Student Theses and Dissertations

Date of Award

2004

Document Type

Thesis

RU Laboratory

Ho Laboratory

Keywords

DNA vaccines, HIV-1 vaccines, salmonella typhimurium

Abstract

The promise of D N A vaccination is limited in part by invasive, untargeted administration. We refined the strategy by incorporating attenuated bacteria to deliver plasmids directly to APCs. Initial studies involved human macrophages, which were infected with aroASalmonella typhimurium transformed with eukaryotic vectors encoding 8- galactosidase. Results of staining with x-gal demonstrated effective transfer of plasmids to the target cells, which expressed the reporter gene themselves. Similarly, immunocytochemical staining of macrophages infected with bacteria bearing a eukaryotic vector encoding HIV-1 env showed expression of the viral gene. Macrophages were also infected with bacteria harboring vectors encoding SIV env and HIV-1 tat, then subjected to lysis, RNA extraction and RT-PCR. In this way, conceptual proof of the system was achieved at the level of transcription. Indeed, the latter case, we recovered a splice product, which specifically confirms eukaryotic expression. Further studies involved human DCs, cultivated from PBMCs in vitro. We first used flow cytometry to show that infection with Salmonella induces maturation, a state ultimately necessary for immunogenicity. Once again, bacteria carrying Bgalactosidase vectors were used initially. DCs were also infected with bacteria carrying a vector encoding HIV-1 gag, then stained for expression of the viral protein. Both immunocytochemistry and flow cytometry confirmed Gag expression. Finally, transduced DCs were co-cultivated with autologous T-cells in studies aimed at detecting interferon-7 expression as a gauge of antigen presentation. Bacteria were first transformed with eukaryotic vectors encoding influenza Ml protein. PBMCs were therefore obtained from donors known to have memory responses to influenza. Co-cultures yielded significant T-cell responses (by ELISpot and flow cytometry) to DCs transduced to express Ml. We next obtained PBMCs from HIV-1-infected donors, setting up co-cultures involving DCs infected with bacteria carrying HIV-1 gag vector. Again, significant T-cell responses were detected when DCs were transduced to express the viral protein using Salmonella. Thus, using attenuated S. typhimurium, we can deliver vectors encoding foreign antigens directly to human APCs. DCs transduced to express viral proteins can present antigen to autologous T-cells in vitro. As a vaccine tool, this strategy represents a potential way to achieve non-invasive, targeted delivery of DNA vaccines.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/200

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