eprintid: 10023 rev_number: 6 userid: 6 dir: disk0/00/01/00/23 datestamp: 2011-11-10 20:08:00 lastmod: 2016-12-19 14:37:58 status_changed: 2011-11-10 20:08:00 type: thesis_degree metadata_visibility: show contact_email: nmm23@pitt.edu item_issues_count: 0 eprint_status: archive creators_name: Melhem, Nada M. creators_email: nmm23@pitt.edu creators_id: NMM23 title: DENDRITIC CELLS TRANSFECTED WITH AUTOLOGOUS SIV RNA: POTENTIAL AIDS VACCINE ispublished: unpub divisions: sch_gsph_infectiousdiseasesmicrobiology full_text_status: public keywords: dominant and subdominant epitopes; genetic variability; Therapeutic vaccine abstract: The need for a therapeutic human immunodeficiency virus (HIV) vaccine is urgent for the control of the acquired immunodeficiency syndrome (AIDS) epidemic. The variability of the virus as well as its ability to undergo escape mutations in T cell epitopes are important obstacles facing the development of an AIDS vaccine. Consequently, a powerful strategy would be the induction of robust antigen specific T cell responses targeting patient-specific virus sequences expressed during the course of infection. An attractive vaccine approach to achieve this is the use of dendritic cells (DC) transfected with in vitro transcribed mRNA encoding autologous virus sequences. The rhesus macaque model provides an ideal preclinical setting to test the therapeutic potential of DC-based vaccination. We hypothesize that optimal antigen presentation and stimulation of potent T cell responses could be achieved by loading DC from SIV-infected macaques with mRNA encoding virus-derived sequences isolated during the course of infection. This represents a powerful strategy for the generation of a potential therapeutic AIDS vaccine. In support of our hypothesis, we generated the following evidence: (1) nucleofection is a superior method for efficient transfection of human and monkey monocyte-derived DC with DNA and mRNA to conventional electroporation and lipofection; (2) nucleofection of DC with mRNA led to better protein expression and DC maturation as compared to DNA transfection; (3) mRNA nucleofection of DC resulted in rapid and sustained gene expression, a critical factor in DC-based immunotherapy for durable antigen presentation; (4) nucleofection of monkey monocyte-derived DC with wild-type non codon-optimized gag mRNA was efficiently expressed and induced strong antigen-specific T cell responses whereas DNA transfection led to non-specific T cell stimulation; (5) enhanced CD4+ T cell responses were observed when Gag was redirected to the lysosomal pathway via the targeting signal of the lysosome-associated membrane protein (LAMP-1) following nucelofection of DC with mRNA; (6) rhesus DC transfected with lysosome-targeted gag mRNA encoding an escape mutation in an immunodominant CTL epitope stimulated CD4+ and CD8+ T cell responses of almost equivalent magnitude directed towards undefined epitopes outside of the mutated region; (7) gag or env mRNA transfected-DC from SIV-infected macaques stimulated significant antigen-specific T cell responses in an entirely autologous system; (8) DC cotransfected with gag mRNA as well as mRNA encoding CD70 or OX40L did not result in enhanced immunostimulatory functions. HIV/AIDS is a significant public health problem demanding action. This work demonstrates that mRNA-transfected DC expressing SIV antigen from infected monkeys stimulate broad and relevant T cell responses, thus supporting this approach for the generation of a therapeutic HIV vaccine to decrease the burden associated with the infection. date: 2008-01-31 date_type: completed institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Barratt-Boyes, Simon M etdcommittee_name: Gambotto, Andrea etdcommittee_name: Rinaldo, Charles etdcommittee_name: Murphey-Corb, Michael etdcommittee_name: Kalinski, Pawel etdcommittee_email: smbb@pitt.edu etdcommittee_email: gambottoa@upmc.edu etdcommittee_email: rinaldo@pitt.edu etdcommittee_email: mcorb@pitt.edu etdcommittee_email: kalinskip@upmc.edu etdcommittee_id: SMBB etdcommittee_id: AGAMB etdcommittee_id: RINALDO etdcommittee_id: MCORB etdcommittee_id: PAK5 etd_defense_date: 2007-09-20 etd_approval_date: 2008-01-31 etd_submission_date: 2007-12-04 etd_access_restriction: immediate etd_patent_pending: FALSE assigned_doi: doi:10.5195/pitt.etd.2011.10023 thesis_type: dissertation degree: PhD committee: Simon M. Barratt-Boyes (smbb@pitt.edu) - Committee Chair committee: Andrea Gambotto (gambottoa@upmc.edu) - Committee Member committee: Charles Rinaldo Jr. (rinaldo@pitt.edu) - Committee Member committee: Michael Murphey-Corb (mcorb@pitt.edu) - Committee Member committee: Pawel Kalinski (kalinskip@upmc.edu) - Committee Member etdurn: etd-12042007-204943 other_id: http://etd.library.pitt.edu/ETD/available/etd-12042007-204943/ other_id: etd-12042007-204943 citation: Melhem, Nada M. (2008) DENDRITIC CELLS TRANSFECTED WITH AUTOLOGOUS SIV RNA: POTENTIAL AIDS VACCINE. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/10023/1/MELHEM_121207.pdf