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Gag-positive reservoir cells can be identified by a new technique and can be cleared by CTL ? Una O’Doherty June 29, 2013 Our experimental system HLADR-, CD69-, CD25- CD4+ T Or CD3C28 activated Infect (X4 or R5 HIV) Culture 3-4d Measure total & integrated HIV DNA O’Doherty U J. Virol. 2000 O’Doherty U J.Virol. 2002 Swiggard WJ J. Virol. 2005 Measure Gag expression Gag expression in resting cells without spreading expression Activated Resting cells produce Gag but little Env May explain lack of spreading infection Activations with CD3/28 increase both Gag & Env Pace PLoS Pathogen 2012 Can Gag expression lead to clearance of latently infected cells? in vitro model for reservoir clearance CD4+T + HIV +/Integrase inhibitor HIV granules CD8+T Migueles Immunity 2008 Gag expression leads to clearance of latently infected cells Do Gag expressing resting CD4+T cell exist in vivo? Gag expressing resting T exist in vivo Calculation of the fraction of integrated HIV DNA expressing Gag Patient Gag+/all Correction sorted cells for live cells 0.0000083 0.877 RU5/ rCD4 0.131 Integrated HIV/PBMC 0.000073 Gag+/ INT (%) 2% TP1 Years VL<50 3 TP2 4 0.0000313 0.3524 0.052 0.0018 0.3% TP3 2 0.0000010 0.975 1.143 0.00082 0.1% TP4 2 0.0000027 0.986 2.163 0.00133 0.4% Calculation assumes all integrated HIV DNA is in resting CD4+ T cells so % expressing Gag is likely an underestimate Is there any evidence for CTL clearance of reservoir in vivo? CTL activity against latent cells correlates with reservoir size in vivo In vivo p < 0.05 for blue (CP) and red (EC) In vitro Integration correlates with IUPM Menodoza Blood 2012 Eriksson PLoS Pathogen 2013 Summary of in vitro and in vivo findings Latently infected cells can express HIV proteins – continuum of latency CTL clear latently infected cells A fraction of the reservoir in rCD4T express Gag in vivo in patients on ART CTL activity against latently infected CD4+ T cells may be a determinant of integration levels Implications for functional cure CTL may have activity against GPR - suggests expanded targets of CTL against latent cells and potential for therapeutic vaccines Integrated HIV DNA measurements are useful to evaluate therapies that target HIV reservoirs Superinfected resting CD4+ T cells provide a model of latency and immune clearance Acknowledgements Erin Graf Matt Pace Luis Agosto Jenny Yu Lindsay Lynch Laura DeMaster NIH: Michele Di Mascio Stephen Migueles Mark Connors UCSF: Steve Deeks Hiroyu Hatano Rick Hecht Penn: Jim Riley Carl June Don Siegel Drew Weissman Mike Betts Penn CFAR: Farida Shaheen Funding: NIH, Merck, amfAR, PKC pharmaceuticals Sorting for Gag + resting T in vivo (except CD4) PBMCs from ART suppressed patients and stained with anti-Gag Gag expressing resting T exist in vivo Gag expressing resting T in vivo