An In Vitro Microneutralization Assay for SARS‐CoV‐2 Serology and Drug Screening

Amanat, Fatima; White, Kris M.; Miorin, Lisa; Strohmeier, Shirin; McMahon, Meagan; Meade, Philip; Liu, Wen Chun; Albrecht, Randy A.; Simon, Viviana; Martínez-Sobrido, Luis; Moran, Thomas M.; Garcı́a-Sastre, Adolfo; Krammer, Florian

doi:10.1002/cpmc.108

Cited by 176 publications

(230 citation statements)

References 14 publications

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“…All neutralization assays were performed in the biosafety level 3 (BSL-3) facility following institutional guidelines as described previously (19,22). Briefly, serum samples were heat-inactivated at 56°C for 60 minutes prior to use.…”

Section: Micro-neutralization Assaymentioning

confidence: 99%

A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine

Sun

McCroskery

Liu

et al. 2020

Preprint

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A successful SARS-CoV-2 vaccine must be not only safe and protective but must also meet the demand on a global scale at low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent COVID-19 vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections or significantly attenuated SARS-CoV-2 induced disease. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.

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Section: Micro-neutralization Assaymentioning

confidence: 99%

A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine

Sun

McCroskery

Liu

et al. 2020

Preprint

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“…In the case of the peroxidase staining, a separate assay is needed to assess cell toxicity of NAbs or antiviral compounds. Both assays rely on the use of the SARS-CoV-1 cross-reactive NP mAb, 1C7 (Amanat et al, 2020) but any other mAb targeting a SARS-CoV-2 viral antigen can be used in the peroxidase and the infrared staining described below.…”

Section: Development Of the Prmnt Assaysmentioning

confidence: 99%

Rapidin vitroassays for screening neutralizing antibodies and antivirals against SARS-CoV-2

Park

Oladduni

Chiem

et al. 2020

Preprint

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Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

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“…Vero.E6 cells (ATCC CRL-1586-clone E6) were maintained in culture using Dulbecco's Modified Eagle Medium (DMEM; Gibco) which was supplemented with Antibiotic-Antimycotic (100 U/ml penicillin-100 μg/ml streptomycin-0.25 ug/ml Amphotericin B) (Gibco; 15240062) and 10% fetal bovine serum (FBS; Corning). SARS-CoV-2 (isolate USA-WA1/2020 BEI Resources, NR-52281) was grown in Vero.E6 cells as previously described and was used for the in vivo challenge (20). A viral seed stock for a non-replicating human adenovirus type-5 (HAdV-C5) vector expressing the human ACE2 receptor was obtained from the Iowa Viral Vector Core Facility.…”

Section: Discussionmentioning

confidence: 99%

“…While all groups showed good reactivity, a similar pattern emerged in which ΔCS and ΔCS-PP groups showed higher reactivity than WT and PP groups (Figure 2E). Finally, we performed microneutralization assays with authentic SARS-CoV-2 (20). Here, the WT, PP and ΔCS groups showed similar levels of neutralization while the ΔCS-PP group animals had higher serum neutralization titers ( Figure.…”

Section: All Versions Of the Recombinant Spike Protein Induce Robust mentioning

confidence: 95%

Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model

Amanat

Strohmeier

Rathnasinghe

et al. 2020

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The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the PP mutations completely protected from challenge in this mouse model.ImportanceA vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validates the choice of antigens that contain the PP mutation and suggests that deletion of the polybasic cleavage site could lead to a further optimized design.

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An In Vitro Microneutralization Assay for SARS‐CoV‐2 Serology and Drug Screening

Cited by 176 publications

References 14 publications

A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine

A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine

Rapidin vitroassays for screening neutralizing antibodies and antivirals against SARS-CoV-2

Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model

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