Novel linear B-cell epitopes mapping with monoclonal antibodies to VP3 protein from bluetongue virus serotype 1.

Abstract

Bluetongue is an infectious disease affecting domestic and wild ruminants that is caused by bluetongue virus (BTV). VP3 is the major component of the BTV inner capsid and a structural protein that is essential for viral maturation. The B-cell epitopes on BTV-1 VP3 that are recognized by the humoral immune response remain largely uncharacterized. In this study, we expressed and purified recombinant BTV-1 VP3 in Escherichia coli and used it to immunize BALB/c mice. Hybridoma technology was used to generate eight monoclonal antibodies (mAbs): 3A5, 3B2, 3E6, 3H5, 3B4, 3C5, 3D3, and 3E1. The B-cell epitopes recognized by these mAbs were mapped using a series of overlapping truncated peptides expressed as glutathione S-transferase fusion proteins. Two linear epitopes were identified: 7QRPERIKTT15, recognized by mAbs 3B4, 3C5, 3D3, and 3E1, and 119TEDEPEKFYS128, recognized by mAbs 3A5, 3B2, 3E6, and 3H5. Cross-reactivity analysis confirmed that these mAbs were specific to BTV1 VP3 under the test conditions, with the limitation that cross-reactivity validation was performed only against EHDV-2 VP3. This study identified key antigenic epitopes on BTV-1 VP3 under test conditions, providing structural studies, diagnostic tool development, or viral assembly research. These findings expand the available repertoire of BTV-1 VP3-specific monoclonal antibodies and define two linear B-cell epitope regions within VP3, providing a foundation for future structural studies, diagnostic tool development, and investigations into BTV core assembly.