In vitro neutralization of nervous necrosis virus by a nanobody binding to the protrusion domain of capsid protein

Abstract

Viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), has infected more than 177 marine and freshwater species belonging to 30 families, posing a devastating threat to aquaculture worldwide. Neutralizing antibodies can be used for both viral diagnosis and therapy, besides, analysis of the binding regions of virions with neutralizing antibody can facilitate the design of highly effective vaccines and drugs. As the smallest antibody with complete antigen binding function, nanobody is an ideal raw material for development of neutralizing antibody. In the study, the potential application of nanobody in neutralization of NNV was explored in vitro, and the binding regions of NNV capsid protein with nanobody were checked. An immunized phage-displayed nanobody library was firstly constructed by immunization alpaca with purified NNV. Twenty-two phage clones with strong binding activity to NNV were acquired by affinity panning, and six of them were selected for prokaryotic expression and purification. The recognition and binding ability of the selected nanobodies to NNV were verified by yeast two-hybrid and indirect ELISA, and data showed that the nanobody, NNV-Nb2, had the strongest affinity to NNV. NNV-Nb2 and NNV-Nb5 had strong NNV neutralizing activities with the 90% inhibitory concentrations of 12.30 and 27.82 mg/L, respectively. Moreover, NNV-Nb2 can bind to the protrusion domain of NNV capsid protein as analysis by yeast two-hybrid and western blot. Results so far indicated that NNV-Nb2 is a promising tool for diagnosis and treatment of VER, and the protrusion domain of NNV capsid protein is a key target for the design of highly effective vaccines and drugs.