Session 51Poster Presentations Novel Vaccine Approaches Session Day and Time: Thursday 1:30 - 3:30 pm Room: Hall D
428 Transduction of Human Dendritic Cells Using Attenuated Salmonella typhimurium as a Vaccine Strategy against HIV-1 D. Gurner*, Y. Huang, D. D. Ho Aaron Diamond AIDS Res Ctr, Rockefeller Univ, New York, NY
Background: The promise of genetic immunization is limited in part because of its invasive, untargeted administration. Here we describe successful refinement of the strategy by incorporating attenuated bacteria that deliver DNA plasmids directly to antigen presenting cells. Specifically, we show that human dendritic cells (DCs) can be effectively transduced using Salmonella typhimurium.
Methods: Attenuated (aro A-) S. typhimurium were transformed with vectors expressing the Beta-galactosidase gene under the control of either a prokaryotic (pTrc) or eukaryotic (pCMV) promoter. Additional bacteria were either left untransformed or transformed with irrelevant plasmids. Immature DCs were cultivated from PBMCs, then infected with the bacteria. DCs were ultimately analyzed by staining and FACS.
Results: Incubation with x-gal revealed no staining in the control DCs, and minimal (< 1%), punctate staining in those infected with bacteria bearing the pTrc vector. In contrast, DCs infected with bacteria bearing the pCMV expression vector showed widespread (30%-50%) staining, with a distinct, diffusely intracellular morphology. FACS analysis using FDG, a fluorescent Beta-galactosidase substrate, revealed an increase in expression from a background of 11%-43% (with a MOI of 25), or 7%-66% (with a MOI of 50). FACS also confirmed that exposure to the bacteria efficiently matures the DCs.
Conclusions: Using transformed attenuated bacteria, we can directly transduce human DCs with vectors encoding foreign antigens. As a vaccine tool, this strategy represents a way to achieve noninvasive, targeted delivery of DNA vaccines. Ongoing and future work involves evaluation of antigen presentation as a gauge of immunogenic potential using bacteria bearing viral gene vectors.
