Real-time cell analysis: A high-throughput approach for testing SARS-CoV-2 antibody neutralization and escape

Suryadevara, Naveenchandra; Gilchuk, Pavlo; Zost, Seth J.; Mittal, Nikhil; Zhao, Li Leyna; Crowe, James E.; Carnahan, Robert H.

doi:10.1016/j.xpro.2022.101387

Cited by 11 publications

(10 citation statements)

References 23 publications

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“…We next sought to identify neutralization-resistant viral variants for each of the seven nAbs described in this study. To do this, we employed two different methods: (1) a high-throughput escape mutant generation assay using real-time cellular analysis based on similar assays previously described ( Gilchuk et al, 2020 ; Greaney et al, 2021 ; Suryadevara et al, 2022 ) and (2) serial passaging of virus in increasing concentrations of neutralizing antibodies ( Figure 2a ). Previously, we demonstrated that our mAbs showed similar neutralization potencies for VSV-pseudotyped viruses and authentic viruses ( Engdahl et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

“…The RTCA method described above was modified to generate escape mutant viruses after a single passage under saturating neutralizing antibody concentrations ( Greaney et al, 2021 ; Suryadevara et al, 2022 ; Gilchuk et al, 2021 ). 0.5, 1, 5, 10, or 20 µg/mL (depending on the potency of the antibody to the given virus) of the selection antibody was mixed 1:1 in 2% FBS-supplemented DMEM with VSV/ANDV (~10,000 IU per well) or VSV/SNV (~12,000 IU per well) and incubated for 1 hr at 37 °C and then added to cells.…”

Section: Methodsmentioning

confidence: 99%

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Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Engdahl

Binshtein

Brocato

et al. 2023

eLife

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Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While infections in humans are relatively rare, mortality rates range from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the human humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity to authentic hantaviruses. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Engdahl

Binshtein

Brocato

et al. 2023

eLife

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“…To determine neutralizing activity of IgG proteins, we used real-time cell analysis (RTCA) assay on an xCELLigence RTCA MP Analyzer (ACEA Biosciences Inc.) that measures virus-induced cytopathic effect (CPE) (Suryadevara, Gilchuk et al 2022). Briefly, 50 μL of cell culture medium (DMEM supplemented with 2% FBS) was added to each well of a 96-well E-plate using a ViaFlo384 liquid handler (Integra Biosciences) to obtain background reading.…”

Section: Elisamentioning

confidence: 99%

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody

Johnson,

Wall,

Kramer

et al. 2024

Preprint

Self Cite

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Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

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“…We next sought to identify neutralization-resistant viral variants for each of the seven nAbs described in this study. To do this, we used a recombinant vesiculostomatitis virus (VSV) pseudotyped with the ANDV or SNV glycoproteins and performed a high throughput escape mutant generation assay using real-time cellular analysis based on similar assays previously described [21][22][23] . We tested each antibody at saturating neutralization conditions and evaluated escape based on delayed cytopathic effect (CPE) in each individual replicate well.…”

Section: Low Likelihood Of Viral Escape For Neutralizing Hantavirus A...mentioning

confidence: 99%

“…RTCA escape mutant screening. The RTCA method described above was modified to generate escape mutant viruses after a single passage under saturating neutralizing antibody concentrations 22,23,69 . 0.5, 1, 5, 10, or 20 µg/mL (depending on the potency of the antibody to the given virus) of the selection antibody was mixed 1:1 in 2% FBS-supplemented DMEM with VSV/ANDV (~10,000 IU per well) or VSV/SNV (~12,000 IU per well) and incubated for 1 hour at 37°C and then added to cells.…”

Section: Flow Cytometric Binding Analysis To Mutant M Segments a Flow...mentioning

confidence: 99%

Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Engdahl

Binshtein

Brocato

et al. 2022

Preprint

View full text Add to dashboard Cite

Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While sporadic in North and South America, many infections occur in Europe and Asia, with mortality ranging from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity across hantavirus species. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines.

show abstract

Real-time cell analysis: A high-throughput approach for testing SARS-CoV-2 antibody neutralization and escape

Cited by 11 publications

References 23 publications

Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody

Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

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