Omicron RBD expressed in E. coli outperforms mammalian-expressed S1 spike protein in generating highly neutralizing anti-SARS-CoV-2 antibodies in mice.

SARS-CoV-2 variants are characterized by high transmissibility, rapid mutations, and the ability to evade immune responses, underscoring the urgent need for affordable and easily scalable vaccine platforms. We previously reported that the E. coli-expressed receptor-binding domain (RBD) from both the Omicron BA.5 and the Wuhan variants, administered at 30 μg x 4 doses, induces robust humoral and cellular immune responses in mice. Here, using Omicron BA.5 as a model, we demonstrate that a much lower 5 μg x 2 doses of E. coli-expressed RBD elicited a strong anti-SARS-CoV-2 response even without adjuvant. In contrast, the mammalian-expressed S1 spike protein (S1) failed to do so under identical conditions. With TiterMax Gold adjuvant, both RBD and S1 induced T-cell memory and balanced immune responses; however, RBD consistently elicited stronger responses. In addition, anti-RBD sera neutralized SARS-CoV-2 in a pseudovirus neutralization assay. The superior immunogenicity of RBD is likely attributable to its high purity (RP-HPLC purified), native-like conformation, including all four native disulfide bonds and the enrichment of key neutralizing epitopes by focusing on the ACE2 receptor-binding region. Collectively, these results highlight E. coli-expressed RBD as a promising, safe, and cost-effective antigen for next-generation subunit vaccine development, and suggest that it may serve as a valuable tool for serological testing.