A dual-glycoprotein-targeting bispecific nanoantibody confers potent neutralization against Nipah virus

Nipah virus (NiV) is a biosafety level 4 pathogen that has consistently posed a significant threat to public health. The two envelope proteins on the viral surface, the receptor-binding protein (RBP) and the fusion (F) protein, are the primary targets of neutralizing antibodies. However, the use of antibodies targeting a single viral antigen often leads to the emergence of resistant strains. Here, by panning phage display library, we obtained a C-type nanoantibody (C-Nab), G-3-1, targeting the head domain of RBP. The half-life-extended G-3-1-HLE effectively protected hamsters against authentic NiV challenge. Furthermore, we constructed a fusion protein, 2A1-3-8-(AP)₁₂-G-3-1, by linking G-3-1 to the F-targeting nanobody 2A1-3-8 via a rigid (AP)₁₂ linker. This fusion protein exhibited improved and balanced neutralizing activity. In addition, the in vivo prophylactic efficacy of 2A1-3-8-(AP)₁₂-G-3-1 was validated in a mouse model of NiV pseudovirus infection. Collectively, this study on a dual-envelope-protein-targeting bispecific antibody not only provides a promising antiviral candidate, but also offers an important design strategy for next-generation antibody optimization.