T-cell Responses to Concurrent HIV and Herpesvirus Infections

using separated PBMCs, HIV-1 specific T-cells will be labeled with major histocompatibility complex (MHC) I tetramers previously described to be specific for HIV-1 [14-18]. Labeled cells will be subjected to flow cytometry for identity confirmation via established antibody cell marker staining, counting and sorting into single cells. The TCR genes of the individually sorted HIV-1 tetramer positive T-cells will then be sequenced, establishing a repertoire of TCR sequences. Diversities of the TCR gene repertoires obtained from each study group will be assessed using the established algorithm for Simpson's diversity index [9].

process as described above, with use of newly identified and previously published herpesvirus specific MHC I tetramers, including those for HSV-1, HSV-2, EBV and CMV, [19-25]. Comparisons of TCR repertoires will be made between newly acquired and latent herpesvirus infections. Diversities of the TCR gene repertoires obtained from each study group will be assessed using the established algorithm for Simpson's diversity index [9].

Intracellular and cell surface proteins will be assessed to determine if T-cells that have entered the exhaustion pathway. These markers include, but are not limited to, PD-1, Tim3, Tcf7, Tbet, and Tox. Marker expression will be measured at the transcript or protein level [1, 2]; T-cells will be concomitantly labeled/detected with T-cell specific markers and HIV-1 MHC I tetramers to confirm identity. Using this information, a binary value of "exhausted' or "not exhausted" will be assigned.

presence (or lack of) of HIV infection will be determined by HIV antigen/antibody testing. Control will be assessed by CD4+ T-cell counts, HIV viral titers, infections acquired likely due to immunodeficiency. The main outcome measure will be viral load. HIV related laboratory results including HIV screen, viral titers, T-cell counts will all be disclosed to patients as per standard of care.

at first visit, all patients will have IgM and IgG levels in the clinical laboratory for each named virus to establish presence of chronic vs. newly acquired herpesvirus infections. At each visit all patients will have serological testing via HSV-1, HSV-2, CMV, EBV and specific IgG direct antibody detection with quantitative enzymatic immune assay (EIA). Patients with active mucosal (oral or genital) or skin lesions consistent with primary or secondary HSV infections will be swabbed and sent for PCR and/or direct fluorescence antibody assay (DFA) in the clinical laboratory. Control will be assessed by quantitative EIA results and frequency of herpesvirus specific symptoms of flare (acute or reactivated infections).

peripheral blood mononuclear cells (PBMCs) will be separated from patient's whole blood specimens. DNA will be extracted and specific HLA loci will be amplified using PCR, and yielded products will be sequenced. This will guide appropriate use of TCR tetramers.