Pre-fusion RSV F Polypeptide Stabilization via Cysteine Disulfide Bonds
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current vaccine candidates targeting the respiratory syncytial virus (RSV) F glycoprotein face challenges with stability, purity, reproducibility, and potency, particularly in eliciting neutralizing antibodies due to the metastable pre-fusion conformation's instability and the burial of dominant neutralizing epitopes.
Innovation Solution
Development of pre-fusion RSV F polypeptides and chimeric polypeptides with introduced cysteine residues to form disulfide bonds, post-fusion modifications such as deletion of helices or addition of glycosylation sites to stabilize the pre-fusion conformation, and use of chimeric proteins with neutralizing epitopes from other viruses to induce protective immunity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If pre-fusion RSV F protein is used as vaccine antigen, then neutralizing antibody response is improved, but protein stability deteriorates due to metastable conformation
Solution Approach 1:
The patent introduces cysteine mutations at specific positions (e.g., positions 164, 190, 296, 396, 483) to create disulfide bonds that stabilize the pre-fusion conformation. This chemical modification changes the protein's structural parameters, locking it in the desired metastable state without requiring continuous energy input or special conditions.
Solution Approach 2:
The patent creates chimeric F proteins by combining RSV F sequences with stabilizing elements from other paramyxoviruses (such as parainfluenza virus or Newcastle disease virus). This composite approach borrows stable structural features from related viruses to reinforce the pre-fusion conformation while retaining RSV-specific neutralizing epitopes.
2Stability of the object's composition
If pre-fusion conformation is stabilized through cysteine mutations, then protein stability is improved, but manufacturing complexity increases
Solution Approach 1:
The introduced cysteine residues automatically form disulfide bonds under physiological conditions during protein expression and folding. The system self-stabilizes the pre-fusion conformation through spontaneous disulfide bond formation, eliminating the need for external stabilization agents or complex post-translational modification steps in the manufacturing process.
3Reliability
If dominant neutralizing epitopes are exposed, then immunogenicity is improved, but conformational stability deteriorates
Solution Approach 1:
The patent applies local stabilization through targeted cysteine mutations at specific positions that form disulfide bonds in proximity to neutralizing epitopes. This localized approach stabilizes the conformational regions containing dominant epitopes (such as the HRA and HRB heptad repeat regions) without requiring global structural changes, thereby maintaining immunogenicity while preventing unwanted conformational transitions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The approach stabilizes the pre-fusion conformation, enhances the immunogenicity by exposing neutralizing epitopes, and potentially increases the efficacy of RSV vaccines by inducing neutralizing antibodies and protective immunity.
Implementation Method 1
The pre-fusion RSV F polypeptide comprises at least two introduced cysteine residues that are in close proximity to one another, and form a disulfide bond that stabilizes the pre-fusion RSV F polypeptide
Data Source
AI summary
The invention relates to pre-fusion RSV F protein and polypeptides that contain one or more amino acid mutations that stabilize the pre-fusion conformation or destabilize the post-fusion conformation. The invention also relates to methods for inducing an immune response to pre-fusion RSV F.