A novel TK/gE-deleted pseudorabies virus vaccine expressing CCL3L1 enhances humoral immunity by stimulating the dendritic cell-T follicular helper-germinal center axis.
Pseudorabies virus (PRV) remains a major threat to the swine industry, particularly due to the emergence of highly virulent PRV variants that have caused severe outbreaks in Asia in recent years. These emerging strains exhibit enhanced pathogenicity and immune evasion, resulting in insufficient protection by traditional inactivated or attenuated vaccines derived from classical strains such as Bartha. Therefore, novel antiviral vaccine strategies with improved immunogenicity are urgently needed. In this study, an attenuated recombinant PRV (rPRV) was constructed from the emerging HNX strain by removing the TK and gE loci and introducing a CCL3L1 expression cassette (HNX-ΔTK/ΔgE-CCL3L1). The immunogenicity and protective efficacy of this recombinant virus were systematically evaluated. HNX-ΔTK/ΔgE-CCL3L1 significantly promoted the activation of bone marrow-derived dendritic cells (DCs) in vitro and enhanced DC activation in lymph nodes (LNs) in vivo. Vaccination with HNX-ΔTK/ΔgE-CCL3L1 induced robust humoral immune responses, including increased virus-neutralizing and glycoprotein B-specific antibody levels observed from the second week post-immunization onward. Furthermore, rPRV-expressed CCL3L1 enhanced T cell-dependent germinal center (GC) responses, resulting in improved protection against lethal PRV challenge. Our results demonstrate that CCL3L1 acts as an effective molecular adjuvant by potentiating humoral immunity through activation of the conventional DC-T follicular helper cell-GC B cell axis, supporting HNX-ΔTK/ΔgE-CCL3L1 as a potential vaccine for the control of emerging PRV variants.