NB-1

African swine fever (ASF) is a highly infectious and lethal swine disease, leading to enormous losses in the pig industry. K205R, a non-structural protein of ASF virus (ASFV), is abundantly expressed at the early stages of viral infection and induces a strong immune response. In our previous study, five strains of K205R-specific nanobodies (Nbs) were screened through phage display technology, among which Nb1, Nb14, Nb35, and Nb82 exhibited good affinity. In the present study, the above four Nbs were successfully expressed in HEK293T cells and exhibited strong reactivity. Four Nbs recognized linear B-cell epitopes of K205R in both prokaryotic and eukaryotic expression systems. Besides, four Nbs specifically reacted with the K205R protein of ASFV-infected cells. Two epitopes 1 MVEPR 5 and 188 RTQF 191 were further identified, with highly conserved in different ASFV strains, and could interact with inactivated ASFV-positive sera, indicating that the two epitopes were natural linear B-cell epitopes. Moreover, structural analysis indicated that both epitopes were exposed on the surface of the K205R molecule. Notably, the identified epitope 188 RTQF 191 was first reported. Overall, these findings provide valuable insights for K205R as an effective diagnostic tool and vaccine development.

African swine fever (ASF) is the number one killer affecting the pig industry, and there are no effective strategies for prevention. The ASFV K205R protein is prominently expressed in the early stages of viral infection, triggering a robust immune response. The full understanding of K205R protein epitopes provides a theoretical basis for the development of vaccine-candidate proteins. Nanobodies exhibit superior capability in detecting concealed epitopes of antigens compared with traditional antibodies. Here, we identify two epitopes 1 MVEPR 5 and 188 RTQF 191 based on nanobodies as a tool. Notably, the epitope 188 RTQF 191 is being reported for the first time. These epitopes are highly conserved in different ASFV strains and represent natural linear B-cell epitopes. This study opens up nanobodies as a new tool for the identification of epitopes and also provides a direct material basis for the development of ASFV vaccines.