(173o) Production of Novel Sars-Cov-2 Spike Truncations in Chinese Hamster Ovary Cells Leads to High Expression and Binding to Antibodies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus infectious disease 2019 (COVID-19), has led to over 6 million deaths to date. Containment efforts such as surveillance and herd immunity require generating large quantities of viral glycoproteins. Therefore, the COVID-19 pandemic has highlighted the critical need for rapid, scalable, and cost-effective production of recombinant glycoproteins for use as antigens in clinical and research applications.

For SARS-CoV-2, scalable production of Spike glycoprotein is important for diagnosis and vaccination strategies. Spike is a major antigen that is responsible for entry into cells, and it is the primary target for antibody binding. Thus, immunoassays to assess immunity of individuals commonly utilize the Spike protein. Protein-based SARS-CoV-2 vaccines also rely on delivering Spike protein (Heath et al., 2021).

A major challenge to scaling these immunoassays and protein-based vaccines is producing large amounts of Spike protein in a cost-effective manner. Different forms of full-length Spike have been produced in mammalian cell lines, including mutations to increase stability, but Spike is difficult to express, with titers typically at approximately 5-30 mg/L (Amanat et al., 2020; Hsieh et al., 2020). Alternatively, the Spike RBD has been expressed, which can have titers that are an order of magnitude above those of Spike. However, RBD is less sensitive than Spike in serological assays (Amanat et al., 2020). Modifications to Spike and RBD have been made to increase stability and expression, such as through rational structure-based approaches and mutational scanning (Hsieh et al., 2020; Smaoui & Yahyaoui, 2021; Starr et al., 2020). Nonetheless, conserving the sequence of Spike and RBD is important for accurate comparisons with existing variants.

Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We optimize the production period of Spike and demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg/L at day 7. In comparison, RBD titers peak at 54 mg/L at day 3. We also develop 8 Spike truncations (T1-T8) to determine if a truncation can balance the high expression of RBD and high binding capabilities of Spike. The truncations T1 and T4 have high expression at 130 mg/L and 73 mg/L, respectively, which are higher than RBD titers. We also evaluate antibody binding via ELISA against antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against an anti-Spike monoclonal antibody and even outperforms Spike for an anti-Spike polyclonal antibody. T4 has overall lower sensitivity than full-length Spike to the antibodies, but still has higher sensitivity than RBD.

Using molecular dynamics simulations, we find that T1 has more structural similarity to full-length Spike than does T4. The computational results are corroborated by circular dichroism analysis for secondary structure composition, which also show higher similarity between Spike and T1 than T4. Taken together, these results suggest T1 is a promising Spike alternative for use in various applications. Future investigations include serological tests using the truncations.

References

Amanat, F., Stadlbauer, D., Strohmeier, S., Nguyen, T. H. O., Chromikova, V., McMahon, M., Jiang, K., Arunkumar, G. A., Jurczyszak, D., Polanco, J., Bermudez-Gonzalez, M., Kleiner, G., Aydillo, T., Miorin, L., Fierer, D. S., Lugo, L. A., Kojic, E. M., Stoever, J., Liu, S. T. H., ... Krammer, F. (2020). A serological assay to detect SARS-CoV-2 seroconversion in humans. Nature Medicine, 26(7), 1033–1036. https://doi.org/10.1038/s41591-020-0913-5

Heath, P. T., Galiza, E. P., Baxter, D. N., Boffito, M., Browne, D., Burns, F., Chadwick, D. R., Clark, R., Cosgrove, C., Galloway, J., Goodman, A. L., Heer, A., Higham, A., Iyengar, S., Jamal, A., Jeanes, C., Kalra, P. A., Kyriakidou, C., McAuley, D. F., ... Toback, S. (2021). Safety and Efficacy of NVX-CoV2373 Covid-19 Vaccine. New England Journal of Medicine, 0(0), null. https://doi.org/10.1056/NEJMoa2107659

Hsieh, C.-L., Goldsmith, J. A., Schaub, J. M., DiVenere, A. M., Kuo, H.-C., Javanmardi, K., Le, K. C., Wrapp, D., Lee, A. G., Liu, Y., Chou, C.-W., Byrne, P. O., Hjorth, C. K., Johnson, N. V., Ludes-Meyers, J., Nguyen, A. W., Park, J., Wang, N., Amengor, D., ... McLellan, J. S. (2020). Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science (New York, N.y.), eabd0826. https://doi.org/10.1126/science.abd0826

Smaoui, M. R., & Yahyaoui, H. (2021). Unraveling the stability landscape of mutations in the SARS-CoV-2 receptor-binding domain. Scientific Reports, 11(1), 9166. https://doi.org/10.1038/s41598-021-88696-5

Starr, T. N., Greaney, A. J., Hilton, S. K., Ellis, D., Crawford, K. H. D., Dingens, A. S., Navarro, M. J., Bowen, J. E., Tortorici, M. A., Walls, A. C., King, N. P., Veesler, D., & Bloom, J. D. (2020). Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell, 182(5), 1295-1310.e20. https://doi.org/10.1016/j.cell.2020.08.012