The Virology Core of this program project will conduct the virologic and serologic assessments outlined in Projects I-III. The Core Virology Laboratory will: 1) preserve plasma, PBMC, lymph node tissue as specified in the protocol for the immunologic, molecular, molecular, and virologic studies defined in the protocol, including shipment of samples to appropriate collaborators, 2) perform the virological and serological assessments outlined in the protocol, 3) assess the extent to which the therapeutic strategies outlined in Projects 1 and 2 affect the virological course of recent and chronic HIV infection, 4) provide the samples of plasma, PBMC, lymphoid tissue to the Mullins' laboratory for the genetic studies outlined in Project III, 5) perform the assays to quantify neomycin labeled T cells in PBMC during the course of therapy, 6) develop and perform assays to quantify adoptively infused T cells in PBMC or other tissues that are gene protected or have unique TCR regions, 7) use in situ PCR methods to determine the location of adoptively infused T cells in lymphoid organs and to define the relationship of these cells to HIV infected cells in lymph node tissue, and 8) measure whether adoptively transferred CD4+ T cells affect antibody responses to HIV and other unrelated organisms (e.g. HSV/CMV, enteroviruses). Much of the background and work of the core have been provided in the narratives to Projects I- III. The Core will be directed by Dr. L. Corey and the studies will be centered within the combined medical virology division of the University of Washington and Fred Hutchinson Cancer Research Center. Extensive interactions including shared laboratory meetings, and laboratory space are currently in place between the Core Virology Laboratory and the laboratories of Drs. Greenberg, Riddell and Mullins. These facilitate the extensive interactions required for the detailed virologic studies on the patients enrolled in the trials. A wide variety of laboratory techniques will be utilized to meet the specific aims of the proposal. These include assays to detect low copy numbers of HIV-1 RNA, in situ hybridization to detect HIV-1 RNA in tissue samples and novel in situ PCR techniques combined with flow cytometry or in situ hybridization to quantitate and localize adoptive infused T cells in PBMC, lymph node, and other tissue sites. Proposed studies include the development of high throughput real time quantitative PCR based assays to measure HIV RNA and to quantify gene protected CD4+ T cells containing the polyTAR/polyTAR-RRE and anti-integrase genes. A listing of the assays to be performed in the Core is given in Table 1 of the narrative.
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