An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model

• Published in: International journal of molecular sciences Vol. 23; no. 24; p. 15744
• Main Authors: Brindha, Subbaian, Yoshizue, Takahiro, Wongnak, Rawiwan, Takemae, Hitoshi, Oba, Mami, Mizutani, Tetsuya, Kuroda, Yutaka
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 12.12.2022; MDPI
• Subjects: Animals; Antibodies; Antibodies, Neutralizing; Antibodies, Viral; Antigens; Chemical bonds; Coronaviruses; COVID-19; COVID-19 Vaccines; E coli; Enzymes; Escherichia coli; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Proteins; SARS-CoV-2; Severe acute respiratory syndrome coronavirus 2; Spectrum analysis; Vaccines; Vaccines, Subunit; Viral infections; Viral Vaccines; disulfide bond; oxidative refolding; subunit antigen; pseudovirus neutralization; Escherichia coli expression; immunogenicity; cell surface markers
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms232415744

A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD’s eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell’s receptor, with a binding constant of 7.9 × 10−9 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.