tetano
Editor, Senior Moderator
PLoS Pathog
. 2024 Aug 30;20(8):e1012487.
doi: 10.1371/journal.ppat.1012487. eCollection 2024 Aug. Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris
Tongxin Zhao[SUP] 1 [/SUP], Sheng Liu[SUP] 2 [/SUP], Pengyan Wang[SUP] 1 [/SUP], Yanfang Zhang[SUP] 1 [/SUP], Xinrui Kang[SUP] 3 [/SUP], Xiaoqian Pan[SUP] 3 [/SUP], Linjie Li[SUP] 1 [/SUP], Dedong Li[SUP] 1 [/SUP], Ping Gao[SUP] 1 [/SUP], Yaling An[SUP] 1 [/SUP], Hao Song[SUP] 4 [/SUP], Kefang Liu[SUP] 1 [/SUP], Jianxun Qi[SUP] 1 [/SUP], Xin Zhao[SUP] 1 [/SUP], Lianpan Dai[SUP] 1 [/SUP], Peipei Liu[SUP] 5 [/SUP], Peiyi Wang[SUP] 2 [/SUP], Guizhen Wu[SUP] 5 [/SUP], Taicheng Zhu[SUP] 6 [/SUP], Kun Xu[SUP] 4 [/SUP], Yin Li[SUP] 6 [/SUP], George F Gao[SUP] 1 4 5 [/SUP]
Affiliations
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 Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 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 P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris 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.
. 2024 Aug 30;20(8):e1012487.
doi: 10.1371/journal.ppat.1012487. eCollection 2024 Aug. Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris
Tongxin Zhao[SUP] 1 [/SUP], Sheng Liu[SUP] 2 [/SUP], Pengyan Wang[SUP] 1 [/SUP], Yanfang Zhang[SUP] 1 [/SUP], Xinrui Kang[SUP] 3 [/SUP], Xiaoqian Pan[SUP] 3 [/SUP], Linjie Li[SUP] 1 [/SUP], Dedong Li[SUP] 1 [/SUP], Ping Gao[SUP] 1 [/SUP], Yaling An[SUP] 1 [/SUP], Hao Song[SUP] 4 [/SUP], Kefang Liu[SUP] 1 [/SUP], Jianxun Qi[SUP] 1 [/SUP], Xin Zhao[SUP] 1 [/SUP], Lianpan Dai[SUP] 1 [/SUP], Peipei Liu[SUP] 5 [/SUP], Peiyi Wang[SUP] 2 [/SUP], Guizhen Wu[SUP] 5 [/SUP], Taicheng Zhu[SUP] 6 [/SUP], Kun Xu[SUP] 4 [/SUP], Yin Li[SUP] 6 [/SUP], George F Gao[SUP] 1 4 5 [/SUP]
Affiliations
- PMID: 39213280
- PMCID: PMC11364227
- DOI: 10.1371/journal.ppat.1012487
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 Pichia pastoris. We found that wild-type P. pastoris produced hyperglycosylated RBD-dimer protein containing four N-glycosylation sites in P. pastoris. Therefore, we engineered the wild type P. pastoris (GS strain) into GSΔOCH1pAO by deleting the OCH1 gene (encoding α-1,6-mannosyltransferase enzyme) to decrease glycosylation, as well as by overexpressing the HIS4 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 P. pastoris. Several homogeneous RBD-dimer proteins were produced in the GSΔOCH1pAO strain, demonstrating the feasibility of using the P. pastoris 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.