A neuraminidase-targeted nanobody confers broad protection against influenza B virus

Abstract

ABSTRACT Influenza B virus (IBV) exhibits antigenic diversity and significantly contributes to the annual burden of influenza-related disease, posing substantial impacts on global public health and the economy. In this study, we focused on the neuraminidase (NA) of IBV, immunized an alpaca with tetrameric IBV NA, and identified a series of nanobodies with a common feature of the “DYR” motif in CDR3, exhibiting broad inhibitory activity against diverse NAs of IBV. Among these, the most potent VHH5 broadly inhibits NA from multiple representative strains across ancestral, B/Yamagata/16/88-like, and B/Victoria/2/87-like lineages. Moreover, fusing VHH5 to a human immunoglobulin G1 Fc domain significantly increased its antiviral potency in vitro, with the 50% inhibitory concentration in the low nanomolar range. Structural modeling and molecular dynamics simulations revealed that the “DYR” motif within the elongated CDR3 loop of VHH5 penetrates the catalytic pocket and engages the highly conserved catalytic site of IBV NA. Experimental mutagenesis analysis of the predicted interface further supported this binding model. Furthermore, the VHH5-Fc protects mice from the lethal challenge with a high dose of the IBV-2024 virus in both prophylactic and therapeutic settings. Our findings highlight the potential of the Fc-fused VHH5 antibody in controlling influenza B virus infections. IMPORTANCE Although influenza B viruses (IBVs) account for a substantial proportion of seasonal influenza virus infections, they have received comparatively less research attention. The small size and flexible binding modes of nanobodies enable them to access cryptic or recessed epitopes, supporting their development as potent clinical therapeutics. Here, we report the identification of VHH5, a nanobody targeting conserved residues within the neuraminidase active site of the influenza B virus, conferring broad inhibitory activity. Moreover, Fc-fused VHH5 enhanced antiviral potency in vitro and provided robust protection against lethal IBV challenge in mice. Together, these findings establish VHH5-Fc as a promising therapeutic candidate with potent and broad-spectrum activity against antigenically diverse IBV strains. Although influenza B viruses (IBVs) account for a substantial proportion of seasonal influenza virus infections, they have received comparatively less research attention. The small size and flexible binding modes of nanobodies enable them to access cryptic or recessed epitopes, supporting their development as potent clinical therapeutics. Here, we report the identification of VHH5, a nanobody targeting conserved residues within the neuraminidase active site of the influenza B virus, conferring broad inhibitory activity. Moreover, Fc-fused VHH5 enhanced antiviral potency in vitro and provided robust protection against lethal IBV challenge in mice. Together, these findings establish VHH5-Fc as a promising therapeutic candidate with potent and broad-spectrum activity against antigenically diverse IBV strains.