Wzz Proteins Control O-Polysaccharide Chain Length
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Solution Overview
Problem
The production of bacterial conjugate vaccines is hindered by the natural variability in O-polysaccharide chain length and molecular weight of Gram-negative bacteria, leading to challenges in achieving uniform product synthesis and high immunogenicity, as longer chains are more immunogenic but harder to produce in sufficient quantities and at a lower cost.
Innovation Solution
Overexpressing specific Wzz family proteins in Gram-negative bacteria to control and increase the production of longer O-polysaccharide chains, shifting the production from short to intermediate or long chain lengths, thereby enhancing the yield and immunogenicity of lipopolysaccharides for use in conjugate vaccines.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If size fractionation is used to obtain desired saccharide size, then immunogenicity is improved, but production cost increases and process complexity increases
Solution Approach 1:
The patent applies preliminary action by genetically engineering the bacterial strain before production to constitutively express Wzz proteins that control O-polysaccharide chain length. This pre-configures the production system to naturally generate uniformly sized saccharides with desired immunogenicity, eliminating the need for post-production size fractionation steps and reducing manufacturing complexity and cost
2Reliability
If longer O-polysaccharide chains are produced to enhance immunogenicity, then vaccine effectiveness is improved, but production yield decreases
Solution Approach 1:
The patent applies parameter changes by modifying the expression levels of Wzz proteins through genetic engineering. By optimizing the concentration and activity of Wzz chain-length regulators, the system produces longer O-polysaccharide chains with enhanced immunogenicity while maintaining high production yields through constitutive expression in engineered bacterial strains
3Manufacturing precision
If multiple wzz genes are expressed to control chain length variability, then product uniformity is improved, but genetic complexity increases
Solution Approach 1:
The patent applies merging by combining multiple wzz genes into a single genetic construct or operon that is co-expressed from one regulatory element. This approach achieves coordinated expression of multiple Wzz proteins needed for uniform chain length control while simplifying genetic manipulation and reducing the complexity of maintaining multiple separate expression systems
Data Source
AI summary
Longer chain antigenic O-polysaccharide chains for use as a hapten in conjugate vaccines can be produced in a controlled manner using recombinant Gram-negative bacteria that overexpress native or heterologous genes of the wzz family, for example wzzB. Bacteria expressing a chosen wzz gene have modified O-polysaccharide chain lengths, allowing the bacteria to produce lipopolysaccharides having the longer O-polysaccharides. The LPS produced by the bacteria can be hydrolyzed to form core-O-polysaccharide molecules that can be conjugated to a carrier molecule, for example flagellin, to produce a vaccine. The invention also provides recombinant bacteria producing the longer chain O-polysaccharides, the polysaccharide molecules, themselves, conjugated vaccines comprising the O-polysaccharides, pharmaceutical compositions and kits.