Coronavirus Spike Protein Stabilization via Peptide Linkers and Proline Mutations
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current SARS-CoV-2 spike protein vaccines face challenges with stability during storage, manufacturing, freeze/thaw cycles, and lyophilization/resuspension, limiting their effectiveness and durability, especially with emerging variants.
Innovation Solution
A mutant coronavirus spike protein with modifications such as short or rigid peptide linkers at the furin cleavage site, additional disulfide bonds, and proline mutations for enhanced trimeric stability, expressed in various cell types, forming dimers, trimers, or nanoparticles for improved expression and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the wild-type SARS-CoV-2 spike protein is used, then the protein can bind to ACE2 and initiate infection, but the protein exhibits poor stability during storage, manufacturing, freeze/thaw cycles, and lyophilization/resuspension
Solution Approach 1:
The spike protein is divided into S1 and S2 subunits connected by a peptide linker at the furin cleavage site. This segmentation prevents premature cleavage and maintains structural integrity during storage and processing, while still allowing functional conformational changes when needed for ACE2 binding.
Solution Approach 2:
Proline mutations are introduced at specific positions (e.g., K986P, V987P) to alter the local structural parameters and conformational dynamics of the spike protein. These parameter changes stabilize the prefusion conformation, improving reliability during storage and manufacturing without compromising ACE2 binding capability.
2Stability of the object's composition
If proline mutations are introduced to stabilize the prefusion conformation, then trimeric stability is improved, but the complexity of protein design and characterization increases
Solution Approach 1:
Proline mutations are introduced at specific local positions (e.g., K986P, V987P, N501P) rather than throughout the entire protein. This localized modification approach stabilizes the prefusion conformation and improves trimeric stability while minimizing the overall complexity of design and characterization.
Solution Approach 2:
The spike protein is pre-stabilized in the prefusion conformation through proline mutations and peptide linker design before encountering destabilizing conditions during storage or processing. This preliminary stabilization action prevents conformational drift and maintains trimeric integrity throughout the vaccine lifecycle.
3Reliability
If peptide linkers are used to connect S1 and S2 subunits, then furin cleavage is prevented and stability is improved, but the flexibility and natural processing of the protein are reduced
Solution Approach 1:
A peptide linker is introduced as an intermediary element at the furin cleavage site to connect the S1 and S2 subunits. This linker prevents premature furin cleavage and improves manufacturing stability, while still allowing controlled conformational changes and ACE2 binding when needed, thus balancing stability with functional flexibility.
Data Source
AI summary
The present invention includes a mutant coronavirus spike protein, methods of making and using, vaccines, vectors and nucleic acids, comprising at least one of the following modifications: a short flexible peptide linker or a rigid peptide linker in place of the furin cleavage site loop to genetically link an 51 and S2 subunit; at least one additional disulfide bond; or 1, 2, 3, 4, or 5 proline mutations for greater trimeric stability, wherein the resulting mutant coronavirus spike protein has at least one of: a higher stability or a higher level of expression when compared to a non-modified coronavirus spike protein. In one example, the coronavirus is SARS, MERS, 229E (alpha), NL63 (alpha), OC43 (beta), HKU1 (beta), SARS-CoV-2, or an emerging variant thereof. Current SARS-CoV-2 variants include, e.g., B.1.1.7, B.1.1.7 with E484K, B.1.135, B.1.351, P.1, B.1.427, D614G, B.1.1351, or B.1.429, Lambda (i.e., C.37), Mu (i.e. B.1.621), and others.