peptide concentrations using Bio-Layer Interferometry (BLI). The formation of the S1-ACE2 complex was inhibited when the S1 protein was preincubated with the peptide, however, no significant inhibitory effect was observed in the absence of preincubation. Dissociation kinetics revealed that the peptide remained bound to the S1-ACE2 complex and stabilized this complex. This suggestion was confirmed by computational mapping of the S1 protein surface for peptide binding that revealed two additional sites, located at some distance from the receptor binding domain of S1. These additional binding sites may affect the interaction between the peptide, the S1 protein, and ACE2.

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Longitudinal analysis of the humoral response to SARS-CoV-2 spike RBD in convalescent plasma donors

10.1101/2020.07.16.206847 ◽

2020 ◽

Cited By ~ 5

Author(s):

Josée Perreault ◽

Tony Tremblay ◽

Marie-Josée Fournier ◽

Mathieu Drouin ◽

Guillaume Beaudoin-Bussières ◽

...

Keyword(s):

Longitudinal Analysis ◽

Plasma Cells ◽

De Novo ◽

Disease Onset ◽

Humoral Response ◽

Spike Protein ◽

Strongly Correlated ◽

Receptor Binding Domain ◽

Specific Antibodies ◽

Plasma Collection

ABSTRACTHéma-Québec, the blood supplier in the Province of Quebec, Canada, collects and tests convalescent plasma used in a clinical trial to determine the clinical efficacy of this product for the treatment of hospitalized COVID-19 patients. So far, we have collected 1159 plasma units from 282 COVID-19 convalescent donors. The presence of antibodies to the receptor binding domain (RBD) of SARS-CoV-2 spike protein in convalescent donors was established at the first donation. Seropositive donors were asked to donate additional plasma units every six days. Until now, 15 donors have donated at least four times and, in some cases, up to nine times. This allowed us to perform a longitudinal analysis of the persistence of SARS-CoV-2 RBD-specific antibodies in these repeat donors, with the first donation occurring 33-77 days after symptoms onset and donations up to 71-114 days after symptoms onset thereafter. In all donors, the level of antibodies remained relatively stable up to about 76 days after symptoms onset but then started to decrease more rapidly to reach, in some convalescent donors, a seronegative status within 100-110 days after symptoms onset. The decline in anti-RBD antibodies was not related to the number of donations but strongly correlated with the numbers of days after symptoms onset (r = 0.821). This suggests that de novo secretion of SARS-CoV-2 RBD antibodies by short-lived plasma cells stopped about 2-3 months after disease onset, an observation that has important implications for convalescent plasma collection and seroprevalence studies undertaken several months after the peak of infection.

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