Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris

Zhao, Tongxin1; Liu, Sheng2; Wang, Pengyan1; Zhang, Yanfang1; Kang, Xinrui3; Pan, Xiaoqian3; Li, Linjie1; Li, Dedong1; Gao, Ping1; An, Yaling1; Song, Hao4; Liu, Kefang1; Qi, Jianxun1; Zhao, Xin1; Dai, Lianpan1; Liu, Peipei5; Wang, Peiyi2; Wu, Guizhen5; Zhu, Taicheng6; Xu, Kun4; Li, Yin6; Gao, George F.1,4,5

2024-08-01

10.1371/journal.ppat.1012487

PLOS PATHOGENS   影响因子和分区

1553-7366

1553-7374

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 GS Delta OCH1pAO by deleting the OCH1 gene (encoding alpha-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 GS Delta OCH1pAO 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.

SCI

英语

其他

National Key Research and Development Program of China["2021YFC2301300","2020YFA0907100"] ; Excellent Young Scientist Program from National Natural Science Foundation of China (NSFC)[82122031]

Microbiology ; Parasitology ; Virology

Microbiology ; Parasitology ; Virology

WOS:001304121000001

PUBLIC LIBRARY SCIENCE

Web of Science

1.Chinese Acad Sci, Inst Microbiol, CAS Key Lab Pathogen Microbiol & Immunol, Beijing, Peoples R China
2.Southern Univ Sci & Technol, Cryo EM Ctr, Shenzhen, Peoples R China
3.Univ Chinese Acad Sci UCAS, Med Sch, Beijing, Peoples R China
4.Chinese Acad Sci, Beijing Inst Life Sci, Res Network Immun & Hlth RNIH, Beijing, Peoples R China
5.Chinese Ctr Dis Control & Prevent, Natl Inst Viral Dis Control & Prevent, NHC Key Lab Biosafety, Beijing, Peoples R China
6.Chinese Acad Sci, Inst Microbiol, Dept Microbial Physiol & Metab Engn, State Key Lab Microbial Resources, Beijing, Peoples R China

Zhao, Tongxin,Liu, Sheng,Wang, Pengyan,et al. Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris[J]. PLOS PATHOGENS,2024,20(8).

Zhao, Tongxin.,Liu, Sheng.,Wang, Pengyan.,Zhang, Yanfang.,Kang, Xinrui.,...&Gao, George F..(2024).Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris.PLOS PATHOGENS,20(8).

Zhao, Tongxin,et al."Protective RBD-dimer vaccines against SARS-CoV-2 and its variants produced in glycoengineered Pichia pastoris".PLOS PATHOGENS 20.8(2024).