Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice. MethodThe RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice. Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 {+/-} 3.0 mg/L of protein in the fermentation supernatant. 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 protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.

Receptor-binding domain recombinant protein RBD219-N1C1 on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

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. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. 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. 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. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Alum Induces Protective Immunity and Reduces Immune Enhancement

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.Competing Interest StatementThe authors have declared no competing interest.View Full Text