SARS-CoV-2 Antibody Neutralization Assay Platforms Based on Epitopes Sources: Live Virus, Pseudovirus, and Recombinant S Glycoprotein RBD

The unusual cases of pneumonia outbreak were reported from Wuhan city in late December 2019. Serological testing provides a powerful tool for the identification of prior infection and for epidemiological studies. Pseudotype virus neutralization assays are widely used for many viruses and applications in the fields of serology. The accuracy of pseudotype neutralizing assay allows for its use in low biosafety lab and provides a safe and effective alternative to the use of wild-type viruses. In this study, we evaluated the performance of this assay compared to the standard microneutralization assay as a reference. The lentiviral pseudotype particles were generated harboring the Spike gene of SARS-CoV-2. The generated pseudotype particles assay was used to evaluate the activity of neutralizing antibodies in 300 human serum samples from a COVID-19 sero-epidemiological study. Testing of these samples resulted in 55 positive samples and 245 negative samples by pseudotype viral particles assay while microneutralization assay resulted in 64 positive and 236 negative by MN assay. Compared to the MN, the pseudotyped viral particles assay showed a sensitivity of 85.94% and a specificity of 100%. Based on the data generated from this study, the pseudotype-based neutralization assay showed a reliable performance for the detection of neutralizing antibodies against SARS-CoV-2 and can be used safely and efficiently as a diagnostic tool in a biosafety level 2 laboratory.

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A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

10.21203/rs.3.rs-858163/v1 ◽

2021 ◽

Author(s):

Syed Hani Abidi ◽

Kehkeshan Imtiaz ◽

Akbar Kanji ◽

Shama Qaiser ◽

Erum Khan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Vero Cells ◽

Neutralization Assay ◽

Dilution Method ◽

Rt Pcr ◽

Serum Samples ◽

Live Virus ◽

Viral Neutralization ◽

Microneutralization Assay ◽

Infected Cells

Abstract Background Individuals recovering from COVID-19 are shown to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability and some may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays that allow the determination of virus neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike receptor binding domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of the cytopathic effect, then lysed and RNA RT-PCR of SARS-CoV-2. The Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, while 4 samples gave neutralization at lower dilution, while one sample did not give any neutralization. The correlation between RBD OD and neutralization potential was found to be statistically correlated. Conclusion We describe a rapid RT-PCR based SARS-CoV-2 microneutralization assay for detection of neutralizing antibodies. This can effectively be used to test anti-viral activity of serum antibodies for investigation of both disease-driven and vaccine-induced responses.

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Antibody neutralization assay with SARS-CoV-2 and SARS-CoV pseudovirus v1 (protocols.io.biztkf6n)

protocols.io ◽

10.17504/protocols.io.biztkf6n ◽

2020 ◽

Author(s):

Bei Wang ◽

Wen Hsin ◽

Cheng I

Keyword(s):

Neutralization Assay ◽

Antibody Neutralization

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Similar antibody responses against SARS-CoV-2 in HIV uninfected and infected individuals on antiretroviral therapy during the first South African infection wave

Clinical Infectious Diseases ◽

10.1093/cid/ciab758 ◽

2021 ◽

Author(s):

Jumari Snyman ◽

Shi-Hsia Hwa ◽

Robert Krause ◽

Daniel Muema ◽

Tarylee Reddy ◽

...

Keyword(s):

South Africa ◽

Neutralization Assay ◽

Sub Saharan Africa ◽

People Living With Hiv ◽

Live Virus ◽

Antibody Secreting Cell ◽

Virus Neutralization Assay ◽

Time To Peak ◽

Domain Specific ◽

Sub Saharan

Abstract Background There is limited understanding of SARS-CoV-2 pathogenesis in African populations with a high burden of infectious disease comorbidities such as HIV. The kinetics, magnitude and duration of virus-specific antibodies and the underlying B cell responses in people living with HIV (PLWH) in sub-Saharan Africa have not been fully characterized. Methods We longitudinally followed SARS-CoV-2 infected individuals in Durban, KwaZulu-Natal, South Africa and characterized SARS-CoV-2 receptor binding domain-specific IgM, IgG and IgA antibodies weekly for a month, and then at 3 months post diagnosis. 7/30 (41.7%) were PLWH, 83% (25/30) of which were on ART and with full HIV suppression. Potency of convalescent plasma neutralization was determined using a live virus neutralization assay and antibody secreting cell population frequencies were determined by flow cytometry. Results Similar seroconversion rates, time to peak antibody titer, peak magnitude and durability of anti-SARS-CoV-2 IgM, IgG, IgA, were observed in HIV uninfected and PLWH with complete HIV suppression on ART. In addition, similar neutralization potency against an isolate of SARS-CoV-2, circulating at the time of sampling in the first wave of SARS-CoV-2 infections in South Africa was observed in both groups. Loss of IgA was significantly associated with age (p=0.023) and a previous diagnosis of TB (p=0.018). Conclusions Similar antibody response kinetics and neutralization potency in HIV negative and PLWH on stable ART in an African setting suggests that COVID-19 natural infections may confer comparable antibody immunity in these groups. This provides hope that COVID-19 vaccines will be effective in PLWH on stable ART.

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Serology in Children with Multisystem Inflammatory Syndrome (MIS-C) associated with COVID-19

10.1101/2020.07.10.20150755 ◽

2020 ◽

Cited By ~ 1

Author(s):

Christina A. Rostad ◽

Ann Chahroudi ◽

Grace Mantus ◽

Stacey A. Lapp ◽

Mehgan Teherani ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Limit Of Detection ◽

Geometric Mean ◽

Neutralization Assay ◽

Adverse Outcomes ◽

Igg Antibody ◽

Live Virus ◽

Lengths Of Stay ◽

Antibody Titers ◽

Inflammatory Syndrome

Objectives: We aimed to measure SARS-CoV-2 serologic responses in children hospitalized with multisystem inflammatory syndrome (MIS-C) compared to COVID-19, Kawasaki Disease (KD) and other hospitalized pediatric controls. Methods: From March 17, 2020 - May 26, 2020, we prospectively identified hospitalized children at Children's Healthcare of Atlanta with MIS-C (n=10), symptomatic PCR-confirmed COVID-19 (n=10), KD (n=5), and hospitalized controls (n=4). With IRB approval, we obtained prospective and residual blood samples from these children and measured SARS-CoV-2 spike (S) receptor binding domain (RBD) IgM and IgG binding antibodies by quantitative ELISA and SARS-CoV-2 neutralizing antibodies by live-virus focus reduction neutralization assay. We statistically compared the log-transformed antibody titers among groups and performed correlation analyses using linear regression. Results: All children with MIS-C had high titers of SARS-CoV-2 RBD IgG antibodies, which correlated strongly with neutralizing antibodies (R2=0.667, P<0.001). Children with MIS-C had significantly higher SARS-CoV-2 RBD IgG antibody titers (geometric mean titer [GMT] 6800, 95%CI 3495-13231) than children with COVID-19 (GMT 626, 95%CI 251-1563, P<0.001), children with KD (GMT 124, 95%CI 91-170, P<0.001) and other hospitalized pediatric controls (GMT 85 [all below assay limit of detection], P<0.001). All children with MIS-C also had detectable RBD IgM antibodies, indicating recent SARS-CoV-2 infection. RBD IgG titers correlated with erythrocyte sedimentation rate (ESR) (R2=0.512, P<0.046) and with hospital and ICU lengths of stay (R2=0.590, P=0.010). Conclusion: Quantitative SARS-CoV-2 RBD antibody titers may have a role in establishing the diagnosis of MIS-C, distinguishing it from other similar clinical entities, and stratifying risk for adverse outcomes.

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Antibody Response of BNT162b2 and CoronaVac Platforms in Recovered Individuals Previously Infected by COVID-19 against SARS-CoV-2 Wild Type and Delta Variant

Vaccines ◽

10.3390/vaccines9121442 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1442

Author(s):

Ruiqi Zhang ◽

Ka-Wa Khong ◽

Ka-Yi Leung ◽

Danlei Liu ◽

Yujing Fan ◽

...

Keyword(s):

Antibody Response ◽

Geometric Mean ◽

Neutralizing Antibody ◽

Fold Increase ◽

Neutralization Assay ◽

Wild Type ◽

Vaccine Platform ◽

Live Virus ◽

Virus Neutralization Assay ◽

Geometric Mean Titre

Vaccinating recovered patients previously infected by COVID-19 with mRNA vaccines to boost their immune response against wild-type viruses (WT), we aimed to investigate whether vaccine platform and time of vaccination affect immunogenicity against the SARS-CoV-2 WT and Delta variant (DV). Convalescent patients infected by COVID-19 were recruited and received one booster dose of the BNT162b2 (PC-B) or CoronaVac (PC-C) vaccines, while SARS-CoV-2 naïve subjects received two doses of the BNT162b2 (CN-B) or CoronaVac (CN-C) vaccines. The neutralizing antibody in sera against the WT and DV was determined with live virus neutralization assay (vMN). The vMN geometric mean titre (GMT) against WT in recovered individuals previously infected by COVID-19 reduced significantly from 60.0 (95% confidence interval (CI), 46.5–77.4) to 33.9 (95% CI, 26.3–43.7) at 6 months post recovery. In the PC-B group, the BNT162b2 vaccine enhanced antibody response against WT and DV, with 22.3-fold and 20.4-fold increases, respectively. The PC-C group also showed 1.8-fold and 2.2-fold increases for WT and DV, respectively, after receiving the CoronaVac vaccine. There was a 10.6-fold increase in GMT in the CN-B group and a 1.3-fold increase in the CN-C group against DV after full vaccination. In both the PC-B and PC-C groups, there was no difference between GMT against WT and DV after vaccination. Subjects in the CN-B and CN-C groups showed inferior GMT against DV compared with GMT against WT after vaccination. In this study, one booster shot effectively enhanced the pre-existing neutralizing activity against WT and DV in recovered subjects.

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A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

PLoS ONE ◽

10.1371/journal.pone.0259551 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0259551

Author(s):

Syed Hani Abidi ◽

Kehkashan Imtiaz ◽

Akbar Kanji ◽

Shama Qaiser ◽

Erum Khan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Vero Cells ◽

Neutralization Assay ◽

Dilution Method ◽

Rt Pcr ◽

Serum Samples ◽

Live Virus ◽

Viral Neutralization ◽

Microneutralization Assay ◽

Infected Cells

Background Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro‐neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

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Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera

10.1101/2020.08.13.20157222 ◽

2020 ◽

Cited By ~ 8

Author(s):

Kasopefoluwa Y. Oguntuyo ◽

Christian S Stevens ◽

Chuan-Tien Hung ◽

Satoshi Ikegame ◽

Joshua A. Acklin ◽

...

Keyword(s):

Viral Entry ◽

High Throughput Screening ◽

Neutralizing Antibodies ◽

Neutralization Assay ◽

Virus Neutralization ◽

Strong Positive Correlation ◽

Plasma Therapy ◽

Live Virus ◽

Virus Neutralization Assay ◽

Marked Variability

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

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A simple and safe antibody neutralization assay based on polio pseudoviruses

Human Vaccines & Immunotherapeutics ◽

10.1080/21645515.2018.1526553 ◽

2018 ◽

Vol 15 (2) ◽

pp. 349-357 ◽

Cited By ~ 3

Author(s):

Zheng Jiang ◽

Guixiu Liu ◽

Liao Guo-yang ◽

Mingbo Sun ◽

Kangwei Xu ◽

...

Keyword(s):

Neutralization Assay ◽

Antibody Neutralization

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mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 Omicron variant

10.1101/2021.12.20.473557 ◽

2021 ◽

Author(s):

Venkata-Viswanadh Edara ◽

Kelly E Manning ◽

Madison Ellis ◽

Lilin Lai ◽

Kathryn M Moore ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Antibody Responses ◽

Spike Protein ◽

Live Virus ◽

Neutralizing Activity ◽

Virus Neutralization Assay ◽

Fold Reduction

The BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines generate potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the global emergence of SARS-CoV-2 variants with mutations in the spike protein, the principal antigenic target of these vaccines, has raised concerns over the neutralizing activity of vaccine-induced antibody responses. The Omicron variant, which emerged in November 2021, consists of over 30 mutations within the spike protein. Here, we used an authentic live virus neutralization assay to examine the neutralizing activity of the SARS-CoV-2 Omicron variant against mRNA vaccine-induced antibody responses. Following the 2nd dose, we observed a 30-fold reduction in neutralizing activity against the omicron variant. Through six months after the 2nd dose, none of the sera from naive vaccinated subjects showed neutralizing activity against the Omicron variant. In contrast, recovered vaccinated individuals showed a 22-fold reduction with more than half of the subjects retaining neutralizing antibody responses. Following a booster shot (3rd dose), we observed a 14-fold reduction in neutralizing activity against the omicron variant and over 90% of boosted subjects showed neutralizing activity against the omicron variant. These findings show that a 3rd dose is required to provide robust neutralizing antibody responses against the Omicron variant.

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