RSV F Protein Trimer Stabilization via T4 Fibritin Fusion
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Solution Overview
Problem
Current methods for producing RSV F protein compositions face challenges in achieving stability and homogeneity due to the complexity of RSV F protein processing, structure, and refolding, making it difficult to obtain purified, homogeneous, immunogenic preparations.
Innovation Solution
The development of an immunogenic composition comprising a soluble recombinant RSV F protein in trimer form, with a first domain comprising the RSV F ectodomain and a second domain with a heterologous oligomerization domain from bacteriophage T4 fibritin, which is stable and consistent, allowing for a predictable immune response.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If RSV F protein is expressed as soluble ectodomain, then solubility is improved, but protein stability and homogeneity deteriorate due to complex processing and refolding
Solution Approach 1:
The patent uses a heterologous oligomerization domain (such as GCN4 leucine zipper or T4 fibritin foldon) as an intermediary element fused to the RSV F ectodomain. This intermediary domain promotes stable trimer formation and prevents aggregation during expression and purification, thereby achieving both solubility and stability simultaneously.
Solution Approach 2:
The patent creates a composite protein structure by fusing the RSV F ectodomain with a heterologous oligomerization domain. This composite construction combines the immunogenic RSV F ectodomain with a stable, soluble oligomerization module, resulting in a protein that maintains both solubility and structural stability.
2Reliability
If RSV F protein is cleaved into subunits, then immunogenicity is improved, but homogeneity and consistency between batches deteriorate
Solution Approach 1:
The patent performs proteolytic cleavage as a preliminary action during the protein expression and purification process, rather than after. By planning for controlled cleavage at specific sites (such as furin cleavage sites) during manufacturing, the process ensures consistent and reproducible generation of immunogenic subunits while maintaining batch homogeneity.
3Reliability
If RSV F protein forms trimers, then immunogenicity is improved, but purification difficulty and heterogeneity worsen
Solution Approach 1:
The heterologous oligomerization domain acts as a mediator that facilitates trimer formation while providing a consistent structural framework that simplifies purification. The domain's ability to promote stable trimer assembly reduces heterogeneity and makes the purification process more predictable and manageable.
Solution Approach 2:
The patent utilizes parameter changes in the oligomerization domain (such as mutations in the GCN4 leucine zipper or T4 fibritin foldon) to optimize trimer stability and solubility. These parameter modifications enhance the protein's tendency to form homogeneous trimers, thereby simplifying purification while maintaining high immunogenicity.
Data Source
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AI summary
The present invention relates to immunogenic compositions comprising RSV F protein, methods for preparing compositions that contain RSV F protein ecto-domain polypeptides, and to certain engineered RSV F proteins and nucleic acids that encode the engineered RSV F proteins. Compositions prepared using the methods can contain RSV F protein ecto-domain polypeptides in a predominant or single desired form and conformation. The invention also relates to methods for inducing an immune response to RSV F.