In a recent study published on the bioRxiv* preprint server, an international team of researchers developed a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antigen (RBD-J6 2) second generation through molecular engineering. with two additional amino acid (aa) substitutions (S383D and L518D mutations) in a core hydrophobic cryptic RBD epitope to improve stability and expression against SARS-CoV-2 (COV) variants of concern.
Study: Molecular engineering of a cryptic epitope in Spike RBD improves manufacturability and neutralizing breadth against SARS-CoV-2 variants. Image credit: Design_Cells / Shutterstock
Sarbecovirus vaccines that can be produced and distributed among low- and middle-income countries are required. Subunit protein vaccines have been cost-effectively manufactured on a large scale with convenient thermal requirements, of which several vaccines have demonstrated anti-SARS-CoV-2 efficacy.
The study authors previously performed molecular engineering experiments to improve RBD production and stability in yeast. As a result, they developed an engineered variant of the SARS-CoV-2 spike (S) protein RBD antigen (RBD-J) with improved immunogenicity and manufacturability compared to the ancestral strain (Wuhan-Hu-1) RBD.
About the study
In the present study, the researchers extended their previous analysis by performing further molecular engineering analysis of the engineered SARS-CoV-2 RBD.
A hydrophobic patch in the RBD core proximal to the C-terminus was modified [reduced or eliminated by mutations] to improve the stability, solubility and secretion of RBD. For analysis, 21 aa substitutions previously reported to increase RBD expression in yeast while preserving angiotensin-converting enzyme (ACE2) binding capacity were selected. Each of them was evaluated individually. Each RBD contained the L452K mutation, which the authors had previously shown to enhance RBD stability and expression.
Each RBD variant was transferred into yeast for evaluation of RBD secretion. We evaluated combinations of three aspartic acid mutations, including the L452K and F490W mutations in the receptor-binding motif (RBM) of the hydrophobic patch of the RBD core. The physiological properties of RBD-J6 and RBD-J were compared. Far ultraviolet circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC), static light scattering (SLS), reversed-phase high-performance liquid chromatography (HPLC), and biolayer interferometry experiments were performed ( BLI).
We assessed the binding of RBD-J6 to ACE2 and several nAbs (neutralizing antibodies) targeting different RBD epitopes. The team then assessed the rise of polyclonal Abs in RBD-J-bound mice vaccinated with RBD-J6 and the binding of serological Abs obtained from messenger ribonucleic acid (mRNA) infected by SARS-CoV- 2 Delta VOC and coronavirus disease 2019 (COVID-19). ) evaluated vaccine-immunized convalescents in RBDs designed initially (RBD-J) and subsequently (RBD-J6). The team investigated whether the manufacturability and stability benefits gained by introducing mutations in the hydrophobic patch of RBD-J6 would also benefit RBD antigens containing alpha and beta VOC mutations.
Three Beta VoC RBD mutations (K417N, E484K, and N501Y) were added to RBD-J6 (hereafter, RBD-J6 β). We compared the immunogenicity of RBDs initially designed and subsequently conjugated to HBsAg VLPs (hepatitis B surface antigen virus-like particle) containing Beta VOC mutations. In addition, K18-hACE2 (human ACE2) transgenic mice were intramuscularly administered the alum-adjuvanted VLP-RBD conjugate or the Pfizer-BioNTech mRNA vaccine twice three weeks apart to determine the effects of the RBD-J6 aa substitutions on the immunogenicity of the designed vaccine.
Serological responses were assessed against SARS-CoV-2 VOC RBDs after two weeks, five weeks and seven weeks of vaccination. After seven weeks, K18-hACE2 mice vaccinated with RBD-J β and RBD-J6 β were challenged with Alpha VOC or Beta VOC. In addition, SARS-CoV-2 RNA titers were determined in cranial and lung tissues of mice and SARS-CoV-2 VOC neutralization was assessed.
results
RBD-J6 showed binding to sera from convalescent individuals from Delta and to all nAbs tested except nAbs targeting core class IV epitopes of RBD (EY6A and CR3022). Modification of the hydrophobic patch improved RBD secretion titers by threefold and improved stability; however, the modifications now showed significant differences in RBD immunogenicity or antigenicity.
VLP-RBD conjugate induced cross-reactive immunity in mice against SARS-CoV-2 VOCs such as Alpha and Beta. Additional mutations in RBD-J6 enhanced RBD productivity fourfold compared to the ancestral RBD strain, and three aspartic acid mutations (S383D, R408D, and L518D) most prominently enhanced RBD expression from 60 mg/ L to 173 mg/L. In addition, RBD-J6 showed reduced surface hydrophobicity. The Tm (thermal melting temperature) of RBD-J6 (63 °C) was higher than that of RBD-J in all temperature-based analyses, indicative of higher conformational and colloidal stability of RBD- J6, and the designed RBD was destabilized by aluminum and CpG adjuvants.
Vaccination status did not alter the binding affinity of both the RBDs designed for ACE2, Delta convalescent sera, and the nAbs tested. RBD-J6 β also showed similar ACE2 binding to RBD-J6, and no increase in ACE2 binding was observed by adding Alpha and Beta VOC RBD mutations. SARS-CoV-2 RNA tires were 30% lower in the brain and lungs of mice in the case of RBD-J6 β expression compared with RBD-J6 expression, and the lungs showed a less inflammation after Alpha VOC or Beta VOC challenge. The cross-neutralizing potencies of the VLP – RBD-J6 β conjugate and the Pfizer COVID-19 mRNA vaccine were comparable.
Overall, the study findings highlighted the potential use of RBD-J6 to improve RBD-based subunit vaccine development, with improved manufacturability, stability, and access in low- and middle-income countries.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.