Engineered Low‐Endotoxin Bacterial Biomimetic Vesicles for Enhanced Oral Dual‐Antigen Subunit Vaccine Delivery

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Subunit vaccines are promising for disease prevention because of their safety and cost‐effectiveness. However, their efficacy is limited by low immunogenicity and gastrointestinal degradation after oral administration. To address this issue, low‐endotoxin Salmonella choleraesuis strain SC‐L3 was engineered via lipid A modification to generate bacterial biomimetic vesicles (BBVs) with reduced endotoxin activity. BBVs were functionalized using ClyA‐embedded SpyCatcher and Streptococcus protein G for dual antigen coupling, and further coated with chitosan oligosaccharides (COS) to enhance mucosal penetration and gastrointestinal stability. Using mCherry as a model antigen, we obtained optimized mCherry‐CSS‐BBV@COS that showed high antigen protection rates (83% and 63% in simulated gastric and intestinal fluids, respectively), capacity for lysosomal escape and effective stimulation of M1 macrophage polarization in vitro. Oral administration of mCherry‐CSS‐BBV@COS elicited robust systemic IgG and mucosal sIgA responses in mice. Furthermore, dual‐antigen BBV conjugates (GDH‐gD‐Fc‐CSS‐BBV@COS) co‐delivering Streptococcus suis glutamate dehydrogenase and pseudorabies virus gD‐Fc induced antigen‐specific humoral, mucosal and cellular immunity, conferring complete protection against lethal challenges with the respective pathogens. In summary, we generated a versatile, low‐endotoxin BBV platform for oral combination subunit vaccines, offering a novel strategy for protection against viral and bacterial infections.