Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

Hsieh, Ching-Lin; Goldsmith, Jory A.; Schaub, Jeffrey M.; DiVenere, Andrea M.; Kuo, Hung-Che; Javanmardi, Kamyab; Le, Kevin C.; Wrapp, Daniel; Lee, Alison G.; Liu, Yutong; Chou, Chia-Wei; Byrne, Patrick O.; Hjorth, Christy K.; Johnson, Nicole V.; Ludes-Meyers, John; Nguyen, Annalee W.; Park, Juyeon; Wang, Nianshuang; Amengor, Dzifa; Lavinder, Jason J.; Ippolito, Gregory C.; Maynard, Jennifer A.; Finkelstein, Ilya J.; McLellan, Jason S.

The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A 3.2 Ã…-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.