Hansenula Polymorpha Expression of CA10 Virus-Like Particles
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Solution Overview
Problem
Current vaccines for Coxsackievirus A10 (CA10) lack effectiveness and safety due to incomplete inactivation risks and batch variability, with no commercially available vaccines, necessitating a more efficient and safer preventive measure for hand-foot-and-mouth disease.
Innovation Solution
Development of a Hansenula polymorpha expression system for high-yield, large-scale production of CA10 virus-like particles (VLPs) using a recombinant vector with optimized P1 and 3CD genes, followed by a purification process involving ultrafiltration and three-step chromatography to achieve high-purity VLPs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If inactivated vaccines are used, then vaccine development is achieved, but incomplete inactivation risk and virulence recovery occur
Solution Approach 1:
The patent extracts and removes the viral nucleic acid from the virus particle, producing virus-like particles (VLPs) that lack genetic material. This extraction eliminates the risk of incomplete inactivation and virulence recovery while preserving the structural integrity and immunogenicity of the viral capsid, thereby resolving the safety concerns of inactivated vaccines.
Solution Approach 2:
The patent creates artificial copies of the viral structure (VLPs) that mimic natural virus particles but do not contain functional viral nucleic acid. These copies replicate the capsid structure and surface epitopes of CA10 virus while being inert, thus providing immunogenicity without the harmful effects of live or inactivated whole viruses.
2Reliability
If inactivated vaccines are used, then vaccine development is achieved, but epitopes are easily destroyed during inactivation
Solution Approach 1:
The patent performs preliminary assembly of the viral capsid structure in the expression system before purification, ensuring that the VLPs are properly folded and configured with intact epitopes. This preliminary structural formation prevents epitope destruction that would occur during subsequent inactivation steps, as the VLPs are already in their final, stable conformation.
Solution Approach 2:
The patent produces artificial VLP copies that inherently possess stable, intact epitopes since they are assembled de novo in the expression system rather than derived from inactivated viruses. This copying approach ensures epitope integrity is maintained from the outset, eliminating the harmful effect of epitope destruction during inactivation.
3Reliability
If inactivated vaccines are used, then vaccine development is achieved, but batch yield is low and batch differences are large
Solution Approach 1:
The patent employs a recombinant expression system to produce VLPs through controlled cellular machinery, enabling precise replication of the viral capsid structure. This molecular copying approach ensures uniformity in VLP size, shape, and epitope configuration across batches, eliminating the batch variability inherent in inactivated virus preparations while maintaining high yield.
Solution Approach 2:
The patent optimizes expression parameters including temperature, pH, induction time, and media composition to maximize VLP production yield and consistency. By controlling these parameters, the system achieves high batch yield and minimal batch differences, resolving the productivity and consistency issues of inactivated vaccines.
4Productivity
If traditional expression systems are used, then VLP production is achieved, but expression efficiency is low
Solution Approach 1:
The patent optimizes multiple expression parameters including temperature (37°C), induction time (4-8 hours), inducer concentration (1-10 mM), and media composition to maximize VLP production. These parameter optimizations enable high expression efficiency and yield, surpassing traditional expression systems.
Solution Approach 2:
The patent utilizes a composite expression system combining Hansenula polymorpha yeast cells with optimized media formulations and induction agents. This composite approach leverages the metabolic capabilities of the host organism along with enhanced media nutrients and inducers to achieve superior VLP production efficiency and quantity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method enables the production of high-purity, immunogenic, and safe CA10 VLPs, suitable for vaccine development, with improved stability and scalability, addressing the limitations of existing vaccines.
Implementation Method 1
The virus particle has an icosahedral spherical structure... The viral protein shell consists of 4 polypeptide chains, namely VP1, VP2, VP3 and VP4. These 4 proteins form a subunit, and 60 subunits constitute the capsid protein of the virus. Studies have shown that the capsid protein of the virus can self-assemble into a VLP in the cell.
Implementation Method 2
followed by a purification process involving ultrafiltration and three-step chromatography to achieve high-purity VLPs
Implementation Method 3
followed by a purification process involving ultrafiltration and three-step chromatography to achieve high-purity VLPs
Data Source
AI summary
The present disclosure provides an engineered Hansenula fungus that efficiently expresses CA10 (Coxsackievirus A10) virus-like particles and uses thereof. The engineered fungus includes a recombinant vector carrying the P1 and 3CD genes of the CA10 virus optimized according to preferred codons of Hansenula. The present disclosure also provides a preparation method for CA10 virus-like particles and vaccines prepared therefrom.