Identification of a conserved neutralizing epitope on the ASFV H240R protein by a nanobody that inhibits viral attachment.

African swine fever virus (ASFV), a highly contagious double-stranded DNA virus, causes a hemorrhagic disease with mortality approaching 100% in domestic pigs. Despite intensive efforts, effective vaccine options remain limited, highlighting the need for continuously identifying viral neutralizing epitopes. Nanobodies can effectively engage cryptic epitopes that are often inaccessible to conventional antibodies due to their small size. Here, we immunized a Bactrian camel with recombinant ASFV H240R expressed in a bacterial system, and an H240R-specific VHH phage-display library was constructed and screened, yielding a nanobody named H240R-Nb82. H240R-Nb82 efficiently neutralized distinct ASFV isolates (genotypes Ⅰ, Ⅱ, and their recombinants) in porcine alveolar macrophages. Mechanistically, H240R-Nb82 inhibited the early stage of ASFV infection by blocking viral attachment to host cells. Epitope mapping using truncation analysis and site-directed alanine mutagenesis showed that H240R-Nb82 binds to a linear, native virion-accessible conserved region within H240R (residues 33GHYPFSFELK42) and His34 made an important contribution to H240R-Nb82 binding. H240R-Nb82 also directly bound purified ASFV virions. Moreover, immunization of mice with the epitope-containing antigen induced sera capable of neutralizing ASFV infection in porcine alveolar macrophages, demonstrating the protective potential of this conserved determinant. Collectively, these findings define a previously unrecognized conserved neutralizing epitope on ASFV H240R and provide a new target protein for developing ASFV subunit vaccines.