Abstract B1 cells differ from B2 cells in their developmental origin, phenotype, and function. They are anatomically located in various tissues of the immune system, and contribute to around 90% of the circulating antibodies in blood. B1 cells are often activated in a T-cell independent manner promoted by either antigens that contain a B-cell mitogen such as LPS, or by molecules that have highly repetitive structures such as bacterial capsular polysaccharides, with both pathways having the potential to undergo class-switch recombination and somatic hypermutation. Previously, we have shown that immunization with bacterial outer membrane vesicles engineered to display defined glycan structures on their surface (glycOMVs) promotes a strong glycan-specific IgG response for a wide range of bacterial and mammalian glycans. Here, we show that the structure of lipid A in these (glycOMVs) displaying the oncofetal antigen polysialic acid (polySia), influences the activation and class-switching of polySia-specific B1 cells from the spleen and peritoneal cavity in mice. We found that structural remodeling of lipid A from its normal hexa-acylated structure to a penta-acylated variant prevents the activation of polySia-specific B1 cells in the spleen and enhances the activation of polySia-specific B2 cells from the peritoneal cavity. Interestingly, we observed no changes in the glycan-specific IgG titers following immunization of wildtype B6 mice with either construct, even though there was no B cell activation in the spleen of mice receiving penta-acylated glycOMVs. However, these same glycOMVs failed to promote a glycan-specific IgG response in T-deficient mice, suggesting that this response was entirely T-cell dependent. This work was supported by Bill and Melinda Gates Foundation grant OPP1217652 (to MPD), NSF grants CBET-1159581, CBET-1264701 and CBET-1936823 (to MPD), the Defense Threat Reduction Agency (DTRA) grants HDTRA1-15-10052 and HDTRA1-20-10004 (to MPD), NIH grants 1R01GM137314 and 1R01GM127578 (to MPD) and the Fleming Scholars Fellowship (to NLB).