CROI 2016 Abstract eBook

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Poster Abstracts

Conclusions: Our data indicate that TSCM are cycling and support productive infection. Moreover, the TSCM pool is dynamic and expands in response to IL15. This cytokine activates several pathways favorable to infection (transcription and chromatin remodeling) and counteracts the antiviral activity of SAMHD1. A global characterization of the features of infected TSCM is a key factor for the development of new strategies to eliminating the reservoir. 245LB Single Cell Transcriptome Sequencing of Human Lymph Node HIV-Infected CD4 Cells Joseph P. Casazza 1 ; Eli Boritz 1 ; David Ambrozak 2 ; Amy R. Henry 1 ; SamW. Darko 2 ; Costantinos Petrovas 2 ; Rebecca M. Lynch 1 ; Gustavo ReyesTeran 3 ; Daniel C. Douek 1 ; Richard A. Koup 1 1 VRC, NIAID, NIH, Bethesda, MD, USA; 2 NIH, Bethesda, MD, USA; 3 Natl Inst of Respiratory Diseases, Mexico City, Mexico Background: Identification of live HIV-infected CD4 cells allows the characterization of individual cells both by flow cytometry and by transcriptomic analysis. Methods: Cervical lymph nodes were obtained from 3 viremic HIV-infected volunteers, median CD4 count 671(range 247-1,288) and median viral load 17,437(14,075-29,923). Approximately 90 individual CD45RO + PD1 hi CD4 dim cells were index sorted into individual wells for each patient. In addition, surface expression of HIV envelope on CD4 T cells was identified using fluorescently labelled PGT121. Whole transcriptome libraries were generated from the purified RNA of sorted single cells using established methods and deep sequenced on an Illumina HiSeq (Trombetta, JJ et al. (2014) Curr Protoc Mol Biol 4.222.1-422.17). Results: The median frequency of HIV-RNA containing PD1 hi CD4 dim cells was 8.0 (4.6-8.0)%. In cells from two of the three lymph nodes the frequency HIV RNA containing cells was higher in cells staining for PGT121, 15 and 18%, compared to 6 and 5%, respectively. There was no difference in HIV RNA containing cells within PGT121 + and PGT121 - populations in the lymph node from the third volunteer. The percentage of total reads containing HIV RNA varied between 0.02-6.0% in individual HIV+ cells. In two of the three volunteers the median frequency of HIV reads was higher in PGT121 + cells than in PGT121 - cells, 0.15 and 0.6%, compared to 0.04 and 0.03% PGT121 - cells. In the other sample there was essentially no difference. Conclusions: These data show that it is possible to identify HIV-infected CD4 T cells at a level which makes whole cell transcriptome analysis of HIV RNA+ and similar HIV RNA- cells possible. 246 BST-2, TRIM22, and RAD51 in Host Susceptibility to HIV-1 Infection and Virus Control Ravesh Singh 1 ;Vivek Naranbhai 2 ; Lise Jamieson 2 ; Nigel Garrett 2 ; Salim A. Karim 2 ;Thumbi P. Ndungu 3 1 HIV Pathogenesis Prog, Univ of Kwa-Zulu Natal, Durban, South Africa; 2 CAPRISA, Univ of KwaZulu-Natal, Durban, South Africa; 3 Univ of KwaZulu-Natal, Durban, South Africa Background: To establish infection and replicate efficiently, HIV-1 must overcome host antiviral restriction factors and utilize host replication cofactors. We hypothesized that expression levels of specific HIV-1 restriction factors or replication co-factors may modulate infection risk among HIV-1 exposed persons or potentiate viral control during early infection. Methods: We measured by quantitative real-time PCR levels of 25 previously validated HIV antiviral factors (IFN-α2b, IFNβ, MxA, TRIM5α, TRIM11, TRIM19, TRIM25, TRIM27, TRIM28, TRIM36, PAF1, CTR9, AP2M1, RTF1, DNM2, MKRN3, HERC5, C3orf63, SETDB1, RPRD2, COX18, RNF19A, BST-2, APOBEC3F and p21) and two replication cofactors (Rab7A and RAD51) in peripheral blood mononuclear cells (PBMCs) of high risk, HIV-1 uninfected participants, and in recently HIV-1 infected participants. Using two well-pedigreed cohorts, the CAPRISA 002 study, (Training cohort) and the antiretroviral-naïve placebo arm of the CAPRISA 004 study, (Validation cohort), we explored in vivo association with with risk of HIV-1 acquisition and biomarkers of disease progression. Generalized Estimating Equations (GEE) models were fitted for expression levels of all HIV-1 restriction factors and replication cofactors, plasma viral loads and CD4+ T cell counts. Differences between groups were evaluated by using Student’s t-test. Results: In both the training set cohort and the validation cohort (CAPRISA 004 study) non-seroconverters had significantly higher pre-infection (baseline) levels of the antiviral factor BST-2 than seroconverters (p < 0.003). Levels of TRIM22, another antiviral factor, correlated negatively with viral load (all p < 0.05) whereas levels of the replication cofactor RAD51 correlated positively with viral load in both cohorts (all p < 0.05). None of the other factors consistently associated with HIV-1 outcomes. Conclusions: These data suggest that high expression of BST-2, an antiviral factor that inhibits efficient HIV-1 release is associated with reduced susceptibility to HIV-1 infection, and that some HIV-1 restriction factors and replication factors may play a role that impacts the level of viral load during early HIV-1 infection. 247 6-Amino-Acid Insertion/Deletion Polymorphism in TIM1 Confers Protections Against HIV1 Irma Saulle 1 ; Michela Masetti 2 ; Mara Biasin 2 ; DariaTrabattoni 2 ; Manuela Sironi 3 ; Christian Brander 4 ; FrancescaVichi 5 ; Sergio Lo Caputo 5 ; Mario Clerici 2 ;Wbeimar Aguilar-Jiménez 6 1 Univ of Milan, Brescia, Italy; 2 Univ of Milan, Milan, Italy; 3 Scientific Inst for Recovery and Care E. Medea, Milan, Italy; 4 IrsiCaixa Inst for AIDS Rsr, Badalona, Spain; 5 S. Maria Annunziata Hosp, Florence, Italy; 6 Univ of Antioquia, Medellin, Colombia Background: TIM-1 (T-cell immunoglobulin and mucin domain 1), a cell surface glycoprotein, facilitates the entry of enveloped virus, such as HIV, into host cells. Because the length of the mucin domain of TIM-1 is a critical factor in modulating viral entry, we assessed whether the TIM-1 18-bp insertion/deletion polymorphismmodulates susceptibility to HIV-1 infection in three independent cohorts of HIV-exposed seronegative (HESN) individuals. Methods: The Tim-1 18-bp insertion/deletion polymorphismwas genotyped in three case/control cohorts of HIV sexually-exposed HESN and their HIV-1-infected partners with different geographic origin (Italy, Peru and Colombia); data from an additional cohort were retrieved from a previous study conducted in Thailand. CD4+ T lymphocytes purified from 34 healthy controls (HC) grouped according to their TIM-1 genotype were infected in vitro with HIV-1 Ba-L and viral replication was assessed after 5 days by measuring viral p24 levels produced by the infected cells. Results: In all comparisons, homozygosity for the short TIM-1 allele was more common in HESN than in HIV-1 infected subjects. A meta-analysis of the four association analyses, revealing no heterogeneity among samples, yielded a p value of 0.005. These results were sustained by a drastic and significant reduction of viral replication in CD4+ T lymphocytes isolated from HC that were homozygous for the short TIM-1 allele compared to subjects carrying at least one long allele (t-test, p= 0.042). Conclusions: The TIM-1 deletion allele protects from infection with a recessive effect. In vitro infection assays on CD4+ T lymphocytes support this conclusion and underscore a complex interaction between enveloped viruses and TIMmolecules that need further investigation. 248 MicroRNA Profile in CD8 T Cells FromHIV-Infected Individuals and Disease Progression Montserrat Plana 1 ; Lander Egaña-Gorroño 1 ; Alberto C. Guardo 1 ; Manel Enric Bargalló 1 ; ElisendaVilaplana 2 ;Tuixent Escribà 3 ; Iñaki Perez 1 ; Josep María Gatell 4 ; Felipe García 4 ; Mireia Arnedo 1 ; for the HIV Controllers Consortium of the AIDS Spanish Network 1 IDIBAPS, Barcelona, Spain; 2 Univ de Vic, Barcelona, Spain; 3 Fundació Clínic, Barcelona, Spain; 4 Hosp Clínic de Barcelona, Barcelona, Spain Background: The relationship between host microRNAs (miRNA), viral control and immune response has not been elucidated in the field of HIV yet. The aim of this study was to assess the differential miRNA expression profile in CD8+ T-cells between HIV-infected individuals who differ in terms of disease progression. Methods: miRNA profile from resting and CD3/CD28-stimulated CD8+ T-cells from uninfected individuals (HIV-, n=11), Elite Controllers (EC, n=15), Viremic Controllers (VC, n=15), Viremic Progressors (VP, n=13) and HIV-infected patients under antiretroviral treatment (ART, n=14) was assessed using Affymetrix miRNA 3.1 strip arrays. After background correction, quantile normalization and median polish summarization, normalized data were fit to a linear model. Multiple test was corrected using 5% false discovery rate (FDR). The analysis comprised: resting samples between groups; stimulated samples between groups; and stimulated versus resting samples within each group. Enrichment analyses of the putative target genes were perfomed using bioinformatical algorithms (TargetScan, miRanda, miRWalk). Results: A downregulated miRNA pattern was observed when resting samples from all infected groups were compared to HIV- (hsa-miR-4734 in VP vs HIV-, hsa-miR-4505 in EC vs HIV- and hsa-miR-4492 and hsa-miR-4508 in ART vs HIV-). A miRNA downregulation was also observed when stimulated samples from EC, ART and HIV- groups were compared to the VP, being hsa-miR-4492 the most downregulated. This miRNA targets 2346 predicted genes including the Linker for Activation of T-Cells (LAT), a key protein in

Poster Abstracts

96

CROI 2016