(27aw) Production and Application of SARS-CoV-2 Pseudotyped Virus for Screening Antiviral Efficacy of Test Compounds on a High-Throughput Platform

Since its emergence in late 2019 in Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a pandemic that has infected over 761 million people and claimed over 6.8 million lives, according to WHO as of March 29 2023. With frequent mutations in the virus’s spike protein, it has become imperative to be able to test drugs for their efficacy in preventing and treating infection and to do so for multiple compounds against multiple strains of the virus that appear or are circulating. There is also concern that new and emerging strains may escape vaccine- or infection-induced immunity. Pseudovirus surrogates are ideal to study interactions of SARS-CoV-2 to study cell-surface receptors for viral entry. The other requirement is developing a high-throughput platform, which would allow quantitative testing multiple variants of concern (VOCs) against multiple drugs.

Pseudoviruses are generated in adherent HEK293T cells by transfecting with a reporter plasmid, a packaging plasmid and the plasmid expressing the SARS-CoV-2 spike glycoprotein (SGP). The cell supernatant is collected 48 and 72 h post transfection, then subjected to a 2-step concentration protocol. This results in high titers of 1.2x10⁶ TU/mL of serum-free pseudovirus particles, which are used in pseudovirus neutralization assays on 96-well plates. For this purpose, a human ACE2 293T cell line is used, which expresses Angiotensin Converting Enzyme-2 (ACE2), a receptor to which the spike protein binds to initiate infection. Pseudovirus particles are incubated with various concentrations of compounds at 37°C for 1 h, then added to cells. After 2 h the medium is changed, and the cells imaged 48 h later. This assay protocol has been used to test entry-level inhibition of pseudovirus particles by antibodies as well as sulfated polysaccharides such as fucoidans, suramin, heparin, and pentosan polysulfate (PPS). Among these, antibody CC12.14, which targets the SGP receptor binding domain (RBD), has been found to exhibit entry-level inhibition of the Wuhan-Hu-1 (wild-type) pseudovirus, with an IC₅₀ value of 5.2 ng/mL. We have also demonstrated that fucoidan extracts from brown alga possess very strong binding to pseudovirus particles. As a result, we are investigating the use of fucoidan extracts as prophylactic nasal spray formulations and have observed protection against entry of wild-type pseudovirus, having an IC₅₀ value of 5.5 ng/mL. We have also shown that a small polysulfated molecule, suramin, with an EC₅₀ of 134 µM for the wild-type pseudovirus (Kwon et al, 2023,) also exhibits entry-level inhibition. PPS showed an IC₅₀ value of 0.45 µg/mL for the wild-type pseudovirus (Zhang et al, 2022). Finally, we are transitioning standard 96-well plate assays to a high-throughput platform, which allows for simultaneous screening of myriad drugs against different VOCs. This platform involves a two-chip microscale system with volumes as low as 1000 nL. As a result of the scaled-down platform, less reagent and virus particles are required, which allows for greater screening efficiency. This will address the need for broad surveillance screening of new variant immune escape and can be extended to study other infectious diseases.

References:

1. Zhang F, He P, Rodrigues AL, Jeske W, Tandon R, Bates JT, Bierdeman MA, Fareed J, Dordick J, Linhardt RJ. Potential Anti-SARS-CoV-2 Activity of Pentosan Polysulfate and Mucopolysaccharide Polysulfate. Pharmaceuticals 15(2):258 (2022). https://doi.org/10.3390/ph15020258.
2. Kwon P, Xu S, Hanseul O, Kwon S, Rodrigues AL, Feroz M, Fraser K, He P, Zhang F, Hong J, Lindhart RJ, Dordick, J. Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions. Commun. Biol. (2023). (Publication forthcoming)