scholarly journals Choosing the right tool for the job: A comprehensive assessment of serological assays for SARS-CoV-2 as surrogates for authentic virus neutralization

2021 ◽  
Author(s):  
Nicholas Wohlgemuth ◽  
Kendall Whitt ◽  
Sean Cherry ◽  
Ericka Kirkpatrick Roubidoux ◽  
Chun-Yang Lin ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Virus Neutralization ◽  
Diagnostic Tools ◽  
Pseudotyped Virus ◽  
Global Pandemic ◽  
Level 3 ◽  
The Right ◽  
Antibody Levels ◽  
Reliable Methods ◽  
Biosafety Level

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside of biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped VSV virus neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase and secreted embryonic alkaline phosphatase (SEAP) expressing pseudotyped virus neutralizations, followed by GFP expressing pseudotyped virus neutralization, and then the ELISAs.

scholarly journals Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2

2020 ◽  
Author(s):  
James Brett Case ◽  
Paul W. Rothlauf ◽  
Rita E. Chen ◽  
Zhuoming Liu ◽  
Haiyan Zhao ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Spike Protein ◽  
Glycoprotein Gene ◽  
Level 3 ◽  
Focus Reduction ◽  
High Throughput Imaging ◽  
Biosafety Level

ABSTRACTAntibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune γ-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.

scholarly journals Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays

2020 ◽  
Author(s):  
Arantxa Valdivia ◽  
Ignacio Torres ◽  
Victor Latorre ◽  
Carla Frances-Gomez ◽  
Eliseo Albert ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Pseudotyped Virus ◽  
S Protein ◽  
Neutralizing Activity ◽  
Virus Neutralization Assay ◽  
Degree Correlation ◽  
Antibody Titers

Background: Whether antibody levels measured by commercially-available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. Objectives: To evaluate the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 Spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Patients and Methods: Ninety sera from 51 hospitalized COVID-19 patients were assayed by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG and the COVID-19 ELISA IgG assays. Results: Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (Rho=0.73) and moderate for the remaining assays (Rho=0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. Conclusions: the suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

scholarly journals Neutralizing antibody response in non-hospitalized SARS-CoV-2 patients

2020 ◽  
Author(s):  
Natalia Ruetalo ◽  
Ramona Businger ◽  
Karina Althaus ◽  
Simon Fink ◽  
Felix Ruoff ◽  
...  
Keyword(s):  
Antibody Response ◽  
Western Blot ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Virus Neutralization ◽  
Surrogate Markers ◽  
High Antibody ◽  
Serological Survey ◽  
Course Of Disease ◽  
Antibody Levels

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2 and two other patients (4%) were only positive in one of the six serological assays employed. For the remainder, antibody response against the S-protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. Regarding neutralization, only six patients (12%) could be classified as highly neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.

scholarly journals 564. SARS-CoV-2 Ferritin Nanoparticle Vaccines Elicit Broad SARS Coronavirus Immunogenicity

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S384-S384
Author(s):  
M G Joyce ◽  
Wei-Hung Chen ◽  
Rajeshwer Sankhala ◽  
Agnes Hajduczki ◽  
Paul Thomas ◽  
...  
Keyword(s):  
Immune Responses ◽  
Neutralizing Antibody ◽  
High Dose ◽  
Pandemic Preparedness ◽  
Virus Stock ◽  
Global Pandemic ◽  
Recent Emergence ◽  
Blocking Activity ◽  
Antibody Levels ◽  
Antigenic Profile

Abstract Background The zoonotic emergence of SARS-CoV-2 quickly developed into a global pandemic. Multiple vaccine platforms have been advanced to clinical trials and emergency use authorization. The recent emergence of SARS-CoV-2 virus variants with Spike receptor-binding domain (RBD) and N-terminal domain (NTD) mutations, highlights the need for next-generation vaccines that can elicit immune responses that are resilient against Spike mutations. Methods Using a structure-based vaccine design approach, we developed multiple optimized SARS-CoV-2 nanoparticle immunogens that recapitulate the structural and antigenic profile of the SARS-CoV-2 prefusion spike. We assessed these immunogens in murine immunogenicity studies and in a K18-hACE2 transgenic mouse model with a SARS-CoV-2 challenge. Immune sera from vaccinated mice were assessed for SARS-CoV-2 binding, and neutralization against SARS-CoV-2, variants of concern, and the heterologous SARS-CoV-1 virus. Results In combination with a liposomal-saponin based adjuvant (ALFQ), these immunogens induced robust binding, ACE2-inhibition, and authentic virus and pseudovirus neutralization. A Spike-Ferritin nanoparticle (SpFN) vaccine elicited neutralizing ID50 titers >10,000 after a single immunization, while RBD-Ferritin (RFN) nanoparticle immunogens elicited ID50 titer values >10,000 values after two immunizations. Purified antibody from SpFN- or RFN-immunized mice was transfused into K18-ACE2 transgenic mice and challenged with a high-dose SARS-CoV-2 virus stock. In order to understand the breadth of vaccine-elicited antibody responses, we analyzed SpFN- and RBD-FN-immunized animal sera against a set of heterologous SARS-CoV-2 RBD variants and SARS-CoV RBD. High binding titers with ACE2-blocking activity were observed against SARS-CoV-2 variants and the heterologous SARS-CoV-1 RBD. Furthermore, both SpFN- and RFN-immunized animal sera showed SARS-CoV-1 neutralizing ID50 titers of >2000. Conclusion These observations highlight the importance of SARS-CoV-2 neutralizing antibody levels in providing protection against emerging SARS-like coronaviruses and provide a robust platform for pandemic preparedness. Structure-based design enables development of a SARS-CoV-2 nanoparticle immunogen. Disclosures All Authors: No reported disclosures

scholarly journals Longitudinal dynamics of the neutralizing antibody response to SARS-CoV-2 infection

2020 ◽  
Author(s):  
Kai Wang ◽  
Quan-Xin Long ◽  
Hai-Jun Deng ◽  
Jie Hu ◽  
Qing-Zhu Gao ◽  
...  
Keyword(s):  
Proinflammatory Cytokines ◽  
Symptom Onset ◽  
Neutralizing Antibodies ◽  
Antiviral Agents ◽  
Neutralizing Antibody ◽  
Peak Time ◽  
Igg Antibody ◽  
Longitudinal Dynamics ◽  
Global Pandemic ◽  
Antibody Levels

Background Coronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of SARS-CoV-2-specific neutralizing antibodies (NAbs) in COVID-19 patients. Methods Blood samples (n=173) were collected from 30 COVID-19 patients over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs, using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. Results SARS-CoV-2-specific NAb titers were low for the first 7-10 d after symtom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 d (IQR 24-59 d) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR 19.6-42.4%). NAb titers increased over time in parallel with the rise in IgG antibody levels, correlating well at week 3 (r = 0.41, p < 0.05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including SCF, TRAIL, and M-CSF. Conclusions These data provide useful information regarding dynamic changes in NAbs in COVID-19 patients during the acute and convalescent phases.

scholarly journals Evaluation of a SARS-CoV-2 surrogate virus neutralization test for detection of antibody in human, canine, cat and hamster sera

2020 ◽  
Author(s):  
Joseph Sriyal Malik Peiris ◽  
Ranawaka APM Perera
Keyword(s):  
Neutralization Test ◽  
Cross Reactivity ◽  
Virus Neutralization Test ◽  
Virus Neutralization ◽  
Level 3 ◽  
Multiple Species ◽  
Biosafety Level

Surrogate neutralization assays for SARS-CoV-2 that can be done without biosafety-level-3 containment and across multiple species are desirable. We evaluate a recently developed surrogate virus neutralization test (sVNT) in comparison to 90% plaque reduction neutralization tests (PRNT90) in human, canine, cat and hamster sera and found excellent concordance between the two assays. Using a panel of immune sera to other coronaviruses, we confirm the lack of cross reactivity in sVNT and PRNT90 assays.

scholarly journals Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus based assay

2020 ◽  
Author(s):  
Jianhui Nie ◽  
Qianqian Li ◽  
Jiajing Wu ◽  
Chenyan Zhao ◽  
Huan Hao ◽  
...  
Keyword(s):  
Small Molecules ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Virus Stock ◽  
Virus Attachment ◽  
Fusion Inhibitors ◽  
Level 2 ◽  
Biosafety Level

Abstract Jianhui Nie, Qianqian Li, and Jiajing Wu contributed equally to this work.Pseudotyped viruses are useful virological tools due to their safety and versatility. Based on a VSV pseudotyped virus production system, we developed a pseudotyped virus-based neutralization assay against SARS-CoV-2 in biosafety level 2 facilities. This protocol includes production, titration of the SARS-CoV-2 S pseudotyped virus and neutralization assay based on it. Various types of samples targeting virus attachment and entry could be evaluated for their potency, including serum samples derived from animals and humans, monoclonal antibodies, fusion inhibitors (peptides or small molecules). If the pseudotyped virus stock has been prepared in advance, it will take 2 days to get the potency data for the candidate samples. Experience of handling cells is needed before implementing this protocol.

scholarly journals Disease Severity, Fever, Age, and Sex Correlate With SARS-CoV-2 Neutralizing Antibody Responses

2021 ◽  
Vol 11 ◽  
Author(s):  
Stephan Schlickeiser ◽  
Tatjana Schwarz ◽  
Sophie Steiner ◽  
Kirsten Wittke ◽  
Nabeel Al Besher ◽  
...  
Keyword(s):  
Logistic Regression ◽  
Odds Ratio ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Proportional Odds ◽  
Factors Affecting ◽  
Elisa Testing ◽  
Level 3 ◽  
Antibody Titers ◽  
Antibody Levels

Clinical trials on the use of COVID-19 convalescent plasma remain inconclusive. While data on safety is increasingly available, evidence for efficacy is still sparse. Subgroup analyses hint to a dose-response relationship between convalescent plasma neutralizing antibody levels and mortality. In particular, patients with primary and secondary antibody deficiency might benefit from this approach. However, testing of neutralizing antibodies is limited to specialized biosafety level 3 laboratories and is a time- and labor-intense procedure. In this single center study of 206 COVID-19 convalescent patients, clinical data, results of commercially available ELISA testing of SARS-CoV-2 spike-IgG and –IgA, and levels of neutralizing antibodies, determined by plaque reduction neutralization testing (PRNT), were analyzed. At a medium time point of 58 days after symptom onset, only 12.6% of potential plasma donors showed high levels of neutralizing antibodies (PRNT50 ≥ 1:320). Multivariable proportional odds logistic regression analysis revealed need for hospitalization due to COVID-19 (odds ratio 6.87; p-value 0.0004) and fever (odds ratio 3.00; p-value 0.0001) as leading factors affecting levels of SARS-CoV-2 neutralizing antibody titers in convalescent plasma donors. Using penalized estimation, a predictive proportional odds logistic regression model including the most important variables hospitalization, fever, age, sex, and anosmia or dysgeusia was developed. The predictive discrimination for PRNT50 ≥ 1:320 was reasonably good with AUC: 0.86 (with 95% CI: 0.79–0.92). Combining clinical and ELISA-based pre-screening, assessment of neutralizing antibodies could be spared in 75% of potential donors with a maximal loss of 10% of true positives (PRNT50 ≥ 1:320).

scholarly journals Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients

2020 ◽  
Vol 58 (12) ◽  
Author(s):  
Larry L. Luchsinger ◽  
Brett P. Ransegnola ◽  
Daniel K. Jin ◽  
Frauke Muecksch ◽  
Yiska Weisblum ◽  
...  
Keyword(s):  
New York ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Pseudotyped Virus ◽  
Antibody Activity ◽  
Plasma Samples ◽  
Serological Tests ◽  
Population Immunity ◽  
Therapeutic Trials ◽  
Antibody Levels

ABSTRACT The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.

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