Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants.
Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Mice were immunized twice intramuscularly at a 21-day interval.
We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. This formulation is equivalent to the CorbevaxTM vaccine that recently received emergency use authorization by the Drugs Controller General of India.
We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. Our findings highlight the importance of elucidating the key interrelationships between antigen-adjuvant interactions, storage stability, and in vivo performance to enable successful formulation development of stable and efficacious subunit vaccines. Interestingly, the loss of in vivo potency associated with heat-stressed RBD-J formulated with AH+CpG after one dose was abrogated by a booster. In contrast, RBD-J formulated with AP+CpG showed minimal antigen-adjuvant interactions, a better stability profile, but suboptimal immune responses. Although AH+CpG-adjuvanted RBD-J was the least stable in vitro, the formulation was most potent at eliciting SARS-CoV-2 pseudovirus neutralizing antibodies in mice. DSC studies demonstrated that aluminum-salt and CpG adjuvants decrease the conformational stability of RBD-J and suggest a direct CpG-RBD-J interaction. ACE2 competition ELISA analyses of formulated RBD-J stored at varying temperatures (4, 25, 37☌) revealed that RBD-J was destabilized by AH, an effect exacerbated by CpG. While RBD-J was 50% adsorbed to AH and <15% to AP, addition of CpG resulted in complete AH binding, yet no improvement in AP adsorption. We assessed the effect of antigen-adjuvant interactions on the stability and mouse immunogenicity of various RBD-J preparations. Here, a yeast-produced recombinant RBD antigen (RBD-L452K-F490W or RBD-J) was formulated with various combinations of aluminum salt (Alhydrogel®, AH AdjuPhos®, AP) and CpG 1018 adjuvants. Antibodies against RBD neutralize SARS-CoV-2 by blocking viral attachment to the host cell receptor, ACE2. To this end, subunit-based approaches targeting the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein remain attractive. Low-cost, refrigerator-stable COVID-19 vaccines will facilitate global access and improve vaccine coverage in low-and middle-income countries. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans. The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice.
After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines.