CROI 2015 Program and Abstracts

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Oral Abstracts

Methods: Paired seminal and blood samples from 45 asymptomatic chronically HIV-infected CMV-seropositive MSM on long term ART and with HIV RNA levels in blood plasma <50 copies/ml were studied. Levels of CMV DNA in semen and blood were measured by RT-PCR, and cell-associated HIV DNA and RNA transcripts (unspliced) were measured in PBMC by droplet digital PCR. Markers of T cell exhaustion (PD-1) and senescence (CD57) were measured in PBMC by flow cytometry for CD4 and CD8 T cells and subsets (naïve [CD45RA + CD27 + CD28 + ], central memory [CD45RA - CD27 + CD28 + ], effectors [CD4 + CD45RA - / + CD27 - CD28 + or CD8 + CD45RA - / + CD27 + CD28 - ] and terminally differentiated [CD45RA + / - CD27 - CD28 - ]). Associations between immunological markers and asymptomatic CMV and HIV replication, HIV DNA, CMV IgG, age, current and nadir CD4 and time on ART were determined using univariate and multivariate analysis. Results: CMV DNA was detected in 42% of seminal samples and 20% of PBMC. Detectable CMV DNA in semen but not blood was associated with increased PD-1 expression on circulating CD4 T cells compared to no CMV (P=0.01), particularly in the effector and terminally differentiated subsets (P<0.05). Similarly, higher levels of cellular HIV RNA (but not HIV DNA) were positively associated with greater PD-1 expression on total CD4 and central memory blood subset (P<0.01). There was no association between CMV DNA (blood and semen) or cellular HIV RNA with CD8 exhaustion or senescence or with markers of CD4 senescence. In multivariate analysis, detection of seminal CMV and higher cellular HIV RNA remained associated with increased PD-1 expression on total CD4 T cells (P<0.05). No other variable contributed significantly to the model. Conclusions: Our data suggest that detection of CMV in the genital tract may contribute to the activation of the PD-1 axis on circulating T cells during suppressive ART. Because increased PD-1 on T cells has been implicated in the maintenance of the HIV reservoir, HIV disease progression and the inability of the immune system to adequately control HIV infection, future studies should examine whether CMV-dependent mechanisms play a role in T cell exhaustion. 301 HIV Myeloid Derived Suppressor Cells Control Cytomegalovirus Inflammation by IL-27 Ankita Garg ; Stephen Spector University of California San Diego, La Jolla, CA, US Background: CMV is associated with persistent inflammation in HIV-infected persons. Here, we studied the effect of HIV expanded myeloid derived suppressor cells (MDSCs) in controlling CMV specific inflammation. Methods: PBMCs from HIV–/CMV-seropositive (CMV+) donors were cultured in presence of heat inactivated HIV. After 5 days, CD11b + CD33 + CD14 + HLA DR hi (DR hi monocytes) and CD11b + CD33 + CD14 + HLA DR -/lo (MDSCs) cell subsets were sorted by flow cytometry. B7H4 (a negative regulator of T cell function) was silenced using siRNA and cultured with/without autologous PBMCs in presence/absence of CMV pp65 peptides (pp65; 1 m g/ml). In some experiments, PBMCs were cultured with HIV/pp65 in presence/absence of neutralizing anti-IL-27 antibody. Enumeration of B7H4 on MDSCs, regulatory T-cells, intracellular IFN γ , activated forms phospho(p)-Zap70 and p-Akt were determined by flow cytometry; IFN γ and IL-27 were quantified by ELISA. Data were analyzed using two-tailed, paired Student’s t test. Results: MDSCs cultured with autologous PBMCs exposed to pp65 caused a decrease in IFN γ production vs. controls or DR hi monocytes (p=0.002). IFN γ release was restored when MDSCs were transfected with B7H4 siRNA and cultured with PBMCs in presence of pp65 (p=0.02). MDSCs cultured with PBMCs did not alter pp65 induced activation of proximal T-cell signaling molecule Zap70 but decreased activation of Akt; this was restored when B7H4 was knocked down in MDSCs cultured with PBMCs. Culture of MDSCs with pp65 produced more IL-27 vs. control MDSCs and DR hi monocytes (p=0.05). Culture of CMV+ PBMCs with pp65 increased the frequency of CD4 + IFN γ + cells and release of IFN γ in supernatants vs. controls (p=0.04). IFN γ was further increased when PBMCs were cultured in presence of anti-IL-27 and stimulated with pp65; CMV– PBMCs did not produce IFN γ when treated with pp65. Furthermore, culture of CMV+ PBMCs with pp65 led to expansion of FoxP3 + Tregs vs. controls (p=0.02) and CMV– (p=0.003); addition of anti-IL-27 had no effect on Treg expansion. Finally, HIV expanded MDSCs had increased expression of B7H4 when compared to DR hi monocytes (p=0.03) which was inhibited in the presence of anti-IL-27 neutralizing antibody (p=0.05). Conclusions: These findings suggest that IL-27 down regulates IFN γ during CMV infection. IL-27 induces B7H4 expression on HIV MDSCs which controls CMV induced T-cell IFN γ production by inhibiting p-Akt. IL-27 and B7H4 provide new therapeutic targets to control inflammation during HIV infection. 302 Persistent Elevation of Inflammation Markers in HIV+ Persons With CMV Disease Melissa Schechter 1 ; Bruno Andrade 2 ; Eleanor M.Wilson 1 ;Virginia Sheikh 2 ; Sonya Krishnan 2 ; Margaret Caplan 1 ; Gregg Roby 2 ; Adam Rupert 3 ; Peter Burbelo 4 ; Irini Sereti 2 1 NIAID, Leidos Biomedical Inc., Fredrick, MD, US; 2 National Institute of Allergy and Infectious Diseases, Bethesda, MD, US; 3 NIAID, Leidos Biomedical Inc., Fredrick, MD, US; 4 National Institute of Dental and Craniofacial Research, Bethesda, MD, US Background: HIV+ individuals have a high prevalence of cytomegalovirus (CMV) co-infection and a minority of them develop CMV-associated end organ disease (EOD). CMV co-infection has also been associated with increased risk for cardiovascular events. The potential contribution of CMV infection or disease in chronic immune activation and non- infectious complications in HIV remains unclear. Methods: In a case control study, HIV+ persons (CD4 <100 cells/ m L pre-ART) with CMV EOD (N=25) or detectable CMV viremia (N=7) pre-ART were matched 1:1 by CD4 T-cell counts and age with HIV+ persons with undetectable CMV viremia by PCR. Participants were evaluated pre-ART (week 0) and at week (W) 12 and 48 after ART initiation. Cryopreserved plasma was used to measure markers of inflammation (CRP, IFN- γ , IL-12 p70, IL-10, IL-1 β , IL-8, IL-6, TNF- α ), monocyte activation and vascular injury (sICAM-3, E-Selectin, sCD14, sTF, MP-TF, CX3CL1, P-Selectin, Thrombomodulin, SAA, MCP-1, Eotaxin-3) and coagulation (D-dimer, Factor X Chromogenic, TF Chromogenic) by ELISA-based assays. Data were analyzed using Mann-Whitney and Spearman rank (correlation) tests. Results: EOD cases were 66%male, with median age of 41 years, CD4 of 11 cells/ m L, plasma HIV RNA of 5.12 log 10 c/mL and CMV PCR 2.93 log 10 c/mL units at W0. EOD cases had higher plasma levels of HIV RNA (p=0.01), CRP (p=0.03), IFN- γ (p=0.02), IL-10 (p=0.01), IL-8 (p=0.01), IL-6 (p=0.03), and SAA (p=0.001) compared to controls at W0. At W12, SAA (p=0.02), IFN- γ (p=0.0002), IL-10 (p=0.0003), IL-8 (p=0.01) and TNF- α (p=0.003) remained higher in EOD cases than in controls. At W48, IFN- γ (p=0.0.01), TNF- α (p=0.02) and IL-12 p70 (p=0.04) were persistently higher in EOD cases than in controls. Baseline, CMV IgG levels inversely correlated with sCD14 (r=-0.3, p=0.04), Fractalkine (r=-0.3, p=0.02) and HIV viral load (r=-0.33, p=0.01) but not with presence of EOD. Conclusions: CMV end organ disease and viremia in HIV+ persons is associated with increased plasma levels of inflammatory and vascular injury biomarkers pre-ART with some persisting after ART. Our data suggest a protracted inflammatory response to CMV that may contribute to HIV pathogenesis and non-infectious complications. Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the National Institute of Allergy and Infectious Diseases.

Oral Abstracts

107

CROI 2015