Ex-vivo rectal tissue infection with HIV-1 to assess time to protection following oral preexposure prophylaxis with tenofovir disoproxil/emtricitabine.

OBJECTIVES: We wished to assess time to protection from HIV-1 infection following oral tenofovir disoproxil and emtricitabine (TDF/FTC) as preexposure prophylaxis (PrEP), using ex-vivo rectal tissue infections and drug concentration measures in blood and rectal tissue. DESIGN/METHODS: Participants from the ANRS PREVENIR study (NCT03113123) were offered this sub-study after a 14-day wash-out. We used an ex-vivo model to evaluate rectal tissue HIV-1 susceptibility before and after PrEP, 2 h after two pills or 7 days of a daily pill of TDF/FTC. PrEP efficacy was expressed by the difference (after-before) of 14-day cumulative p24 antigen levels. TFV-DP and FTC-TP levels were measured in rectal tissue and PBMCs and correlated with HIV-1 infection. RESULTS: Twelve and 11 men were analyzed in the 2 h-double dose and 7 days-single dose groups, respectively. Cumulative p24 differences after-before PrEP were -144 pg/ml/mg (IQR[-259;-108]) for the 2 h-double dose group ( P  = 0.0005) and -179 pg/ml/mg (IQR [-253;-86]) for the 7 days-single dose group ( P  = 0.001), with no differences between groups ( P  = 0.93). Rectal TFV-DP was below quantification after a double dose, but FTC-TP levels were similar to levels at 7 days. There was a significant correlation between rectal FTC-TP levels and p24 changes after a double dose ( R  = -0.84; P  = 0.0001). CONCLUSION: Oral TDF/FTC provided similar protection against HIV-1 infection of rectal tissue 2 h after a double dose or 7 days of a daily dose. At 2 h, this protection seems driven by high FTC-TP concentrations in rectal tissue. This confirms the importance of combining TDF and FTC to achieve early protection.

A Randomized, Open-Label, Crossover Phase 1 Safety and Pharmacokinetic Study of Oral Maraviroc and Maraviroc 1% Gel (the CHARM-03 Study).

The Combination HIV Antiretroviral Rectal Microbicide-3 (CHARM-03) study was a randomized, open-label, crossover Phase 1 safety and pharmacokinetic (PK) study of oral maraviroc (MVC) and MVC 1% gel. At a single site, healthy HIV-uninfected men and women were enrolled and randomized to an open label crossover sequence of eight consecutive daily exposures to MVC 300 mg dosed orally, MCV 1% gel dosed rectally, and MVC 1% gel dosed vaginally. Male participants received oral and rectal dosing and female participants received oral, rectal, and vaginal dosing. Assessments were undertaken at baseline and following each 8-day period and included collection of plasma, rectal/cervical tissue (CT), and rectal/endocervical/vaginal fluids. Eleven men and nine women were enrolled. Two participants withdrew from the study before receiving study product. There were 25 adverse events, of which 24 were Grade 1 (G1) and one was G2 (unrelated). After eight doses, MVC was quantifiable in all samples following oral, rectal, or vaginal product administration. The highest drug concentrations in plasma, rectal tissue (RT), and CT were associated with oral, rectal, and vaginal drug delivery, respectively. There were significant reductions in tissue drug concentrations when rectal and cervical biopsies were incubated in media before tissue processing for PK (p < .0001). Only oral MVC was associated with limited protection in the rectal explant HIV challenge model (p < .05). There were no immunological changes in RT, and all products were acceptable to participants. In conclusion, all products were found to be safe and acceptable and did not induce local inflammation. The lack of ex vivo efficacy demonstrated in study samples may be due to rapid disassociation of MVC from the explant tissue. ClinicalTrials.gov Identifier: NCT02346084.

Tofacitinib inhibits inflammatory cytokines from ulcerative colitis and healthy mucosal explants and is associated with pSTAT1/3 reduction in T-cells.

Poor translatability of animal disease models has hampered the development of new inflammatory bowel disorder (IBD) therapeutics. We describe a preclinical, ex vivo system using freshly obtained and well-characterized human colorectal tissue from patients with ulcerative colitis (UC) and healthy control (HC) participants to test potential therapeutics for efficacy and target engagement, using the JAK/STAT inhibitor tofacitinib (TOFA) as a model therapeutic. Colorectal biopsies from HC participants and patients with UC were cultured and stimulated with multiple mitogens ± TOFA. Soluble biomarkers were detected using a 29-analyte multiplex ELISA. Target engagement in CD3+CD4+ and CD3+CD8+ T-cells was determined by flow cytometry in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMCs) following the activation of STAT1/3 phosphorylation. Data were analyzed using linear mixed-effects modeling, t test, and analysis of variance. Biomarker selection was performed using penalized and Bayesian logistic regression modeling, with results visualized using uniform manifold approximation and projection. Under baseline conditions, 27 of 29 biomarkers from patients with UC were increased versus HC participants. Explant stimulation increased biomarker release magnitude, expanding the dynamic range for efficacy and target engagement studies. Logistic regression analyses identified the most representative UC baseline and stimulated biomarkers. TOFA inhibited biomarkers dependent on JAK/STAT signaling. STAT1/3 phosphorylation in T-cells revealed compartmental differences between PBMCs and MMCs. Immunogen stimulation increases biomarker release in similar patterns for HC participants and patients with UC, while enhancing the dynamic range for pharmacological effects. This work demonstrates the power of ex vivo human colorectal tissue as preclinical tools for evaluating target engagement and downstream effects of new IBD therapeutic agents.NEW & NOTEWORTHY Using colorectal biopsy material from healthy volunteers and patients with clinically defined IBD supports translational research by informing the evaluation of therapeutic efficacy and target engagement for the development of new therapeutic entities. Combining experimental readouts from intact and dissociated tissue enhances our understanding of the tissue-resident immune system that contribute to disease pathology. Bayesian logistic regression modeling is an effective tool for predicting ex vivo explant biomarker release patterns.

A Multiple Dose Phase 1 Assessment of Rilpivirine Long Acting in a Model of Preexposure Prophylaxis Against HIV.

The MWRI-01 study characterized the safety, acceptability, pharmacokinetic (PK), and pharmacodynamic (PD) profile of rilpivirine (RPV) long acting (LA) in a model of preexposure prophylaxis (PrEP). Prospective, open-label Phase 1 study. The safety and acceptability of three repeated doses of RPV LA were monitored. Blood, tissue (rectal, cervical, and vaginal), and biological fluids (vaginal and endocervical) were collected at baseline and at 1- to 2-month intervals throughout the study for PK and PD assessment. Eight women and four men received three intramuscular doses of 1,200 mg of RPV LA given 8 weeks apart. There were a total of 195 adverse events (AEs) reported, of which 138 (70.8%) were Grade 1 and 55 (28.2%) were Grade 2. The most common AE was injection site pain. Geometric mean (90% confidence interval) plasma RPV concentrations at 56 days after the first and third doses were 39 (33-45) ng/mL (female)/29 (17-40) ng/mL (male) and 59 (45-62) ng/mL (female)/40 (30-51) ng/mL (male), respectively. Exposure to RPV LA was associated with significant inhibition of HIV-1BaL viral replication in the ex vivo rectal explant model (p < .0001) that persisted for up to 4 months after the third dose of RPV LA. In contrast, no viral suppression was seen in cervicovaginal tissue. Multiple dose administration of RPV LA was safe and well tolerated, and was associated with prolonged suppression of viral replication in rectal explant tissue.

The Use of Droplet Digital PCR to Quantify HIV-1 Replication in the Colorectal Explant Model.

Ex vivo explant models are used to characterize in vitro efficacy of preexposure prophylaxis (PrEP) agents. Tissue is challenged with virus in culture and HIV-1 p24 levels are quantified with enzyme-linked immunosorbent assay (ELISA) on supernatants collected throughout a 14-21-day incubation. Due to the narrow dynamic range of HIV-1 p24 kits, we evaluated whether droplet digital PCR (ddPCR) provides an alternative method to quantify HIV-1 replication in supernatant samples. We used samples from the MWRI-01 study, which evaluated the pharmacokinetic/pharmacodynamic profile of long-acting rilpivirine using the explant model (McGowan et al. Lancet HIV 2016). HIV-1 pol RNA was measured with ddPCR, either directly with a one-step method or reverse transcribed to cDNA before ddPCR (two-step method) on supernatants from the MWRI-01 study. Previously analyzed HIV-1 p24 antigen levels (Alliance; Perkin-Elmer) were available for comparison purposes. Both ddPCR methods strongly correlated with HIV-1 p24 and displayed similar patterns of HIV-1 suppression before and after rilpivirine. Compared to the p24 ELISA, two-step and one-step ddPCR reduced the amount of hands-on time by approximately one-half and two-thirds, respectively. ddPCR also required less sample and based on p24 versus ddPCR correlation, could potentially reduce the explant culture time from 14 to 10 days (r2 = 0.78, p < .001) due to the increased sensitivity of ddPCR. We demonstrate that ddPCR is a suitable alternative to HIV-1 p24 ELISA to quantify HIV-1 infection in the explant model and has the potential to decrease explant culture time.

Prevalence and determinants of anal human papillomavirus infection in men who have sex with men and transgender women.

Human papillomavirus (HPV) prevalence varies by population. This study investigated anal HPV type detection risk by country in a population of men who have sex with men (MSM) and transgender women (TW) at risk of HIV. Sexually active HIV-1-uninfected MSM and TW were enrolled at eight sites: four in the United States (US), two in Thailand, one in Peru, and one in South Africa. Baseline anal HPV swabs were collected, and DNA typing was performed. One hundred and ninety-five participants, 76 (42%) from the US, had a mean age of 30.9 years (range 18-64). In 182 participants with results available, anal HPV infection was common with 169 (93%) with ≥1 type, 132 (73%) with ≥1 nine-valent vaccine types, and 66 (36%) with HPV 16. Participants in the US had a higher prevalence of HPV 16 (56%, p = 0.004) and HPV 6 (69%, p < 0.001) compared to the other regions. Stimulant drug use was significantly associated with HPV 6 detection. Anal HPV is highly prevalent in this population of MSM and TW sampled from four countries, with HPV 16 the most commonly detected type. The nine-valent HPV vaccine has the potential to provide significant protection if given prior to exposure.

Immunological responsiveness of intestinal tissue explants and mucosal mononuclear cells to ex vivo stimulation.

BACKGROUND: There are limited data on the immunological responsiveness of healthy intestinal tissue when it is cultured and stimulated ex vivo. Such an ex vivo model has the potential to be a valuable tool in understanding disease pathogenesis and as a preclinical tool for the assessment of candidate therapeutic agents used to treat inflammatory bowel disease (IBD). AIM: We undertook a comprehensive study to evaluate ex vivo immunological responses of intestinal tissue and isolated mucosal mononuclear cells (MMC) to a broad range of stimuli. METHODS: Colorectal biopsies (explants) were obtained from healthy participants by flexible sigmoidoscopy and were placed either directly into culture or digested to isolate MMC prior to placement in culture. Explants or MMC were treated with polyinosinic:polycytidylic acid (Poly IC), phytohemagglutinin (PHA), lipopolysacccharides from E Coli (LPS), anti-CD3/CD28 antibodies, or IL-1ß/TNF-α for 24 h. Supernatants were assayed for 40 inflammatory biomarkers using multiplexed enzyme-linked immunosorbent assay (ELISA). The isolated MMCs were further characterized using twelve color flow cytometry. RESULTS: Explants have greater weight adjusted constitutive expression of inflammatory biomarkers than MMCs. Biomarker responses varied as a function of immunogen and use of intact tissue or isolated cells. PHA applied to intact explants was the most effective agent in inducing biomarker changes. Stimulation induced activated and memory cellular phenotypes in both explants and MMCs. CONCLUSIONS: The breadth and magnitude of responses from intact and enzymatically digested intestinal tissue explants stimulated with exogenous immunogens are complex and vary by tissue form and treatment. Overall, PHA stimulation of intact explants produced the most robust responses in normal human colorectal tissue. This system could potentially serve as a preliminary model of the disease state, suitable for small scale screening of new therapeutic agents prior to using IBD patient derived tissue.

Ranpirnase Reduces HIV-1 Infection and Associated Inflammatory Changes in a Human Colorectal Explant Model.

Ranpirnase (RNP) is a low molecular weight type III endoribonuclease, which demonstrates broad antiviral and antitumor properties. We sought to characterize the antiviral activity of RNP against HIV-1 and to determine whether RNP modulates local inflammatory changes associated with HIV infection in the colorectal explant model. Colorectal explants were incubated for 2 h with HIV-1BaL, in the presence of increasing concentrations of RNP (0-60 µg/mL). After washing, explants were cultured for 14 days, with supernatant collected at days 3, 7, 10, and 14. All samples were assayed for HIV-1 p24. Additionally, 30 soluble inflammatory biomarkers were assayed in the day 3 supernatant sample. Other biopsies were stimulated with lipopolysaccharides (LPS) (10 µg/mL) in the presence of RNP and soluble biomarkers assayed at day 3. RNP inhibited productive infection of the colorectal explants with HIV-1BaL and induced a dose-dependent decrease in 15/30 biomarkers. Affected biomarkers included IP-10, MDC, MIP-1α, MIP-1ß, TARC, IL12-p40, IL-15, IL-17, IL-1α, IL-7, IFNγ, IL12-p70, IL-1ß, IL-4, IL-5, and TNF-ß. Similarly, RNP dose-dependent inhibition was demonstrated in 7/30 biomarkers after LPS stimulation, all of which overlapped with HIV-1BaL-induced biomarker changes. The ability of RNP to inhibit both colorectal explant HIV-1BaL infection and inflammatory changes associated with HIV-1 infection makes RPN a promising agent for topical rectal pre-exposure prophylaxis.

A Pilot Study of the Immunologic, Virologic, and Pathologic Consequences of Intra-anal 5% Imiquimod in HIV-1-Infected Men With High-Grade Squamous Intraepithelial Lesions.

BACKGROUND: Imiquimod can be used to treat internal anal high-grade squamous intraepithelial lesions. In HIV-1-infected individuals there is a theoretical concern for increased HIV replication in anorectal tissue secondary to imiquimod-induced mucosal inflammation. OBJECTIVE: The purpose of this study was to assess local virologic, immunologic, and pathologic effects of imiquimod treatment in HIV-infected individuals. DESIGN: This was a pilot study at a single academic center. SETTINGS: The study was conducted at the University of Pittsburgh Anal Dysplasia Clinic. PATIENTS: HIV-1-infected individuals with biopsy-confirmed internal anal high-grade squamous intraepithelial lesions were included. INTERVENTION: Imiquimod cream was prescribed for intra-anal use 3 times per week for 9 weeks. MAIN OUTCOME MEASURES: Anal human papillomavirus typing, anal and rectal tissue HIV-1 RNA and DNA quantification, cytokine gene expression, and anal histology were measured. RESULTS: Nine evaluable participants (1 participant was lost to follow-up) were all white men with a median age of 46 years (interquartile range = 12 y) and a median CD4 T-cell count of 480 cells per cubic millimeter (interquartile range = 835). All were taking antiretroviral therapy, and 7 of 9 had HIV-1 RNA <50 copies per milliliter. The median dose of imiquimod used was 27.0 (interquartile range = 3.5), and there was a median of 11 days (interquartile range = 10 d) from last dose to assessment. There was no progression to cancer, no significant change in the number of human papillomavirus types detected, and no significant change in quantifiable cytokines/HIV-1 RNA or DNA levels in anal or rectal tissue. Seven (35%) of 20 high-grade lesions resolved to low-grade squamous intraepithelial lesions. LIMITATIONS: The study was limited by the small number of participants and variable time to final assessment. CONCLUSIONS: Intra-anal imiquimod showed no evidence of immune activation or increase in HIV-1 viral replication in anal and rectal tissue and confirmed efficacy for intra-anal high-grade squamous intraepithelial lesion treatment morbidity. See Video Abstract at http://links.lww.com/DCR/A498.

Long-acting rilpivirine as potential pre-exposure prophylaxis for HIV-1 prevention (the MWRI-01 study): an open-label, phase 1, compartmental, pharmacokinetic and pharmacodynamic assessment.

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.

Project Gel a Randomized Rectal Microbicide Safety and Acceptability Study in Young Men and Transgender Women.

OBJECTIVES: The purpose of Project Gel was to determine the safety and acceptability of rectal microbicides in young men who have sex with men (MSM) and transgender women (TGW) at risk of HIV infection. METHODS: MSM and TGW aged 18-30 years were enrolled at three sites; Pittsburgh, PA; Boston, MA; and San Juan, PR. Stage 1A was a cross-sectional assessment of sexual health and behavior in MSM and TGW. A subset of participants from Stage 1A were then enrolled in Stage 1B, a 12-week evaluation of the safety and acceptability of a placebo rectal gel. This was followed by the final phase of the study (Stage 2) in which a subset of participants from Stage 1B were enrolled into a Phase 1 rectal safety and acceptability evaluation of tenofovir (TFV) 1% gel. RESULTS: 248 participants were enrolled into Stage 1A. Participants' average age was 23.3 years. The most common sexually transmitted infection (STIs) at baseline were Herpes simplex (HSV)-2 (16.1% by serology) and rectal Chlamydia trachomatis (CT) (10.1% by NAAT). 134 participants were enrolled into Stage 1B. During the 12 week period of follow-up 2 HIV, 5 rectal CT, and 5 rectal Neisseria gonorrhea infections were detected. The majority of adverse events (AEs) were infections (N = 56) or gastrointestinal (N = 46) and were mild (69.6%) or moderate (28.0%). Of the participants who completed Stage 1B, 24 were enrolled into Stage 2 and randomized (1:1) to receive TFV or placebo gel. All participants completed Stage 2. The majority of AEs were gastrointestinal (N = 10) and of mild (87.2%) or moderate (10.3%) severity. CONCLUSIONS: In this study we were able to enroll a sexually active population of young MSM and TGW who were willing to use rectal microbicides. TFV gel was safe and acceptable and should be further developed as an alternative HIV prevention intervention for this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT01283360.

Prevalence of Anal Human Papillomavirus Vaccine Types in the Bangkok Men Who Have Sex With Men Cohort Study.

BACKGROUND: The quadrivalent human papillomavirus (qHPV) and 9 valent (nHPV) vaccine are licensed for males to prevent anal HPV-associated dysplasia and cancer caused by HPV types 6, 11, 16, and 18 (qHPV) and additional types 33, 35, 45, 52, and 58 (nHPV), respectively. Both conditions are common in HIV-infected and HIV-uninfected men who have sex with men (MSM). It is not well documented which anal HPV vaccine types are most prevalent in Southeast Asia. METHODS: A convenience sample of 400 anal swabs were obtained from 200 HIV-infected and 200 HIV-uninfected sexually active Bangkok MSM Cohort Study participants. After swab collection in PreservCyt (Cytyc Corp, Marlborough, MA), the media was stored at -80°C until processing. DNA was extracted, amplified by polymerase chain reaction, denatured, and then hybridized to probes for 37 HPV types and ß-globin. RESULTS: The mean participant age was 25.6 years (range, 18-55 years); the mean CD4 T-cell count was 410 cells/mm in the HIV-infected participants. Among all swab samples, 386 (192 HIV-positive and 194 HIV-negative) had adequate ß-globin for HPV genotype testing. Anal HPV type was detected in 44.3% of participants whose samples underwent genotype testing. Both qHPV and nHPV types were more frequently detected in HIV-infected compared with HIV-uninfected (42.2% vs. 23.2% [P < 0.01], 50.0% vs. 24.2% [P < 0.01]), respectively). There were no significant relationships between social behaviors (alcohol use, drug use) or sexual behaviors (number of partners, condom usage, sexual positioning) and anal HPV prevalence. CONCLUSIONS: The prevalence of anal vaccine HPV types in Thai MSM was similar to that reported in MSM from Western populations and has a similar distribution by HIV status. Targeting young MSM with vaccination could offer protection against HPV vaccine types.

A Phase 1 Randomized, Open Label, Rectal Safety, Acceptability, Pharmacokinetic, and Pharmacodynamic Study of Three Formulations of Tenofovir 1% Gel (the CHARM-01 Study).

OBJECTIVES: The CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study. METHODS: Participants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial. RESULTS: All three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/106 cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection. CONCLUSIONS: All three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection. TRIAL REGISTRATION: ClinicalTrials.gov NCT01575405.