A High-Throughput and Robust Relative Potency Assay Measuring Human Cytomegalovirus Infection in Epithelial Cells for...

Area of Science:

• Virology
• Vaccinology
• Biotechnology

Background:

• Human cytomegalovirus (HCMV) infection and disease prevention requires a vaccine, representing a significant unmet medical need.
• Previous HCMV vaccine candidates, including attenuated viruses and antigen-based approaches, faced development challenges limiting clinical progression.

Purpose of the Study:

• To develop and implement a high-throughput, robust relative potency assay for a live-attenuated HCMV vaccine candidate.
• To measure HCMV infection in ARPE-19 epithelial cells using the novel Imaging of Relative Viral Expression (IRVE) assay.

Main Methods:

• The IRVE assay quantifies HCMV infection by immunostaining for Immediate Early 1 (IE1) protein and counting IE1-positive cells.
• A custom robotic system enabled 384-well plate automation for increased assay throughput.
• Key parameters including cell density, infection time, and cell age were optimized.

Main Results:

• The IRVE assay demonstrated effectiveness in measuring relative potency variations influenced by manufacturing processes and formulation.
• Assay optimization involved evaluating microplate format, cell density, serum concentration, infection time, and cell age.
• Correlation with established lower-throughput assays (plaque and Infectivity of Early Gene Expression) validated IRVE as a potency screening tool.

Conclusions:

• The IRVE assay has been successfully implemented and utilized for several years in HCMV vaccine clinical development.
• This assay provides a valuable tool for supporting the advancement of HCMV vaccine candidates.