Carolina Garrido, N Rallón, N Zahonero, M López, V Soriano, C de Mendoza, and JM Benito

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

3. Background • Switching experiences in HCIII: • ATV • DRV • RAL 700 600 500 THPE0132 CD4 count (cell/mm3) 400 Switch one drug for another in a context of undetectable viremia 300 ATV DRV RAL n 52 25 86 CD4+T cells (cell/mm3) baseline 524 [344,703] 231 [157-493] 555 [429-776] + 6 months 444 [354,606] 291 [176,492] 630 [472,812] Δ CD4 -22 [-127,55] 28 [-36,80] 40 [-31,136] p 0.173 0.104 0.012 Raltegravir immunologic outcomes

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

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

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. Methods Patients and samples Viral load < 50 RNA cop/mL +6 months Baseline Control group Raltegravir group Mantain the same regimen Switch to a RAL-containig regimen • 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

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

9. Results 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

10. Results * 800 600 CD4+T-cell (cell/mm3) 400 200 0 Control Raltegravir CD4+T-cell count • After the 6-month period, only the group who switched to RAL experienced a significant change in CD4 count

11. Results Baseline +6 months Wilcoxon Signed Ranks Test p Control Raltegravir CD4+T-cell maturation 0.117 Effector 0.199 Naive 0.723 Effector memory Central memory 0.133 0.421 Effector 0.014 * Naive * 0.005 Effector memory 0.421 Central memory 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

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

13. Results * * * * + 6 months Baseline p=0.036 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

14. Results Control-RAL baseline Control-RAL + 6 months 100 100 80 80 60 60 % naïve cells expressing CD38 % naïve cells expressing CD38 40 40 20 20 R Sq Linear = 0,51 R Sq Linear = 0,262 0 0 0 20 40 60 80 100 0 20 40 60 80 100 % naïve cells expressing CD31 % naïve cells expressing CD31 Pearson correlation sig (2-tailed) < 0.001 Pearson correlation sig (2-tailed) = 0.001 Association between CD38 and CD31

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+Naivecells. • 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