A mucosal prime–systemic boost strategy using a glycoengineered bacterial membrane vesicle vaccine elicits protective mucosal immunity against pneumococci in mice

Prime-boost vaccination using different administration routes may enhance protective immunity compared with homologous regimens. Here, we evaluated all eight possible three-dose combinations of intranasal (IN) and subcutaneous (SC) immunization using a glycoengineered membrane vesicle vaccine derived from probiotic Escherichia coli and displaying pneumococcal capsular polysaccharide. This self-adjuvanting vaccine platform induced robust humoral responses regardless of administration route. While all regimens elicited serum IgG responses, only IN-IN-IN, IN-IN-SC, and SC-IN-IN induced strong mucosal SIgA responses and enhanced pneumococcal clearance in the nasal cavity. Among these, the heterologous IN-IN-SC regimen generated the balanced mucosal and systemic immunity and exhibited the highest opsonophagocytic activity. These findings support route optimization as a practical strategy for maximizing the efficacy of glycoengineered membrane vesicle vaccines.