Engineered Signal Peptides for Flavivirus E Protein Expression
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Solution Overview
Problem
Current methods for producing flavivirus vaccines face challenges in achieving high levels of envelope (E) protein expression and secretion, which are essential for effective vaccine production and diagnostics, as existing signal peptides result in low expression levels.
Innovation Solution
Engineered signal peptides, such as X1GAX2TSVGIV GLLLTTAMA or X1RSGVX2WTWIFLTMALTMAMAT, are used in fusion polypeptides with flavivirus E and prM proteins to enhance expression and secretion of E protein and virus-like particles (VLPs), along with expression vectors like recombinant replication-inducible vaccinia virus, to facilitate vaccine development.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If existing signal peptides are used in fusion polypeptides with flavivirus E protein, then the vaccine construct can be produced, but the expression level of E protein is low
Solution Approach 1:
The signal peptide sequence is modified by changing specific amino acid residues (e.g., substituting hydrophobic residues at positions 2-6 to enhance membrane insertion and secretion efficiency). These parameter changes in the peptide sequence directly improve E protein expression levels and vaccine production efficiency without requiring complete redesign of the vaccine construct
Solution Approach 2:
The fusion polypeptide is constructed as a composite structure combining an engineered signal peptide with the flavivirus E protein and prM protein. This composite design allows the signal peptide to optimize secretion while the E-prM portion maintains immunogenicity and VLP formation capability, achieving both high expression and vaccine efficacy
2Ease of operation
If existing signal peptides are used in fusion polypeptides with flavivirus E protein, then the vaccine construct can be produced, but the secretion efficiency of E protein is low
Solution Approach 1:
Specific amino acid substitutions in the signal peptide (particularly enhancing hydrophobicity in the membrane-spanning region) optimize the peptide's ability to facilitate protein secretion across cellular membranes. These parameter changes directly improve secretion efficiency and overall vaccine production
3Quantity of substance
If natural signal peptide sequences are used, then the fusion polypeptide can be expressed, but the formation of virus-like particles is insufficient
Solution Approach 1:
The signal peptide sequence is optimized by modifying specific parameters (amino acid composition, hydrophobicity, length) to enhance its ability to direct proper protein folding and VLP assembly. These targeted changes improve VLP formation without requiring complex sequence designs
Solution Approach 2:
The engineered signal peptide performs preliminary actions by facilitating proper co-translational translocation and initial folding of the E-prM fusion protein in the endoplasmic reticulum. This preliminary processing is essential for subsequent VLP assembly and secretion, enabling efficient particle formation
Data Source
AI summary
Disclosed herein are flavivirus signal peptide mutants useful for enhancing the production and secretion of flavivirus envelope (E) viral proteins or virus-like proteins. Also disclosed herein are methods of vaccinating subjects (e.g., human subjects) against a flavivirus comprising administering an expression vector, wherein the expression vector comprises a polynucleotide, and a fusion polypeptide comprising an engineered signal peptide and a flavivirus envelope (E) protein