IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers

Vandergaast, Rianna; Carey, Timothy S.; Reiter, Samantha; Lathrum, Chase; Lech, Patrycja; Gnanadurai, Clement W.; Haselton, Michelle; Buehler, Jason; Narjari, Riya; Schnebeck, Luke; Roesler, Anne; Sevola, Kara; Suksanpaisan, Lukkana; Bexon, Alice; Naik, Shruthi; Brunton, Bethany; Weaver, Scott C.; Rafael, Grace; Tran, Sheryl; Baum, Alina; Kyratsous, Christos A.; Peng, Kah Whye; Russell, Stephen J.

doi:10.1128/msphere.00170-21

Cited by 20 publications

(21 citation statements)

References 49 publications

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“…The Pearson correlation coefficient r values range from .82 to .93, with all P < .0001. 104,[108][109][110] The r values among neutralization assays using HIV pseudovirus and MN are not quite consistent, which range from .69 to .92 (P < .05). 96,105,111 It may be because HIV-1 and VSV pseudoviruses are single-cycle viruses, or their S protein density may be lower than that of authentic SARS-CoV-2.…”

Section: Pseudovirus Neutralization Assaymentioning

confidence: 93%

Advances in Neutralization Assays for SARS‐CoV‐2

Wang

et al. 2021

Scand J Immunol

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The coronavirus disease 2019 (COVID‐19) pandemic has triggered a global health emergency and brought disaster to humans. Tremendous efforts have been made to control the pandemic, among which neutralizing antibodies (NAbs) are of specific interest to researchers. Neutralizing antibodies are generated within weeks after infection or immunization and can protect cells from virus intrusion and confer protective immunity to cells. Thus, production of NAbs is considered as a main goal for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccines and NAbs may be used for patient treatment in the form of monoclonal antibodies. Neutralization assays are capable of quantitatively detecting NAbs against SARS‐CoV‐2, allowing to explore the relationship between the level of NAbs and the severity of the disease, and may predict the possibility of re‐infection in COVID‐19 patients. They can also be used to test the effects of monoclonal antibodies, convalescent plasma and vaccines. At present, wild‐type virus neutralization assay remains the gold standard for measuring Nabs, while pseudovirus neutralization assays, Surrogate virus neutralization test (sVNT) and high‐throughput versions of neutralization assays are popular alternatives with their own advantages and disadvantages. In this review article, we summarize the characteristics and recent progress of SARS‐CoV‐2 neutralization assays. Special attention is given to the current limitations of various neutralization assays so as to promote new possible strategies with NAbs by which rapid SARS‐CoV‐2 serological diagnosis and antiviral screening in the future will be achieved.

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Section: Pseudovirus Neutralization Assaymentioning

confidence: 93%

Advances in Neutralization Assays for SARS‐CoV‐2

Wang

et al. 2021

Scand J Immunol

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“…The PRNT is considered the reference VNT method, but it requires a Biosafety Level 3 (BSL-3) laboratory, takes at least two working days, and is very labor-consuming, making it impossible to use this test for any mass screenings [ 12 ]. The live virus could be substituted to the less dangerous pseudotyped virus, for example, the replication-competent vesical stomatitis virus with the S-protein from SARS-CoV-2 and the reporter gene of the firefly luciferase, resulting in the pseudotyped virus neutralization test (pVNT) [ 14 ]. This substitutive test can be performed in much more common BSL-2 laboratories but still requires a live cell culture, unusual analytical equipment, and 2–3 days to complete, so the routine mass use of pVNT is questionable.…”

Section: Introductionmentioning

confidence: 99%

Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD

et al. 2022

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The humoral response to the SARS-CoV-2 S protein determines the development of protective immunity against this infection. The standard neutralizing antibodies detection method is a live virus neutralization test. It can be replaced with an ELISA-based surrogate virus neutralization test (sVNT), measuring the ability of serum antibodies to inhibit complex formation between the receptor-binding domain (RBD) of the S protein and the cellular ACE2 receptor. There are conflicting research data on the sVNT methodology and the reliability of its results. We show that the performance of sVNT dramatically improves when the intact RBD from the Wuhan-Hu-1 virus variant is used as the plate coating reagent, and the HRP-conjugated soluble ACE2 is used as the detection reagent. This design omits the pre-incubation step in separate tubes or separate microplate and allows the simple quantification of the results using the linear regression, utilizing only 3–4 test sample dilutions. When this sVNT was performed for 73 convalescent plasma samples, its results showed a very strong correlation with VNT (Spearman’s Rho 0.83). For the RBD, bearing three amino acid substitutions and corresponding to the SARS-CoV-2 beta variant, the inhibitory strength was diminished for 18 out of 20 randomly chosen serum samples, and the magnitude of this decrease was not similar to the change in overall anti-RBD IgG level. The sVNT assay design with the ACE2-HRP is preferable over the assay with the RBD-HRP reagent and is suitable for mass screening of neutralizing antibodies titers.

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“…Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible high-throughput antibody assays which can pinpoint people who are at a particular risk. They may also provide information on how often booster vaccinations might be needed and help in developing improved vaccines and immunotherapies [177][178][179]. Nevertheless, there are significant concerns about only using the antibody response in coronavirus infections as a sole metric of protective immunity [180].…”

Section: What Are Strong and Measurable Correlations Between Protection And Disease?mentioning

confidence: 99%

Immune Responses against SARS-CoV-2—Questions and Experiences

et al. 2021

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Understanding immune reactivity against SARS-CoV-2 is essential for coping with the COVID-19 pandemic. Herein, we discuss experiences and open questions about the complex immune responses to SARS-CoV-2. Some people react excellently without experiencing any clinical symptoms, they do not get sick, and they do not pass the virus on to anyone else (“sterilizing” immunity). Others produce antibodies and do not get COVID-19 but transmit the virus to others (“protective” immunity). Some people get sick but recover. A varying percentage develops respiratory failure, systemic symptoms, clotting disorders, cytokine storms, or multi-organ failure; they subsequently decease. Some develop long COVID, a new pathologic entity similar to fatigue syndrome or autoimmunity. In reality, COVID-19 is considered more of a systemic immune–vascular disease than a pulmonic disease, involving many tissues and the central nervous system. To fully comprehend the complex clinical manifestations, a profound understanding of the immune responses to SARS-CoV-2 is a good way to improve clinical management of COVID-19. Although neutralizing antibodies are an established approach to recognize an immune status, cellular immunity plays at least an equivalent or an even more important role. However, reliable methods to estimate the SARS-CoV-2-specific T cell capacity are not available for clinical routines. This deficit is important because an unknown percentage of people may exist with good memory T cell responsibility but a low number of or completely lacking peripheral antibodies against SARS-CoV-2. Apart from natural immune responses, vaccination against SARS-CoV-2 turned out to be very effective and much safer than naturally acquired immunity. Nevertheless, besides unwanted side effects of the currently available vector and mRNA preparations, concerns remain whether these vaccines will be strong enough to defeat the pandemic. Altogether, herein we discuss important questions, and try to give answers based on the current knowledge and preliminary data from our laboratories.

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IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers

Cited by 20 publications

References 49 publications

Advances in Neutralization Assays for SARS‐CoV‐2

Advances in Neutralization Assays for SARS‐CoV‐2

Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD

Immune Responses against SARS-CoV-2—Questions and Experiences

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