CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

investigated whether this molecular signature existed in primary latently-infected resting central memory CD4 + T cells and whether this could be used to selectively target and kill latent HIV harboring cells. Methods: CCL19-treated naïve CD4 + T cells isolated from HIV-uninfected donors were infected with HIV then expanded in the presence of IL2 for 12 d. Memory CD4 + T cells were then isolated and cultured in the presence of IL7 for a further 20 d then analyzed by flow cytometry. HIV integration was analyzed by Alu-LTR qPCR. Expression of XIAP was assessed using Western blotting. HIV p24 antigen was quantified by ELISA. Long-lived, resting memory CD4 + T cells were then treated with the XIAP antagonist GDC-0152, the SMAC mimetic antagonist birinapant or the inhibitor of XIAP embelin. Apoptosis was assessed using annexin-V combined with propidium iodide staining as well as by Western blotting for PARP and caspase 3 cleavage. Data were analyzed using the Student’s t test. Results: After the 32 d infection, CD4 + T cells displayed a resting central memory CD4 + T cell phenotype (CD45R0 + CD62L + CCR7 + CD25 - Ki-67 - ). Alu-LTR qPCR and p24 ELISA demonstrated that these cells contained the equivalent of 1 copy of integrated HIV DNA in the absence of viral release into the culture supernatant. Moreover, XIAP expression was significantly increased compared with uninfected cells ( P < 0.05). Targeting XIAP with birinapant, GDC-0152, and embelin resulted in a significant dose-dependent increase in the number of latently infected resting central memory CD4 + T cells undergoing apoptosis which was not observed in mock-infected cells. Moreover, we did not observe an increase in p24 antigen expression. Conclusions: We have identified XIAP as a molecular signature of latently infected primary long-lived, resting central memory CD4 + T cells. Moreover, these cells are more sensitive to XIAP-agonist-induced apoptosis than uninfected cells. Therefore, by targeting XIAP with selective inhibitors and antagonists we have developed a novel approach that Background: CD4 + T-cells from HIV-infected individuals on antiretroviral therapy (ART) expressing the Immune Checkpoint (IC) marker of T-cell activation PD1, are preferentially infected [Chomont Nat Med 2009]. Characterizing the role of PD1 and other IC in HIV persistence during ART may identify new potential targets for eliminating latently infected T cells. Using an in vitro model of latency, we aimed to define the mechanism of how PD1 and other IC contribute to the establishment and maintenance of latent infection of both proliferating and non-proliferating T-cells. Methods: Resting CD4 + T-cells, isolated from blood of HIV-negative individuals, labelled with the proliferation dye eFluor670, were cultured alone, with autologous blood myeloid dendritic cells (mDC) or monocytes for 24h with staphyloccocal enterotoxin B (SEB). Cells were infected with CCR5-tropic eGFP-reporter virus. Expression of IC ligands including PDL1/PDL2 (ligands for PD1), CD80/CD86 (ligand for CTLA4), Galectin 9 (possible ligand for Tim3) and herpes virus entry mediator; HVEM (ligand for B and T cell attenuator (BTLA)) on mDC and monocytes were measured at baseline and 24h post infection. Non-productively-infected, non-proliferating (eGFP - eFluor670 hi ) and proliferating (eGFP - eFluor670 lo ) T-cells were sorted by flow cytometry day 5 post infection and further sorted on the basis of IC expression, including PD1, Tim3, CTLA4, BTLA, TIGIT or LAG3. Inducible latent infection was quantified by measuring eGFP expression in sorted subsets after α CD3/CD28+IL-7+IL-2 activation and integrase inhibitor L8. Results: Ligands for all IC were expressed, albeit at differing levels, on mDC and monocytes 24h post T-cell co-culture and HIV infection. Post integration latency in non- proliferating CD4 + T-cells was significantly enriched in cells positive for PD1 (mean fold change in eGFP expression compared to PD1 negative (MFC) =39, p=0.02, n=5), Tim3 (MFC=3.4, p=0.04, n=6), CTLA-4 (MFC=4.4, p=0.01, n=4) or BTLA (MFC=4.2, p=0.004, n=6) but not TIGIT (MFC=2.9, p=0.24, n=5) or LAG3 (MFC=2.9, p=0.67, n=3). Post integration latency in proliferating T-cells was significantly enriched in cells expressing PD1 (MFC=2.8, p=0.04, n=5) but not other IC. Conclusions: This in vitro model of HIV latency shows PD1 to be preferentially expressed on both non-proliferating and proliferating latently infected T-cells. Interventions that alter expression or function of PD1 should be explored to eliminate latency. 389LB 2B4+PD1+ Naïve and Memory CD4+ T Cells Are AssociatedWith Residual Viremia on ART Cynitha Klamar; Feiyu Hong; John Bui; Anthony R. Cillo; Arcadio Agudelo-Hernandez; Deborah A. McMahon; Charles R. Rinaldo; JohnW. Mellors; Bernard J. Macatangay University of Pittsburgh, Pittsburgh, PA, US Background: CD4+ T cell expression of inhibitory receptors (IRs) has been reported to be a marker of latently HIV-infected cells. We determined whether co-expression of various IRs on naïve (T N ) and memory (T M ) CD4+ T cells is associated with persistent HIV-1 expression in patients on effective ART. Methods: Expression of PD1, CTLA4, TIM3, 2B4, CD160, and LAG3, in CD4+ T cells from patients on suppressive ART was determined using flow cytometry. Boolean gating using Flowjo was done to evaluate 57 different combinations of inhibitory receptors in naïve (CD45RA+ CCR7+), central memory (T CM; CD45RA-CCR7+), and effector memory (T EM ; CD45RA-CCR7-), CD4+ T cells. PBMC and plasma were tested for cell-associated HIV DNA (CA-DNA) and RNA (CA-RNA) and residual viremia using qPCR targeting 3’ pol. Correlation between markers was assessed using Spearman’s rho. Results: PBMC were analyzed from 30 patients on suppressive ART (<50 copes/ml) for a median of 7.4 years. Of the T N and T M expressing a single IR, frequencies were highest for those expressing TIM3 alone (mean: T N =42.6%, T M =23.9%) followed by PD1 and 2B4. Single-expression of the other 3 IRs was seen in <0.5% of the cells. Correlations between the frequencies of single-expression of TIM3, PD1, or 2B4 on T N , T CM , and T EM and residual viremia were modest (with R values<0.45; p=0.01-0.04). No correlations were seen with CA-DNA or RNA. Evaluation of multiple IR co-expression (i.e. 2-6 IRs expressed) revealed that the combinations PD1/TIM3/2B4+, PD1/2B4+, PD1/TIM3+, PD1/2B4+ had the highest frequencies (mean: T N =3.9% T CM =6.1% T EM =7.9%), whereas the other 53 IR combinations had frequencies <0.1%. Although PD1/TIM3+ cells had the highest frequencies (T N =10.7% T CM =19.1% T EM =20.1%), PD1/TIM3 expression only modestly correlated with residual viremia for T N only (R=0.42; p=0.02), and with CA-DNA in T EM (R=0.40; p=0.03). By contrast, highly significant correlations were observed between residual viremia and PD1/2B4+ T N (R=0.59; p<0.001) and PD1/2B4+T CM (R=0.57, p=0.001), but not with CA-RNA or -DNA. Conclusions: In patients on suppressive ART, a substantial fraction of naïve and memory CD4+ T cells co-express different combinations of PD1, 2B4, and TIM3. The specific combination of PD1 and 2B4 co-expression on naïve and central memory is strongly associated with the level of residual viremia on ART, suggesting that targeting more than one IR could be needed to maximize effects of HIV-1 persistence. 390 Nascent LTR-Driven Transcription Can Lead to Translation of HIV Proteins in Resting CD4+ T Cells Laura DeMaster 1 ; Alexander Pasternak 2 ; Una O’Doherty 1 1 University of Pennsylvania, Philadelphia, PA, US; 2 Academic Medical Center University of Amsterdam, Amsterdam, Netherlands Background: We have previously described a model of direct infection of resting CD4+ T cells and contrasted it with models of activated CD4+ T cell infection. We found that infected resting CD4 cells express low levels of viral protein without releasing infectious virus, raising the possibility that reservoirs may express HIV proteins in vivo and be visible selectively eliminates latently-infected cells that does not require reactivation of HIV gene expression. 388 PD1 Identifies Latently HIV-Infected Nonproliferating and Proliferating CD4 + T Cells Renee M. van der Sluis 1 ; Nitasha A. Kumar 1 ;Vanessa A. Evans 1 ; Rafick P. Sekaly 2 ; Remi Fromentin 2 ; Nicolas Chomont 2 ; Paul U. Cameron 1 ; Sharon R. Lewin 1 1 Doherty Institute, Melbourne University, Melbourne, Australia; 2 VGTI-Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, US

Poster Abstracts

285

CROI 2015