CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

uninfected controls were analyzed by flow cytometry for their expression of immunological markers, cytokines and the HIV capsid protein, p24. Supernatant p24 was also measured by ELISA. Additionally, we used RNA microarrays and confirmatory immunoblots to compare gene expression patterns of cells from different polarizing conditions. Results: T h 17-polarizing cytokines (IL-1b, IL-6, IL-23 and TGF-b) markedly increased HIV infection, relative to IL-2 treated, donor-matched controls (mean %p24 + increased 2.75- fold, t-test p<0.001, n =10). Supernatant p24 from CCR6-sorted, HIV BAL infected cells was highest among donor-matched, T h 17-enriched populations (p<0.001, n =5, left figure panel). HIV infection was significantly higher among T h 17 cells compared with IL-17 - or IFNγ + cells, even upon infecting with HIV AMLV , a replication-defective HIV vector with a pseudotype envelope (%IL-17 + cells were overrepresented among p24+ cells by 2.9-fold, p<0.001, n =6). Further, HIV AMLV -infected T h 17 cells produced more viral capsid protein per cell. In CCR6 + -sorted cells, we observed a 3.0-fold increase in p24 geometric mean fluorescence intensity among IL-17 + cells, compared with IL-17 - cells (p<0.01, right figure panel). Our data also reveal that T h 17-polarized cells have diminished expression of Ribonuclease A superfamily proteins. Conclusions: Here we show that T h 17 cells exhibit heightened permissiveness to productive HIV infection. Our findings link T h 17 polarization to increased HIV replication, suggesting that T h 17 cells might be major sources of viral production during acute HIV infection.

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Spread of HIV-1 Is Delayed in T-Cells in the Absence of Integrin LFA-1 Anika Hain; Rene M. Linka; Arndt Borkhardt; Dieter Häussinger; Carsten Münk Heinrich-Heine-Univ Düsseldorf, Düsseldorf, Germany

Background: LFA-1 (Lymphocyte function-associated antigen 1) is found on all T-cells and is involved in recruitment to the site of infection. It binds to ICAM-1 on antigen- presenting cells and functions as an adhesion molecule. LFA-1/ICAM-1 interaction has been shown to be important for T cell-T cell interactions, leading to T cell proliferation and differentiation. LFA-1 is formed by the integrin alpha L chain ITGAL and beta 2 chain ITGB2. We wanted to study whether LFA-1 is important for HIV-1 replication in T-cells. Methods: Jurkat T-cells were engineered using the CRISPR/CAS9 technology targeting ITGAL to generate LFA-1 knock-out cells. This LFA-1 KO cells showed no ITGAL expression but normal levels of ICAM-1 on the cell surface. Single-round HIV-1 reporter vectors and multiple round (virus spreading) experiments using HIV-1 NL4-3 were applied to compare wild type with LFA-1 KO cells. Results: HEK293T express only very low levels of ICAM-1. Ectopic expression of ICAM-1 in HEK293T cells caused enhanced ICAM-1 incorporation in HIV-1 particles. HIV-1 luciferase reporter viruses - normalized for reverse transcriptase activity- gained infectivity (three to five-fold) by overexpression of ICAM-1 compared to particles made without ICAM-1 co- expression. Wild type and LFA-1 KO cells revealed no differential permissivity to HIV-1 reporter viruses, independent whether authentic Envelope or heterologous VSV glycoprotein was used for pseudotyping. This finding correlated with equal particle binding to wild-type and KO cells. However, replication competent HIV-1 showed a three-to-four day delayed replication kinetic in the LFA-1 KO cells in comparison to regular Jurkat cells. Conclusions: The cellular factors that modulate HIV-1 replication in T-cells are only partially described. The ICAM-1 incorporation into HIV-1 affects the viral infectivity. Our data implicate that in T-cells the spreading replication is enhanced by LFA-1 expression. This data in addition show that LFA-1 is not an essential dependency factor for HIV-1, but might be relevant to describe disease variability in patients. 190 Background: Nef is multifunctional HIV-1 accessory protein that is critical for pathogenesis and infectivity. Nef inhibits T cell receptor (TCR) signaling in infected CD4 T cells, presumably to reduce activation-induced cell death and increase progeny virion production, which may contribute to disease outcome. Elite controllers (EC) are a rare group of individuals who spontaneously maintain undetectable viral load in the absence of treatment. We previously showed that Nef clones derived from EC were impaired for numerous functions compared to clones from chronic progressors (CP). Here, we examined the ability of EC Nef clones to inhibit TCR signaling. Methods: 45 EC and 46 CP Nef isolates were cloned into an expression plasmid. Jurkat cells were co-transfected with a plasmid expressing Nef and a NFAT driven-luciferase reporter plasmid. After 18 hours, cells were stimulated with anti-CD3 antibody to activate calcineurin via a calcium flux and luminescence was measured at 6 hours. The ability of Nef clones to inhibit TCR signaling was normalized to WT Nef (SF2 strain), such that function greater or less than WT is represented as >100% or <100%, respectively. Chimeric Nef constructs between 3 notably defective EC-derived isolates and SF2 Nef, and Nef point mutations, were generated by overlap extension PCR. Results: Nef clones derived from EC displayed significantly lower ability to inhibit TCR signaling (median 87 [interquartile range (IQR) 75-93] %) compared to CP-derived clones (median 95 [IQR 89-98] %) (p<0.001). Codon-function analysis revealed a significant association between Nef R21K and reduced function (median 75% versus 92%) (p<0.001). Point mutations in SF2 Nef confirmed this result, with R21K, R21L and R21T displaying function 15%, 15% and 11% lower than WT SF2, respectively. Chimeric constructs mapped the contributing mutations of 3 notably defective EC clones to the first 32 amino acids of Nef. Rare polymorphisms were observed in these EC clones at highly conserved residues, R21L and P25T. A single amino acid reversion (T25P) in one EC clone restored function from 27% to 70%. Conclusions: EC Nef clones displayed an attenuated ability to inhibit TCR signaling. These results highlight the potential impact of natural sequence variation on this function, especially within the N-terminal arm of Nef. Differences in Nef’s ability to modulate T cell activation status could contribute to improved clinical outcome in some EC. Inhibition of TCR Signaling Is Impaired in Nef Clones Derived From Elite Controllers StevenW. Jin 1 ;Tristan Markle 1 ; Asa Rahimi 1 ; Bruce D.Walker 2 ; Zabrina Brumme 1 ; Mark A. Brockman 1 1 Simon Fraser Univ, Burnaby, BC, Canada; 2 Ragon Inst of MGH, MIT, and Harvard, Cambridge, MA, USA

Poster Abstracts

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CROI 2016