Plasmids used in this study.

<div><p>Protective vaccines are crucial for preventing and controlling coronavirus disease 2019 (COVID-19). Updated vaccines are needed to confront the continuously evolving and circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. These vaccines should be safe, effective, amenable to easily scalable production, and affordable. Previously, we developed receptor binding domain (RBD) dimer-based protein subunit vaccines (ZF2001 and updated vaccines) in mammalian cells. In this study, we explored a strategy for producing RBD-dimer immunogens in <i>Pichia pastoris</i>. We found that wild-type <i>P</i>. <i>pastoris</i> produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in <i>P</i>. <i>pastoris</i>. Therefore, we engineered the wild type <i>P</i>. <i>pastoris</i> (GS strain) into GSΔOCH1pAO by deleting the <i>OCH1</i> gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the <i>HIS4</i> gene (encoding histidine dehydrogenase) to increase histidine synthesis for better growth. In addition, RBD-dimer protein was truncated to remove the R328/F329 cleavage sites in <i>P</i>. <i>pastoris</i>. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the <i>P</i>. <i>pastoris</i> expression system. We further resolved the cryo-EM structure of prototype-Beta RBD-dimer complexed with the neutralizing antibody CB6 to reveal the completely exposed immune epitopes of the RBDs. In a murine model, we demonstrated that the yeast-produced RBD-dimer induces robust and protective antibody responses, which is suitable for boosting immunization. This study developed the yeast system for producing SARS-CoV-2 RBD-dimer immunogens, providing a promising platform and pipeline for the future continuous updating and production of SARS-CoV-2 vaccines.</p></div>