Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

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. 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. The 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 was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of 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 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. 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.

Structural and Biophysical Characterization of the HCV E1E2 Heterodimer for Vaccine Development.

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.

Genetic modification to design a stable yeast-expressed recombinant SARS-CoV-2 receptor binding domain as a COVID-19 vaccine candidate.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. METHOD: We constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. RESULTS AND CONCLUSIONS: These three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. GENERAL SIGNIFICANCE: By genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.

Understanding molecular mechanisms of biologics drug delivery and stability from NMR spectroscopy.

Protein therapeutics carry inherent limitations of membrane impermeability and structural instability, despite their predominant role in the modern pharmaceutical market. Effective formulations are needed to overcome physiological and physicochemical barriers, respectively, for improving bioavailability and stability. Knowledge of membrane affinity, cellular internalization, encapsulation, and release of drug-loaded carrier vehicles uncover the structural basis for designing and optimizing biopharmaceuticals with enhanced delivery efficiency and therapeutic efficacy. Understanding stabilizing and destabilizing interactions between protein drugs and formulation excipients provide fundamental mechanisms for ensuring the stability and quality of biological products. This article reviews the molecular studies of biologics using solution and solid-state NMR spectroscopy on structural attributes pivotal to drug delivery and stability. In-depth investigation of the structure-function relationship of drug delivery systems based on cell-penetrating peptides, lipid nanoparticles and polymeric colloidal, and biophysical and biochemical stability of peptide, protein, monoclonal antibody, and vaccine, as the integrative efforts on drug product design, will be elaborated.