Understanding the in vivo fate of vaccine antigens and adjuvants and their safety is crucial for the rational design of mucosal subunit vaccines. Prime and pull vaccination using the T helper 17-inducing adjuvant CAF01 administered parenterally and mucosally, respectively, has previously been suggested as a promising strategy to redirect immunity to mucosal tissues. Recently, we reported a promising tuberculosis (TB) vaccination strategy comprising of parenteral priming followed by intrapulmonary (i.pulmon.) mucosal pull immunization with the TB subunit vaccine candidate H56/CAF01, which resulted in the induction of lung-localized, H56-specific T cells and systemic as well as lung mucosal IgA responses. Here, we investigate the uptake of H56/CAF01 by mucosal and systemic innate myeloid cells, antigen-presenting cells (APCs), lung epithelial cells and endothelial cells in mice after parenteral prime combined with i.pulmon. pull immunization, and after parenteral or i.pulmon. prime immunization alone. We find that i.pulmon. pull immunization of mice with H56/CAF01, which are parenterally primed with H56/CAF01, substantially enhances vaccine uptake and presentation by pulmonary and splenic APCs, pulmonary endothelial cells and type I epithelial cells and induces stronger activation of dendritic cells in the lung-draining lymph nodes, compared with parenteral immunization alone, which suggests activation of both innate and memory responses. Using mass spectrometry imaging of lipid biomarkers, we further show that (i) airway mucosal immunization with H56/CAF01 neither induces apparent local tissue damage nor inflammation in the lungs, and (ii) the presence of CAF01 is accompanied by evidence of an altered phagocytic activity in alveolar macrophages, evident from co-localization of CAF01 with the biomarker bis(monoacylglycero)phosphate, which is expressed in the late endosomes and lysosomes of phagocytosing macrophages. Hence, our data demonstrate that innate myeloid responses differ after one and two immunizations, respectively, and the priming route and boosting route individually affect this outcome. These findings may have important implications for the design of mucosal vaccines intended for safe administration in the airways.

There is an unmet medical need for new subunit vaccines that induce cytotoxic T-lymphocyte (CTL) responses to prevent infection with a number of pathogens. However, stimulation of CTL responses via clinically acceptable subcutaneous (s.c.) and intramuscular (i.m.) injection is challenging. Recently, we designed a liposomal adjuvant [cationic adjuvant formulation (CAF)09] composed of the cationic lipid dimethyldioctadecylammonium (DDA) bromide, a synthetic monomycoloyl glycerol analog and polyinosinic:polycytidylic acid, which induce strong CTL responses to peptide and protein antigens after intraperitoneal administration. By contrast, CAF09 does not stimulate CTL responses upon s.c. or i.m. injection because the vaccine forms a depot that remains at the injection site. Hence, we engineered a series of nanoemulsions (CAF24a-c) based on the active components of CAF09. The oil phase consisted of biodegradable squalane, and the surface charge was varied systematically by replacing DDA with zwitterionic distearoylphosphoethanolamine. We hypothesized that the nanoemulsions drain to the lymph nodes to a larger extent than CAF09, upon s.c. co-administration with the model antigen chicken egg ovalbumin (OVA). This results in an increased dose fraction that reaches the draining lymph nodes (dLNs) and subsequently activates cross-presenting dendritic cells (DCs), which can prime CTL responses. Indeed, the nanoemulsions induced antigen-specific CD8+ T-cell responses, which were significantly higher than those stimulated by OVA adjuvanted with CAF09. We explain this by the observed rapid localization of CAF24a in the dLNs and the subsequent association with conventional DCs, which promotes induction of CTL responses. Uptake of CAF24a was not specific for DCs, because CAF24a was also detected with B cells and macrophages. No measurable dose fraction of CAF09 was detected in the dLNs within the study period, and CAF09 formed a depot at the site of injection. Importantly, s.c. vaccination with OVA adjuvanted with CAF24a induced significant levels of specific lysis of antigen-pulsed splenocytes were induced after, which was not observed for OVA adjuvanted with CAF09. Thus, CAF24a is a promising adjuvant for induction of CTL responses upon s.c. and i.m. immunization, and it offers interesting perspectives for the design of vaccines against pathogens for which CTL responses are required to prevent infection.