1. Impact of Depot Medroxyprogesterone (DMPA) on Human Vaginal Leukocytes and HIV-1 Target Cells Andrea Ries Thurman MD Neelima Chandra PhD (PI) Gustavo F Doncel MD PhD 23 JULY 2012

2. Background Depot Medroxyprogesterone Acetate (DMPA): – Highly effective long acting reversible contraceptive Prevents Unintended/Mistimed Pregnancies – Only one absolute contraindication – High usage in developing world World Contraceptive Use 2009, UN Population Division – Sole P-only injectable in the US

3. Relevance: DMPA and HIV Acquisition Observational studies AOR 1.73 – 3.90 for increased HIV acquisition with DMPA use Ungchusak K et al. J AIDS Hum Retrovirol 1996 Martin HL et al. J Infect Dis 1998 Baeten JM et al. AIDS 2007 Morrison CS et al. AIDS 2010 Sero-discordant couples: Injectable use ↑↑ HIV-1 acquisition and transmission (AHR 2 – 3) Heffron et al. Lancet Infect Dis 2012 2012 WHO meeting re-consider medical eligibility criteria for DMPA in women at high risk of HIV-1 Category 1*

4. Epithelial Thinning Cell layers, height in µm – Dramatic Atrophy with P/DMPA in NHP models Marx PA et al. Nat Med 1996 Smith SM et al. J InfectDis 2000 Jiang Y et al. J Med Primatol 2009 Pal R et al. Virology 2009 Salle B et al. J Infect Dis 2010 Veazey RS et al. Nat Med 2003 – Humans Bahamondes L et al. Contraception 2000 Mauck CK et al. Contraception 1999 Miller L et al. ObstetGynecol 2000 Ildgruben AK et al. ObstetGynecol 2003

5. Epithelial Integrity Other markers of epithelial integrity E Cadherin and Zona Occludin 1 (ZO-1) Epithelial Cell Proliferation (Ki67) E Cadherin density altered by vaginal estrogen therapy Gorodeski GI. Endocrinology 2007

6. Alteration of Mucosal Immune Cells Small cohorts support exogenous progesterone alters mucosal environment – Wieser F et al. Fertil Steril 2001 – Ildgruben AK et al. Obstet Gynecol 2003 Most data on endogenous and exogenous hormones and alteration of mucosal inflammatory/immune cell populations in humans from endometrium – Pudney et al. Biol Reprod 2005 – Wira et al Am J Reprod Immunol 2011

7. Objective Determine if exogenous and endogenous progesterone alters epithelial integrity and the cellular inflammatory status of the vaginal mucosa Secondary analysis – Mauck CK et al. Contraception 1999 Jul;60(1):15-24.

8. Methods 15 women (BTL, no hormonal contraception) Luteal Phase Days 22 - 26 (Confirmed Serum P) Follicular Phase Days 8 - 12 (Serum E2 and P) 12 weeks ± 1 week post DMPA

9. Endpoints Epithelial integrity – Epithelial height in µm (reticles) and # cell layers – Epithelial proliferation (Ki67 cells) – Intracellular adhesion proteins E Cadherin and ZO-1 (Integrated Optical Density - IOD) Immune cell populations by IHC – CD45, CD3, CD4, CD8, CD1a, CD68 bearing leukocytes – HLA-DR, CCR5 (markers of activation and proliferation )

10. Results Demographic data (N = 15) – Mean age 35.9 (± 3.1) years-old – Mean weight 158.5 (± 25.0) pounds – Mean menstrual cycle length 29.0 (± 1.4) days – All with previous pregnancy – Mean gravidity 2.7 (± 1.1) Follicular versus luteal phase – All endpoints NS

11. Epithelial Thickness & Proliferation Epithelial characteristics Mean ± standard deviation Median (25 th, 75 th quantile) Paired T test P value Wilcoxan Signed Rank P Value FollicularLutealDMPA Follicular vs DMPA Luteal vs DMPA Thickness (µm)307.8 ± 96.8269.6 ± 80.1272.3 ± 94.60.330.94 # Cell layers31.2 ± 8.128.2 ± 7.228.9 ± 6.50.420.79 Ki-6735.7 (19.3, 64.3) 17.1 (9.0, 68.4) 72.6 (43.2, 126.9) 0.0040.005

12. Intracellular Adhesion Proteins (NS) Epithelial adhesion proteins Median integrated optical density (IOD) per stained field (X 10 6 ) (25 th, 75 th percentile, quantile)* Wilcoxon signed rank P value FollicularLutealDMPA Follicular vs DMPA Luteal vs DMPA E Cadherin 3.5 (0.4, 4.7) 1.6 (0.7, 8.1) 1.6 (1.0, 3.2) 0.640.42 ZO-10.1 (0.03, 0.93) 0.2 (0.04, 0.65) 0.2 (0.04, 0.3) 0.380.22

13. Luteal DMPA ZO-1 E Cadherin Follicular

14. Immune Cells (Significant Increase with DMPA) Phenotype vaginal epithelial immune cells Median cell count (cells/mm 2 )Wilcoxon signed rank P value Follicular Phase Luteal Phase DMPA Treatment Follicular vs DMPA Luteal vs DMPA CD4570.673.5156.90.030.22 CD340.045.976.50.080.04 CD828.129.152.60.150.01 CD1a26.427.633.90.131.00 CD681.53.37.20.010.07

15. Activated Immune Cells (Significant Increase with DMPA) Phenotype of Immune Cells in the Vaginal Epithelium Median Cell Count (cells/mm 2 ) (25 th, 75 th percentile, quantile) Wilcoxon Signed Rank P value FollicularLutealDMPA Follicular vs DMPA Luteal vs DMPA CCR5 (LP)0.2 (0, 0.5)0.2 (0, 0.6)0.8 (0, 1.2)0.090.05 HLA-DR97.4 (81.0, 120.0) 82.4 (46.5, 102.6) 113.9 (85.1, 184.3) 0.020.35

16. Follicular LutealDMPA HI CD3 CD8 CCR5 HLA-DR

17. CONCLUSIONS Epithelial Thinning (thickness, #cell layers) NS Epithelial Integrity (E Cadherin, ZO-1, Ki67) NS DMPA use resulted in significant increase in: – CD45, CD3, CD8, CD68 leukocytes – CCR5 (LP), HLA-DR, markers

18. Acknowledgments CONRAD Microbicide Development Laboratory Gustavo F Doncel MD PhD (Principal Investigator) Neelima Chandra PhD Sharon Anderson PhD Nazita Yousefieh PhD Nancy Gonyea Dept of Biostatistics Eastern Virginia Medical School Tina D Cunningham PhD CONRAD Clinical Team Christine Mauck MD MPH

19. Epithelial Thickness & Proliferation Epithelial characteristics Mean ± standard deviation Median (25 th, 75 th quantile) Paired T test P value Wilcoxan Signed Rank P Value FollicularLutealDMPA Follicular vs DMPA Luteal vs DMPA Thickness (µm)307.8 ± 96.8269.6 ± 80.1272.3 ± 94.60.330.94 # Cell layers31.2 ± 8.128.2 ± 7.228.9 ± 6.50.420.79 Ki-67+35.7 (19.3, 64.3) 17.1 (9.0, 68.4) 72.6 (43.2, 126.9) 0.0040.005

20. Intracellular Adhesion Proteins (NS) Epithelial adhesion proteins Median integrated optical density (IOD) per stained field (X 10 6 ) (25 th, 75 th percentile, quantile)* Wilcoxon signed rank P value FollicularLutealDMPA Follicular vs DMPA Luteal vs DMPA E Cadherin 3.5 (0.4, 4.7) 1.6 (0.7, 8.1) 1.6 (1.0, 3.2) 0.640.42 ZO-10.1 (0.03, 0.93) 0.2 (0.04, 0.65) 0.2 (0.04, 0.3) 0.380.22

21. Luteal DMPA ZO-1 E Cadherin Follicular

22. Immune Cells (Significant Increase with DMPA) Phenotype vaginal epithelial immune cells Median cell count (cells/mm 2 )Wilcoxon signed rank P value Follicular PhaseLuteal Phase DMPA Treatment Follicular vs DMPA Luteal vs DMPA CD45 70.6 (37.5, 159.6) 73.5 (31.7, 120.3) 156.9 (112.4, 277.3) 0.030.22 CD3 40.0 (23.8, 60.0) 45.9 (11.3, 70.7) 76.5 (47.8, 117.9) 0.080.04 CD4 (LP) 0 (0, 0)0 (max 11.6)0 (max 9.3)NA CD8 28.1 (18.4, 44.9) 29.1 (9.3, 54.4) 52.6 (40.9, 85.6) 0.150.01 CD1a 26.4 (16.7, 60.9) 27.6 (17.8, 41.7) 33.9 (26.0, 54.9) 0.131.00 CD68 1.5 (0, 5.1)3.3 (0, 5.4)7.2 (3.9, 22.0)0.010.07

23. Activated Immune Cells (Significant Increase with DMPA) Phenotype of Immune Cells in the Vaginal Epithelium Median Cell Count (cells/mm 2 ) (25 th, 75 th percentile, quantile) Wilcoxon Signed Rank P value FollicularLutealDMPA Follicular vs DMPA Luteal vs DMPA CCR5 (LP)0.2 (0, 0.5)0.2 (0, 0.6)0.8 (0, 1.2)0.090.05 HLA-DR97.4 (81.0, 120.0) 82.4 (46.5, 102.6) 113.9 (85.1, 184.3) 0.020.35

24. Follicular LutealDMPA HI CD3 CD8 CCR5 HLA-DR