SARS-CoV-2 Spike Pseudoviruses: A Useful Tool to Study Virus Entry and Address Emerging Neutralization Escape Phenotypes

  1. Raj Kalkeri
  2. Zhaohui Cai
  3. Shuling Lin
  4. John Farmer
  5. Yury V. Kuzmichev
  6. Fusataka Koide

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Abstract

SARS-CoV-2 genetic variants are emerging around the globe. Unfortunately, several SARS-CoV-2 variants, especially variants of concern (VOCs), are less susceptible to neutralization by the convalescent and post-vaccination sera, raising concerns of increased disease transmissibility and severity. Recent data suggests that SARS-CoV-2 neutralizing antibody levels are a reliable correlate of vaccine-mediated protection. However, currently used BSL3-based virus micro-neutralization (MN) assays are more laborious, time-consuming, and expensive, underscoring the need for BSL2-based, cost-effective neutralization assays against SARS-CoV-2 variants. In light of this unmet need, we have developed a BSL-2 pseudovirus-based neutralization assay (PBNA) in cells expressing the human angiotensin-converting enzyme-2 (hACE2) receptor for SARS-CoV-2. The assay is reproducible (R2 = 0.96), demonstrates a good dynamic range and high sensitivity. Our data suggest that the biological Anti-SARS-CoV-2 research reagents such as NIBSC 20/130 show lower neutralization against B.1.351 SA (South Africa) and B.1.1.7 UK (United Kingdom) VOC, whereas a commercially available monoclonal antibody MM43 retains activity against both these variants. SARS-CoV-2 spike PBNAs for VOCs would be useful tools to measure the neutralization ability of candidate vaccines in both preclinical models and clinical trials and would further help develop effective prophylactic countermeasures against emerging neutralization escape phenotypes.

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  1. Version published to 10.3390/microorganisms9081744
  2. ScreenIT

    SciScore for 10.1101/2021.07.16.452709: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-SARS-CoV-2 RBD Neutralizing Antibody, Human IgG1 (Cat#SAD-S35, Acro Biosystems, Newark, DE), SARS-CoV-2 (2019-nCoV
    Anti-SARS-CoV-2 RBD
    suggested: None
    Human IgG1
    suggested: (Abcam Cat# ab20402, RRID:AB_445563)
    SARS-CoV-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For pseudovirus production, Lenti-X-293T cells were co-transfected with pNL4-3.
    Lenti-X-293T
    suggested: None
    Sera 28-days post-infection from three (3) different species of non-human primates (NHPs) rhesus macaques (RM), cynomolgus monkeys (CM), African Green Monkeys (AGMs) (N=2 each), challenged with SARS-CoV-2 (SARS-CoV-2 USA_WA1/2020 strain at 4.0 x 106 TCID50/mL through intra-tracheal route) were used in the pseudovirus assay. 2.3. Pseudovirus Based Neutralization Assay: HEK293T-ACE2 cells (NR-52511, BEI Resources, Manasas, VA) were grown in Dulbecco’s Minimal Essential Medium (DMEM, Lonza, Walkersville, MD), supplemented with 10% Fetal Bovine Serum (FBS) according to standard culture conditions.
    HEK293T-ACE2
    suggested: None
    HEK293-ACE2 cells were seeded at ~20,000 cells per well into 96-well plates (100 μL per well) DMEM with 10% FBS without antibiotic the day before the assay.
    HEK293-ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    pNL4-3.Luc.R-E-, NIH AIDS Reagent, Catalog Number: 3418 was licensed/obtained from the New York University School of Medicine. Codon Optimized SARS-CoV-2 Spike gene from isolate 2019-nCoV_HKU-SZ-002a_2020 (GenBank: MN938384) was synthesized at GeneWiz, South Plainfield, NJ.
    pNL4-3.Luc.R-E-
    suggested: None
    Spike gene was cloned into eukaryotic expression plasmid pcDNA3.1 to generate plasmid, designated as pSRC332.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    pSRC332
    suggested: None
    For pseudovirus production, Lenti-X-293T cells were co-transfected with pNL4-3.
    pNL4-3
    suggested: None
    Luc.R-E- and pSRC322 using JetPrime transfection reagent (Polyplus Transfection, New York, NY).
    pSRC322
    suggested: None
    The next day cells were co-transfected with 3 μg pSRC332 and 12 μg pNL43. Luc. R. E using JetPrime® Transfection Reagent following the manufacturer’s instruction.
    pNL43
    suggested: None
    Software and Algorithms
    SentencesResources
    pNL4-3.Luc.R-E-, NIH AIDS Reagent, Catalog Number: 3418 was licensed/obtained from the New York University School of Medicine. Codon Optimized SARS-CoV-2 Spike gene from isolate 2019-nCoV_HKU-SZ-002a_2020 (GenBank: MN938384) was synthesized at GeneWiz, South Plainfield, NJ.
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Luminescence signal was normalized to the virus control after background subtraction and analyzed using PRISM software using four parameter logistic curve fitting to calculate 50% pseudovirus based neutralization index (PBNI50).
    PRISM
    suggested: (PRISM, RRID:SCR_005375)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.07.16.452709 on bioRxiv