title: In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine.
abstract: BACKGROUND
  Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively.
  
  
  RESULTS
  We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T(EM)) and IFN-γ release.
  
  
  CONCLUSIONS
  In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.
authors.affiliation: Department of Pharmaceutical Sciences, University of Oklahoma Health Science Center, Oklahoma City, OK 73117, USA.
authors.forename: Prachi
authors.initials: P
authors.lastname: Vilekar
authors.forename: Vibhudutta
authors.initials: V
authors.lastname: Awasthi
authors.forename: Pallavi
authors.initials: P
authors.lastname: Lagisetty
authors.forename: Catherine
authors.initials: C
authors.lastname: King
authors.forename: Nathan
authors.initials: N
authors.lastname: Shankar
authors.forename: Shanjana
authors.initials: S
authors.lastname: Awasthi
mesh_terms: D000281:Administration, Intranasal; D000818:Animals; D001086:Arabinofuranosyluracil / Q000031:analogs & derivatives / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D048430:Cell Shape; D002478:Cells, Cultured; D003045:Coccidioides / Q000276:immunology; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005657:Fungal Vaccines / Q000008:administration & dosage* / Q000276:immunology*; D007114:Immunization / Q000379:methods*; D007156:Immunologic Memory; D016130:Immunophenotyping; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D023421:Models, Animal; D057054:Molecular Imaging; D010957:Plasmids / Q000235:genetics; D013937:Thymidine Kinase / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection; D019076:Transgenes / Q000235:genetics
chemical_list: D016207:Cytokines; D018076:DNA, Complementary; D000939:Epitopes; D005657:Fungal Vaccines; D001086:Arabinofuranosyluracil; C043457:fialuridine; D013937:Thymidine Kinase