1. 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

2. IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Background 2 Dinoso JB et al. PNAs 2009, 3 Buzón MJ et al. Nature Med 2010 1 Chun TW, et al. J Infect Dis 2007; 2 Dinoso JB et al. PNAs 2009, 3 Buzón MJ et al. Nature Med 2010

3. 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

4. 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

5. 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

6. 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

7. IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Methods: Latent reservoir Every week CD8 T cells Day 15 and 21 Results: IUPM 1 2 3 4 5 6

8. 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

9. 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) 1M491169683<50ddI+3TC+NVP124 2M47486673<50FTC+TDF+ATV/r75 3M46796650<503TC+ABC+ATV78 4F467871109<50FTC+TDF+LPV/r57 5M45534784<50AZT+3TC+ABC142 6M58728639<50AZT+3TC+ABC144 7M326941608<50FTC+TDF+ATV/r50 8M3012411414<50FTC+TDF+LPV/r42 9M305891064<50FTC+TDF+EFV38 Median [IQR] 46 [31-48] 711 [547-793] [547-793]784 [673-1109] [673-1109]<50- 75 [38-144] Baseline characteristics of the patients

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

11. Open symbols represent measurements below the limit of detection UIPM

12. 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

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

14. IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA Baseline w4 w12 w24 w36 5.4 [4.7-7.6] 5.4 [3.5-6.5] 2.3 [0.4-2.9] 3.3 [2.3-4.7] 3.5 [0.9-4.6] Baseline w4 w12 w24 w36 3.2 [2.6-3.5] 2.5 [0.9-3.3] 0.8 [0.2-2.4] 1.6 [1.1-2.3] 1.8 [1.4-2.0] CD4 cell activation p<0.001 p=0.115 p=0.03 p=0.05 p=0.755 CD38 + HLA-DR + (%) Follow up (weeks) 0 10 8 6 4 2 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 + (%) 0 10 8 6 4 2 Basalw12w4w24HIV-1 Negative w36 Follow up (weeks) p=0.28 Immune Activation Markers

15. 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 W24 711 [547-793] 686 [589-1005] 680 [601-910] Baselin e W4 W12 W24 42 [22-53] 46 [35-51] p= 0.833 38 [30-58] p= 0.715 51 [18-55] p= 0.917 Baseline Immunological Status

16. 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

17. 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)

18. 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

19. 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

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

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