High-yield production and preclinical protection assessment of a Chinese Hamster Ovary-K1 (CHO-K1) derived PRV gB and gD subunit vaccine against variant pseudorabies virus in pigs.

Pseudorabies virus (PRV) re-emergence in swine highlights the need for safe, scalable vaccines compatible with DIVA surveillance. We developed a CHO - K1 subunit platform expressing PRV gB-N + IV and gD that achieved high and consistent antigen yields (1.12 and 2.0 g/L, respectively) with downstream purity >95 %, supporting a practical manufacturing path for veterinary biologics. Formulated with Montanide ISA 201 VG or aluminum hydroxide, the vaccine was evaluated in piglets challenged with a virulent JK strain and compared with two commercial inactivated vaccines. Under a discriminatory intranasal challenge dose (2 mL of 10^5.0 TCID50/mL) established in a dose-ranging pre-study, the subunit vaccine conferred robust protection comparable to commercial products, significantly reducing clinical scores and mortality. Vaccinated piglets remained clinically healthy without fever or disease symptoms; only transient mild fever (< 41 °C) temperature elevations were observed. Safety was demonstrated in piglets under single-dose, repeated-dose, and overdose regimens and in pregnant sows vaccinated twice at mid-gestation, with no adverse clinical signs, injection-site reactions, or effects on rectal temperature, gestation, litter size, or piglet viability. As a gB/gD - based formulation, the vaccine does not induce anti-gE antibodies and is compatible with gE-based DIVA surveillance, an advantage for field application and eradication programs. Collectively, these data establish the feasibility of high-yield CHO-derived gB/gD antigens and provide preliminary in vivo evidence of clinical protection against a circulating PRV variant, supporting further development and broader strain coverage evaluation.