High-density fermentation of Streptococcus suis serotype 31 for inactivated vaccine production

Streptococcus suis serotype 31 (SS31) is a clinically important swine pathogen for which inactivated vaccine development requires a scalable cultivation process and evidence that the resulting bacterin has protective activity. However, conventional culture processes often yield insufficient biomass, whereas aggressive high-density fermentation may perturb the physiological features that determine vaccine quality. Herein, we demonstrate a Response Surface Methodology (RSM)-guided fermentation-to-vaccine strategy that optimizes the SS31 culture microenvironment and evaluates the protective activity of a bacterin produced under the optimized conditions. Single-factor screening and Box-Behnken modeling identified glucose, organic nitrogen and newborn bovine serum as dominant growth variables, leading to an optimized formulation containing 2.19 g/L glucose, 43.8 g/L organic nitrogen and 5.89% serum. When scaled to a 14-L bioreactor with residual sugar controlled at 1–3 g/L and pH maintained at 7.2, this process achieved 1.51 × 10¹⁰ CFU/mL, approximately seven-fold higher than shake-flask cultivation. The LD₅₀ estimates were 2.50 × 10⁶ CFU/mouse for SS31 cultured in the optimized medium and 2.14 × 10⁷ CFU/mouse for SS31 cultured in Martin medium, indicating a difference in the virulence-associated phenotype under the experimental conditions. These estimates do not demonstrate the conservation or enhancement of vaccine antigens. Among the four vaccine formulations tested, the aluminum-adjuvanted vaccine containing 3 × 10⁹ pre-inactivation CFU equivalents/mL yielded the highest observed survival (10/10 mice) after challenge with 2 LD₅₀. The organ bacterial-load and histopathological findings were evaluated descriptively. These findings demonstrate the technical feasibility of the SS31 cultivation process at the 14-L scale and support further evaluation of the resulting inactivated vaccine against S. suis .