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.

E. coli-expressed proteins could provide a rapid, cost-effective, and safe antigen for subunit vaccines, provided we can produce them in a properly folded form inducing neutralizing antibodies. Here, we use an E. coli-expressed SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as a model to examine whether it yields neutralizing antisera with effects comparable to those generated by the S1 subunit of the spike protein (S1 or S1 subunit, thereafter) expressed in mammalian cells. We immunized 5-week-old Jcl-ICR female mice by injecting RBD (30 µg) and S1 subunit (5 µg) according to four schemes: two injections 8 weeks apart with RBD (RBD/RBD), two injections with S1 (S1/S1), one injection with RBD, and the second one with S1 (RBD/S1), and vice versa (S1/RBD). Ten weeks after the first injection (two weeks after the second injection), all combinations induced a strong immune response with IgG titer > 105 (S1/RBD < S1/S1 < RBD/S1 < RBD/RBD). In addition, the neutralization effect of the antisera ranked as S1/RBD~RBD/S1 (80%) > S1/S1 (56%) > RBD/RBD (42%). These results indicate that two injections with E. coli-expressed RBD, or mammalian-cell-produced spike S1 subunit alone, can provide some protection against SARS-CoV-2, but a mixed injection scheme yields significantly higher protection.