Cell-Free IpaB Antigen Synthesis with Chaperone and Non-Natural Amino Acids
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Solution Overview
Problem
Current methods for preventing and treating Shigella dysentery are inadequate, as there is a need for effective compositions and methods to address this significant public health issue.
Innovation Solution
The development of immunogenic compositions comprising IpaB polypeptide antigens with non-natural amino acids incorporated at specific positions, conjugated to O-antigen Shigella polysaccharides, and expressed using cell-free protein synthesis in the presence of an exogenous IpgC chaperone protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods are used to prevent and treat Shigella dysentery, then existing treatments can be applied, but they are inadequate and fail to provide effective prevention and treatment
Solution Approach 1:
The patent modifies the IpaB antigen by incorporating non-natural amino acids at specific positions (such as K241, K262, K269, K283, K289, K299, C309, K312, S329, S333, D347, E360, K368, E372, K376, D380, K384, E387, D392, K394, K395, K397, K424, K429, K436, K440, K448, K451, K470, and K482) to enhance immunogenicity. This chemical modification of the antigen structure creates a more effective vaccine candidate that overcomes the limitations of conventional prevention and treatment methods
2Productivity
If IpaB antigen is synthesized using traditional protein expression methods, then production can proceed, but yield and efficiency are insufficient
Solution Approach 1:
The patent replaces traditional cellular protein expression systems with cell-free protein synthesis (CFPS) technology. This substitution eliminates the complexities of cellular metabolism and regulation, enabling direct in vitro translation of the IpaB gene into protein with higher yield and easier process control. The CFPS system uses purified cellular components (ribosomes, tRNAs, enzymes, and energy sources) to synthesize the antigen without requiring living cells
Solution Approach 2:
The patent optimizes CFPS reaction conditions including nucleotide composition, amino acid concentrations, energy source levels, and incubation parameters to maximize IpaB production. By adjusting these biochemical parameters, the system achieves high-yield antigen synthesis that is both productive and manufacturable
3Stability of the object's composition
If IpaB antigen is produced without chaperone proteins, then synthesis can occur, but protein folding and stability are compromised
Solution Approach 1:
The patent introduces IpgC chaperone proteins into the CFPS system to assist in the proper folding and stabilization of the IpaB antigen. These chaperones act as intermediary molecules that prevent misfolding and aggregation, ensuring the antigen achieves its correct three-dimensional structure and maintains stability without requiring complex purification or stabilization protocols
Data Source
AI summary
The present disclosure provides a cell-free method for synthesizing an Invasion Plasmid Antigen B (IpaB) antigen associated with a Shigella bacterium comprising exogenous addition of the purified chaperone protein IpgC to the cell-free synthesis mixture. The disclosure further provides IpaB antigen mutants comprising non-natural amino acids incorporated during cell-free synthesis, enabling covalent conjugation to a Shigella O-antigen polysaccharide. Further provided are Ipa B antigens and conjugates thereof, as well as immunogenic compositions prepared with the synthesized IpaB antigens and conjugates thereof and methods of use.