An advanced, highly sensitive and versatile Cypridina luciferase-based HPV pseudovirus system for broad-spectrum neutralization and non-invasive In vivo monitoring.

INTRODUCTION: Evaluating next-generation prophylactic human papillomavirus (HPV) vaccines requires high-throughput and scalable neutralization assays spanning a wide array of genotypes. Standard secreted alkaline phosphatase (SEAP)-based platforms, however, are limited by tedious protocols, such as mandatory serum heat inactivation, and restricted sensitivity. To address these bottlenecks, we developed an advanced pseudovirus (PsV) platform using Cypridina luciferase (cLuc), a versatile dual-reporter system that facilitates both extracellular secretion and intracellular monitoring.

METHODS: A comprehensive library of cLuc-engineered PsVs was constructed, covering 28 distinct mucosal and cutaneous HPV genotypes. We verified their structural integrity and antigenic presentation via neutralization profiling with L2 N-terminus-specific monoclonal antibodies (JWW-1/JWW-2).

RESULTS: The cLuc-PsVs demonstrated robust infectivity along with excellent analytical linearity. Critically, this system eliminated the need for thermal inactivation of sera while maintaining an exceptional correlation (R² > 0.99) with traditional SEAP assays during evaluations of hyperimmune mouse sera. Beyond in vitro screens, the platform's translational value was confirmed in a murine cervicovaginal challenge model, where real-time bioluminescence imaging enabled non-invasive tracking of mucosal infection across all 28 genotypes.

DISCUSSION: This dual-purpose cLuc-PsV platform provides a highly efficient, sensitive alternative to conventional neutralization assays. By unifying high-throughput in vitro quantification with longitudinal in vivo imaging, this system offers a robust tool to accelerate the preclinical screening of multivalent HPV vaccine candidates.