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Introduction: The majority of new HIV infections occur through mucosal transmission. The availability of readily applicable and accessible platforms for anti-retroviral (ARV) delivery is critical for the prevention of HIV acquisition through sexual transmission in both women and men. There is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. The silk fibroin (SF) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. The objective of this study was to test safety and efficacy of SF for anti-HIV drug delivery to mucosal sites and for viral prevention., Methods: We formulated a potent HIV inhibitor Griffithsin (Grft) in a mucoadhesive silk fibroin (SF) drug delivery platform and tested the application in a non-human primate model in vivo and a pre-clinical human cervical and colorectal tissue explant model. Both vaginal and rectal compartments were assessed in rhesus macaques (Mucaca mulatta) that received SF (n = 4), no SF (n = 7) and SF-Grft (n = 11). In this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo SHIV challenge., Results: Effective Grft release and retention in mucosal tissues from the SF-Grft platform resulted in protection against HIV in human cervical and colorectal tissue as well as against SHIV challenge in both rhesus macaque vaginal and rectal tissues. Mucoadhesion of SF-Grft inserts did not cause any inflammatory responses or changes in local microbiota., Conclusions: We demonstrated that in vivo delivery of SF-Grft in rhesus macaques fully protects against SHIV challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. Our study provides support for the development of silk fibroin as a highly promising, user-friendly HIV prevention modality to address the global disparity in HIV infection., (© 2020 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of the International AIDS Society.)

Objective: HIV Prevention Trials Network 069/AIDS Clinical Trials Group A5305 was a study of 48-week oral pre-exposure prophylaxis (PrEP) regimens in MSM and transgender women. A rectal substudy was included to evaluate drug concentrations in rectal compartment vs. blood, gut-associated lymphoid tissue (GALT) responses to four antiretroviral PrEP regimens [maraviroc (MVC), MVC + emtricitabine (FTC), MVC + tenofovir (TFV) disoproxil fumarate, and TFV disoproxil fumarate + FTC], and to determine whether ARV exposure was associated with ex-vivo suppression of HIV infection in colorectal explants., Methods: C-C chemokine receptor type 5 (CCR5) genotype was characterized using PCR. At baseline and at Weeks 24, 48, and 49, GALT phenotype was characterized by flow cytometry, rectal biopsies were challenged with HIV-1BaL, and tissue and plasma pharmacokinetics were measured via mass spectrometry., Results: Exposure to MVC was not associated with increased expression of CD4+/CCR5+ HIV target T cells. Significant ex-vivo viral suppression compared with baseline was seen at Weeks 24 and 48, ranging from 1.4 to 1.8 log10 for all study regimens except the MVC-alone arm which did not show statistically significant viral suppression at Week 48. Tissue concentrations of TFV, TFV-diphosphate, and FTC were correlated with viral suppression., Conclusion: MVC-containing HIV PrEP regimens did not increase GALT CD4+ T-cell activation or the CD4+/CCR5+ phenotype. No virologic suppression was seen with MVC-alone at Week 48 compared with combination regimens, suggesting MVC monotherapy might be less effective than combination antiretroviral PrEP regimens.

Objectives: Despite plasma virologic suppression with antiretroviral therapy (ART), HIV persists in gut tissue. The objectives of this study were to compare plasma and rectal tissue HIV RNA dynamics and to assess relationships with dolutegravir (DTG) plasma and tissue concentrations., Design: A longitudinal cohort study of HIV-infected treatment-naïve individuals initiating DTG-based ART was conducted over 12 weeks with plasma and rectal tissue sampling (Clinicaltrials.gov:NCT02924389)., Methods: HIV RNA and DTG concentrations were quantified in plasma and rectal tissue samples collected pre-ART (baseline) and post-ART at weeks 2, 6, and 12 using Abbott Real-Time HIV-1 assays and high-performance liquid chromatography tandem mass spectroscopy, respectively. Relationships between rectal tissue RNA and DTG concentrations were modeled using binary logistic regression, controlling for repeated measures., Results: Twelve participants were enrolled: six (50.0%) women, nine (75.0%) black, median age 42.0 years (Q1 31.2, Q3 52.0). All attained plasma virologic suppression by week 6. 11 of 12 (91.7%) had detectable rectal tissue HIV RNA at baseline, and only three of 11 (27.3%) achieved rectal tissue virologic suppression at any time-point. Compared with rectal tissue nonsuppressors, three of three (100.0%) of rectal tissue suppressors were women, had higher BMI, 35.9 kg/m (range 24.9-38.5) versus 20.6 (17.7-29.9), P = 0.05, and lower baseline log plasma HIV RNA: 3.7 copies/ml (range 3.6-4.4) versus 4.7 (3.8-5.4), P = 0.02. No significant relationships between rectal tissue RNA suppression and DTG concentrations were seen., Conclusion: Rectal tissue HIV RNA persisted in most participants and was not predicted by DTG concentrations. Impact of host factors, particularly sex, on tissue HIV viral dynamics warrants further exploration.

Background: Long-acting injectable antiretroviral agents are being developed for HIV-1 prevention. The MWRI-01 study was done to characterise the safety, acceptability, and pharmacokinetic and pharmacodynamic profile of long-acting rilpivirine., Methods: We did a phase 1 open-label study at the University of Pittsburgh. We enrolled healthy individuals (aged 18-45 years) who were seronegative for HIV-1. Participants were assigned alternately one intramuscular dose of either 1200 mg or 600 mg long-acting rilpivirine, beginning with the 1200 mg dose. We obtained plasma specimens, genital and rectal fluids, and tissue samples (rectal, cervical, and vaginal) before and after exposure to long-acting rilpivirine for assessment of pharmacokinetics and ex-vivo biopsy challenge with HIV-1. Our primary objective was to characterise product safety, and the analysis included all enrolled participants. This trial is registered with ClinicalTrials.gov, number NCT01656018., Findings: 36 participants were enrolled into the study, of whom 24 were women and 12 men. 12 women and six men received each dose. 204 adverse events were reported among the 36 participants, of which 200 (98%) were grade 1-2. The most common adverse event was injection site reaction. All grade 3 and 4 adverse events were deemed not related to rilpivirine. Geometric mean (90% CI) concentrations in plasma of rilpivirine at day 28 post dose were 53 ng/mL (38-67) in women and 43 ng/mL (23-63) in men for the 1200 mg dose and 28 ng/mL (19-37) in women and 17 ng/mL (9-24) in men for the 600 mg dose. The tissue-to-plasma ratio for rilpivirine in rectal tissue was about two-fold higher than in vaginal and cervical tissue (1·10-1·53 vs 0·61-0·72 and 0·50-0·71, respectively). Exposure to long-acting rilpivirine suppressed viral replication significantly in rectal tissue (p<0·0001), and this suppression persisted for up to 4 months. By contrast, no viral suppression was seen in cervical or vaginal tissue., Interpretation: Ongoing research will characterise longer term safety and acceptability of multiple injections and help ascertain whether long-acting rilpivirine should advance to assessment of efficacy in preventing HIV-1 infection., Funding: Bill & Melinda Gates Foundation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The pharmacokinetics (PK) of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP), the active anabolites of tenofovir disoproxil fumarate (TDF), and emtricitabine (FTC) in blood, genital, and rectal compartments was determined in HIV-positive and seronegative adults who undertook a 60-day intensive PK study of daily TDF/FTC (plus efavirenz in HIV positives). Lymphocyte cell sorting, genital, and rectal sampling occurred once per subject, at staggered visits. Among 19 HIV-positive (3 female) and 21 seronegative (10 female) adults, TFV-DP in peripheral blood mononuclear cells (PBMC) accumulated 8.6-fold [95% confidence interval (CI): 7.2-10] from first-dose to steady-state concentration (Css) versus 1.7-fold (95% CI: 1.5-1.9) for FTC-TP. Css was reached in ∼11 and 3 days, respectively. Css values were similar between HIV-negative and HIV-positive individuals. Css TFV-DP in rectal mononuclear cells (1,450 fmol/10 6 cells, 898-2,340) was achieved in 5 days and was >10 times higher than PBMC (95 fmol/10 6 cells, 85-106), seminal cells (22 fmol/10 6 cells, 6-79), and cervical cells (111 fmol/10 6 cells, 64-194). FTC-TP Css was highest in PBMC (5.7 pmol/10 6 cells, 5.2-6.1) and cervical cells (7 pmol/10 6 cells, 2-19) versus rectal (0.8 pmol/10 6 cells, 0.6-1.1) and seminal cells (0.3 pmol/10 6 cells, 0.2-0.5). Genital drug concentrations on days 1-7 overlapped with estimated Css, but accumulation characteristics were based on limited data. TFV-DP and FTC-TP in cell sorted samples were highest and achieved most rapidly in CD14 + compared with CD4 + , CD8 + , and CD19 + cells. Together, these findings demonstrate cell-type and tissue-dependent cellular pharmacology, preferential accumulation of TFV-DP in rectal mononuclear cells, and rapid distribution into rectal and genital compartments., Competing Interests: Author Disclosure Statement Peter Anderson receives contract work and grant support from Gilead Sciences, paid to his institution. All other authors report no competing financial interests exit.

Objectives: Pharmacokinetic studies in animal models are important for assessing the prophylactic potential of antiretroviral drugs for HIV prevention. This study sought to identify clinically relevant doses of the marketed integrase inhibitors raltegravir, elvitegravir and dolutegravir in macaques and investigate drug penetration and antiviral activity in mucosal secretions., Methods: Macaques received one oral dose of raltegravir, elvitegravir or dolutegravir alone or in combination with emtricitabine and tenofovir disoproxil fumarate followed by drug level measurements in blood and rectal and vaginal secretions. Antiviral activity was investigated in TZM-bl cells exposed to SHIV162p3 in the presence of rectal secretions collected from treated animals., Results: Plasma drug concentrations with 50 mg/kg raltegravir or elvitegravir were within the range seen in humans receiving 400-800 mg of raltegravir or 800 mg of unboosted elvitegravir but lower than with 150 mg of elvitegravir boosted with cobicistat. AUC0-24 values for dolutegravir increased proportionally with the dose, with a calculated human-equivalent dose of 20 mg/kg. Elvitegravir showed the highest penetration in rectal and vaginal fluids despite the absence of pharmacological boosting, followed by raltegravir and dolutegravir. Rectal secretions collected at 24 h from treated macaques blocked infection of TZM-bl cells by 50% at dilutions of 1/1000 (raltegravir), 1/800 (dolutegravir) and >1/30 000 (elvitegravir)., Conclusions: We defined macaque doses of HIV integrase inhibitors that recapitulate human clinical doses, which will facilitate efficacy and dose escalation studies in macaques. High and sustained drug concentrations and activity in mucosal secretions suggest that integrase inhibitors are promising candidates for HIV prevention., (Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: This study estimated the number of daily tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) doses required to achieve and maintain (after discontinuation) intracellular drug concentrations that protect against human immunodeficiency virus (HIV) infection for men who have sex with men (MSM)., Methods: Tenofovir diphosphate (TFV-DP) concentrations in peripheral blood mononuclear cells (PBMCs) and rectal mononuclear cells from an intensive pharmacokinetic study ("Cell-PrEP" [preexposure prophylaxis]) of 30 days of daily TDF/FTC followed by 30 days off drug were evaluated. A regression formula for HIV risk reduction derived from PBMCs collected in the preexposure prophylaxis initiative study was used to calculate inferred risk reduction. The time required to reach steady state for TFV-DP in rectal mononuclear cells was also determined., Results: Twenty-one HIV-uninfected adults participated in Cell-PrEP. The inferred HIV risk reduction, based on PBMC TFV-DP concentration, reached 99% (95% confidence interval [CI], 69%-100%) after 5 daily doses, and remained >90% for 7 days after stopping drug from steady-state conditions. The proportion of participants reaching the 90% effective concentration (EC90) was 77% after 5 doses and 89% after 7 doses. The percentage of steady state for natural log [TFV-DP] in rectal mononuclear cells was 88% (95% CI, 66%-94%) after 5 doses and 94% (95% CI, 78%-98%) after 7 doses., Conclusions: High PrEP activity for MSM was achieved by approximately 1 week of daily dosing. Although effective intracellular drug concentrations persist for several days after stopping PrEP, a reasonable recommendation is to continue PrEP dosing for 4 weeks after the last potential HIV exposure, similar to recommendations for postexposure prophylaxis., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Combination microbicide vaginal rings may be more effective than single microbicide rings at reducing/preventing sexual transmission of HIV. Here, we report the pre-clinical development and macaque pharmacokinetics of matrix-type silicone elastomer vaginal rings containing dapivirine and darunavir., Methods: Macaque rings containing 25 mg dapivirine, 100 mg dapivirine, 300 mg darunavir or 100 mg dapivirine+300 mg darunavir were manufactured and characterized by differential scanning calorimetry. In vitro release was assessed into isopropanol/water and simulated vaginal fluid. Macaque vaginal fluid and blood serum concentrations for both antiretrovirals were measured during 28 day ring use. Tissue levels were measured on day 28. Ex vivo challenge studies were performed on vaginal fluid samples and IC50 values were calculated., Results: Darunavir caused a concentration-dependent reduction in the dapivirine melting temperature in both solid drug mixes and in the combination ring. In vitro release from rings was dependent on drug loading, the number of drugs present and the release medium. In macaques, serum concentrations of both microbicides were maintained between 10(1) and 10(2) pg/mL. Vaginal fluid levels ranged between 10(3) and 10(4) ng/g and between 10(4) and 10(5) ng/g for dapivirine and darunavir, respectively. Both dapivirine and darunavir showed very similar concentrations in each tissue type; the range of drug tissue concentrations followed the general rank order: vagina (1.8 × 10(3)-3.8 × 10(3) ng/g)  > cervix (9.4 × 10(1)-3.9 × 10(2) ng/g)  > uterus (0-108 ng/g)  > rectum (0-40 ng/g). Measured IC50 values were >2 ng/mL for both compounds., Conclusions: Based on these results, and in light of recent clinical progress of the 25 mg dapivirine ring, a combination vaginal ring containing dapivirine and darunavir is a viable second-generation HIV microbicide candidate., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Antiretroviral therapy has become a central component of combination in HIV prevention efforts. Defining the individual exposure of commercially available antiretroviral therapy in genital secretions and vulnerable mucosal tissues is paramount to designing future prevention interventions., Methods: A pharmacokinetic (PK) study was performed in 12 HIV-negative men receiving 600 mg of darunavir, 100 mg of ritonavir, and 200 mg of etravirine orally, twice daily for 8 days. Seven blood plasma (BP) samples were collected over 12 hours on day 1 (PK1) and days 7 and 8 (PK2). One rectal tissue (RT) sample from each subject was collected during PK1 and PK2. During PK1, 2 seminal plasma (SP) samples were collected from each subject. During PK2, 6 SP samples were collected from each subject over 2 days., Results: Antiretrovirals were detected in SP and RT within 1 hour after a single dose. Over PK1 and PK2, SP exposures were lower than BP by 80%-92% (DRV), 89-95% (RTV), and 83-88% (ETR). However, protein binding in SP (14% for darunavir, 70% for ritonavir, and 97% for etravirine) was lower than in BP. Rectal tissue exposures were higher than BP by 39- to 155-fold for darunavir, 12- to 61-fold for ritonavir, and 20- to 40-fold for etravirine., Conclusions: Lower SP protein binding resulted in higher pharmacologically active darunavir and etravirine concentrations compared with BP. High RT concentrations may also be favorable for suppressing viral replication in the gastrointestinal mucosa. The high protein-unbound exposures in SP and total exposures in RT support further investigations of darunavir plus ritonavir and etravirine in secondary prevention.