Immunogenicity studies of recombinant RBD SARS-CoV-2 as a COVID-19 vaccine candidate produced in Escherichia coli

Abstract

The severe acute respiratory syndrome coronavirus 2 -related global COVID-19 pandemic has been impacting millions of people since its outbreak in 2020. COVID-19 vaccination has proven highly efficient in reducing illness severity and preventing infection-related fatalities. The World Health Organization has granted emergency use approval to multiple, including protein subunit technology-based, COVID-19 vaccines. Foreseeably, additional COVID-19 subunit vaccine development would be essential to meet the accessible and growing demand for effective vaccines, especially for Low-Middle-Income Countries (LMIC). The SARS-CoV-2 spike protein receptor binding domain (RBD), as the primary target for neutralizing antibodies, holds significant potential for future COVID-19 subunit vaccine development. In this study, we developed a recombinant Escherichia coli-expressed RBD (rRBD) as a vaccine candidate and evaluated its immunogenicity and preliminary toxicity in BALB/c mice. The rRBD induced humoral immune response from day 7 post-vaccination and, following the booster doses, the IgG levels increased dramatically in mice. Interestingly, our vaccine candidate also significantly induced cellular immune response, indicated by the incrased IFN-ɣ-producing cell numbers. We observed no adverse effect or local reactogenicity either in control or treated mice. Taken together, our discoveries could potentially support efficient and cost-effective vaccine antigen production, from which LMICs could particularly benefit.

Keywords: COVID-19; Escherichia coli; RBD; SARS-CoV-2; Vaccine candidate.

Fig. 1

Plasmid map and rRBD expression profile. A. pET16b-RBD plasmid containing the rRBD gene. B. SDS-PAGE analysis of rRBD production and purification. Lane 1: insoluble fraction (IB) containing rRBD; lane 2: lysate; lane 3: solubilized IB-rRBD; lane 4: NiNTA purified unfolded rRBD; lane 5: refolded rRBD.

Fig. 2

Comparative antigenicity evaluation of rRBD with commercial RBD using serum from three convalescent COVID-19 patients. Serum was diluted to 1:1000. The data is presented as the mean value of triplicate samples, with error bars indicating the standard error of the means.

Fig. 3

Mouse immunization with rRBD. A. Immunization and sample collection schedule in BALB/c mice. (B and C) Humoral immune responses against rRBD SARS-CoV-2 in female and male mice (n = 6 for each group) B and C. IgG and IgM levels were significantly higher both in male and female mice in rRBD-Alum group than in the PBS-Alum, respectively. **p < 0.001, ****p < 0.0001.

Fig. 4

T-cell responses against rRBD SARS-CoV-2 in mice. A. Male and female mice IFN-γ secreted cells was significantly higher in RBD-Alum group than in PBS-Alum group with *p < 0.05 and ****p < 0.001. B. Both female and male mouse group of RBD-Alum displayerd higher number of IL-4 producing cells than PBS-Alum group with statistical significances for male and female of *p < 0.05 and ****p < 0.0001. SFC: Spot Forming Cells.