Vaccine Engineering in the Time of COVID: Rapid and Cost-Effective Development of Stable Clones for the Production of Sars-Cov-2 Vaccine Candidates in HEK293T Cells (Industry Candidate)

The COVID-19 pandemic has crudely demonstrated the value of enabling platforms to develop effective vaccines in a short time. Currently, there are no specific treatments or vaccines approved against COVID-19.

In this work, we describe the development of HEK293T cells engineered for the transient and stable expression of antigens of SARS-CoV-2, the causal agent of COVID-19.

We transfected the HEK293T cells with vectors engineered to produce different versions of antigens inspired in different SARS-CoV-2 proteins (i.e., Spike (S) and Nucleoprotein (NP)) and intracellular Green Fluorescent Protein (GFP) using Lipofectamine 3000. We then selected the transfected cells using puromycin pressure, dilution cloning, and cloning disks. This integrated strategy generated antigen-producing cells in 7 days with a transient expression of ~1 mg/L. Stable pools were produced after 4 weeks, with expression levels of ~1 mg/L. Stable clones with expression levels of ~2.0 mg/L were obtained within 10 weeks. The produced antibodies exhibited the expected functionality; they were selectively bound by anti-SARS-CoV-2 antibodies in ELISA experiments both using commercial reagents and convalescent sera from COVID (+) patients. In addition, we developed perfusion processes for the continuous production of antigens from adherent cultures.

By the combined use of GFP and the set of selection techniques here described, we drastically reduced the time from transfection to stable clone generation without resorting to costly equipment. In outbreaks or emergencies, this platform can remarkably shorten the development of new vaccines and biopharmaceuticals.