A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

Muruato, Antonio E.; Fontes-Garfias, Camila R.; Ren, Ping; Garcia-Blanco, Mariano A.; Menachery, Vineet D.; Xie, Xuping; Shi, Pei–Yong

doi:10.1038/s41467-020-17892-0

Cited by 295 publications

(311 citation statements)

References 21 publications

(17 reference statements)

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“…Fifty percent virus neutralisation GMTs, measured by an authentic SARS-CoV-2 neutralisation assay 25 , were detectable in rhesus macaque sera by Day 21 after Dose 1 and peaked at a GMT of 962 (Day 35, 14 days after Dose 2 of 30 μg) or 1,689 (Day 28, 7 days after Dose 2 of 100 μg; Fig. 3b).…”

Section: Resultsmentioning

confidence: 98%

“…The SARS-CoV-2 neutralisation assay used a previously described strain of SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics and engineered by the insertion of an mNeonGreen (mNG) gene into open reading frame 7 of the viral genome 25 . This reporter virus generates similar plaque morphologies and indistinguishable growth curves from wild-type virus.…”

Section: Methodsmentioning

confidence: 99%

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A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

Vogel

Kanevsky

Che

et al. 2020

Preprint

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126

151

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To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD ‘up’, two-RBD ‘down’ state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFNγ+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).

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Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

Vogel

Kanevsky

Che

et al. 2020

Preprint

Self Cite

126

151

View full text Add to dashboard Cite

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“…Consequently, there is a strong demand to expand currently applied acute serological diagnostics measuring the overall immune response against SARS-CoV-2, towards a more NeutrobodyPlex can be performed fully automatable and in high-throughput and can readily be applied for large cohort screening. As it requires only non-living and non-infectious viral material, costs and safety conditions can be substantially decreased 19,29 . Furthermore, the NeutrobodyPlex is highly sensitive as low serum dilutions (tested dilution: 1:400) are sufficient for analysis which significantly reduces patient material compared to standard assays.…”

Section: Discussionmentioning

confidence: 99%

“…However, most available serological tests measure the full immune response and can therefore not differentiate between total binding and neutralizing antibodies 4,17,18 . Detection of the latter is still mostly performed by conventional virus neutralization tests (VNTs), which are both time consuming (2 -4 days) and require work with infectious SARS-CoV-2 virions in a specialized biosafety level 3 (BSL3) facility 19 . To overcome these limitations, we aimed to employ Nbs as antibody surrogates and developed a competitive binding approach to screen for neutralizing antibodies on a high-throughput basis in samples from patients or vaccinated individuals.…”

Section: Introductionmentioning

confidence: 99%

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

Wagner

Kaiser

Gramlich

et al. 2020

Preprint

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Facing the worldwide disease progression of COVID-19 caused by the SARS-CoV-2 virus, the situation is highly critical and there is an unmet need for effective vaccination, reliable diagnosis and therapeutic intervention. Neutralizing binding molecules such as antibodies or derivatives thereof have become important tools for acute treatment of COVID-19. Additionally, such binders provide the unique possibility to monitor the emergence and presence of a neutralizing immune response in infected or vaccinated individuals. Here we describe a set of 11 unique nanobodies (Nbs), originated from an immunized alpaca which bind with high affinities to the glycosylated SARS-CoV-2 Spike receptor domain (RBD). Using a multiplex in vitro binding assay we showed that eight of the selected Nbs effectively block the interaction between RBD, S1-domain and homotrimeric Spike protein with the angiotensin converting enzyme 2 (ACE2) as the viral docking site on human cells. According to competitive binding analysis and detailed epitope mapping, we grouped all Nbs blocking the RBD:ACE2 interaction in three distinct Nb-Sets and demonstrated their neutralizing effect with IC50 values in the low nanomolar range in a cell-based SARS-CoV-2 neutralization assay. Tested Nb combinations from different sets showed substantially lower IC50 values in both functional assays indicating a profound synergistic effect of Nbs simultaneously targeting different epitopes within the RBD. Finally, we applied the most potent Nb combinations in a competitive multiplex binding assay which we termed NeutrobodyPlex and detected a neutralizing immune response in plasma samples of infected individuals. We envisage that our Nbs have a high potential for prophylactic as well as therapeutic options and provide a novel approach to screen for a neutralizing immune response in infected or vaccinated individuals thus helping to monitor the immune status or to guide vaccine design.

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“…Fluorescence‐based assays use an engineered viral particle with a fluorescent protein gene expressed upon viral infection of eukaryotic cells 10 . One recently developed fluorescence microwell assay correlated well with plaque reduction but was faster and could be readily automated 7 . In contrast, live SARS‐CoV‐2 neutralization assays remain available only in specialized laboratories and are not widely used to screen convalescent plasma due to complexity of implementation, high cost, low throughput, and biosafety concerns.…”

mentioning

confidence: 99%

SARS‐CoV‐2 neutralization and serology testing of COVID‐19 convalescent plasma from donors with nonsevere disease

et al. 2020

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Background: The transfer of passive immunity with convalescent plasma is a promising strategy for treatment and prevention of COVID-19, but donors with a history of nonsevere disease are serologically heterogenous. The relationship between SARS-Cov-2 antigen-binding activity and neutralization activity in this population of donors has not been defined. Study Design and Methods: Convalescent plasma units from 47 individuals with a history of nonsevere COVID-19 were assessed for antigen-binding activity of using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. These results were compared with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. Results: Positive correlations of varying strength (Spearman r = 0.37-0.52) between antigen binding and viral neutralization were identified. Donors age 48 to 75 years had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75%-82%) for those with the highest levels of neutralization. Conclusion: The strength of the relationship between antigen-binding activity and neutralization varies depending on the clinical assay used. Units in the highest tertile of binding activity for each assay are predominantly comprised of those with the greatest neutralization activity.

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A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

Cited by 295 publications

References 21 publications

A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

SARS‐CoV‐2 neutralization and serology testing of COVID‐19 convalescent plasma from donors with nonsevere disease

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