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.