Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

By K. H. Crawford, Rachel T. Eguia, A. Dingens, Andrea N. Loes, Keara D. Malone, C. Wolf, H. Chu, M. A. Tortorici, D. Veesler, M. Murphy and 4 more. bioRxiv, 2020

780 citations recorded by the source corpus.

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Subfields: Genetics, Infectious Diseases

View Paper: https://www.semanticscholar.org/paper/266bcde0c6bf0c0e92347e6d403388f09dfbed3a

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Genetics

1. Tyler N. Starr
2. K. H. Crawford
3. M. A. Tortorici
4. Allison J. Greaney
5. Jesse D. Bloom
6. A. Walls
7. D. Veesler

In Infectious Diseases

1. D. Veesler
2. A. Walls
3. M. A. Tortorici
4. Jesse D. Bloom

Citations in the corpus

• Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies (2020, positive): “In vitro neutralization assays for SARS-CoV-2 include pseudoviruses based on HIV lentiviral particles (Chen et al., 2020; Crawford et al., 2020; Ou et al., 2020; Robbiani et al., 2020; Wu et al., 2020a), murine leukemia virus retroviral particles (Pinto et al.”
• Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition (2020, positive): “The resulting viruses were titered as previously described (Crawford et al., 2020a) .”
• Title: Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice (2020, neutral): “lentiviral particles were prepared as described (18, 56) using genes encoding S protein”
• Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding (2020, positive): “Finally, we validated the deep mutational scanning measurements in the context of spike-pseudotyped lentiviral particles (Figures 4H and S4J; Crawford et al., 2020).”
• A human coronavirus evolves antigenically to escape antibody immunity (2020, methodology): “We deleted the last 19 amino acids of the spike’s C-terminus (in the cytoplasmic tail) as this modification has been reported (Crawford et al., 2020a; Kawase et al., 2009; Rogers et al., 2020) and validated in our hands (Figure S3) to improve titers of virions pseudotyped with spike.”
• mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant (2021, positive): “neutralization assay that we and others have previously validated (Crawford et al., 2020; GarciaBeltran et al., 2021b; Ju et al., 2020; Moore et al., 2004; Pinto et al., 2020; Wang et al., 2020; Yang et al., 2020) to assess SARS-CoV-2 variant neutralization (Figure 2B).”
• Deep mutational scanning of SARS-CoV-2 receptor binding domain reveals constraints on folding and ACE2 binding (2020, positive): “Finally, we tested how some mutations affected spike-mediated entry of pseudotyped lentiviral particles into ACE2-expressing target cells (Figure 4E) (Crawford et al., 2020).”
• Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity (2021, positive): “Vaccine-elicited neutralizing responses to circulating SARS-CoV-2 strains We accrued a cohort of 99 individuals who received either one or two full doses of the BNT162b2 or mRNA-1273 vaccines and used a luminescence-based lentiviral pseudovirus neutralization assay that we and others have previously”
• COVID-19 neutralizing antibodies predict disease severity and survival (2020, positive): “Using these spike modifications, we developed a CoV pseudovirus neutralization assay compatible with high-throughput liquid handling instrumentation in 384-well plate format using our previously published lentiviral vector system expressing both luminescent and fluorescent marker transgenes (Figure ”
• Circulating SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity (2021, positive): “…measured against pseudovirus and live SARS-CoV-2 cultures (Wang et al. 2020; Ju et al. 2020; Pinto et al. 2020; Yang et al. 2020; Moore et al. 2004; Crawford et al. 2020 In summary, our data highlights the challenges facing all vaccines whose designs were finalized early in the pandemic and based…”
• Efficient evolution of human antibodies from general protein language models (2023, neutral): “A five-plasmid system was used for viral production, as described in Crawford et al. 64 .”
• Comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human serum antibodies (2021, background): “For most of the 17 individuals, both the RBD binding and the neutralizing activity decreased moderately from one to four months post-infection (Crawford et al., 2020a) (Supplementary Table 1).”