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The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris , and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections. [Display omitted] • Nanobodies to Spike proteins neutralize recent SARS-CoV-2 variants. • Nanobody-spike structures explain mutation tolerance. • Vaporizing technology allows nanobody application as aerosol. • Prophylactic inhalation suppresses infection below detectability. • Nanobodies diminish weight loss even when applied 24 h after infection. [ABSTRACT FROM AUTHOR]

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration. [Display omitted] • Uncoordinated-5 receptor D (Unc5D) and glypican-3 (GPC3) form an octameric complex • Anti-GPC3 nanobodies (Nanoglue, Nanobreak) enhance or weaken Unc5-GPC3 binding • Unc5/GPC3 signaling causes cell contact-repulsion in vitro • In vivo , Unc5/GPC3 modulate the migration of cortical neurons and neuroblastoma cells Detailed structural analysis and modulation of binding between uncoordinated-5 receptor D (Unc5D) and morphogen receptor glypican-3 (GPC3) reveal how finely balanced Unc5-GPC3 interactions regulate the migration of neuronal and cancer cells. [ABSTRACT FROM AUTHOR]