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

Nie, Jianhui; Li, Qianqian; Wu, Jiajing; Zhao, Chenyan; Hao, Huan; Liu, Huan; Zhang, Li; Nie, Lingling; Qin, Haiyang; Wang, Meng; Lu, Qiong; Li, Xiaoyü; Sun, Qiyu; Liu, Junkai; Fan, Chen; Huang, Weijin; Xu, Miao; Wang, Youchun

doi:10.1038/s41596-020-0394-5

Cited by 370 publications

(331 citation statements)

References 43 publications

Supporting

Mentioning

310

Contrasting

Order By: Relevance

“…To confirm that the four linear B-cell epitopes generated antibodies against SARS-CoV-2, we immunized six- In SARS-CoV and MERS-CoV 60 , pseudovirus neutralization assay is a sensitive and quantitative method. We therefore tested the concentration of neutralizing antibodies in immune sera from wild type mice 7 days after the fifth vaccination against SARS-CoV-2 pseudovirus using a pseudotyped virus-based neutralization assay developed recently for SARS-CoV-2 61 ; mean EC50 is 154 for 'CVNFNFNGL') ( Fig. 3F), whereas no neutralizing antibodies were found in the two control group.…”

Section: Antibodies Against the Four Linear B-cell Epitopes Neutralizmentioning

confidence: 99%

See 1 more Smart Citation

Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

Zhao

Yang

et al. 2020

Preprint

View full text Add to dashboard Cite

SARS-CoV-2 unprecedentedly threatens the public health at worldwide level. There is an urgent need to develop an effective vaccine within a highly accelerated time. Here, we present the most comprehensive S-protein-based linear B-cell epitope candidate list by combining epitopes predicted by eight widely-used immune-informatics methods with the epitopes curated from literature published between Feb 6, 2020 and July 10, 2020. We find four top prioritized linear B-cell epitopes in the hotspot regions of S protein can specifically bind with serum antibodies from horse, mouse, and monkey inoculated with different SARS-CoV-2 vaccine candidates or a patient recovering from COVID-19. The four linear B-cell epitopes can induce neutralizing antibodies against both pseudo and live SARS-CoV-2 virus in immunized wild-type BALB/c mice. This study suggests that the four linear B-cell epitopes are potentially important candidates for serological assay or vaccine development.

show abstract

Section: Antibodies Against the Four Linear B-cell Epitopes Neutralizmentioning

confidence: 99%

“…A pseudotyped virus-based neutralization assay against SARS-CoV-2 in biosafety level 2 facilities was performed as previously described 61 . Briefly, serial dilutions of the samples to be tested were mixed with 325-…”

Section: Sars-cov-2 Pseudovirus Neutralization Assaymentioning

confidence: 99%

Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

Zhao

Yang

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Current antiviral drug screening and quantification of neutralizing antibodies rely on the use of SARS-CoV-2 pseudoviruses [7][8][9][10][11][12][13][14] . The use of live virus requires biosafety level (BSL) 3 facility and practice, which limits the use of wild-type virus in common laboratories.…”

Section: Introductionmentioning

confidence: 99%

“…The use of live virus requires biosafety level (BSL) 3 facility and practice, which limits the use of wild-type virus in common laboratories. Both lentivirus and vesicular stomatitis virus (VSV), pseudotyped with the SARS-CoV-2 S protein, are used in cellbased neutralization assays and in antiviral drug screening [8][9][10][11] . SARS-CoV-2 contains four structural proteins: the spike protein (S), the membrane protein (M), the envelope protein (E), and the nucleocapsid protein (N) 22,23 .…”

Section: Introductionmentioning

confidence: 99%

“…The VSV-based pseudovirus is generated by transfection of a producer cell line with a SARS-CoV-2 S expression vector, and then followed by infection of the cell with a VSV reporter virus that lacks the VSV major spike glycoprotein (G). The VSV-based pseudovirus can generate a higher signal-to-noise ratio and the neutralization assay takes about 24 hours, whereas the lentivirus-based pseudovirus produces less robust signal and takes 48-72 hours [7][8][9][10][11] . However, a major issue for the VSV-based pseudovirus is the likely presence of residual VSV virus, which may result in high rates of false-positive results 27 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Development of a novel hybrid alphavirus-SARS-CoV-2 particle for rapid in vitro screening and quantification of neutralization antibodies, viral variants, and antiviral drugs

Hetrick

Chilin

et al. 2020

Preprint

View full text Add to dashboard Cite

SUMMARYTimely development of vaccines and antiviral drugs are critical to control the coronavirus disease 2019 (COVID-19) global pandemic 1–6. Current methods for validation of vaccine efficacy involve the use of pseudoviruses, such as the SARS-CoV-2 spike protein (S) pseudotyped lentivirus or vesicular stomatitis virus (VSV), to quantify neutralizing antibodies for blocking viral infection 7–14. The process of pseudovirus infection and quantification is time consuming and can take days to complete. In addition, pseudoviruses contain structural proteins not native to SARS-CoV-2, which may alter particle properties in receptor binding and responses to antibody neutralization 15. Here we describe the development of a new hybrid alphavirus-SARS-CoV-2 particle (Ha-CoV-2) for rapid screening and quantification of neutralization antibodies and antiviral drugs. Ha-CoV-2 is a non-replicating SARS-CoV-2 virus-like particle, composed of only SARS-CoV-2 structural proteins (S, M, N, and E) and a RNA genome derived from a fast expressing alphavirus vector 16. We demonstrate that Ha-CoV-2 can rapidly and robustly express reporter genes in target cells within 3-5 hours following viral entry. We further validate the Ha-CoV-2 system for rapid quantification of neutralization antibodies and antiviral drugs. In addition, we assembled a Ha-CoV-2 particle bearing the D614G mutant spike protein, and found that the mutation led to an approximately 200% increase in virion infectivity. These results demonstrate that Ha-CoV-2 can also be applied for rapid monitoring and quantification of viral mutations for effects on neutralizing antibodies induced by vaccines.

show abstract

Assessment of Broadly Reactive Responses in Patients With MERS-CoV Infection and SARS-CoV-2 Vaccination

et al. 2023

View full text Add to dashboard Cite

ImportanceIn the ongoing COVID-19 pandemic, there remain unanswered questions regarding the nature and importance of the humoral immune response against other coronaviruses. Although coinfection of the Middle East respiratory syndrome coronavirus (MERS-CoV) with the SARS-CoV-2 has not been documented yet, several patients previously infected with MERS-CoV received the COVID-19 vaccine; data describing how preexisting MERS-CoV immunity may shape the response to SARS-CoV-2 following infection or vaccination are lacking.ObjectiveTo characterize the cross-reactive and protective humoral responses in patients exposed to both MERS-CoV infection and SARS-CoV-2 vaccination.Design, Setting, and ParticipantsThis cohort study involved a total of 18 sera samples collected from 14 patients with MERS-CoV infection before (n = 12) and after (n = 6) vaccination with 2 doses of COVID-19 mRNA vaccine (BNT162b2 or mRNA-1273). Of those patients, 4 had prevaccination and postvaccination samples. Antibody responses to SARS-CoV-2 and MERS-CoV were assessed as well as cross-reactive responses to other human coronaviruses.Main Outcomes and MeasuresThe main outcomes measured were binding antibody responses, neutralizing antibodies, and antibody-dependent cellular cytotoxicity (ADCC) activity. Binding antibodies targeting SARS-CoV-2 main antigens (spike [S], nucleocapsid, and receptor-binding domain) were detected using automated immunoassays. Cross-reactive antibodies with the S1 protein of SARS-CoV, MERS-CoV, and common human coronaviruses were analyzed using a bead-based assay. Neutralizing antibodies (NAbs) against MERS-CoV and SARS-CoV-2 as well as ADCC activity against SARS-CoV-2 were assessed.ResultsA total of 18 samples were collected from 14 male patients with MERS-CoV infection (mean [SD] age, 43.8 [14.6] years). Median (IQR) duration between primary COVID-19 vaccination and sample collection was 146 (47-189) days. Prevaccination samples had high levels of anti-MERS S1 immunoglobin M (IgM) and IgG (reactivity index ranging from 0.80 to 54.7 for IgM and from 0.85 to 176.3 for IgG). Cross-reactive antibodies with SARS-CoV and SARS-CoV-2 were also detected in these samples. However, cross-reactivity against other coronaviruses was not detected by the microarray assay. Postvaccination samples showed significantly higher levels of total antibodies, IgG, and IgA targeting SARS-CoV-2 S protein compared with prevaccination samples (eg, mean total antibodies: 8955.0 AU/mL; 95% CI, −5025.0 to 22936.0 arbitrary units/mL; P = .002). In addition, significantly higher anti-SARS S1 IgG levels were detected following vaccination (mean reactivity index, 55.4; 95% CI, −9.1 to 120.0; P = .001), suggesting potential cross-reactivity with these coronaviruses. Also, anti-S NAbs were significantly boosted against SARS-CoV-2 (50.5% neutralization; 95% CI, 17.6% to 83.2% neutralization; P &lt; .001) after vaccination. Furthermore, there was no significant increase in antibody-dependent cellular cytotoxicity against SARS-CoV-2 S protein postvaccination.Conclusions and RelevanceThis cohort study found a significant boost in cross-reactive NAbs in some patients exposed to MERS-CoV and SARS-CoV-2 antigens. These findings suggest that isolation of broadly reactive antibodies from these patients may help guide the development of a pancoronavirus vaccine by targeting cross-reactive epitopes between distinct strains of human coronaviruses.

show abstract

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

Cited by 370 publications

References 43 publications

Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

Development of a novel hybrid alphavirus-SARS-CoV-2 particle for rapid in vitro screening and quantification of neutralization antibodies, viral variants, and antiviral drugs

Assessment of Broadly Reactive Responses in Patients With MERS-CoV Infection and SARS-CoV-2 Vaccination

Contact Info

Product

Resources

About