Development of flow cytometry bead-based opsonophagocytic assays to dissect cell-mediated functional antibody...

Malaria remains a major global health burden. Vaccines targeting the Circumsporozoite protein represent a significant advancement in malaria control and have demonstrated meaningful public health impact. However, no validated correlate of protection has yet been established, making it critical to assess antibody functionality alongside antigen-specific antibody quantitation to evaluate vaccine candidates. Antibody-dependent phagocytosis is one such mechanism that contributes to parasite clearance, which can be modelled using cell lines. While THP-1 (monocyte-like) assays have been described, HL-60 (neutrophil-like) models are less established in the malaria field. Here, we aimed to develop and characterize a PfCSP repeat region (NANP6) bead-based opsonophagocytic assay using differentiated HL-60 cells, to adapt a THP-1 assay, and to assess IgG subclass- and FcγR-dependent differences between these in vitro models. Ultimately, our goal is to demonstrate the potential application of these assays for exploratory assessment of human clinical samples.

Fluorescent NANP6-coated beads were opsonized with recombinant IgG1-2-3-4 monoclonal antibodies (identical binding domain, different Fc) or human sera from an RTS, S/AS01E trial. Opsonized beads were incubated with model effector cells, phagocytosis was quantified by flow cytometry, and EC50 values were derived using four-parameter logistic regression. FcγR-blocking experiments and assay performance (precision, dilution linearity, sensitivity) were evaluated.

Both assays discriminated phagocytic potency across IgG subclasses, revealing that FcγR dependence differs by subclass and between the two cell models. Both assays were successfully adapted to human sera, demonstrating robust performance with high precision, strong dilution linearity, and high sensitivity across samples with varying anti-CSP antibody levels. These characteristics support their suitability for reproducible assessment of functional antibody responses in clinical samples.

Two opsonophagocytic assays recapitulating monocyte-like and neutrophil-like effector pathways were developed and characterized. Both demonstrated precise, sensitive, and quantitative measurement of antibody-dependent phagocytosis in human sera, while capturing distinct functional differences between the models, supporting their use as exploratory tools to evaluate functional antibody responses in clinical samples.