Recombinant Influenza HA Trimers With Stable Native Epitope Folding
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Solution Overview
Problem
Current influenza vaccines face challenges in predicting dominant strains, antiviral resistance, immune escape, and egg-based production methods that can cause hypersensitivity and disrupt supply, while recombinant HA proteins struggle with maintaining immunogenicity and quaternary structure.
Innovation Solution
Development of recombinant influenza A hemagglutinin (HA) polypeptides with specific amino acid mutations at positions 355, 380, and 432, allowing for stable trimer formation without heterologous domains, and production in mammalian cells to induce an immune response.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If egg-based methods are used to produce influenza vaccines, then production capacity is increased, but suboptimal protection and hypersensitivity issues occur
Solution Approach 1:
The patent extracts the hemagglutinin antigen production from the egg-based system and transfers it to a cell culture-based recombinant expression system. This removes the harmful factors associated with egg-based production (hypersensitivity risks and suboptimal protection) while maintaining production capacity through scalable cell culture methods.
Solution Approach 2:
The patent introduces recombinant DNA technology as an intermediary between the vaccine design and production stages. By expressing hemagglutinin antigens in cell cultures through recombinant expression vectors, the system achieves controlled, high-yield production of immunogenic antigens without the limitations of egg-based methods.
2Ease of manufacture
If recombinant HA is produced in cell cultures, then production control is improved, but maintaining immunogenicity and quaternary structure becomes difficult
Solution Approach 1:
The patent optimizes expression parameters in cell cultures, including codon optimization of the HA gene sequence, adjustment of expression conditions (temperature, pH, nutrient composition), and selection of appropriate cell lines. These parameter changes enable controlled production while maintaining proper protein folding and quaternary structure.
Solution Approach 2:
The patent produces trimeric HA complexes by co-expressing multiple HA subunits (HA1 and HA2) in the cell culture system. The recombinant expression system facilitates the assembly of these subunits into the native trimeric quaternary structure, preserving immunogenicity while enabling controlled production.
3Quantity of substance
If high yields of trimeric HA are required, then vaccine effectiveness is improved, but production complexity increases
Solution Approach 1:
The patent performs preliminary optimization of the expression system before large-scale production. This includes selecting optimal cell lines, optimizing transfection conditions, and pre-validating the expression system's ability to produce trimeric HA. These preliminary actions simplify subsequent high-yield production by establishing a robust, standardized platform.
Solution Approach 2:
The patent utilizes the cell culture system's endogenous machinery for protein folding, post-translational modifications, and quaternary structure assembly. By leveraging the cell's natural capabilities rather than requiring complex external intervention, the system achieves high yields of properly folded trimeric HA with reduced production complexity.
Data Source
AI summary
The invention provides recombinant influenza A hemagglutinin (HA) polypeptides, comprising an HA1 and a HA2 domain of an influenza A virus HA, and comprising an amino acid sequence wherein:(a) the amino acid at position 355 is W; and(b) the amino acid at position 432 is I and/or the amino acid at position 380 is I;and wherein the numbering of the amino acid positions in the amino acid sequence of the HA polypeptide is according to the numbering of amino acids in the amino acid sequence of HA from a reference H3N2 influenza strain, in particular the reference strain H3N2 A/Aichi/2/68 (SEQ ID NO: 1), immunogenic fragments thereof, nucleic acid molecules encoding said polypeptides or immunogenic fragments, and uses thereof.