A Phase 1 Clinical Trial to Assess the Safety and Pharmacokinetics of a Tenofovir Alafenamide/Elvitegravir Insert Administered Rectally for HIV Prevention.

BACKGROUND: On-demand topical products could be an important tool for human immunodeficiency virus (HIV) prevention. We evaluated the safety, pharmacokinetics, and ex vivo pharmacodynamics of a tenofovir alafenamide/elvitegravir (TAF/EVG, 20 mg/16 mg) insert administered rectally. METHODS: MTN-039 was a phase 1, open-label, single-arm, 2-dose study. Blood, rectal fluid, and rectal tissue were collected over 72 hours following rectal administration of 1 and 2 TAF/EVG inserts for each participant. RESULTS: TAF/EVG inserts were safe and well tolerated. EVG and tenofovir (TFV) were detected in blood plasma at low concentrations: median peak concentrations after 2 inserts were EVG 2.4 ng/mL and TFV 4.4 ng/mL. Rectal tissue EVG peaked at 2 hours (median, 2 inserts = 9 ng/mg) but declined to below limit of quantification in the majority of samples at 24 hours, whereas tenofovir diphosphate (TFV-DP) remained high >2000 fmol/million cells for 72 hours with 2 inserts. Compared to baseline, median cumulative log10 HIV p24 antigen of ex vivo rectal tissue HIV infection was reduced at each time point for both 1 and 2 inserts (P < .065 and P < .039, respectively). DISCUSSION: Rectal administration of TAF/EVG inserts achieved high rectal tissue concentrations of EVG and TFV-DP with low systemic drug exposure and demonstrable ex vivo inhibition of HIV infection for 72 hours. Clinical Trials Registration . NCT04047420.

Extended Postexposure Protection Against Vaginal Simian/Human Immunodeficiency Virus Infection With Tenofovir Alafenamide Fumarate/Elvitegravir Inserts in Macaques.

Vaginal inserts that can be used on demand before or after sex may be a desirable human immunodeficiency virus (HIV) prevention option for women. We recently showed that inserts containing tenofovir alafenamide fumarate (TAF, 20 mg) and elvitegravir (EVG, 16 mg) were highly protective against repeated simian/human immunodeficiency virus (SHIV) vaginal exposures when administered to macaques 4 hours before or after virus exposure (93% and 100%, respectively). Here, we show in the same macaque model that insert application 8 hours or 24 hours after exposure maintains high efficacy (94.4% and 77.2%, respectively). These data extend the protective window by TAF/EVG inserts and inform their clinical development for on-demand prophylaxis in women.

Preferences for Long-Acting PrEP Products Among Women and Girls: A Quantitative Survey and Discrete Choice Experiment in Eswatini, Kenya, and South Africa.

While oral pre-exposure prophylaxis (PrEP) can substantially reduce HIV risk, there are important barriers to uptake and adherence. We explored preferences for long-acting injectable and implantable PrEP among women and girls in Eswatini, Kenya, and South Africa. We conducted an online quantitative survey and discrete choice experiment (DCE) among adolescent girls (15-17), young women (18-29), and adult women (30-49). Participants completed a survey about their demographics and behavior and a DCE with 5 attributes (format, insertion location, number of insertions, dual-protection, and palpability). We recruited 1236 respondents (Eswatini = 420; Kenya = 350; South Africa = 493) in May 2022. Most participants were sexually active (72%), nearly 29% of whom reported recently engaging in transactional sex. 46% had heard of oral PrEP, but of those, only 16% reported having ever used it. Product format and dual-protection were significant predictors of product choice. Relative to a 2-month injection, participants had 1.76 times the odds (95% CI 1.08-2.04) of choosing a 6-month injectable, and 1.70 the odds (95% CI 1.06-1.92) of choosing a 12-month removable implant. Compared to a single-indication product, respondents had 2.46 times the odds (95% CI 1.04-2.68) of preferring a product also protecting against pregnancy, and 2.81 the odds (95% CI 1.04-3.05) of choosing a product that also protected against STIs. Adolescent girls and women in these countries showed strong preferences for longer-acting PrEP product formats, as well as those offering dual-protection. Introduction of long-acting options could improve PrEP uptake and reduce HIV burdens in east and southern African settings.

History of Rectal Product Use and Country of Residence Influence Preference for Rectal Microbicide Dosage Forms Among Young Sexual and Gender Minorities: A Multi-country Trial Comparing Placebo Douche, Suppository, and Insert Products.

The DESIRE Study (MTN-035) explored product preference among three placebo rectal microbicide (RM) formulations, a rectal douche (RD), a suppository, and an insert, among 210 sexually active transgender people and men who have sex with men in five counties: the United States, Peru, Thailand, South Africa, and Malawi. Participants used each product prior to receptive anal sex (RAS) for 1 month, following a randomly assigned sequence, then selected their preferred product via computer assisted self-interview. In-depth interviews examined reasons for preference. We compared product preference and prior product use by country to explore whether geographic location and experience with the similar products impacted preference. A majority in the United States (56%) and Peru (58%) and nearly half in South Africa (48%) preferred the douche. Most in Malawi (59%) preferred the suppository, while half in Thailand (50%) and nearly half in South Africa (47%) preferred the insert. Participants who preferred the douche described it as quick and easy, already routinized, and serving a dual purpose of cleansing and protecting. Those who preferred the insert found it small, portable, discreet, with quick dissolution. Those who preferred the suppository found the size and shape acceptable and liked the added lubrication it provided. Experience with product use varied by country. Participants with RD experience were significantly more likely to prefer the douche (p = 0.03). Diversifying availability of multiple RM dosage forms can increase uptake and improve HIV prevention efforts globally.

RESUMEN: El estudio DESIRE (MTN-035) exploró la preferencia de producto entre tres formulaciones de microbicida rectal (MR) de placebo, una ducha rectal, un supositorio y un inserto, entre 210 personas transgénero y hombres que tienen sexo con hombres en cinco países: los Estados Unidos, Perú., Tailandia, Sudáfrica y Malawi. Los participantes utilizaron cada producto antes del sexo anal receptive (SAR) durante un mes, siguiendo una secuencia asignada al azar, luego seleccionaron su producto preferido mediante una autoentrevista asistida por computadora. Las entrevistas en profundidad examinaron los motivos de preferencia. Comparamos la preferencia de producto y el uso previo del producto por país para explorar si la ubicación geográfica y la experiencia con la forma farmacéutica impactaron la preferencia. Una mayoría en los Estados Unidos (56%) y Perú (58%) y casi la mitad en Sudáfrica (48%) prefirieron la ducha rectal. La mayoría en Malawi (59%) prefirió el supositorio, mientras que la mitad en Tailandia (50%) y casi la mitad en Sudáfrica (47%) prefirió el inserto. Los participantes que prefirieron la ducha rectal la describieron como rápida y fácil, ya parte de su rutina y que tenía el doble propósito de limpiar y proteger. Los que prefirieron el inserto lo consideraron pequeño, portátil, discreto y de rápida disolución. Los que prefirieron el supositorio encontraron que tenía un tamaño y forma aceptables y proveía lubricación adicional. La experiencia con el uso del producto varió según el país. Los participantes con experiencia con duchas rectales tenían significativamente más probabilidades de preferir la ducha rectal (p = 0,03). Diversificar la disponibilidad de múltiples formas farmacéuticas de MR puede aumentar la aceptación y mejorar los esfuerzos de prevención del VIH a nivel mundial.

Cervical and systemic innate immunity predictors of HIV risk linked to genital herpes acquisition and time from HSV-2 seroconversion.

OBJECTIVES: To examine innate immunity predictors of HIV-1 acquisition as biomarkers of HSV-2 risk and biological basis for epidemiologically established HIV-1 predisposition in HSV-2 infected women. METHODS: We analysed longitudinal samples from HIV-1 negative visits of 1019 women before and after HSV-2 acquisition. We measured cervical and serum biomarkers of inflammation and immune activation previously linked to HIV-1 risk. Protein levels were Box-Cox transformed and ORs for HSV-2 acquisition were calculated based on top quartile or below/above median levels for all HSV-2 negative visits. Bivariate analysis determined the likelihood of HSV-2 acquisition by biomarker levels preceding infection. Linear mixed-effects models evaluated if biomarkers differed by HSV-2 status defined as negative, incident or established infections with an established infection cut-off starting at 6 months. RESULTS: In the cervical compartment, two biomarkers of HIV-1 risk (low SLPI and high BD-2) also predicted HSV-2 acquisition. In addition, HSV-2 acquisition was associated with IL-1ß, IL-6, IL-8, MIP-3α, ICAM-1 and VEGF when below median levels. Systemic immunity predictors of HSV-2 acquisition were high sCD14 and IL-6, with highest odds when concomitantly increased (OR=2.23, 1.49-3.35). Concomitant systemic and mucosal predictors of HSV-2 acquisition risk included (1) serum top quartile sCD14 with cervical low SLPI, VEGF and ICAM-1, or high BD-2; (2) serum high IL-6 with cervical low VEGF and ICAM-1, SLPI, IL-1ß and IL-6; and (3) serum low C reactive protein with cervical high BD-2 (the only combination also predictive of HIV-1 acquisition). Most cervical biomarkers were decreased after HSV-2 acquisition compared with the HSV-2 negative visits, with incident infections associated with a larger number of suppressed cervical biomarkers and lower serum IL-6 levels compared with established infections. CONCLUSIONS: A combination of systemic immunoinflammatory and cervical immunosuppressed states predicts HSV-2 acquisition. A persistently suppressed innate immunity during incident HSV-2 infection may add to the increased HIV-1 susceptibility.

Single-Administration Long-Acting Microarray Patch with Ultrahigh Loading Capacity and Multiple Releases of Thermally Stable Antibodies.

Current antibody (Ab) therapies require development of stable formulations and an optimal delivery system. Here, we present a new strategy to create a single-administration long-lasting Ab-delivery microarray (MA) patch, which can carry high doses of thermally stabilized Abs. The MA fabricated by an additive three-dimensional manufacturing technology can be fully embedded into the skin via a single application to deliver doses of Abs at multiple programmable time points, thus sustaining Ab concentrations in systemic circulation. We developed an MA formulation that stabilized and delivered human immunoglobulins (hIg) in a time-controlled manner while maintaining their structure and functionality. As an example, the b12 Ab─a broadly neutralizing Ab against HIV-1─maintained antiviral activity in vitro after MA manufacturing and heat exposure. Pharmacokinetic studies of MA patch-delivered hIg in rats successfully provided a proof of concept for concurrent and time-delayed Ab delivery. These MA patches codeliver different Abs, providing a tool for expanded protection against viral infections or combination HIV therapy and prevention.

Changes in concentrations of cervicovaginal immune mediators across the menstrual cycle: a systematic review and meta-analysis of individual patient data.

BACKGROUND: Hormonal changes during the menstrual cycle play a key role in shaping immunity in the cervicovaginal tract. Cervicovaginal fluid contains cytokines, chemokines, immunoglobulins, and other immune mediators. Many studies have shown that the concentrations of these immune mediators change throughout the menstrual cycle, but the studies have often shown inconsistent results. Our understanding of immunological correlates of the menstrual cycle remains limited and could be improved by meta-analysis of the available evidence. METHODS: We performed a systematic review and meta-analysis of cervicovaginal immune mediator concentrations throughout the menstrual cycle using individual participant data. Study eligibility included strict definitions of the cycle phase (by progesterone or days since the last menstrual period) and no use of hormonal contraception or intrauterine devices. We performed random-effects meta-analyses using inverse-variance pooling to estimate concentration differences between the follicular and luteal phases. In addition, we performed a new laboratory study, measuring select immune mediators in cervicovaginal lavage samples. RESULTS: We screened 1570 abstracts and identified 71 eligible studies. We analyzed data from 31 studies, encompassing 39,589 concentration measurements of 77 immune mediators made on 2112 samples from 871 participants. Meta-analyses were performed on 53 immune mediators. Antibodies, CC-type chemokines, MMPs, IL-6, IL-16, IL-1RA, G-CSF, GNLY, and ICAM1 were lower in the luteal phase than the follicular phase. Only IL-1α, HBD-2, and HBD-3 were elevated in the luteal phase. There was minimal change between the phases for CXCL8, 9, and 10, interferons, TNF, SLPI, elafin, lysozyme, lactoferrin, and interleukins 1ß, 2, 10, 12, 13, and 17A. The GRADE strength of evidence was moderate to high for all immune mediators listed here. CONCLUSIONS: Despite the variability of cervicovaginal immune mediator measurements, our meta-analyses show clear and consistent changes during the menstrual cycle. Many immune mediators were lower in the luteal phase, including chemokines, antibodies, matrix metalloproteinases, and several interleukins. Only interleukin-1α and beta-defensins were higher in the luteal phase. These cyclical differences may have consequences for immunity, susceptibility to infection, and fertility. Our study emphasizes the need to control for the effect of the menstrual cycle on immune mediators in future studies.

Recurrent bacterial vaginosis following metronidazole treatment is associated with microbiota richness at diagnosis.

BACKGROUND: Bacterial vaginosis-a condition defined by a shift from Lactobacillus dominance to a polymicrobial, anaerobic bacterial community-increases the risk of acquiring sexually transmitted infections and other complications of the female reproductive tract. Antibiotic treatment frequently fails to return the microbiome to an optimal Lactobacillus-dominated state. No criteria currently exist to identify the patients likely to experience treatment failure. OBJECTIVE: We sought to identify the pretreatment community signatures associated with treatment failure through 16S ribosomal RNA gene analysis. STUDY DESIGN: Twenty-eight women who were enrolled in an oral metronidazole treatment trial of bacterial vaginosis were studied. Cervicovaginal lavage samples were collected before metronidazole treatment and at 7 and 30 days posttreatment. Cervicovaginal lavage DNA was amplified and sequenced using a paired-end, V4 region 2×150 MiSeq run. RESULTS: Of the 28 women, 25% failed to clear bacterial vaginosis; 35.7% demonstrated a transient clearance, shifting to community-type 2 (Lactobacillus iners dominant) at visit 2 only; 7.1% demonstrated a delayed clearance, reaching community-type 2 at the final visit only; and 32.1% of patients experienced sustained bacterial vaginosis clearance. Examination of the community composition and structure demonstrated that both the richness and the evenness were significantly lower for the women who experienced sustained clearance, whereas the women who failed to clear bacterial vaginosis possessed the highest median levels of richness, evenness, and diversity pretreatment. Soluble immune factors in the lower reproductive tract improved significantly following a shift from community-type 4 to a Lactobacillus-dominant microbiome, with the samples categorized as community-type 2 possessing significantly higher levels of secretory leukocyte protease inhibitor, growth-regulated alpha protein, and macrophage inflammatory protein-3 and significantly lower levels of intercellular adhesion molecule-1. Although the shifts to Lactobacillus dominance improved the markers of mucosal tissue health, these gains were only temporary among the women who experienced recurrence. CONCLUSION: Assemblies of highly diverse microbiota are associated with the enhanced resilience of bacterial vaginosis to standard metronidazole treatment. These communities may be foundational to treatment resistance or simply an indication of a well-established community made possible by canonical biofilm-forming taxa. Future studies must target the transcriptional activity of these communities under the pressure of antibiotic treatment to resolve the mechanisms of their resistance.

HIV Pre-exposure Prophylaxis Implant Stated Preferences and Priorities: Results of a Discrete Choice Experiment Among Women and Adolescent Girls in Gauteng Province, South Africa.

For adolescent girls (AG) and young women (YW), adherence barriers may limit the effectiveness of daily oral HIV pre-exposure prophylaxis (PrEP). Due to its low-burden and long-lasting product attributes, PrEP implants could remove some of the critical adherence barriers of oral PrEP products for individuals at risk of HIV. To explore stated preferences for a long-acting PrEP implant, we conducted a quantitative survey and discrete choice experiment with AG (ages 15-17), YW (18-34), and female sex workers (FSW; ≥ 18) in Gauteng Province, South Africa. We completed 600 quantitative surveys across the three subgroups of women. Respondents stated preference for an implant that provided longer HIV protection (24 months versus 6 months) and required a single insertion. They stated that they preferred a biodegradable implant that could be removed within 1 month of insertion. Respondents had no preference for a particular insertion location. Overall, 78% of respondents said they would be likely (33%) or very likely (45%) to use a PrEP implant were one available, with the majority (82%) stating preference for a product that would provide dual protection against HIV and unintended pregnancies. To reduce their risk of HIV, AG, YW, and FSW in our survey reported a strong willingness to use long-acting, highly-effective, dissolvable PrEP implants.

RESUMEN: Las niñas adolescentes (NA) y mujeres jóvenes (MJ), pueden enfrentar barreras de adherencia que limitan la eficacia de la profilaxis oral previa a la exposición al VIH (PrEP). Ya que el implante de PrEP es un producto que requiere de poca intervención de la usuaria y es de larga duración, podría eliminar algunas de las barreras de adherencia más importantes en el uso de los productos orales de PrEP para aquellas personas en riesgo de infección de VIH. Para explorar las preferencias declaradas en cuanto al implante de PrEP de acción prolongada, llevamos a cabo una encuesta cuantitativa y un experimento de elección discreta (DCE) con NA (de 15 a 17 años), MJ (de 18 a 34 años) y mujeres trabajadoras del sexo (MTS; ≥ 18 años) en la provincia de Gauteng, Sudáfrica. Administramos 600 encuestas cuantitativas en los tres subgrupos de mujeres. Los resultados indican la preferencia por un implante que proporciona una protección contra el VIH más prolongada (24 meses a comparación con 6 meses) y que requiere de una única inserción. Las participantes afirmaron que prefieren un implante biodegradable que puede retirarse un mes después de su inserción. Las participantes no tenían preferencia por un sitio específico de inserción. En general, el 78% de las participantes indicaron que probablemente (33%) o muy probablemente (45%) utilizarían un implante de PrEP si estuviera disponible, y la mayoría (82%) manifestó su preferencia por un producto que proporcionaba una doble protección contra el VIH y el embarazo no deseado. Para reducir el riesgo de contraer el VIH, las NA, MJ y MTS participantes se mostraron muy dispuestas a utilizar implantes de PrEP de larga duración, altamente eficaces y disolubles.

End-user research in support of long-acting systemic antiretroviral delivery systems: insights from qualitative research with providers and target users in South Africa.

BACKGROUND: While oral pre-exposure prophylaxis (PrEP) has been shown to reduce the risk of HIV, challenges such as adhering to a daily-dosing regimen and persistence have emerged as barriers for at-risks populations in South Africa. This qualitative research sought to investigate perceptions of and preferences for a long-acting, biodegradable implantable PrEP product designed to address these barriers. METHODS: To identify and understand motivators, barriers, and preferences for the PrEP implant, we conducted qualitative in-depth interviews (IDIs) among health care providers (HCPs) and target end-users (young women, adolescent girls, and female sex workers) in urban and rural/peri-urban regions of Gauteng Province, South Africa. The IDIs focused on defining values, beliefs, habits, lifestyles, influencers, and information channels for potential PrEP implant end-users. RESULTS: We conducted 36 IDIs across health care providers and target end-user respondent segments. Respondents had generally positive reactions to the PrEP implant. Most end-users felt that some undesirable aspects of the implant (e.g., side effects, pain during insertion, potential scarring, and inability to remove implant) would be offset by having a highly effective, and long-lasting HIV prevention product. Although some HCPs believed the implantable PrEP would lead to increases in promiscuity and risky sexual behavior, most HCPs saw value in the PrEP implant's long duration of protection, its biodegradability, and the likelihood of higher adherence relative to oral PrEP. CONCLUSIONS: This study is a first step toward further research needed to demonstrate the demand for a biodegradable, long-acting implantable PrEP and suggests such a product would be accepted by end-users and HCPs in South Africa. This study indicates the need to develop more convenient, discreet, long-acting, and highly effective biomedical HIV prevention options for at-risk populations.

Synthesis and Evaluation of Anti-HIV Activity of Mono- and Di-Substituted Phosphonamidate Conjugates of Tenofovir.

The activity of nucleoside and nucleotide analogs as antiviral agents requires phosphorylation by endogenous enzymes. Phosphate-substituted analogs have low bioavailability due to the presence of ionizable negatively-charged groups. To circumvent these limitations, several prodrug approaches have been proposed. Herein, we hypothesized that the conjugation or combination of the lipophilic amide bond with nucleotide-based tenofovir (TFV) (1) could improve the anti-HIV activity. During the current study, the hydroxyl group of phosphonates in TFV was conjugated with the amino group of L-alanine, L-leucine, L-valine, and glycine amino acids and other long fatty ester hydrocarbon chains to synthesize 43 derivatives. Several classes of derivatives were synthesized. The synthesized compounds were characterized by 1H NMR, IR, UV, and mass spectrometry. In addition, several of the synthesized compounds were evaluated as racemic mixtures for anti-HIV activity in vitro in a single round infection assay using TZM-bl cells at 100 ng/mL. TFV (1) was used as a positive control and inhibited HIV infection by 35%. Among all the evaluated compounds, the disubstituted heptanolyl ester alanine phosphonamidate with naphthol oleate (69), pentanolyl ester alanine phosphonamidate with phenol oleate (62), and butanolyl ester alanine phosphonamidate with naphthol oleate (87) ester conjugates of TFV were more potent than parent drug TFV with 79.0%, 76.5%, 71.5% inhibition, respectively, at 100 ng/mL. Furthermore, two fatty acyl amide conjugates of tenofovir alafenamide (TAF) were synthesized and evaluated for comparative studies with TAF and TFV conjugates. Tetradecanoyl TAF conjugate 95 inhibited HIV infection by 99.6% at 100 ng/mL and showed comparable activity to TAF (97-99% inhibition) at 10-100 ng/mL but was more potent than TAF when compared at molar concentration.

Synthesis and Biological Evaluation of 5'-O-Fatty Acyl Ester Derivatives of 3'-Fluoro-2',3'-dideoxythymidine as Potential Anti-HIV Microbicides.

A number of 5'-O-fatty acyl derivatives of 3'-fluoro-2',3'-dideoxythymidine (FLT, 1) were synthesized. These conjugates were evaluated for their potential as topical microbicides with anti-HIV activity against cell-free (X4 and R5), cell-associated, and multidrug-resistant viruses. Compared to FLT and 3'-azido-2',3'-dideoxythymidine (AZT), 5'-O-(12-azidododecanoyl) (5), 5'-O-myristoyl (6), and 5'-O-(12-thioethyldodecanoyl) (8) derivatives of FLT were found to be more active against both cell-free viruses (lymphocytotropic and monocytotropic strains) with EC50 values of 0.4 µM, 1.1 µM, and <0.2 µM, respectively, as well as cell-associated virus with EC50 values of 12.6, 6.4, and 2.3 µM, respectively. Conjugates 5, 6, and 8 exhibited >4 and >30 times better antiviral index than FLT and AZT, respectively. Conjugates 5 and 8 were significantly more potent than FLT against many multidrug-resistant strains. A comparison of the anti-HIV activity with the corresponding non-hydrolyzable ether conjugates suggested that ester hydrolysis to FLT and fatty acids is critical to enable anti-HIV activity. Cellular uptake studies were conducted using fluorescent derivatives of FLT attached with 5(6)-carboxyfluorescein through either ß-alanine (23) or 12-aminododecanoic acid (24) spacers. The lipophilic fluorescent analog with a long chain (24) showed more than 12 times higher cellular uptake profile than the fluorescent analog with a short chain (23). These studies further confirmed that the attachment of fatty acids improved the cellular uptake of nucleoside conjugates. In addition, 5, 6, and 8 were the least cytotoxic and did not alter vaginal cell and sperm viability compared to the positive control, a commercial topical spermicide (N-9), which significantly decreased sperm and vaginal cell viability inducing the generation of proinflammatory cytokines.

Efficacy is Not Everything: Eliciting Women's Preferences for a Vaginal HIV Prevention Product Using a Discrete-Choice Experiment.

As new female-initiated HIV prevention products enter development, it is crucial to incorporate women's preferences to ensure products will be desired, accepted, and used. A discrete-choice experiment was designed to assess the relative importance of six attributes to stated choice of a vaginally delivered HIV prevention product. Sexually active women in South Africa and Zimbabwe aged 18-30 were recruited from two samples: product-experienced women from a randomized trial of four vaginal placebo forms and product-naïve community members. In a tablet-administered survey, 395 women chose between two hypothetical products over eight choice sets. Efficacy was the most important, but there were identifiable preferences among other attributes. Women preferred a product that also prevented pregnancy and caused some wetness (p < 0.001). They disliked a daily-use product (p = 0.002) and insertion by finger (p = 0.002). Although efficacy drove preference, wetness, pregnancy prevention, and dosing regimen were influential to stated choice of a product, and women were willing to trade some level of efficacy to have other more desired attributes.

Seminal exosomes and HIV-1 transmission.

Exosomes are endosomal-derived membrane-confined nanovesicles secreted by many (if not all) cell types and isolated from every human bodily fluid examined up to now including plasma, semen, vaginal secretions and breast milk. Exosomes are thought to represent a new player in cell-to-cell communication pathways and immune regulation, and be involved in many physiological and pathological processes. Susceptibility to HIV-1 infection can be impacted by exosomes, while HIV-1 pathogenesis can alter exosomal function and composition. Exosomes isolated from semen and vaginal fluid of healthy individuals can inhibit HIV-1 infection and/or potently block viral transfer in vitro. However, the role of exosomes in HIV-1 transmission and progression is not fully understood yet and some studies show conflicting results, mainly for exosomes isolated from plasma and breast milk. Determining the composition of exosomes from infected donors and studying their interaction with HIV-1 in vitro compared to exosomes isolated from uninfected donors will provide insights into the role exosomes play in HIV-1 transmission during sexual intercourse and breastfeeding.

Synthesis and anti-HIV activities of unsymmetrical long chain dicarboxylate esters of dinucleoside reverse transcriptase inhibitors.

A series of 11 unsymmetrical dicarboxylate conjugates of dinucleoside reverse transcriptase inhibitors were synthesized. Three dicarboxylic acids, succinic acid, suberic acid and 1,14-tetradecandioc acid, were diesterified with either 3'-azido-2',3'-dideoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine (FLT), 2',3'-dideoxy-3'-thiacytidine (3TC), or 5-fluoro-2',3'-dideoxy-3'-thiacytidine (FTC). The anti-HIV activity of synthesized compounds was evaluated against HIV-1 X4 (IIIB) and R5 (BaL) viral strains in single-round infection assays. Results indicated that the tetradecandioate esters of nucleosides were more active against HIV than the corresponding parent nucleosides and nucleoside conjugates. The tetradecandioate conjugate of FLT and FTC (5) was found to be the most potent compounds with EC50 values of 47 and 75nM against X4 and R5 HIV-1 strains, respectively, while the EC50 values for the parent analogs, FLT and FTC, ranged from 700 to 3300nM.

Multipurpose Prevention Approaches with Antiretroviral-Based Formulations.

We compared the preclinical safety and efficacy of tenofovir (TFV) 1% gel with that of MZC gel [containing 50 µM MIV-150, 14 mM Zn(O2CCH3)2(H2O)2, and 3% carrageenan] through a series of in vitro, ex vivo, and in vivo assays. The two gels showed good antiviral therapeutic indexes (50% cytotoxic concentration/50% effective concentration ratios; range, >25 to 800). MZC showed greater anti-simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) activity than TFV 1% gel in rhesus macaque vaginal explants. MZC protected mice from vaginal herpes simplex virus 2 (HSV-2) challenge (P < 0.0001), but the TFV 1% gel did not.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1ß, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1ß, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

Pharmacokinetic and safety analyses of tenofovir and tenofovir-emtricitabine vaginal tablets in pigtailed macaques.

Vaginal rapidly disintegrating tablets (RDTs) containing tenofovir (TFV) or TFV and emtricitabine (FTC) were evaluated for safety and pharmacokinetics in pigtailed macaques. Two separate animal groups (n = 4) received TFV (10 mg) or TFV-FTC (10 mg each) RDTs, administered near the cervix. A third group (n = 4) received 1 ml TFV gel. Blood plasma, vaginal tissue biopsy specimens, and vaginal fluids were collected before and after product application at 0, 0.5, 1, 4, and 24 h. A disintegration time of <30 min following vaginal application of the RDTs was noted, with negligible effects on local inflammatory cytokines, vaginal pH, and microflora. TFV pharmacokinetics were generally similar for both RDTs and gel, with peak median concentrations in vaginal tissues and vaginal secretions being on the order of 10(4) to 10(5) ng/g (147 to 571 µM) and 10(6) ng/g (12 to 34 mM), respectively, at 1 to 4 h postdose. At 24 h, however, TFV vaginal tissue levels were more sustained after RDT dosing, with median TFV concentrations being approximately 1 log higher than those with gel dosing. FTC pharmacokinetics after combination RDT dosing were similar to those of TFV, with peak median vaginal tissue and fluid levels being on the order of 10(4) ng/g (374 µM) and 10(6) ng/g (32 mM), respectively, at 1 h postdose with levels in fluid remaining high at 24 h. RDTs are a promising alternative vaginal dosage form, delivering TFV and/or FTC at levels that would be considered inhibitory to simian-human immunodeficiency virus in the macaque vaginal microenvironment over a 24-h period.

Comparison of visual and ultraviolet light inspection versus DNA/protein biomarkers to assess product adherence with vaginal microbicide applicators.

BACKGROUND: Objective biomarkers of product use and protocol compliance are urgently needed. We compared the sensitivity and specificity of DNA and protein-based biomarkers, obtained from used vaginal gel applicators, to visual inspection of those applicators under ambient light (visual inspection of returned applicator [VIRA]) and ultraviolet light (UVL). METHODS: Forty women inserted hydroxyethylcellulose placebo gel vaginal applicators under direct observation. Applicators were evaluated by VIRA, UVL, and DNA/protein-based methods at 2 time points: within 7 days of the visit and after storing applicators for approximately 30 days. Semen biomarkers were assayed from vaginal swabs and returned applicators. RESULTS: The overall sensitivity and specificity of DNA and protein-based biomarkers in determining vaginal insertion versus sham handling of returned applicators were 98.3% and 100%, respectively, at both 7- and 30-day evaluations. The overall sensitivity and specificity of VIRA at 7 and 30 days after collection were significantly lower than those of DNA and protein-based biomarkers. Ultraviolet light inspection also had significantly lower overall sensitivity and overall specificity compared with DNA and protein biomarkers. The sensitivity of DNA and protein-based biomarkers for detecting insertion of wiped applicators was 95%, whereas the sensitivity of VIRA (range of 24%-28%) and UVL inspection (range, 38%-84%) was low for this subset. It was feasible to obtain semen biomarkers from vaginal swabs and returned used applicators. CONCLUSIONS: DNA and protein-based biomarkers offer significantly higher sensitivity and specificity compared with VIRA and UVL assessment. The accuracy of these objective biomarkers is maintained despite storage of returned products for approximately 30 days and under conditions potentially modeling field use.

Preclinical and Early Clinical Development of Tenofovir Alafenamide/Elvitegravir Topical Inserts for Effective On-Demand Vaginal and Rectal HIV Prevention.

HIV/AIDS remains a global public health issue, and products available for the prevention of HIV infections are limited, especially those for short-acting, on-demand, user-controlled applications. Topical inserts are products that can be applied vaginally or rectally and have been explored as drug delivery systems. To fill the gap in the HIV prevention product pipeline, CONRAD has developed a topical insert containing tenofovir alafenamide fumarate (TAF) and elvitegravir (EVG), two potent and synergistic antiretrovirals, as a simple, low-cost, and discreet option that can be self-administered vaginally and/or rectally, before and after coitus. In this review, we have described the development path of the TAF/EVG insert up to its current point in clinical testing, highlighting findings from acceptability, preclinical safety, pharmacokinetics, and efficacy evaluations and early clinical studies. In summary, the TAF/EVG inserts are stable, easy to manufacture, low-cost, acceptable, and show highly promising preclinical and clinical results for on-demand topical pre- or post-exposure HIV prevention.