Receptor-Binding Domain (RBD) Antibodies Contribute More to SARS-CoV-2 Neutralization When Target Cells Express High Levels of ACE2

Farrell, Ariana Ghez; Dadonaite, Bernadeta; Greaney, Allison J; Eguia, Rachel; Loes, Andrea N; Franko, Nicholas; Logue, Jennifer K.; Carreño, Juan Manuel; Abbad, Anass; Matreyek, Kenneth A.; Bloom, Jesse D.

doi:10.3390/v14092061

Cited by 29 publications

(31 citation statements)

References 44 publications

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“…The target cell line for different antibodies is determined by whether an antibody is able to neutralize pseudovirus on that cell line. We have previously described this phenomena in (Farrell et al, 2022) where we show that non-ACE2 competing antibodies do not fully neutralize pseudovirus on ACE2 overexpressing cells. While testing antibodies for the current study we also noticed that some S2-targeting antibodies are also not affected by ACE2 overexpression.…”

Section: Methodsmentioning

confidence: 79%

“…HEK-293T were acquired from ATCC (CRL3216), HEK-293T-ACE2 cells are described in (Crawford et al, 2020), generation and characterization of HEK-293T-ACE2-medium cells is described in (Farrell et al, 2022) (referred to ‘medium’ cells in the reference), generation of HEK-293T-rtTA cells is described below. All cells were grown in D10 media (Dulbecco’s Modified Eagle Medium with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin).…”

Section: Methodsmentioning

confidence: 99%

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A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

Dadonaite

Crawford

Radford

et al. 2022

Preprint

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A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations in the viral spike protein. Here we describe a new deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers of spike mutations impact antibody neutralization and pseudovirus infection. We demonstrate this new platform by making libraries of the Omicron BA.1 and Delta spikes. These libraries each contain ~7000 distinct amino-acid mutations in the context of up to ~135,000 unique mutation combinations. We use these libraries to map escape mutations from neutralizing antibodies targeting the receptor binding domain, N-terminal domain, and S2 subunit of spike. Overall, this work establishes a high-throughput and safe approach to measure how ~'10 to the 5' combinations of mutations affect antibody neutralization and spike-mediated infection. Notably, the platform described here can be extended to the entry proteins of many other viruses.

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Section: Methodsmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

Dadonaite

Crawford

Radford

et al. 2022

Preprint

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“… 26 Preclinical findings also observed that two doses of mRNA-1283 administered at lower dose levels than mRNA-1273 elicited higher nAb titers and bAb responses in mice, which may be due to greater antigen expression with mRNA-1283 at lower dose levels. 18 In addition, antibodies to the RBD and NTD regions are the primary contributors to the neutralization response observed in pseudovirus assays, 27 , 28 and the design of mRNA-1283 to focus the immune response to these 2 regions may shift the dose-dependent response of the vaccine to dose levels lower than 10 µg. Whether these findings extend to T-cell responses in humans is currently being assessed.…”

Section: Discussionmentioning

confidence: 99%

Interim analysis of a phase 1 randomized clinical trial on the safety and immunogenicity of the mRNA-1283 SARS-CoV-2 vaccine in adults

Yassini

Hutchens

Paila

et al. 2023

Human Vaccines & Immunotherapeutics

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This interim analysis of an ongoing phase 1 randomized clinical trial evaluated the safety, reactogenicity, and immunogenicity of mRNA-1283, a next-generation SARS-CoV-2 messenger RNA (mRNA)-based vaccine encoding two segments of the spike protein (i.e. receptor binding and N-terminal domains). Healthy adults aged 18–55 years ( n = 104) were randomized (1:1:1:1:1) to receive two doses of mRNA-1283 (10, 30, or 100 µg) or mRNA-1273 (100 µg) administered 28 days apart, or a single dose of mRNA-1283 (100 µg). Safety was assessed and immunogenicity was measured by serum neutralizing antibody (nAb) or binding antibody (bAb) responses. At the interim analysis, no safety concerns were identified and no serious adverse events, adverse events of special interest, or deaths were reported. Solicited systemic adverse reactions were more frequent with higher dose levels of mRNA-1283 than with mRNA-1273. At day 57, all dose levels of the 2-dose mRNA-1283 regimen (including the lowest dose level [10 µg]) induced robust nAb and bAb responses that were comparable to those of mRNA-1273 (100 µg). mRNA-1283 was generally safe in adults, with all dose levels of the 2-dose regimen (10, 30, and 100 µg) eliciting similar immunogenicity as the 2-dose mRNA-1273 regimen (100 µg). Clinical Trials Registration : Clinicaltrials.gov, NCT04813796.

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“…The viral titer was determined by infecting HEK293T-ACE2 cells and measuring relative luciferase units (RLUs). The HEK293T-ACE2 cells used express high levels of ACE2 as described previously (55, 99).…”

Section: Methodsmentioning

confidence: 99%

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection

Guenthoer

Lilly

Starr

et al. 2022

Preprint

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The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs, including recently circulating BA.4/BA.5, in both pseudovirus-based and live virus assays, and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are novel in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

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Receptor-Binding Domain (RBD) Antibodies Contribute More to SARS-CoV-2 Neutralization When Target Cells Express High Levels of ACE2

Cited by 29 publications

References 44 publications

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

Interim analysis of a phase 1 randomized clinical trial on the safety and immunogenicity of the mRNA-1283 SARS-CoV-2 vaccine in adults

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection

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