1. Genital secretions from HIV-1 infected women on effective antiretroviral therapy contain high drug concentrations and low amounts of cell-free virus Anandi N. Sheth, MD Emory University School of Medicine, Division of Infectious Diseases Emory Center for AIDS Research Atlanta, USA

2. Background Antiretroviral therapy (ART) is associated with reduced HIV transmission Genital HIV viral shedding is associated with sexual transmission HIV RNA found in female genital tract (FGT) secretions from women on ART with undetectable plasma HIV RNA

3. SQV (ND)LPV (8%) FGT Antiretroviral Drug Penetration Modified from Cohen et al, Ann Int Med 2007 and Taylor and Davies, Curr Opin HIV/AIDS 2010. Data: Min (JAIDS 2004), Dumond (AIDS 2007), Kwara (CID 2008) Patterson (AAC 2011), Clavel (AAC 2011) 500%400%300%200%100% 75%50%25% 0% FGT Concentration or AUC (% of Plasma) Blood exposure = FGT exposure NRTIsNNRTIsPIs Entry inhibitors Integrase inhibitors 3TC (411%) FTC (395%) ZDV (235%) TFV (75-110%) ddI (21%) ABC (8%) d4T (5%) ETR (130%) NVP (83%) DLV (48%) EFV (0.4%) IDV (200%)DRV (150%)APV (52%)RTV (26%)ATV (18%) MVC (273%) RAL (230%)

4. Study Objectives Characterize FGT HIV shedding over one menstrual cycle among women on one ART regimen – Tenofovir (TFV), emtricitabine (FTC), atazanavir / ritonavir (ATV/r) Evaluate factors associated with genital viral shedding Describe FGT drug penetration over menstrual cycle Investigate relationship between FGT drug penetration and viral shedding

5. Study Design Menses1–3 d2–4 d Screening visit Study Visit 1 Study Visit 2 Study Visit 3 Study Visit 4 Study Visit 5 Study Visit 6 2628242220181614121086420 Follicular phase Menstrual cycle day Luteal phase 20 HIV-infected women on ART Undetectable plasma HIV-1 RNA within 90 days Reported adherence Regular menses Excluded if active vaginal infection Instructed to avoid sexual intercourse and douching 6 paired blood and FGT samples HIV RNA, proviral DNA, antiretroviral drug concentrations

6. Methods Cervicovaginal fluid (CVF) collected by two methods – Lavage (10mL PBS) for HIV RNA and DNA – TearFlo strips (3) for antiretroviral drug concentrations HIV-1 detection – Ultrasensitive Roche Amplicor RNA and DNA assays – RNA limit of quantification (LOQ): 50 copies/mL, detectable below LOQ reported – DNA limit of detection: 10 copies/sample Antiretroviral drug concentrations – 24 hours after previous dose (C 24h ) – HPLC-MS-MS (lower LOQ: 5 ng/mL) Data analysis: generalized estimating equations and mixed-effects linear models

7. Results

8. Demographic and Clinical Characteristics (N=20) Characteristicn (%) or median (range) Age in years African American race 36 (26 – 48) 19 (95) HIV risk category – heterosexual sex19 (95) Sexually active past 6 months17 (85) One sexual partner16 (94) Partner HIV negative12 (71) Years of HIV diagnosis 9 (1 – 17) Nadir CD4 (cells/mm 3 )110 (2 – 320) Current CD4 (cells/mm 3 )412 (71 – 1189) Months since first ART 90 (9 – 155) Months on current ART14 (3 – 41)

9. HIV RNA and DNA Detection Outcome #Visits (%) * (N=119) #Patients (%) (N=20) BLOOD HIV RNA detected 69 (58)16 (80) HIV RNA ≥50 copies/mL 13 (11) 8 (40) HIV proviral DNA detected 119 (100) 20 (100) *119 (99%) visits completed, 8 (7%) semen contamination

10. HIV RNA and DNA Detection Outcome #Visits (%) * (N=119) #Patients (%) (N=20) BLOOD HIV RNA detected 69 (58)16 (80) HIV RNA ≥50 copies/mL 13 (11) 8 (40) HIV proviral DNA detected 119 (100) 20 (100) FGT HIV RNA detected 19 (16) 9 (45) HIV RNA ≥500 copies/10mL lavage 0 ( 0) HIV proviral DNA detected 42 (36)14 (70) *119 (99%) visits completed, 8 (7%) semen contamination

11. HIV Detection by Visit Number in Menstrual Cycle Visit number HIV RNA or DNA detected (% of participants, 95% CI) Blood RNA FGT RNA FGT DNA

12. Factors Associated with FGT HIV Detection Factor HIV RNA Detection Rate Ratio (95%CI) HIV DNA Detection Rate Ratio (95%CI) CVF leuks ≥200 cells/µL2.38 (1.03–5.51)2.41 (1.52–3.80) CVF blood ≥200 cells/µL2.29 (0.86 –6.05)1.50 (0.98–2.30) Proviral DNA in CVF2.81 (1.39–5.64)--- HIV RNA in plasma0.92 (0.33–2.52)2.01 (0.99–4.09) Follicular phase0.89 (0.43–1.89)1.07 (0.71–1.62)

13. Factors Associated with FGT HIV Detection Factor HIV RNA Detection Rate Ratio (95%CI) HIV DNA Detection Rate Ratio (95%CI) CVF leuks ≥200 cells/µL2.38 (1.03–5.51)2.41 (1.52–3.80) CVF blood ≥200 cells/µL2.29 (0.86 –6.05)1.50 (0.98–2.30) Proviral DNA in CVF2.81 (1.39–5.64)--- HIV RNA in plasma0.92 (0.33–2.52)2.01 (0.99–4.09) Follicular phase0.89 (0.43–1.89)1.07 (0.71–1.62)

14. Antiretroviral Drug C 24h (N=119) FGT: Plasma C 24h Geometric Mean Ratio, 95%CI 12.2 (8.71–17.0) 3.42 (2.17– 5.39) 2.49 (1.80–3.44) Mean log 10 concentration (ng/mL), 95% CI Plasma FGT 1428 ng/mL (1035–1970) 560 ng/mL (433–724) 236 ng/mL (157–354) 67 ng/mL (53–85) 909 ng/mL (644–1283) 75 ng/mL (58–96)

15. Antiretroviral Drug C 24h by Visit Number in Menstrual Cycle FTC TFV ATV/r Visit number Mean log 10 drug concentration, 95% CI Plasma FGT

16. FGT Drug C 24h by HIV Detection Status FGT mean log 10 C 24h, 95%CI HIV RNAHIV DNA FTC ATV/r HIV RNA HIV DNA HIV RNAHIV DNA TFV

17. Limitations Analysis of impact of vaginal infections occurring during study is ongoing Dilution of CVF by lavage may underestimate genital HIV RNA levels Drug concentrations represent troughs, not entire dosing interval Only extracellular concentrations measured Comparison of different CVF collection methods for drug concentrations is needed

18. Conclusions ART resulted in adequate FGT penetration of all drugs throughout menstrual cycle Compared to previous reports, FTC and TFV penetration similar, but ATV/r higher In presence of ART, unable to detect HIV RNA at a quantifiable amount in FGT Detection of low-level genital HIV RNA suggests local viral replication not completely inhibited

19. Implications High genital drug concentrations reassuring – Consistent with success seen with oral PrEP – Higher-than-expected FGT concentrations of ATV/r should be explored further Presence of low-level FGT HIV RNA and proviral DNA suggests ART reduces, but may not completely eliminate, sexual transmission

20. Acknowledgements CDC Division of HIV/AIDS Prevention Laboratory Branch Clyde Hart Chou-Pong Pau Tammy Evans-Strickfaden Adebola Adesoye L. Davis Lupo Michael Omondi Richard Haaland Amy Martin Ron Ballard John Papp Christi Phillips Emory Center for AIDS Research Igho Ofotokun Kirk Easley Chelsea Gatcliffe Wendy Armstrong Angela Caliendo Jeff Lennox Carlos del Rio Shenique Harmon Maria Rivas Eva Williams Tanisha Sullivan Tammera Byrd Aswani Vunnava Sara Sanford Nancy Sawyer Ericka Patrick Grady Infectious Diseases Program Gina Bailey-Herring Study participants and their providers This research was supported in part by the Emory Center for AIDS Research (P30 AI050409)