Proteomic Approach for the Identification of PAI-induced PBMC and CD8+ T Cells anti-HIV Mechanism 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. We have previously observed that a polyantigenic immunomodulator, known as PAI, which consists of a mixture of inactivated influenza and bacterial vaccine, induces MHC non restricted non-cytolytic anti-HIV-1 activity. Based on our preliminary data documenting viral suppression, we propose to test the hypothesis that PAI induced antiviral activity can be differentially determined by a proteomic approach. To address this hypothesis, our specific aims are:
Specific Aim #1 : To determine differential protein expression in intracellular lysates from target T cells treated with supernatant from effector cells (PBMC, CD8+and CD8+ depleted T cells) by performing two dimensional gel electrophoresis (2D-GE) and image analysis.
Specific Aim #2 : To identify and compare differentially expressed proteins from target T cells treated with PAI by performing peptide mass finger printing using mass spectrometry (MS), stable-isotope labeling by amino acids in cell culture (SILAC) analysis and database search.
Specific Aim #3 : To validate by Western blots and/or qRT-PCR the identity of differentially expressed proteins identified by mass spectrometry. Preliminary findings identified PBMC proteins responsive to PAI treatment. Master maps were compared to assess differences in protein expression. This revealed 47 differentially expressed spots in PAI treated PBMC. The altered proteins were analyzed by tandem MS (MS/MS) for protein identification. After querying the MS/MS data against the NCBInr protein database, we have identified 11 differentially expressed protein spots. We believe that the identified proteins will generate a proteomic signature of the PAI-anti HIV-1 response. We will make the proteome reference map of PAI treatment in different cell type with their respective related HIV mechanisms available for others to use for comparative studies.
To explore the non-cytolytic immune response as an alternative treatment of HAART it is crucial to identify new anti- HIV-1 cellular mechanism. The identification of these anti-HIV-1 activity produced by PBMC and CD8+ T ceils could have a major therapeutic potential to prevent or treat HIV-1 infections and/or help in elucidating HIV pathogenic mechanism.
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