Closing the gate on SARS-CoV-2: A VSV pseudotype neutralization assay targeting the key to viral entry

To address the urgent and immediate need for R&D tools for the fight against the public health threat from SARS-CoV-2, IBT Bioservices now offers a pseudotype virus system to assay inhibition of infectivity in a BSL-2 environment. Briefly, Vesicular Stomatitis virus (VSV) glycoprotein gene (G) has been substituted with SARS-CoV-2 Spike Protein (rVSV pseudotyped SARS-CoV-2 Spike). The recombinant rVSV-DG SARS-CoV-2 Spike has been incorporated into a convenient luciferase-based neutralization assay for evaluating the efficacy of drug candidates that target Spike-mediated infection (Figure 1). This system is similar to previously published and validated VSV pseudotype platform for Ebolavirus and Marburgvirus1,2,3.

Key advantages: The heavily glycosylated Spike proteins on its surface give Coronavirus a crown-like appearance. These Spike proteins recognize the ACE2 receptor on the host cell surface to facilitate viral entry and fusion (inset). Anti-Spike antibodies have been detected in convalescent patient plasma suggesting the S is immunogenic. It is a key determinant of host and cell tropism, antigenicity and immunogenicity, interspecies transmission as well as pathogenesis4 therefore an attractive candidate for antiviral inhibitors and vaccine development5,6,7.

BLS-2 containment: Virus attachment and entry have been a target for antiviral therapies against major human pathogens such as HIV, Dengue and Ebola virus as well as for Middle Eastern Respiratory Syndrome virus (MERS-CoV) and SARS-CoV. The need for BSL3 containment for such highly pathogenic viruses creates challenges for large-scale screening of therapeutic candidates; the pseudotyped virus platform makes it possible for scientists to safely handle and study such pathogens under BSL2 conditions.

Assay Specifics: Serial semi-log dilutions of each test article and control are mixed with the VSV-pseudotype virus in a 1:1 ratio for 1h at RT followed by incubation over Vero cells at 37°C. The cells are lysed the following day and luciferase activity is measured to assess the potency of each candidate to block viral entry into the cells. Neutralization is determined relative to the untreated virus controls. In dose-response experiments, the concentration of test article causing 50% neutralization (IC50) is determined.

IBT Bioservices is a BSL2 pre-clinical CRO supporting infectious disease R&D. Our mission is to deliver high quality R&D support to advance the fight against infectious diseases. We offer a range of discovery tools and testing services to help progress your project toward your developmental milestone and secure your next round of funding. We share your goals of bringing new treatments to market. If you have a test candidate you would like to evaluate in our in vitro assays or infection model contact us

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References:

  1. Zhao, X., Howell, K., He, S., et al. (2017). Immunization-elicited broadly protective antibody reveals Ebolavirus fusion loop as a site of vulnerability. Cell.169(5):891‐904.e15. doi:10.1016/j.cell.2017.04.038
  2. Howell, K., Brannan, J., Bryan, C. et al. (2017). Cooperativity enables non-neutralizing antibodies to neutralize Ebolavirus. Cell Rep. (19) 2: 413-424 doi: 10.1016/j.celrep.2017.03.049
  3. Millet, J., Goldstein, M., Labitt,R. et al. (2016). A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties, Emerging Microbes & Infections, 5:1, 1-9, doi: 10.1038/emi.2016.125
  4. Dimitrov, D., (2004). Virus entry: molecular mechanisms and biomedical applications. Nat Rev Microbiol2, 109–122. Doi:10.1038/nrmicro817
  5. White, J., Delos, S., Brecher, E. et al. (2008). Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme. Critical Reviews in Biochemistry and Molecular Biology, 43:3, 189-219, doi: 10.1080/10409230802058320
  6. Li, F. (2016). Structure, function, and evolution of Coronavirus Spike Proteins Annual Review of Virology Vol. 3:237-261 doi:10.1146/annurev-virology-110615-042301
  7. Tai, W., He, L., Zhang, X. et al.(2020). Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol17, 613–620, doi: 10.1038/s41423-020-0400-4 

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