Monoclonal antibodies used in this study.

The SARS-CoV-2 Spike (S) protein is essential for viral entry and serves as the primary immunogen in most COVID-19 vaccines. While its role in adaptive immunity is well defined, its potential to contribute directly to innate immune activation remains incompletely understood. Neutrophils, in particular, are prominent effectors in COVID-19 severity, yet how they respond directly to the S protein presented in a multivalent format is unclear. Here, we investigated whether the S protein can directly activate human neutrophils ex vivo using two biologically relevant models: nanoparticles displaying multivalent stabilized prefusion trimeric S glycoprotein, and purified β-propiolactone-inactivated SARS-CoV-2 virions. Neutrophils were exposed to nanoparticles or inactivated virus, either alone or pre-coated with monoclonal or polyclonal anti-S antibodies. Nanoparticles displaying Respiratory Syncytial Virus (RSV) Fusion (F) protein and purified β-propiolactone-inactivated RSV served as comparators. Across all models and conditions tested, the S protein did not induce significant neutrophil responses. No consistent effects were observed on cell viability, surface marker expression, reactive oxygen species production, neutrophil extracellular trap formation, cytokine release, or inflammatory gene expression—even in the presence of anti-S antibodies mimicking immune complexes. Results with F-nanoparticles and inactivated RSV were similarly modest. These findings indicate that the trimeric prefusion S protein, whether displayed multivalently on nanoparticles or in the context of inactivated viral particles, is insufficient to trigger robust neutrophil activation. This work provides insight into the innate immune profile of the S protein and suggests that its use in vaccine platforms is unlikely to directly provoke neutrophil-mediated inflammatory responses.