Effect of 24 Week Intensification with a CCR5-Antagonist on the Decay of the HIV-1 Latent Reservoir IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Laura Díaz 1, Carolina Gutiérrez 2, Carmen Page 2, Raquel Lorente 1, Beatriz Hernández-Novoa 2, Alejandro Vallejo 2, Ester Domínguez 2, María Abad 2, Nadia Madrid 2, Ana Moreno 2, María Jesús Pérez-Elías 2, Rafael Rubio 3, María Ángeles Muñoz-Fernández 1 and Santiago Moreno 2. 1 Immunobiology Department, Hospital General Universitario Gregorio Marañón, Madrid, Spain, 2 Infectious Diseases Service, Hospital Universitario Ramón y Cajal, Madrid, 3 Infectious Diseases Service Hospital Universitario Doce de Octubre, Madrid, Spain

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Background 2 Dinoso JB et al. PNAs 2009, 3 Buzón MJ et al. Nature Med Chun TW, et al. J Infect Dis 2007; 2 Dinoso JB et al. PNAs 2009, 3 Buzón MJ et al. Nature Med 2010

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA  To evaluate the influence of treatment intensification with a CCR5 coreceptor antagonist (maraviroc) on: Objectives Primary endpoint - Latent HIV-1 reservoir Secondary endpoints - Residual viremia measured by ultrasensitive PCR (SCA, <1 copy HIV RNA/mL) - Episomal DNA with 2 LTRs - Immune activation markers

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA ART + Maraviroc Week 0 Week 24 Baseline Study (3 months) Week 48 ARTART Laboratory Determinations Week 12 Week 36 Study Design

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Inclusion criteria Inclusion criteria  Over 18 years of age  Chronic HIV infection  Successful antiretroviral therapy with at least 3 drugs with viral load below 50 copies HIV RNA/mL for at least 2 years  CD4 + T lymphocyte count above 350 cells/mm 3  Demonstration of R5 viral tropism by phenotyping plasma samples stored before antiretroviral therapy Methods

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Laboratory Determinations 2-Residual viremia 3-Episomal DNA with 2 LTRs 1-Latent reservoir 4-Immune activation markers 450ml

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Methods: Latent reservoir Every week CD8 T cells Day 15 and 21 Results: IUPM

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Methods Immune activation markers: CD38 + and HLA-DR + on CD4 and CD8 cells Single Copy Assay (Dr. Sarah Palmer) Episomal DNA with 2 LTRs

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNAPatientGenderAge CD4 count (cells/mm 3 ) CD8 count (cells/mm 3 ) HIV Viral load (copies/mL)ART Duration of ART (months) 1M <50ddI+3TC+NVP124 2M <50FTC+TDF+ATV/r75 3M <503TC+ABC+ATV78 4F <50FTC+TDF+LPV/r57 5M <50AZT+3TC+ABC142 6M <50AZT+3TC+ABC144 7M <50FTC+TDF+ATV/r50 8M <50FTC+TDF+LPV/r42 9M <50FTC+TDF+EFV38 Median [IQR] 46 [31-48] 711 [ ] [ ]784 [ ] [ ]< [38-144] Baseline characteristics of the patients

HIV-1 Latent Reservoir Open symbols represent measurements below the limit of detection IUPM

Open symbols represent measurements below the limit of detection UIPM

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA HIV-1 Residual Viremia (Single Copy Assay) (Single Copy Assay) HIV-1 RNA copies/mL Basal Week 12 * * Failure of internal standard in 2 samples Detection limit < 0,3 cop/mL Detection limit < 2 cop/mL Detection limit <0,6 and < 0,4 cop/mL Week 24

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Episomal DNA with 2 LTRs Follow up (weeks) Number of patients

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Baseline w4 w12 w24 w [ ] 5.4 [ ] 2.3 [ ] 3.3 [ ] 3.5 [ ] Baseline w4 w12 w24 w [ ] 2.5 [ ] 0.8 [ ] 1.6 [ ] 1.8 [ ] CD4 cell activation p<0.001 p=0.115 p=0.03 p=0.05 p=0.755 CD38 + HLA-DR + (%) Follow up (weeks) Basalw12w4w24HIV-1 Negative w36 p=0.031 CD8 cell activation p<0.001 p=0.009 p=0.04 p=0.19 p=0.731 CD38 + HLA-DR + (%) Basalw12w4w24HIV-1 Negative w36 Follow up (weeks) p=0.28 Immune Activation Markers

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA w12 Baseline w12 w4 CD4 cell count Cell/mm 3 w24 Naive CD4 cells CD45RA + CD62L + (%) w24 Baseline W12 W [ ] 686 [ ] 680 [ ] Baselin e W4 W12 W24 42 [22-53] 46 [35-51] p= [30-58] p= [18-55] p= Baseline Immunological Status

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA  After 48 weeks of intensification with MVC, a decrease in the size of the latent reservoir is observed in most patients evaluated. The quantification of the reduction is limited by the sensitivity of the method.  In an apparent paradox, an increase in the residual viremia (SCA) and the episomal DNA with 2 LTRs is found in some of the patients evaluated.  A reduction in the proportion of activated CD4 and CD8 cells was observed.  Overall, we feel that intensification with a CCR5 antagonist may have an impact on the reservoirs and deserves further evaluation. Conclusions

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Discussion The impact on residual replication and viremia is similar to previous reports: no impact/increase (Dinoso et al. PNAs 2009; Buzón et al. Nature Medicine, 2010) The impact on residual replication and viremia is similar to previous reports: no impact/increase (Dinoso et al. PNAs 2009; Buzón et al. Nature Medicine, 2010) Only one previous study has measured episomal DNA with 2 LTRs: a transient increase was found during intensification with raltegravir (Buzón et al. Nature Medicine, 2010) Only one previous study has measured episomal DNA with 2 LTRs: a transient increase was found during intensification with raltegravir (Buzón et al. Nature Medicine, 2010) No previous studies have evaluated the consequences of intensification on the latent reservoir in resting CD4 T cells in chronically infected patients No previous studies have evaluated the consequences of intensification on the latent reservoir in resting CD4 T cells in chronically infected patients -Only one study has measured the effect of initial therapy with a 4 drug regimen, including enfuvirtide (Gandhi et al.JID 2009) -Only one study has measured the effect of initial therapy with a 4 drug regimen, including enfuvirtide (Gandhi et al.JID 2009) -One study evaluated the impact in patients treated during acute infection (Chun TW et al.JID 2007) -One study evaluated the impact in patients treated during acute infection (Chun TW et al.JID 2007)

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Discussion - Inhibition of viral replication in extra plasma sites (e.g. Gut) - Activation of transcription by the mechanism of action of MVC - Evaluation of these mechanisms are currently in progress

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Hospital University Ramón y Cajal Service of Infectious Diseases Madrid, Spain Santiago Moreno Guillén Carolina Gutiérrez Beatriz Hernández Alejandro Vallejo Carmen Page Nadia Madrid María Abad Ester Dominguez José Luis Casado Fernando Dronda Ana Moreno Enrique Navas María Jesús Pérez-Elías Paloma Martí-Belda Maria Pumares Javier Zamora. Statistician Belén Capuz. Radiophysic Hospital University Gregorio Marañón Laboratory of Immunovirology Madrid, Spain María Angeles Muñoz-Fernández Laura Díaz Raquel Lorente D. Pedrero. Radiophysic University of Córdoba. Spain Eduardo Muñoz Karolinska Institute. Stockholm, Sweden Viktor Dahl Sarah Palmer Hospital 12 de Octubre. Madrid, Spain Rafael Rubio Thanks to all participating patients and their families

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Spanish Medical Research Fund (FIS) PI08/0958& The Spanish AIDS Research Network (RIS) RD06/006

IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Discussion Wu Y et al. Plos Patoghens 2009