Mucosal surfaces of the lungs represent a major site of entry for airborne pathogens, and pulmonary administration of vaccines is an attractive strategy to induce protective mucosal immunity in the airways. Recently, we demonstrated the potential of pulmonary vaccination with the tuberculosis subunit antigen H56 adjuvanted with the cationic liposomal adjuvant formulation CAF01, which consists of the cationic lipid dimethyldioctadecylammonium (DDA) bromide and the synthetic cord factor trehalose-6,6′-dibehenate. However, the cationic charge of DDA represents a major safety challenge. Hence, replacing DDA with a safer zwitterionic or anionic phospholipid is an attractive approach to improve vaccine safety, but the effect of liposomal surface charge on the induction of mucosal immunity after airway immunization is poorly understood. Here, we investigated the effect of surface charge by replacing the cationic DDA component of CAF01 with zwitterionic dipalmitoylphosphatidylcholine (DPPC) or anionic dipalmitoylphosphatidylglycerol (DPPG), and we show that charge modification enhances antigen-specific pulmonary T-cell responses against co-formulated H56. We systematically replaced DDA with either DPPC or DPPG and found that these modifications resulted in colloidally stable liposomes that have similar size and morphology to unmodified CAF01. DPPC- or DPPG-modified CAF01 displayed surface charge-dependent protein adsorption and induced slightly higher follicular helper T cells and germinal center B cells in the lung-draining lymph nodes than unmodified CAF01. In addition, modified CAF01 induced significantly higher levels of H56-specific Th17 cells and polyfunctional CD4+ T cells in the lungs, as compared to unmodified CAF01. However, the strong H56-specific humoral responses induced by CAF01 in the lungs and spleen were not influenced by surface charge. Hence, these results provide insights into the importance of surface charge for liposomal adjuvant function and can also guide the design of safe pulmonary subunit vaccines against other mucosal pathogens.

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