A HiBiT-tagged pseudovirus-like particle platform for safe, rapid quantification of virus neutralization and antibody-dependent enhancement.

Accurate quantification of virus neutralization is essential for evaluating the efficacy of vaccines and antibody-based therapies. However, conventional neutralization assays require strict biocontainment or utilize surrogates of infection that increase biosafety but reduce biological relevance. These limitations create critical bottlenecks for the timely development of antiviral immunotherapeutics. Here, we describe a neutralization platform using non-replicating, HiBiT-tagged pseudovirus-like particles (HiBiT-PsVLPs) for safe yet biologically relevant assessment of neutralization. These HiBiT-PsVLPs package the HiBiT protein tag internally and incorporate glycoproteins from pathogenic viruses to recapitulate entry at reduced biosafety levels. Unlike traditional pseudoviruses, HiBiT-PsVLPs lack reporter genes and employ the NanoBiT split luciferase system for rapid, complementation-based luminescent readout of entry and neutralization. Using the SARS-CoV-2 Spike protein as proof-of-concept, we demonstrate efficient pseudotyping, entry, and neutralization of HiBiT-PsVLPs. HiBiT-PsVLP neutralization is specific, reproducible, and reflective of antibody potency and stability. Assay results align closely with an established surrogate virus neutralization test (sVNT), with HiBiT-PsVLPs capturing more diverse mechanisms-of-action (MoAs). Moving beyond SARS-CoV-2, we adapt the HiBiT-PsVLP platform for other clinically relevant viruses, including HIV-1 and emerging pathogens, such as Ebola, Marburg, Lassa, and Nipah viruses. By pairing HiBiT-PsVLPs with Fc gamma receptor (FcγR)-expressing cells, we also demonstrate the capability of this system to detect antibody-dependent enhancement (ADE) of virus entry as an important safety consideration for immunotherapies. Combined, these data establish the utility of the HiBiT-PsVLP platform for safely and rapidly measuring critical antibody activities across diverse viruses and drug development stages. IMPORTANCE: Standard neutralization assays are often slow, labor-intensive, and restricted to high-containment facilities, thus complicating and delaying the development of vaccines and antibody-based treatments. Here, we present a novel neutralization assay system using HiBiT-tagged pseudovirus-like particles (HiBiT-PsVLPs). These particles incorporate entry proteins from diverse pathogenic viruses but are non-replicating and lack viral nucleic acids, thus mitigating the biosafety risks of conventional assays. The particles encapsulate the HiBiT peptide, enabling rapid, luminescent quantitation of entry and neutralization. We demonstrate that this platform accurately measures neutralizing activity of monoclonal antibodies across development stages and sensitively detects antibody-dependent enhancement, a critical safety consideration. Altogether, HiBiT-PsVLPs offer a safe, rapid, and scalable platform to accelerate the development of vaccines and antibody therapeutics targeting a broad range of viruses.