Characterization of in vitro viral neutralization targets of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in alveolar macrophage and evaluation of protection potential against HP-PRRSV challenged based on combination of HP-PRRSV-structure proteins in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global pork industry, resulting in substantial economic losses. Current control measures rely on modified live virus (MLV) vaccines with safety concerns. However, the lack of consensus on protective PRRSV antigens is impeding the development of effective and safety subunit vaccines. In this study, we conducted in vitro virus neutralization (VN) assays in MARC-145 and CRL-2843 cell lines and primary porcine alveolar macrophages (PAMs) to systemically identify PRRSV structural proteins (SPs) recognized by virus-neutralizing antibodies in hyperimmune serum collected from piglets infected with highly pathogenic PRRSV (HP-PRRSV). Additionally, piglets immunized with different combinations of recombinant PRRSV-SPs were challenged with HP-PRRSV to evaluate their in vivo protection potential. Intriguingly, different in vitro VN activities of serum antibodies elicited by each PRRSV SP were observed depending on the cell type used in the VN assay. Notably, antibodies specific for GP3, GP4, and M exhibited highest in vitro VN activities in PAMs, correlating with complete protection (100% survival) against HP-PRRSV challenge in vivo after immunization of piglets with combination of GP3, GP4, M and N (GP3/GP4/M/N). Further analysis of lung pathology, weight gain, and viremia post-challenge revealed that the combination of GP3/GP4/M/N provided superior protective efficacy against severe infection. These findings underscore the potential of this SP combination to serve as an effective PRRSV subunit vaccine, marking a significant advancement in pork industry disease management.