Bacterial HA Polypeptide Refolding for Avian Influenza Vaccine
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Solution Overview
Problem
Current methods for producing influenza vaccines based on the HA protein in bacterial expression systems face challenges in achieving effective refolding and oligomerization, which are crucial for inducing broad immune responses, particularly against highly pathogenic avian influenza virus (HPAIV) H5N1, due to the protein's hydrophobic sequences and cleavage sites that lead to subunit degradation.
Innovation Solution
A 17-522 aa hemagglutinin (HA) polypeptide is produced in E. coli, specifically designed to lack immunologically inert regions like the signal sequence and basic amino acids, allowing for efficient expression and refolding into oligomeric forms that retain neutralizing epitopes, using optimized expression vectors and chromatographic methods to purify and refold the protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If the complete HA protein including signal sequence and basic amino acids is expressed in bacterial system, then the full-length protein is produced, but the protein degrades at cleavage sites and forms subunits instead of functional oligomers
Solution Approach 1:
The invention removes the signal sequence and basic amino acids from the HA protein sequence used for bacterial expression. This extraction of problematic regions prevents degradation at cleavage sites and eliminates interference with proper folding and oligomerization, allowing functional oligomers to form reliably in the bacterial expression system
Solution Approach 2:
The invention modifies specific local regions of the HA protein (signal sequence and basic amino acid regions) while preserving the rest of the protein structure. This localized modification approach maintains the immunogenic epitopes and functional domains while eliminating only the problematic segments that cause degradation and misfolding
2Manufacturing precision
If HA-1 subunit based vaccines are used, then serotype-specific immunity is induced, but cross-immunity against other serotypes is limited due to masking of stalk region epitopes
Solution Approach 1:
The invention divides the HA protein into functional segments by including both the HA-1 subunit (globular domain) and the HA-2 subunit (stalk region) in the expressed polypeptide. This segmentation allows both serotype-specific epitopes in HA-1 and conserved cross-reactive epitopes in HA-2 to be presented to the immune system, achieving dual specificity
Solution Approach 2:
The invention merges the HA-1 and HA-2 subunits into a single expressed polypeptide construct. This combination ensures that both the globular domain (for serotype specificity) and the stalk region (for cross-reactivity) are co-expressed and properly assembled into functional oligomers, providing broad immune protection
3Quantity of substance
If longer HA fragments including HA-2 subunit are used, then conserved neutralizing epitopes are included, but the globular domain masks stalk region recognition by immune cells
Solution Approach 1:
The invention removes the signal sequence and basic amino acids that interfere with proper protein folding and oligomerization. This extraction allows the HA-2 stalk region to be properly exposed and accessible to the immune system, eliminating the masking effect while retaining the conserved neutralizing epitopes
Solution Approach 2:
The invention changes the physical-chemical parameters of the expressed protein by modifying the amino acid sequence (removing basic residues and signal sequence). This parameter change affects the protein's folding, stability, and oligomerization properties, resulting in proper exposure of the stalk region epitopes for immune recognition
Data Source
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AI summary
The invention relates to an influenza viruses hemagglutinin protein as a vaccine antigen against influenza viruses, in particular to a protein being a fragment of H5 hemagglutinin from highly pathogenic avian influenza virus (HPAIV) H5N1 strain, expressed in a bacterial expression system. The protein according to the invention is a potential ingredient of an anti-HPAIV H5N1 vaccine, administered to chickens parenterally or through mucous membranes in the presence of adjuvants. The object of the invention is also a method of obtaining the vaccine protein.