[Expression,Purification,Structure Determination and Immunogenicity Assay of Hepatitis E Virus Capsid Protein p495...

Area of Science:

• Virology
• Structural Biology
• Immunology

Background:

• Hepatitis E virus (HEV) poses a significant global health challenge.
• The commercial HEV vaccine (Hecolin) utilizes p239 particles.
• Developing improved and scalable methods for HEV antigen production is crucial.

Purpose of the Study:

• To establish a robust baculovirus-based insect cell expression system for hepatitis E virus (HEV) p495 protein.
• To characterize the properties and determine the cryo-electron microscopy (cryo-EM) structure of the resulting HEV p495 virus-like particles (VLPs).
• To compare the immunogenicity of HEV p495 VLPs with the p239 antigen found in the Hecolin vaccine.

Main Methods:

• Cloning of HEV ORF2 (amino acids 112-606) into a baculovirus vector for recombinant p495 protein expression.
• Characterization using ELISA, analytical ultracentrifugation, size-exclusion chromatography, and negative-staining transmission electron microscopy (TEM).
• 3D structure determination of p495 VLPs via cryo-EM and comparison with existing crystal structures (PDB ID: 2ZZQ).
• Immunogenicity assessment through mouse vaccination experiments.

Main Results:

• Successful expression and purification of recombinant HEV p495 VLPs with >95% purity and 15 mg/L yield.
• Homogeneous nature of the recombinant p495 protein in solution.
• Determination of an icosahedral T=1 structure for p495 VLPs, consistent with prior structural data.
• Comparable immunogenicity of p495 VLPs to the p239 antigen in mouse models.

Conclusions:

• A robust, scalable method for producing homogeneous and immunogenic HEV p495 VLPs has been established.
• The characterized p495 VLPs hold potential for future HEV vaccine improvements.
• This work supports further research into HEV receptors, epitope mapping, and vaccine design.