Development of a Thermostable Freeze-Dried Live Pseudorabies Vaccine Based on the PRV Bartha-K61 Strain: Formulation Optimization, Stability Evaluation, and Preliminary Immunogenicity in Piglets

Background: Pseudorabies vaccines based on the live attenuated PRV Bartha-K61 strain remain essential for controlling pseudorabies in swine, but poor thermal stability during storage and transport limits field use. Objectives: This study aimed to develop a thermostable freeze-dried live pseudorabies vaccine through the integrated optimization of formulation and lyophilization and to preliminarily assess its humoral immunogenicity in piglets. Methods: Trehalose, mannitol, and glycine were screened as candidate lyoprotectants by single-factor experiments, followed by Box–Behnken response surface optimization using viral titer retention as the response. Critical thermal parameters were determined to establish a formulation-specific lyophilization cycle. Product quality was evaluated by post-lyophilization titer, cake appearance, residual moisture, and electron microscopy, and stability was compared with a commercial freeze-dried vaccine at 2–8 °C, 25 °C, 37 °C, and 45 °C. Conclusions: The optimized formulation, ST005, contained 9.5% trehalose, 2.0% mannitol, and 1.5% glycine. It showed a collapse-related critical temperature of approximately −34.0 °C and a dried-product glass transition temperature of 69.1 °C, produced an intact cake with residual moisture below 3.0%, and preserved viral morphology after lyophilization. Titer loss remained below 1.0 log10 TCID50/mL for 24 months at 2–8 °C, 9 months at 25 °C, 21 days at 37 °C, and 9 days at 45 °C, outperforming the commercial comparator. In PRV-seronegative piglets, stored ST005 induced robust gB-specific and neutralizing antibody responses after storage under refrigerated, ambient, and accelerated conditions.