Nanobodies® specific for respiratory syncytial virus fusion protein protect against infection by inhibition of fusion.

Despite the medical importance of respiratory syncytial virus (RSV) infections, there is no vaccine or therapeutic agent available. Prophylactic administration of palivizumab, a humanized monoclonal RSV fusion (F) protein-specific antibody, can protect high-risk children. Previously, we have demonstrated that RSV can be neutralized by picomolar concentrations of a camelid immunoglobulin single-variable domain that binds the RSV protein F (F-VHHb nanobodies). Here, we investigated the mechanism by which these nanobodies neutralize RSV and tested their antiviral activity in vivo. We demonstrate that bivalent RSV F-specific nanobodies neutralize RSV infection by inhibiting fusion without affecting viral attachment. The ability of RSV F-specific nanobodies to protect against RSV infection was investigated in vivo. Intranasal administration of bivalent RSV F-specific nanobodies protected BALB/c mice from RSV infection, and associated pulmonary inflammation. Moreover, therapeutic treatment with these nanobodies after RSV infection could reduce viral replication and reduced pulmonary inflammation. Thus, nanobodies are promising therapeutic molecules for treatment of RSV.

CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells.

The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.