CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

365 Rapid Construction of HIV-1-Specific T Cell Receptor Gene Therapy Lentiviral Vectors Christian Hofmann ; Christian-Raul Aguilera-Sandoval; Arumugam Balamurugan; Priya Patel; Brian Diep; Edward Martin; Sangeun Park; DianaY. Chen; Hwee Ng; Otto O.Yang University of California Los Angeles, Los Angeles, CA, US Background: HIV-1-specific CD8 + cytotoxic T lymphocytes (CTL) can play an important role in the long-term control of HIV-1 replication in vivo . Unfortunately, most HIV-1-infected persons are unable to control the virus with their T cell receptor (TCR) repertoire, due to inadequate coverage of epitope variation. A potential immunotherapeutic strategy for a functional cure could be the adoptive transfer of gene-modified T cells, equipped with a combination of TCRs covering all common variants of their targeted epitope. We developed a rapid methodology to identify epitope-specific TCR sequences and insert them into lentiviral vectors without need for HLA tetramers, cell sorting, or cell cloning. Methods: We focused on five HIV-1 Gag epitopes based on common or protective HLA type and epitope conservation: KRWIILGLNK 263-272 (KK10, HLA-B*2705), KAFSPEVIPMF 162-172 (KF11, B*5701), GLNKIVRMY 269-277 (GY9, B*1501), RQANFLGKI 429-437 (RI9, B*1302), and WASRELERF 36-44 (WF9, B*3501). Using a quantitative spectratyping method, we directly identified Gag-specific TCR sequences by expansion after epitope stimulation. Both TCR α and β chains were cloned in a one step reaction into a lentiviral vector. The functionality of these TCRs was examined through a Jurkat cell NFAT-dependent GFP reporter assay and also chromium release assays with transduced primary CD8 + T cells against HIV-1- infected T1 cells transduced with the relevant HLA genes. Panels of HIV-1 NL4-3 with common epitope variants were generated by point mutagenesis. Results: We cloned and functionally confirmed sixteen TCRs (seven for KK10, four for KF11, three for GY9, one for RI9, and one for WF9). Several TCRs have been screened for their recognition of epitope variants and functional avidity using synthetic peptides, and will be screened for their antiviral activity against cells infected with HIV-1 containing these variants. Conclusions: Our data demonstrate proof-of-concept for rapid TCR cloning into vectors suitable for gene therapy and capacity to screen these TCRs for ability to recognize epitope variants. This sets the foundation for combination TCR gene therapy to cover epitope variation. 366 The Role of Exosomes in Semen in Suppressing Natural and Vaccine-Induced Immunity Lucia Vojtech ; Sean Hughes; Claire Levy; Florian Hladik University of Washington, Seattle, WA, US Background: Exposure to semen is the primary route of transmission for many sexually transmitted infections, including HIV. Accumulating evidence suggests that components in semen directly impair leukocytes, which could compromise the protective efficacy of vaccine-induced immune responses in the mucosa. Seminal plasma contains large numbers of extracellular microvesicles or exosomes (SE). Exosomes in general have been identified as important mediators of intercellular communication and immunoregulation. Thus, we explored the effects of SE on recipient blood and mucosal immune responses. Methods: SE isolated from semen donated by healthy men were pooled frommultiple donors. Dendritic cells (DCs) were derived from peripheral blood mononuclear cell (PBMC) monocytes. DCs and PBMC cultures were exposed to SE and assayed for SE uptake, immune function, and cytokine production by confocal microscopy, flow cytometry, Luminex, and quantitative PCR. Results: Extracellular vesicles from semen are present at an average concentration of 2.2 x 10 13 particles per ejaculate (n = 18 donors). These SE rapidly and efficiently entered peripheral and vaginal dendritic cells (DCs), more slowly entered B and CD4+ T cells, and only poorly entered CD8+ T cells. In PBMC cultures, SE impaired memory T cell function, reducing the production of TNF-alpha and/or IFN gamma in response to CMV, EBV, or influenza-derived peptides an average of 73% for CD4+ T cells and 55% for CD8+ T cells, in a dose-responsive manner (n = 4 donors). SE also impaired vaginal T cell responses to a superantigen. Exposing only DCs, as opposed to bulk PBMCs, to SE also blocked subsequent CD8+memory T cell activation, reducing the proportion of cells making cytokines by 51% (n = 3 donors). This effect occurred even when only a fraction of DCs (as low as 20%) were exposed to SE. SE also impaired the innate immune response of monocytic THP-1 cells stimulated by bacterial lipopolysaccharide: cytokine production was reduced at both the mRNA level and the protein level. Conclusions: SE inhibit a broad range of both innate and adaptive immune responses. CD4+ T cells appear to be directly affected, while CD8+ T cell function is impaired by affecting the co-stimulatory capacity of antigen-presenting cells. Understanding how programmed immune responses are altered by the presence of semen is important to developing the next generation of vaccines and preventative treatments against sexually transmitted diseases. 367 IFN-alpha Stimulated NK Lysis of HIV-Infected CD4 + T Cells Requires NKp46 and NKG2D Costin Tomescu 1 ; Domenico Mavilio 2 ; Luis J. Montaner 1 1 The Wistar Institute, Philadelphia, PA, US; 2 Humanitas Clinical and Research Center, Milan, Italy Background: Interferon-alpha (IFN-alpha) is a potent clinical immuno-modulatory agent with the capacity to limit viral replication and stimulate NK activity against virally infected target cells. We have previously shown that NK lysis of HIV-1 infected autologous CD4 + primary T cells requires exogenous IFN-alpha stimulation. Here, we identify the specific activating receptor(s) involved in HIV-1 infected target cell lysis by autologous NK cells activated endogenously with IFN-alpha. Methods: Purified CD4 + primary T cells were HIV-1 infected and used to stimulate IFN-alpha secretion from autologous PBMC. NK cell-mediated cytotoxicity was measured with a 4 hour chromium release assay in the presence or absence of neutralizing monoclonal antibodies against 2B4, NTB-A, NKG2D, NKp30, NKp44, and NKp46. Comparisons of three or more paired groups was carried out using a Friedman test adjusted with post-hoc analyses of two groups by a Wilcoxon signed-rank test. Results: Direct recognition of HIV-1 infected target cells by autologous PBMC led to the secretion of significant amounts ( p<0.001 ) of IFN-alpha by Plasmacytoid Dendritic Cells. Endogenous IFN-alpha stimulation led to strong NK activation as detected by CD69 upregulation and triggered NK lysis of HIV-1 infected autologous CD4 + primary T cells ( p<0.01 ). IFN-stimulated NK cell lysis of HIV-1 infected CD4 + primary T cells was significantly decreased in the presence of neutralizing antibodies against NKp46 ( p<0.01 ) and NKG2D ( p<0.05 ) while NKp30, NKp44, 2B4 and NTB-A were not required. Conclusions: The NKp46 and NKG2D activating NK cell receptors are required for NK lysis of HIV-1 infected autologous CD4 + primary T cells following IFN-alpha stimulation. The anti-viral mechanisms of action of IFN-alpha against HIV-1 may in part be due to NK-mediated clearance of HIV-1 infected target cells via the NKp46 and NKG2D receptors.

Poster Abstracts

275

CROI 2015