B-cell epitope mapping of VP5 protein of bluetongue virus serotype 1 using monoclonal antibodies

Bluetongue (BT) is a severe infectious disease affecting ruminants, caused by the bluetongue virus (BTV), an Orbivirus transmitted by Culicoides midges. VP5 is a major component of the outer capsid of BTV, and a key constituent of BTV subunit and virus-like particle vaccines. However, to date, the B-cell epitopes on VP5 recognized by humoral immune responses remain unidentified. In this study, recombinant VP5Δ1-79aa was expressed in Escherichia coli (E. coli) and purified for mouse immunization. Five monoclonal antibodies (mAbs) were identified through hybridoma fusion, clonal selection, and immunological assays. B-cell epitopes were recognized by mAbs 4G9, 6C9, 3A1, 3C8 and 6B1, these epitopes were mapped using a series of truncated overlapping peptides expressed as glutathione S-transferase fusion proteins. 4G9 recognized the ¹⁴⁴DEKQFDILNK¹⁵³ sequence, 6C9 recognized ¹⁵⁴AVTSYNKILT¹⁶³, 3A1 and 3C8 recognized ²⁰⁷VERDGMQEEA²¹⁶, and 6B1 recognized ³¹²ENHKELMHIK³²¹. These specific mAbs and their corresponding B-cell epitopes provide valuable insights into the structure and function of VP5, contributing to the advancement of serological diagnostic methods and development of epitope-based vaccines for BTV.