Imaging murine NALT following intranasal immunization with flagellin-modified circumsporozoite protein malaria vaccines.

Intranasal (IN) immunization with a Plasmodium circumsporozoite (CS) protein conjugated to flagellin, a Toll-like receptor 5 agonist, was found to elicit antibody-mediated protective immunity in our previous murine studies. To better understand IN-elicited immune responses, we examined the nasopharynx-associated lymphoid tissue (NALT) in immunized mice and the interaction of flagellin-modified CS with murine dendritic cells (DCs) in vitro. NALT of immunized mice contained a predominance of germinal center (GC) B cells and increased numbers of CD11c+ DCs localized beneath the epithelium and within the GC T-cell area. We detected microfold cells distributed throughout the NALT epithelial cell layer and DC dendrites extending into the nasal cavity, which could potentially function in luminal CS antigen uptake. Flagellin-modified CS taken up by DCs in vitro was initially localized within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT antigen-presenting cell populations will advance development of an efficacious needle-free vaccine for the 40% of the world's population at risk of malaria.

Efficient phagosomal maturation and degradation of Plasmodium-infected erythrocytes by dendritic cells and macrophages.

Dendritic cells (DC) and macrophages phagocytose pathogens and degrade them in their phagosomes to allow for proper presentation of foreign antigens to other cells of the immune system. The Plasmodium parasite, causative agent of malaria, infects RBC that are phagocytosed by DC and macrophages during the course of infection. Under specific conditions, the functionality of these cells can be affected by phagocytosis of Plasmodium-infected RBC. We investigated whether phagosomal maturation and degradation of Plasmodium yoelii-infected RBC in phagosomes is affected in DC and macrophages. We show that recruitment of the phagolysosomal marker Lamp-1 and of MHC-II, as well as acidification of phagosomes, was achieved in a timely manner. Using P. yoelii-infected RBC labelled with a fluorescent dye or transgenic green fluorescent protein (GFP)-expressing parasites, we found a gradual, rapid decrease in the phagosome fluorescence signal, indicating that P. yoelii-infected RBC are efficiently degraded in macrophages and DC. We also observed that pre-incubation of DC with infected RBC did not affect phagosomal maturation of newly internalized P. yoelii-infected RBC. In conclusion, after phagocytosis, Plasmodium-infected RBC are degraded by DC and macrophages, suggesting that the process of phagosomal maturation is effectively completed in malaria.