Identification of novel epitopes targeting non-structural protein 2 of PRRSV using monoclonal antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV) is leading to huge losses in the swine industry worldwide. Its nonstructural protein 2 (Nsp2), with a cysteine protease domain (PL2), is crucial for virus replication and as a trigger to host innate immune regulation. In this study, three monoclonal antibodies (mAbs) to Nsp2, designated 4A12, 4G8, and 8H11, were generated. Subsequently, a sequence of recombinant peptides with partial overlap was utilized to determine the epitopes using these mAbs. We found three novel minimal linear Nsp2 B cell epitopes, ¹⁸⁸ELSDDSNRPV¹⁹⁷, ⁴²HLKRYSPPAE⁵¹, and ⁵⁴CGWHCISA⁶¹, which were identified by the antibodies 4A12, 4G8, and 8H11, respectively. Structure analysis indicates that ⁴²HLKRYSPPAE⁵¹ and ¹⁸⁸ELSDDSNRPV¹⁹⁷ are located separately in hypervariable region 1 and hypervariable region 2 of Nsp2. Interestingly, ⁵⁴CGWHCISA⁶¹ is located in the PL2 region, which is highly conserved in all arteriviruses, particularly at the expected conserved catalytic site at Cys54. Importantly, ⁵⁴CGWHCISA⁶¹ is located in the inner region of the expected 3D structure of Nsp2, which reveals that the epitope is cryptic. These findings not only provide valuable insight for vaccine design and hold diagnostic potential for the identified epitopes, but also reveal a protective mechanism against variation under selective pressure in an important epitope.