The murine NALT resembles other non-encapsulated lymphoid organs, such as the Peyer’s Patches (PP) of the gut-associated lymphoid tissue (GALT). DC morphological alterations. Murine BMDC were incubated for 30 min on snow with (A) no peptide, (B) 100 g/ml of STF2.(T1BT*)4, or (C) 100 g/ml STF2.CS protein. Cells were then allowed to internalize the proteins for 30 min at 37C. After fixation and permeabilization, intracellular CS was recognized by staining with mAb 2A10, specific for CS protein repeats, followed by biotinylated goat anti-mouse IgG and streptavidin-Qdots 655 (reddish). Nuclei were stained with Hoechst (blue). (D FLJ12894 C G) Confocal microscopic 3D data were imported into Imaris for generation of isosurfaces representing the intracellular distribution of CS protein (reddish) in relation to the nucleus (blue). Representative images show that BMDC containing no flagellin-modified CS constructs (D) or exhibiting a vesicular or CS protein distribution (E) are well spread, while cells having a vesicular/cytosolic CS protein labeling pattern (F) appear rounded. Cells having a predominantly cytosolic CS protein distribution (G) are shrunken, harbor a small and condensed nucleus, and appear to detach. Level bars = 10 m. NIHMS490514-supplement-Supplementary_Numbers_1S_and_2S.pdf (12M) GUID:?FECAE12D-E4A1-448D-A651-3B0508301668 Abstract Intranasal (IN) immunization having a circumsporozoite (CS) protein conjugated to flagellin, a TLR5 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 cells (NALT) in immunized mice and the conversation of flagellin-modified CS with murine dendritic cells (DC) was initially localized Nifenalol HCl within intracellular vesicles followed by a cytosolic distribution. Vaccine modifications to enhance delivery to the NALT and specifically target NALT APC populations will progress development of an efficacious needle-free vaccine for the 40% of the world’s human population at risk of malaria. will require a multifaceted approach to control/eradicate malaria. Optimism for development of vaccines as part of this multipronged approach has been raised by a recent phase III medical trial of a vaccine based on a major sporozoite surface antigen, the circumsporozoite (CS) protein [1, 2]. This CS-based vaccine, termed RTS,S, focuses on Nifenalol HCl the parasite at its pre-erythrocytic phases with the goal of avoiding development of the erythrocytic phases responsible for medical disease. The pre-erythrocytic phases are attractive defense focuses on as sporozoites inoculated from the mosquito vector and the exoerythrocytic forms that consequently develop in the liver can be inhibited by antibody and by cellular Nifenalol HCl immune responses, respectively. Successful prevention of the intra-erythrocytic cycle also will prevent development of parasite lovemaking stages responsible for transmission to the mosquito vector. A key requirement for induction of potent humoral and cellular immunity is the dendritic cell (DC), which bridges the innate and adaptive immune response. Toll-like receptor (TLR) agonists that can Nifenalol HCl be linked to antigens, such as the TLR 5 agonist flagellin, function as strong adjuvants that induce maturation of DC and upregulation of costimulatory molecules required for initiation of adaptive immunity . Viral and bacterial antigens linked to flagellin have shown promise as parenteral and mucosal vaccines in murine studies and clinical tests [3-8]. In a recent murine study, we exhibited the immunogenicity, as well as the and protecting efficacy of antibodies elicited by a recombinant circumsporozoite (CS) protein modified with the TLR5 ligand flagellin when delivered either subcutaneously (SC) or intranasally (IN) (Carapau, Mitchell, Nacer, Shaw, Othoro, Frevert et al, unpublished). The vaccine was comprised of an indicated recombinant Typhimurium flagellin B protein, either full size (STF2) or truncated to remove the hinge region (STF2), fused with T and B cell epitopes of CS protein or a nearly full size CS protein (Physique 1). Sera of mice immunized IN or SC with the flagellin-modified CS constructs experienced similar levels of predominantly IgG1 antibodies to CS or to flagellin. Antibody responses were dependent on flagellin as minimal or no antibodies to either CS or flagellin were acquired in ?/? mice. Immune sera from your IN immunized mice neutralized sporozoite infectivity when pre-incubated with viable transgenic sporozoites expressing CS repeats [9]. Consistent with the findings, IN immunized mice challenged by exposure to the bites of and CS and support the potential of developing a scalable, low cost, needle-free malaria vaccine for the 40% of the world’s human population currently at risk of malaria. Open in a separate window Physique 1 Diagram of CS.
