Structure and immunogenicity of an engineered soluble prefusion-stabilized EBV gB antigen

Area of Science:

• Virology
• Structural Biology
• Vaccine Development

Background:

• Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to significant cancer-related deaths and increased multiple sclerosis risk.
• Over 90% of U.S. adults are EBV-seropositive, yet no approved EBV vaccines or therapeutics are available.
• EBV's envelope glycoprotein, gB, is crucial for epithelial and B cell infection.

Purpose of the Study:

• To engineer a stabilized prefusion variant of the EBV gB protein.
• To determine the structural basis of EBV gB's prefusion conformation.
• To assess the immunogenicity of the engineered EBV gB variant.

Main Methods:

• Utilized AlphaFold-guided modeling, rational design, and ThermoMPNN optimization to engineer a stabilized EBV gB variant (C3-GT).
• Incorporated disulfide bonds and cavity-filling substitutions to enhance stability.
• Performed cryo-electron microscopy (Cryo-EM) to resolve the prefusion structure.
• Conducted murine immunizations and human serum depletion studies to evaluate immunogenicity.

Main Results:

• Successfully engineered a stabilized prefusion EBV gB variant, C3-GT, with a melting temperature of 54°C.
• Determined the high-resolution cryo-EM structure of EBV gB in its prefusion state.
• Observed a trend towards improved functional immunogenicity of C3-GT compared to postfusion gB in preliminary studies.

Conclusions:

• Established engineering principles for stabilizing class III fusion proteins.
• Provided valuable reagents for studying the human antibody response to EBV gB.
• Laid the groundwork for developing EBV gB-based vaccine candidates.