OA21.06 LBCryptic Multiple HIV-1 Infection Revealed by Early,Frequent, and Deep Sampling during Acute Infection

Gustavo Hernan Kijak1,2, Eric Sanders-Buell1,2, Agnes-Laur-ance Chenine1,2, Michael Eller1,2, Nilu Goonetilleke3, RasmiThomas1,2, Sivan Leviyang4, Elizabeth Harbolick1,2, MeeraBose1,2, Phuc Pham1,2, Celina Oropeza1,2, Kultida Poltavee1,2,Anne Marie O’Sullivan1,2, Melanie Merbah1,2, Margaret Cost-anzo1,2, Hui Li5, Will Fischer6, Feng Gao7, Leigh Anne Eller1,2,Robert J. O’Connell8, Samuel Sinei9, Lucas Maganga10, Han-nah Kibuuka11, Sorachai Nitayaphan8, Morgane Rolland1,2,Bette Korber6, Francine McCutchan12, George Shaw5, NelsonMichael1, Merlin Robb1,2, Sodsai Tovanabutra1,2, Jerome Kim1

1U.S. Military HIV Research Program (MHRP), Walter ReedArmy Institute of Research, Silver Spring, MD, United States,2U.S. Military HIV Research Program (MHRP)/ Henry M.Jackson Foundation, Silver Spring, MD, United States, 3Schoolof Medicine, The University of North Carolina at Chapel Hill,Chapel Hill, NC, United States, 4Department of Mathematicsand Statistics, Georgetown University, Washington, DC, UnitedStates, 5Perelman School of Medicine, University of Pennsyl-vania, Philadelphia, PA, United States, 6Theoretical Biology,Los Alamos National Laboratory, Los Alamos, NM, UnitedStates, 7Duke Human Vaccine Institute, Duke UniversityMedical Center, Durham, NC, United States, 8Armed ForcesResearch Institute of Medical Sciences, Bangkok, Thailand,9Walter Reed Project, Kericho, Kenya, 10Mbeya Medical Re-search Programme, Mbeya, Tanzania, United Republic of,11Makerere University-Walter Reed Project, Kampala, Uganda,12Independent Consultant, Silver Spring, MD, United States

Background: In acute HIV-1 infection (AHI) single genomesequencing (SGS) revealed a strong bottleneck at transmission,with 60–90% of sexual infections being established by a singletransmitted/founder (T/F) virus. We combined early and fre-quent sampling with targeted deep sequencing (TDS) to studyviral evolution during AHI.Methods: We studied 7 HIV(-) at entry high-risk RV217 vol-unteers (2 M and 5 F, all with sexual risk) with documented HIVnucleic acid (NA) conversion after twice-weekly testing. Start-ing at d2-7 (d0: first NA + date), we studied 8–9 consecutiveplasma samples (mean sampling interval: 4.1d; peak viremia:d10-18; 1–5 samples were from pre-peak viremia) by HIV SGSand TDS (Ion Torrent; limit of detection: 0.5%).Results: 6/7 persons had pre-peak viremia SGS profiles consistentwith infection by a single T/F virus. However, in 4 persons, addi-tional variants were detected by TDS: in 3 persons at d2-7 (fre-quency: 0.5–4.3%), and in one at d21. Viral populations evolved atdramatic rates, but with different patterns. In #1, the minor variantcirculated at < 5% until d17, then increased to 57% by d31. In #2, theminor variant increased from 3.5% (d7) to 93% (d21), and thendecreased to < 0.5% by d42. In #3 the minor variant was at 0.5–1%between d7-16, then became undetectable, but was 53% at d181. Inparticipant #4, 2 minor variants were detected at d21 (0.5–1.4%),increasing by d28 to 16–36%, respectively. Full length geneticdistances between cognate major and minor variants were 1.0–2.2%,consistent with acquisition of multiple viruses from the same donor.Inter-variant recombinants were detected from d21 onward. Duringearly AHI, both major and minor variants acquired CTL-escapemutations.Conclusions: We show that in apparent single infections minorvariants can occur at levels not detectable by SGS (i.e., crypticmultiple infection). Furthermore these variants contribute to

viral evolution, which may have profound implications for HIVpathogenesis, cure, treatment, and vaccine design.

Cell and Tissue Models of ARVs for Prevention

OA22.01Oral Maraviroc and Tenofovir for HIV Prevention inWomen: An Ex Vivo and Translational Approach

Melanie R. Nicol1, Heather MA Prince2, Cindi W. Emerson1,Julie AE Nelson2, Kristine B. Patterson2, Elizabeth J. Geller2,Myron S. Cohen2, Angela D.M. Kashuba1,2

1University of North Carolina at Chapel Hill, Eshelman Schoolof Pharmacy, Chapel Hill, NC, United States, 2University ofNorth Carolina at Chapel Hill, School of Medicine, Chapel Hill,NC, United States

Background: Our previous studies demonstrate the protectiveconcentration of tenofovir diphosphate (TFVdp) in vaginal ex-plants is > 10-fold higher than in TZM-bl cells. Here we in-vestigate maraviroc’s (MVC) efficacy in cells and vaginalexplants, and determine the explant’s prediction potential of adose-challenge study from biopsies of volunteers given an oraldose of MVC + tenofovir disoproxil fumurate (TDF).Methods: TZM-bl cells (n = 3) and vaginal explants (n = 5 donors)were incubated 24h in MVC 0.01-500ug/mL prior to challengewith HIV-1 JR-CSF. Combination MVC + tenofovir (TFV) wasalso used in cells to define the effects of drugs combined. Com-pared to undosed controls, efficacy was assessed using a luciferasereporter assay in cells, and spliced RNA 24-72h post-inoculationin explants. A dose-challenge study was performed in 6 HIV-, pre-menopausal women administered a single 600mg MVC + 600mgTDF dose. 24h post-dose, 4 vaginal + cervical biopsies were col-lected for viral challenge and evaluated for infection in the samemanner as explant tissue. HIV protection was defined as splicedRNA within one standard deviation of background.Results: In vaginal explants, MVC protective efficacy wanedafter 24h. Within 24h, MVC EC50 was 9.7ug/mL, which was> 1000-fold higher than the EC50 in TZM-bl cells (0.006 ug/mL).Additivity of MVC + TFV was confirmed for HIV protection. TheTZM-bl model and the explant model predicted 100% and 16%efficacy, respectively, 24h after a 600mg MVC + TDF dose. Inthe healthy volunteers, protection was observed in 50% (3/6) ofvaginal biposies and 67% (4/6) of cervical biopsies, with 50% (3/6)of women having complete protection against HIV challenge.Conclusions: Similar to TFVdp, cell models overestimated theefficacy of MVC in vaginal explants. Data from TZM-bl cellmonolayers over predicted, and tissue explants under predicted,efficacy in a healthy volunteer dose-challenge study. Tissueconcentrations at 24h after single high-dose of MVC + TDF weremoderately protective against HIV infection.

OA22.02Pharmacodynamic Activity in Ectocervical and ColonicTissue of Dapivirine, Maraviroc, and CombinationTopical Gels for HIV Prevention

Charlene Dezzutti1,2, Sarah Yandura2, Lin Wang2, Brid Devlin3,Jeremy Nuttall3, Lisa C. Rohan1,2

1University of Pittsburgh, Pittsburgh, PA, United States, 2Ma-gee-Womens Research Institute, Pittsburgh, PA, United States,3IPM, Silver Springs, MD, United States

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Background: Dapivirine (DPV), a non-nucleoside reversetranscriptase inhibitor, and maraviroc (MVC), a CCR5 antago-nist, were formulated into aqueous gels to prevent mucosal HIVtransmission. We hypothesize the combination gel will havemore potency against HIV infection of mucosal tissue as com-pared to either single drug gel.Methods: Dilutions of 0.05% DPV, 0.1% MVC, and 0.05%DPV/0.1% MVC gels were evaluated on polarized ectocervicaland colonic mucosal explant cultures exposed to HIV-1BaL. Afteran overnight culture, the explants were washed and medium re-plenished in the basolateral compartment. Every 3 to 4 days, su-pernatant was collected and replenished for up to 21 days. HIV-1replication was monitored in culture supernatant by p24 ELISA.Results: Dilutions of the gels for ectocervical tissue began at1:20 for DPV concentrations of *75900 nM and MVC con-centrations of *97500 nM, while dilutions for colonic tissuebegan at 1:2000; a 100-fold more dilute than what was used forthe ectocervical tissue. For ectocervical tissue, 7590 nM of DPVresulted in complete tissue protection while 97500 nM of MVCwas partially protective (6 of 8 explants showed no HIV repli-cation). The combination gel at 7590 nM of DPV/9750 nM ofMVC completely protected the tissue, while 759 nM of DPV/975 nM of MVC was partially protective (6 of 8 explants showedno HIV replication). For colonic explants, DPV gel diluted to759 nM completely protected the tissue, higher dilutions showedno protection. MVC gel diluted to 975 nM showed no substantialprotection of the colonic tissue. The combination gel diluted to759 nM of DPV/975 nM of MVC completely protected the co-lonic tissue while the 10-fold higher dilution was partially pro-tective (6 of 8 explants showed no HIV replication).Conclusions: Combining both drugs in a single formulationdemonstrated modest synergy. Collectively, these data provide arationale for further testing of these products as dual compart-ment microbicides.

OA22.03Expression, Activity, and Regulationof Phosphorylating Enzymes in Genital and ColorectalTissues and Immune Cells

Minlu Hu1,2, Tian Zhou1,2, Charlene S. Dezzutti1,3, Sharon L.Hillier1,3, Lisa C. Rohan1–3

1Magee-Womens Research Institute, Pittsburgh, PA, UnitedStates, 2University of Pittsburgh School of Pharmacy, Depart-ment of Pharmaceutical Sciences, Pittsburgh, PA, United States,3University of Pittsburgh, Department of Obstetrics, Gynecology,and Reproductive Sciences, Pittsburgh, PA, United States

Background: Studies of oral tenofovir (TFV) have revealed thatTFV and tenofovir diphosphate (TFV-DP) levels are100 · higher in colorectal tissue than in cervical/vaginal tissueafter a single oral dose. Multiple phosphorylating enzymes (PEs)play a role in TFV activation. However, limited data is availableregarding the expression and activity of these enzymes in thefemale genital and colorectal tissue relative to immune cells.Methods: mRNA expression for 7 PEs (AK2, AK4, NME1,NME2, CKMT1, CKMT2, CKB) in fresh surgical human tissuesamples (cervical n = 6, vaginal n = 5, colorectal n = 5), a vaginalepithelial cell line (VK2), and a T cell line (PM1), was evaluatedusing qRT-PCR. Intracellular TFV-DP formation was tested in VK2and PM1 cells with or without medroxyprogesterone acetate (MPA)and progesterone (P4) using an LC-MS/MS method. Differences inTFV-DP conversion were assessed using Student’s t-test.

Results: Vaginal, ectocervical, and colorectal tissues had similarexpression of PEs except for AK2, which was present at 15-28 · higher levels in colorectal tissue than in ectocervical orvaginal tissues (p < 0.05). The vaginal epithelial cell line wasshown to have 10-10,000 · higher expression of CKB, CKMT1,CKMT2, AK2, and AK4 as compared to levels found for theT cell line (p < 0.05). MPA treatment resulted in a 3-fold in-crease in TFV-DP in the epithelial cell line (p < 0.01) and a 30%decrease in the T cell line (p < 0.01) as compared to controls. P4treatment resulted in a nearly 4-fold increased TFV-DP level inthe epithelial cell line (p < 0.01) and no change in the T cell line.Conclusions: The increased levels of AK2 in colorectal tissuesuggest that AK2 may contribute to the increased levels of TFV-DP observed in colorectal tissues. The higher level of PEs ob-served in vaginal epithelial cell line compared to T cell line,suggests that TFV-DP found in tissues may be predominantlyassociated with epithelial cells. The impact of reproductivehormones on PEs warrants further investigation.

OA22.04Transport and Transport Properties of Tenofovir fromMicrobicide Gels into Vaginal Tissue: Analysis UsingRaman Spectroscopy

Oranat Chuchuen1, Marcus H. Henderson1, Marinella G.Sandros2, Angela D.M. Kashuba3,4, David F. Katz1,5

1Duke University, Biomedical Engineering, Durham, NC,United States, 2University of North Carolina at Greensboro,Nanoscience, Greensboro, NC, United States, 3University ofNorth Carolina at Chapel Hill, Eshelman School of Pharmacy,Chapel Hill, NC, United States, 4University of North CarolinaSchool of Medicine, Department of Infectious Diseases, ChapelHill, NC, United States, 5Duke University, Department of Ob-stetrics and Gynecology, Durham, NC, United States

Background: Common drug release assays use a liquid sink re-ceptor compartment. Permeability assays measure net transportthrough tissue specimens of varying thickness. These do not giveconcentration vs. depth in tissue, nor distinguish drug partitioningat the vehicle-tissue interface from rate of transport in tissue. Wedeveloped a rapid, non-contact method using confocal Ramanspectroscopy to measure drug partitioning and concentration vs.depth in intact tissue layers (epithelium vs. stroma) and to trans-late such data to drug diffusion coefficients. We report here onresults for Tenofovir released from its clinical gel.Methods: Fresh porcine vaginal tissue specimens were treatedwith 1% Tenofovir gel in a Transwell assay for 2-8 hr at 37�C.Gel was applied to either epithelial or stromal tissue surfaces.Results for spatio-temporal concentration profiles were fit to adrug diffusion model to obtain diffusion coefficients in epithe-lium and stroma. To determine partition coefficients, tissuespecimens were incubated by submersion in 1% Tenofovir geland equilibration over 6 h.Results: Tenofovir concentrations exhibited diffusion-like time-and depth-dependent distributions in tissue. Diffusion and par-tition coefficients in epithelium ranged 7x10 - 9 - 3x10 - 8 cm2/s,and 0.5 - 0.8, respectively. Initial measurements gave ‡ 1 logincrease in diffusion coefficient in stroma. Measurements werereferenced to classical permeability data.Conclusions: This standardizable label-free method characterizesdrug concentration distributions in tissue and gel vehicles, deter-mining the fundamental gel-tissue partition coefficient and diffusioncoefficients in gel, epithelium and stroma. Results suggest that the

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