Introduction Antibodies mediate a wide range of antiviral functions, including neutralization and diverse Fc-dependent effector activities. Among these, antibody-dependent cellular phagocytosis (ADCP) has emerged as an important mechanism contributing to pathogen clearance, including during HIV-1 infection. Conventional bead-based ADCP assays typically rely on recombinant envelope glycoprotein (Env), which offers practical advantages but fails to fully recapitulate the native structural, conformational, and glycan features of virion-associated Env. This limitation reduces the physiological relevance of these assays for evaluating antibody function in vivo . Methods We developed a virus particle-based ADCP assay designed to preserve the native membrane-embedded conformation and glycosylation of HIV-1 Env. The assay uses sucrose-purified, inactivated HIV-1 virions coupled to fluorescent beads as phagocytic targets, and the THP-1 human monocytic cell line as effector cells. The assay was optimized for sensitivity, reproducibility, and high‑throughput compatibility, and was applied to evaluate ADCP responses mediated by both monoclonal and polyclonal antibodies across multiple species. Results The virus particle-based ADCP assay enabled robust and reproducible measurement of antibody-mediated phagocytosis in a biologically relevant antigen format. Using this system, we observed Env isolate-specific differences in Fc‑mediated activity that were not fully captured using recombinant gp120‑based assays. Direct comparison revealed that ADCP readouts obtained with recombinant Env proteins did not consistently mirror those measured using native virion-associated Env, highlighting qualitative differences in antibody engagement and Fc effector function depending on antigen presentation. Discussion These findings demonstrate that preservation of native Env structure and glycosylation is critical for accurate assessment of Fc-dependent effector functions such as ADCP. The virus particle-based assay described here provides a practical and scalable framework for functional profiling of antibody responses in the context of HIV-1 infection and vaccination. By revealing antigen context-dependent differences in Fc‑mediated activity, this approach reinforces the importance of using antigenically authentic, virion-based systems to better reflect in vivo antibody function and inform vaccine and immunotherapeutic strategies.
Upadhyay et al. (Fri,) studied this question.