EV71 Virus-Like Particles via Hansenula Polymorpha Expression
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Solution Overview
Problem
Current vaccines for Hand, Foot, and Mouth Disease (HFMD) caused by Enterovirus 71 (EV71) lack effectiveness and safety, with inactivated vaccines showing poor immunogenicity, incomplete inactivation risks, and potential neurologic symptoms, while existing expression systems for EV71 virus-like particles (VLPs) face challenges in large-scale production due to protein denaturation, low yield, and high production costs.
Innovation Solution
The use of the Hansenula polymorpha expression system, where the P1 and 3CD genes of EV71 are codon-optimized and expressed using a recombinant vector (PMV-P1-3CD) to produce EV71 VLPs, which are then purified using a method involving high-pressure homogenization, centrifugation, ultrafiltration, and chromatography, enabling high-level expression and large-scale production of safe and immunogenic VLPs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If inactivated EV71 whole virus vaccine is used, then vaccine coverage is provided, but immunogenicity is poor and inactivation completeness is doubtful
Solution Approach 1:
The patent extracts only the structural proteins (P1 and 3CD genes) from the complete EV71 virus, eliminating the viral genome and other non-essential components. This extraction approach produces VLPs that retain immunogenicity while removing the risk of incomplete inactivation and viral reversion, directly resolving the contradiction between vaccine safety and inactivation completeness.
Solution Approach 2:
The patent creates a copy of the virus's structural proteins that self-assemble into VLPs mimicking the native virus morphology and antigenic determinants. These VLP copies provide the necessary immunogenicity without containing functional viral genetic material, thereby ensuring complete safety while maintaining protective immunity.
2Quantity of substance
If traditional expression systems are used for EV71 VLPs, then vaccine antigen is produced, but protein denaturation occurs and yield is low
Solution Approach 1:
The patent optimizes multiple parameters including codon optimization for the host expression system, adjustment of cultivation conditions, and control of purification parameters. These parameter changes enable high-level expression of P1 and 3CD proteins that correctly fold and self-assemble into stable VLPs, simultaneously achieving high yield and maintaining protein conformational integrity.
3Reliability
If inactivated vaccine is used, then virus coverage is achieved, but dosage is excessive and cost-effectiveness is poor
Solution Approach 1:
The patent concentrates the essential immunogenic components (structural proteins VP1, VP2, VP3, and VP4) into highly purified VLPs with localized antigenic determinants. This localized concentration of quality antigens in VLPs provides superior protective effect at lower doses compared to whole inactivated virus vaccines, improving cost-effectiveness while maintaining reliability.
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
This approach results in high-level expression of EV71 VLPs with good stability, safety, and immunological properties, suitable for industrial production and vaccine development, overcoming the limitations of existing systems by achieving efficient and cost-effective large-scale production of EV71 VLPs.
Implementation Method 1
the P1 and 3CD genes of EV71 are codon-optimized and expressed using a recombinant vector (PMV-P1-3CD) to produce EV71 VLPs
Implementation Method 2
The use of the Hansenula polymorpha expression system, where the P1 and 3CD genes of EV71 are codon-optimized and expressed using a recombinant vector (PMV-P1-3CD) to produce EV71 VLPs
Implementation Method 3
These structural proteins have an ability of native self assembly, and they can form a steric configuration and an antigenic epitope similar to those of the naturally occurring virus particle
Implementation Method 4
purified using a method involving high-pressure homogenization, centrifugation, ultrafiltration, and chromatography
Implementation Method 5
purified using a method involving high-pressure homogenization, centrifugation, ultrafiltration, and chromatography
Implementation Method 6
purified using a method involving high-pressure homogenization, centrifugation, ultrafiltration, and chromatography
Implementation Method 7
purified using a method involving high-pressure homogenization, centrifugation, ultrafiltration, and chromatography
Data Source
AI summary
The present invention provides EV71 virus-like particles and a preparation method and application thereof. The method comprises: connecting a P1 protein gene and a 3CD protease gene of an EV71 virus with a PMV plasmid to construct a PMV-P1-3CD recombinant expression plasmid; then transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression plasmid to obtain an AU-PMV-P1-3CD recombinant expression strain; fermenting and culturing the recombinant expression strain, and inducing the recombinant expression strain to express the EV71 virus-like particle protein with methanol; centrifuging and collecting mycelia for homogeneous breakage at a high pressure; and purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography, so as to obtain EV71 virus-like particles.