Screening, Expression and Identification of Nanobody Against Monkeypox Virus A35R

Introduction: The monkeypox (Mpox) virus epidemic presents a significant risk to global public health security. A35R, a crucial constituent of EEV, plays a pivotal role in virus transmission, serves as a vital target for vaccine development, and has potential for serological detection. Currently, there is a dearth of research on nanobodies targeting A35R. The purpose of this study is to identify specific nanobodies target A35R, so as to provide new antibody candidates for Mpox vaccine development and diagnostic kit development. Methods: Three nanobodies specific to the monkeypox virus protein A35R were screened from a naïve phage display library. After four rounds of panning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA). Further, the nanobody fusion protein was constructed in pNFCG1-IgG1-Fc vector and expressed in HEK293F cells and purified by affinity chromatography. The specificity and affinity of the nanobodies were identified by ELISA. The binding kinetics of the VHH antibody to A35R were assessed via employment of a bio-layer interferometry (BLI) apparatus, thereby determining the nanobodies affinity. Results: The three purified nanobodies showed specific high-affinity binding MPXV A35R, of them, VHH-1 had the best antigen binding affinity (EC50 = 0.010 ug/mL). In addition, VHH-1 on Protein A biosensor can bind Mpox virus A35R, with an affinity constant of 54 nM as determined in BLI assay. Conclusion: In sum, we has obtained three nanobody strains against Mpox virus A35R with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of Mpox virus. Keywords: monkeypox virus, A35R, nanobody, phage display library