Reduced serum neutralization capacity against SARS-CoV-2 variants in a multiplex ACE2 RBD competition assay

As global vaccination campaigns against SARS-CoV-2 proceed, there is emerging interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. To assess neutralizing capacity, we developed a bead-based multiplex ACE2 RBD competition assay as a large scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and RBD, this assay detects the presence of Nabs against SARS-CoV2 in serum. Using this multiplex approach allows the simultaneous analysis of Nabs against all SARS-CoV-2 variants of concern and variants of interest in a single well. Following validation, we analyzed 325 serum samples from 186 COVID-19 patients of varying severity. Neutralization capacity was reduced for all variants examined compared to wild-type, especially for those displaying the E484K mutation. The neutralizing immune response itself, while highly individualistic, positively correlates with IgG levels. Neutralization capacity also correlated with disease severity up to WHO grade 7, after which it reduced.

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Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity

10.1101/2021.08.03.454782 ◽

2021 ◽

Author(s):

Akiko Koide ◽

Tatyana Panchenko ◽

Chan Wang ◽

Sara A Thannickal ◽

Larizbeth A Romero ◽

...

Keyword(s):

Machine Learning ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Machine Learning Algorithms ◽

Two Dimensional ◽

Serum Samples ◽

Antibody Isotype ◽

Neutralization Capacity ◽

Binding Interface ◽

Neutralizing Capacity

Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype-antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.

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Evaluation of a Pseudovirus Neutralization Assay for SARS-CoV-2 and Correlation with Live Virus-Based Micro Neutralization Assay

Diagnostics ◽

10.3390/diagnostics11060994 ◽

2021 ◽

Vol 11 (6) ◽

pp. 994

Author(s):

Ahmed Majdi K. Tolah ◽

Sayed S. Sohrab ◽

Khaled Majdi K. Tolah ◽

Ahmed M. Hassan ◽

Sherif A. El-Kafrawy ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Epidemiological Studies ◽

Neutralization Assay ◽

Serum Samples ◽

Live Virus ◽

Spike Gene ◽

Microneutralization Assay ◽

Viral Particles ◽

Reliable Performance ◽

Prior Infection

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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Immunogenicity of the Ad26.CoV2.S vaccine in people living with HIV

10.1101/2021.10.08.21264519 ◽

2021 ◽

Author(s):

Khadija Khan ◽

Gila Lustig ◽

Mallory Bernstein ◽

Derseree Archary ◽

Sandile Cele ◽

...

Keyword(s):

South African ◽

Neutralizing Antibodies ◽

People Living With Hiv ◽

Open Label ◽

Live Virus ◽

Neutralization Capacity ◽

Risk Of Death ◽

Increased Risk ◽

Significant Difference ◽

Living With Hiv

Background People living with HIV (PLWH) have been reported to have an increased risk of more severe Covid-19 disease outcome and an increased risk of death relative to HIV-uninfected individuals. Here we assessed the ability of the Johnson and Johnson Ad26.CoV2.S vaccine to elicit neutralizing antibodies to the Delta variant in PLWH relative to HIV-uninfected individuals. Methods We enrolled 26 PLWH and 73 HIV-uninfected participants from the SISONKE phase 3b open label South African clinical trial of the Ad26.CoV2.S vaccine in health care workers (HCW) in a prospective observational cohort study. Enrollment was a median 56 days (range 19-98 days) post-vaccination. HCW PLWH had well suppressed HIV viremia. As a comparison, we also enrolled unvaccinated participants previously infected with SARS-CoV-2. This group consisted of 34 PLWH and 28 HIV-uninfected individuals. We used the presence of SARS-CoV-2 nucleocapsid antibodies and any previous record of SARS-CoV-2 infection to differentiate the vaccinated participants into participants who were previously infected with SARS-CoV-2 and those not previously infected. Neutralization capacity was assessed using participant plasma in a live virus neutralization assay of the Delta SARS-CoV-2 variant currently dominating infections in South Africa. This study was approved by the Biomedical Research Ethics Committee at the University of KwaZulu-Natal (reference BREC/00001275/2020). Findings Unvaccinated PLWH showed 6-fold reduced neutralization of the Delta variant relative to HIV-uninfected participants (GMT=105 for HIV-uninfected, 15 for PLWH, p=0.001). The majority (68%) of Ad26.CoV2.S vaccinated HCW were found to be previously infected with SARS-CoV-2. In this group, Delta variant neutralization was 9-fold higher compared to the infected only group (GMT of 306 versus 36) and 26-fold higher relative to the vaccinated only group (GMT=12). There was no significant difference in Delta variant neutralization in vaccinated and previously SARS-CoV-2 infected PLWH relative to vaccinated and previously SARS-CoV-2 infected, HIV-uninfected participants (GMT of 300 for PLWH versus 307 for HIV-uninfected). Vaccinated only participants showed a low neutralization of the Delta variant, with a stronger response in PLWH (GMT=73, for PLWH, 6 for HIV-uninfected, p=0.02). Interpretation While PLWH showed reduced neutralization of the Delta variant following SARS-CoV-2 infection, the neutralization response following Ad26.CoV2.S vaccination was not inferior to HIV-uninfected study participants. Funding South African Medical Research Council, The Bill & Melinda Gates Foundation.

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Evaluation of SARS-CoV-2 Neutralizing Antibodies Using a Vesicular Stomatitis Virus Possessing SARS-CoV-2 Spike Protein

10.21203/rs.3.rs-86916/v1 ◽

2020 ◽

Author(s):

Hideki Tani ◽

Long Tan ◽

Miyuki Kimura ◽

Yoshihiro Yoshida ◽

Hiroshi Yamada ◽

...

Keyword(s):

Virus Infection ◽

Whole Blood ◽

Neutralizing Antibodies ◽

High Specificity ◽

Immunofluorescence Assay ◽

Hospitalized Patients ◽

Pseudotyped Virus ◽

Serum Samples ◽

Live Virus ◽

Specificity And Sensitivity

Abstract Background:SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. Methods:To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an immunofluorescence assay (IFA). Results:The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. Conclusions:In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

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Limited window for donation of convalescent plasma with high live-virus neutralizing antibodies for COVID-19 immunotherapy

10.1101/2020.08.21.261909 ◽

2020 ◽

Author(s):

Abhinay Gontu ◽

Sreenidhi Srinivasan ◽

Eric Salazar ◽

Meera Surendran Nair ◽

Ruth H. Nissly ◽

...

Keyword(s):

Symptom Onset ◽

Neutralizing Antibodies ◽

Igg Antibody ◽

Plasma Therapy ◽

Live Virus ◽

Viral Neutralization ◽

Knowledge Deficit ◽

Neutralizing Capacity ◽

Optimal Window

ABSTRACTThe optimal timeframe for donating convalescent plasma to be used for COVID-19 immunotherapy is unknown. To address this important knowledge deficit, we determined in vitro live-virus neutralizing capacity and persistence of IgM and IgG antibody responses against the receptor-binding domain and S1 ectodomain of the SARS-CoV-2 spike glycoprotein in 540 convalescent plasma samples obtained from 175 COVID-19 plasma donors for up to 142 days post-symptom onset. Robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 persist, in the aggregate, for at least 100 days post-symptom onset. However, a notable acceleration in decline in virus neutralization titers ≥160, a value suitable for convalescent plasma therapy, was observed starting 60 days after first symptom onset. Together, these findings better define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor, including age and COVID-19 disease severity score.One Sentence SummaryEvaluation of SARS-CoV-2 anti-spike protein IgM, IgG, and live-virus neutralizing titer profiles reveals that the optimal window for donating convalescent plasma for use in immunotherapy is within the first 60 days of symptom onset.

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SARS-CoV-2 variants of concern exhibit reduced sensitivity to live-virus neutralization in sera from CoronaVac vaccinees and naturally infected COVID-19 patients

10.1101/2021.07.10.21260232 ◽

2021 ◽

Author(s):

Vimvara Vacharathit ◽

Pakorn Aiewsakun ◽

Suwimon Manopwisedjaroen ◽

Chanya Srisaowakarn ◽

Thanida Laopanupong ◽

...

Keyword(s):

Low Income ◽

Neutralizing Antibodies ◽

Healthcare Workers ◽

Mitigation Strategies ◽

Low Income Countries ◽

Live Virus ◽

Neutralizing Capacity ◽

Need To Evaluate ◽

Beijing China ◽

Spike Sequence

Recent surges in SARS-CoV-2 variants of concern (VOCs) call for the need to evaluate levels of vaccine- and infection- induced SARS-CoV-2 neutralizing antibodies (NAbs). CoronaVac (Sinovac Biotech, Beijing, China) is currently being used for mass vaccination in Thailand as well as other low-income countries. Three VOCs currently circulating within Thailand include the B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta) strains. We assessed NAb potency against the prototypic strain containing the original spike sequence (WT) compared to that against the 3 VOCs using sera derived from a cohort of healthcare workers who received a full 2-dose regimen of CoronaVac. Sera from two other cohorts consisting of COVID-19 patients who had been hospitalized in 2020 and 2021 were evaluated for comparison. We found that, despite equally robust production of S1-RBD-binding IgG and 100% seropositivity, sera from both CoronaVac vaccinees and naturally infected individuals had significantly reduced neutralizing capacity against all 3 VOCs compared to WT. Strikingly, NAb titers against Alpha and Beta were comparable, but Delta appears to be significantly more refractory to NAbs in all groups. Our results may help inform on CoronaVac NAb-inducing capacity, which is a proxy for vaccine efficacy, in the context of the WT strain and 3 VOCs. Our results also have critical implications for public health decisionmakers who may need to maintain efficient mitigation strategies amid a potentially high risk for infection with VOCs even in those who have been previously infected.

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A high-throughput cell- and virus-free assay shows reduced neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma

Science Translational Medicine ◽

10.1126/scitranslmed.abi8452 ◽

2021 ◽

pp. eabi8452

Author(s):

Craig Fenwick ◽

Priscilla Turelli ◽

Céline Pellaton ◽

Alex Farina ◽

Jérémy Campos ◽

...

Keyword(s):

High Throughput ◽

Neutralizing Antibodies ◽

Competitive Inhibition ◽

Natural Infection ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Spike Protein ◽

Serum Samples ◽

Live Virus ◽

Protein Variants

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies in the serum of an individual indicates prior infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral spike protein are more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, inflexible, and potentially biohazardous. Here, we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 spike protein binding to the angiotensin converting enzyme 2 (ACE2) receptor. This high-throughput method matches the performance of the gold standard live virus infection assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific IgG titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 spike protein variants of concern. We used our assay to profile serum samples from 59 patients hospitalized with coronavirus disease 2019 (COVID-19). We found that, although most sera had high activity against the 2019-nCoV parental spike protein and, to a lesser extent, the α (B.1.1.7) variant, only 58% of serum samples could efficiently neutralize a spike protein derivative containing mutations present in the β (B.1.351) variant. Thus, we have developed an assay that can evaluate effective neutralizing antibody responses to SARS-CoV-2 spike protein variants of concern after natural infection and that can be applied to characterize vaccine-induced antibody responses or to assess the potency of monoclonal antibodies.

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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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Recombinant subunits of SARS-CoV-2 spike protein as vaccine candidates to elicit neutralizing antibodies

10.21203/rs.3.rs-414366/v1 ◽

2021 ◽

Author(s):

Faezeh Noorabad ghahroodi ◽

Saeed Khalili ◽

Mohammad Javad Rasaee

Keyword(s):

Recombinant Proteins ◽

In Silico ◽

Neutralizing Antibodies ◽

Spike Protein ◽

Serum Samples ◽

Strong Immune Response ◽

Live Virus ◽

Vaccine Candidates ◽

Antibody Titers

Abstract The spike protein has been reported as one of the most critical targets for vaccine design strategies against the SARS-COV-2 infection. Hence, we have designed, produced, and evaluated the potential use of recombinant proteins derived from spike protein as vaccine candidates capable of neutralizing SARS-COV-2 virus. In silico tools were used to design spike-based subunit recombinant proteins (P1, P2, and P3). These proteins were checked for their ability to be identified by the anti-SARS-COV-2 antibodies by exposing them to Covid-19 serum samples. The proteins were then injected into mice and rabbits and the antibody titers were measured for 170 days. The virus neutralization test (VNT) was performed to analyze the obtained antibodies for their neutralization efficiency. The antibodies that existed in the serum of COVID-19 patients have identified the designed proteins. The anti-spike antibody titer was increased in the animals injected with recombinant proteins. The VNT results revealed that the produced antibodies could neutralize the cultured live virus. The long-lasting antibody titers (130 and 170 days for rabbit and mouse groups) indicated the elicitation of a strong immune response by the recombinant proteins. Subunit vaccines could also be considered as robust tools for effective vaccination against COVID-19. Using a combination of in silico, in vitro, and in vivo experiments, it was shown that the injection of spike-based recombinant proteins could stimulate long-lasting and neutralizing antibody responses. Further evaluation of the recombinant proteins examined in our studies in higher primates and human would elucidate their true potentials.

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