This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It is now accepted that restoration of the immune function using only highly active antiretroviral therapy (HAART) is incomplete. Because of the emergence of drug-resistant strains of HIV-1, therapeutic immunization strategies are needed to reinforce HAART in the treatment of HIV disease. It has been reported that Toll like receptor agonist 7/8 (R-848) induces anti-HIV-1 activity. Toll like receptors (TLR) are evolutionary conserved pathogen receptors that play a key role in innate and adaptive immune system. The TLR have the ability to induce antiviral factors producing cytokines and B-chemokines and triggering IFN (IFN-alpha and Beta) production. We propose to test the hypothesis that TLR agonist 7/8 induced antiviral activity can be differentially determined by a proteomic approach.
Specific Aims 1. To determine the differential anti-HIV1 activity in culture fluids from PBMC, CD8+ T cells and CD8+ T cells depleted PBMC treated with TLR agonist R-848 2. To identify and compare differentially expressed proteins in culture fluids with and without antiviral activity using proteomics 3. To validate the antiviral activity of the identified factors stimulated by TLR agonist R-848 using recombinant protein expression technique We are currently performing dose-response assays to determine the optimal TLR agonist concentration. After 9 days of TLR agonist exposure, culture fluids were collected for Quantitative Real Time PCR and proteomics analysis. Preliminary results reveal that 5 ug/mL is the optimal TLR agonist concentration. Two Dimensional Gel Electrophoresis (2D-GE) experiments from culture fluids at 5 ug/mL and 1 ug/mL were performed in triplicates and differentially expressed proteins were evaluated as compared to the control. We believe that the identified proteins will generate a proteomic signature of the TLR agonist-anti HIV-1 response.
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