by Melhem, Nada M.

Abstract (Summary)
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.
Bibliographical Information:

Advisor:Michael Murphey-Corb; Charles Rinaldo Jr.; Pawel Kalinski; Andrea Gambotto; Simon M. Barratt-Boyes

School:University of Pittsburgh

School Location:USA - Pennsylvania

Source Type:Master's Thesis

Keywords:infectious diseases and microbiology


Date of Publication:01/31/2008

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