A low-cost, highly effective, molecularly engineered COVID vaccine

In a recent study published in bioRxiv* Prepress server, an international team of researchers developed a second generation SARS-CoV-2 (RBD-J6) second-generation (RBD-J6) SARS-CoV-2 receptor-binding antigen by molecular engineering with two amino acids Additional (aa) substitutions (S383D and L518D mutants) in a hydrophobic cryptic RBD core loop enhance stability and expression against SARS-CoV-2 variants of concern (VOCs).

Study: Spike RBD hidden loop molecular engineering improves manufacturability and amplitude neutralization against SARS-CoV-2 variants.  Image Credit: Design_Cells / Shutterstock

Stady: Spike RBD hidden loop molecular engineering improves manufacturability and amplitude neutralization 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 in demand. Unitary protein vaccines have been cost-effectively manufactured on a large scale with adequate thermal requirements, and several vaccines have been shown to be effective against SARS-CoV-2.

The study authors previously conducted molecular engineering experiments to improve RBD production and yeast stability. As a result, they developed an engineered variant of the RBD protein antigen (RBD-J) of SARS-CoV-2 spike(S) with improved immunogenicity and synthetic ability compared to the ancestral strain (Wuhan-Hu-1) RBD.

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In this study, the researchers extend their previous analysis by performing further molecular engineering analyzes of SARS-CoV-2 RBD.

A hydrophobic patch has been modified on the RBD core proximal to the C-terminus. [reduced or eliminated by mutations] To improve RBD stability, solubility and excretion. For the analysis, 21 variants previously reported to enhance RBD expression in yeast while maintaining angiotensin-converting enzyme (ACE2)-binding capacity were selected. Each of them was evaluated separately. Each RBD contained the L452K mutation, which has been shown to improve RBD stability and expression by the authors previously.

Each RBD variant was transfected into yeast to assess RBD secretion. groups of three Aspartic acid Mutations, including the L452K and F490W mutations, were evaluated in the receptor binding (RBM) form of the hydrophobic patch in the RBD core. The physiological properties of RBD-J6 and RBD-J were compared. Far-ultraviolet circular dichroism (CD) spectroscopy, differential calorimetry (DSC), fixed light scattering (SLS), high-performance reverse phase liquid chromatography (HPLC), and bilayer interferometry (BLI) were performed.

RBD-J6 binds to ACE2 and several nAbs (neutralizing antibodies) Targeting different episodes of RBD was evaluated. Next, the team evaluated the polyclonal Ab increase in RBD-J associated with RBD-J6-immunized mice and correlated the serum uptake obtained from SARS-CoV-2 Delta VOC infected with the coronavirus and coronavirus disease 2019 (COVID-19) messenger ribonucleic acid (mRNA) vaccine-immunized convalescents were assayed to (RBD-J) and then (RBD-J6) engineered. The team investigated whether the manufacturability and stability advantages obtained from introducing mutants into the hydrophobic RBD-J6 patch would also benefit RBD. Antigens Contains alpha and beta mutations VOCs.

Three VoC RBD beta mutations (K417N, E484K, and N501Y) were added to RBD-J6 (hereafter RBD-J6). The immunogenicity of betaVOC mutant initially and subsequently engineered was compared with HBsAg VLP (hepatitis B surface virus-like particle). Furthermore, K18-hACE2 (human ACE2) transgenic mice were administered intramuscularly with either alum-enriched VLP-RBD conjugate or Pfizer-BioNTech mRNA vaccine twice every three weeks apart to determine the effects of RBD-J6 aa substitutions on immunity. Engineering vaccine.

Serological responses against SARS-CoV-2 VOC RBDs were assessed two, five and seven weeks after vaccination. Seven weeks later, K18-hACE2 mice inoculated with RBD-Jβ and RBD-J6 were challenged with Alpha VOC or Beta VOC. In addition, SARS-CoV-2 RNA titer was determined in rat cranial and pulmonary tissues, and SARS-CoV-2 VOC neutralization was evaluated.


RBD-J6 showed an association with serum of convalescent delta individuals and all nAbs tested except for RBD core class IV targeting epitope nAbs (EY6A and CR3022). Modification of the hydrophobic patch improved the RBD secretion titer threefold and enhanced stability; However, alterations have now shown significant differences in RBD immunogenicity or antigenicity.

Conjugated VLP-RBD causes cross-reactive immunity in mice against SARS-CoV-2 VOCs such as Alpha and Beta. Additional mutations in RBD-J6 improved RBD yield fourfold compared to the ancestral strain RBD, and three aspartic acid mutations (S383D, R408D, and L518D) prominently improved RBD expression from 60 mg/L to 173 mg/L. Furthermore, 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, indicating a higher conformational and colloidal stability of RBD-J6, and RBD was destabilized Engineered with aluminum and CpG auxiliary materials.

Vaccination status did not change the binding affinity of the ACE2-engineered RBDs, convalescent delta sera, and nAbs tested. RBD-J6β also showed similar binding to ACE2 as RBD-J6, and no increase in ACE2 binding was observed by adding the Alpha and Beta VOC RBD mutants. SARS-CoV-2 RNA frames were 30% lower in the brain and lungs of mice with RBD-J6 expression than in RBD-J6 expression, and the lungs showed less inflammation after the Alpha VOC or Beta VOC test. The cross-neutralizing powers of the VLP-RBD-J6 conjugate and Pfizer’s COVID-19 mRNA vaccine were comparable.

Overall, the results of the study highlight the potential use of RBD-J6 to improve the development of RBD-based subunit vaccines, while improving manufacturability, stability, and access to low- and middle-income countries.

*Important note

bioRxiv It publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behaviour, or be treated as established information.

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