Nanobodies targeting the conserved post-fusion six-helix bundle core provide protective effects against SARS-CoV-2 infection and immunopathology

The post-fusion six-helix bundle (6-HB) of the SARS-CoV-2 spike protein is an evolutionarily constrained structural machinery essential for viral and host membrane fusion, representing a critical vulnerability for antiviral development. While peptide-based fusion inhibitors have been extensively explored, the application of nanobodies, which offer superior stability and the unique capacity to penetrate sterically restricted or cryptic epitopes, remains largely untapped for targeting this conserved fusion core. In this study, we utilized yeast surface display to engineer and isolate high-affinity nanobodies specifically directed against the post-fusion 6-HB. Two lead candidates, ML01 and ML13, exhibited nanomolar to low-micromolar binding affinities and distinct paratope specificity, as confirmed by biophysical characterization and epitope mapping. Functional evaluation demonstrated that these nanobodies exert potent neutralization of pseudotyped and authentic SARS-CoV-2 variants in vitro. Crucially, in vivo validation using a K18-hACE2 neonatal mouse model revealed that ML01 and ML13 administration significantly attenuated pulmonary transcription- and replication-competent virus-like particle (trVLP) entry, suppressed the induction of hyper-inflammatory cytokines (including TNF-α, IL-1β, IL-6, IFN-γ, and CXCL1), and mitigated severe histopathological lung injury. These findings provide a proof-of-concept that the post-fusion 6-HB is a promising target for nanobody-based therapeutic development. ML01 and ML13 emerge as resilient therapeutic candidates capable of providing a potential defense against the evolving SARS-CoV-2 landscape. Whether these nanobodies can cross-react with other coronaviruses remains to be determined.