Development of human single-domain antibodies against influenza based on NA-targeting IgG

The neuraminidase (NA) of influenza A virus (IAV) is an important antiviral target. However, antibodies require precise recognition of the catalytic pocket to exert antiviral effects, making them difficult to obtain through conventional screening. Traditional IgG antibodies also suffer from poor aerosolization efficiency, limiting their effectiveness for respiratory delivery. Human single-domain antibodies (sdAbs) with only the variable domain of the heavy chain (VH) show great potential in combating respiratory viral infections because of their small molecular weight and superior inhalable properties; however, sdAbs often lose stability or binding activity when isolated directly from parental IgG antibodies. To overcome these barriers and accelerate the acquisition of fully human sdAbs targeting the NA active pocket, we extracted the VH domain from broadly neutralizing anti-NA IgG and applied scaffold grafting followed by affinity recovery. Ultimately, we obtained two fully human sdAbs with favorable physicochemical properties that inhibited H5N8 NA enzymatic activity, with IC₅₀ values of 0.82 and 0.59 μg mL^-1. This work enabled the originally insoluble VH fragment of IgG to be expressed in a stable and soluble form and restored its NA-binding activity from undetectable levels to the nanomolar range, providing insights into engineering fully human sdAbs capable of targeting structurally constrained viral enzymes and suitable for inhalable influenza therapeutics.