1. Impact of Raltegravir on Immune Reconstitution and Thymopoiesis in HIV-1 Infected Patients with Undetectable Viremia Carolina Garrido, N Rallón, N Zahonero, M López, V Soriano, C de Mendoza, and JM Benito Hospital Carlos III, Madrid

2. HIV infection and CD4 recovery HIV-infection CD4 + T-cells Immune deficiency Opportunistic infections HAART HIV Viral Load CD4 + T-cells Raltegravir First-in-class integrase inhibitorHas proven potent antiviral activity but also showed good immunologic recovery Background

3. Raltegravir immunologic outcomes Background THPE0132 700 600 500 400 300 CD4 count (cell/mm 3 ) ATVDRVRAL n baseline + 6 months Δ CD4 p CD4+T cells (cell/mm 3 ) 52 524 [344,703] 444 [354,606] -22 [-127,55] 0.173 25 231 [157-493] 291 [176,492] 28 [-36,80] 0.104 86 555 [429-776] 630 [472,812] 40 [-31,136] 0.012 Switching experiences in HCIII: - ATV - DRV - RAL Switch one drug for another in a context of undetectable viremia

4. Immunologic recovery Background  The CD4 + T-cell recovery after HAART can be due to: Recent thymic emigrants (RTEs) The expansion of peripheral T cells Thymus Supply of new lymphocytes to the periphery. Impaired during HIV-infection Kohler and Thiel, Blood, 2009

5. Thymic function T-cell receptor excision circles (TRECs) Stable DNA episomes formed during T-cell receptor gene rearrangement within the thymus ↑ % cells TREC+ = RTEs ↓ % cells TREC+ = cells after several divisions TRECs are lost upon cell division Background CD31 Surface marker Present in TREC-rich T-cells Indicator of recent thymic emigrants

6. Objective  The aim of the study was to characterize the immunologic recovery of HIV-infected patients under suppressed viremia after switching to a RAL containig regimen

7. Patients and samples Viral load < 50 RNA cop/mL Baseline +6 months Switch to a RAL-containig regimenMantain the same regimen Control group Raltegravir group  For each patient, two blood samples were collected: one at baseline one 6 months later  PBMCs were obtained from peripheral blood by density gradient centrifugation  Cells were criopreserved until use Methods

8. Immunologic characterization CD4+T-Lymphocyte effectornaive effector memory central memory - CD4-PC7 - CD45RA-ECD - CD27-PE - CD38-PC5 - CD31-FITC  PBMCs were stained with fluorescent-conjugated monoclonal antibodies specific for cell surface markers:  Expression of these surface markers was analyzed by flow cytometry using a 5- color-flow-cytometer FC500 (Coulter, Miami, FL) Activation marker Recent thymic emigrants (RTE) marker Maturation markers Methods

9. Study population  Baseline characteristics of the study population did not differ among groups  Control group: 84% PI (67% ATV, 17% LPV); 11% NNRTIs; 5% NRTIs  RAL group: 53% PI; 47% 2NRTIs Results

10. CD4 + T-cell count  After the 6-month period, only the group who switched to RAL experienced a significant change in CD4 count Control 800 600 400 200 0 Raltegravir CD4 + T-cell (cell/mm 3 ) * Results

11. CD4 + T-cell maturation Effector Naive Effector memory Central memory Effector Naive Effector memory Central memory 0.421 0.014 0.005 0.421 0.117 0.199 0.723 0.133 * * Wilcoxon Signed Ranks Test p After the 6-months period, significant changes in the population subsets distribution only occurred in the RAL group, where the subset of naive cells increased its proportion +6 monthsBaseline Control Raltegravir Results

12. CD31 expression  CD31 expression did not vary significantly in any of the study groups neither in the whole population or in particular cellular subsets Results Baseline + 6 months p=0.811 p=0.306 p=0.191 p=0.420 p=0.679 p=0.277 p=0.306 p=0.872 p=0.314 p=0.117

13. CD38 expression  In the control group there was a slightly significant decrease in the CD38 expression level of both naive and effector subsets  In the RAL group, we observed a significant decrease in the effector population but a significant increase in the level of CD38 expression of naive cells Results ** * * p=0.036 + 6 months Baseline

14. Association between CD38 and CD31 Results

15. Conclusions  Switching to a RAL-containing regimen in a context of suppressed viremia induced a significant gain in CD4+T-cell count.  This improvement was mainly due to an increase in the subset of CD4+Naive cells.  The increase in CD4+naive cells could not be clearly associated to recent thymic emigrants, as there was no significant rise in the expression of CD31.  However, the increase of CD38 expression on naive CD4+T-cells and the significant association between CD38 and CD31 markers on this subset of CD4+ cells, suggest that the immune recovery observed in patients switching to RAL may be due, at least in part, to newly produced cells in the thymus.

16. Acknowledgments Infectious Diseases Department of Hospital Carlos III (Molecular Biology Lab. + Clinical Section) Norma Rallón Mariola López Jose Miguel Benito Natalia Zahonero Carmen de Mendoza Vicente Soriano