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.

严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）刺突（Spike, S）蛋白是病毒入侵宿主的关键因子，同时也是多数新冠疫苗的核心免疫原。尽管其在适应性免疫中的作用已得到明确阐释，但该蛋白能否直接介导固有免疫激活的潜力仍未完全阐明。中性粒细胞作为新冠重症进程中的主要效应细胞，目前学界对其如何直接响应多价形式呈现的S蛋白仍不甚明晰。本研究采用两种符合生物学相关性的模型，探究S蛋白能否在体外直接激活人源中性粒细胞：一是展示多价稳定融合前三聚体S糖蛋白的纳米颗粒，二是纯化的β-丙内酯灭活SARS-CoV-2病毒颗粒。实验中将中性粒细胞分别与上述纳米颗粒或灭活病毒单独孵育，或与预先包被单克隆/多克隆抗S抗体的样本共孵育。本研究同时设置了呼吸道合胞病毒（Respiratory Syncytial Virus, RSV）融合（Fusion, F）蛋白纳米颗粒与纯化β-丙内酯灭活RSV病毒作为对照。在所有测试模型与实验条件下，S蛋白均未诱导出显著的中性粒细胞应答。无论是否存在模拟免疫复合物的抗S抗体，均未观察到对细胞活力、表面标志物表达、活性氧生成、中性粒细胞胞外陷阱形成、细胞因子释放或炎症基因表达的一致性影响。针对F蛋白纳米颗粒与灭活RSV病毒的实验结果同样较为微弱。上述结果表明，无论是以多价形式展示于纳米颗粒表面，还是存在于灭活病毒颗粒中，三聚体融合前S蛋白均不足以触发强效的中性粒细胞激活。本研究揭示了S蛋白的固有免疫特征，提示其在疫苗平台中应用时，不太可能直接引发中性粒细胞介导的炎症应答。