An Antiretroviral/Zinc Combination Gel Provides 24 Hours of Complete Protection against Vaginal SHIV Infection in Macaques

Repeated use, coitus-independent microbicide gels that do not contain antiretroviral agents also used as first line HIV therapy are urgently needed to curb HIV spread. Current formulations require high doses (millimolar range) of antiretroviral drugs and typically only provide short-term protection in macaques. We used the macaque model to test the efficacy of a novel combination microbicide gel containing zinc acetate and micromolar doses of the novel non-nucleoside reverse transcriptase inhibitor MIV-150 for up to 24 h after repeated gel application.Rhesus macaques were vaginally challenged with SHIV-RT up to 24 h after repeated administration of microbicide versus placebo gels. Infection status was determined by measuring virologic and immunologic parameters. Combination microbicide gels containing 14 mM zinc acetate dihydrate and 50 µM MIV-150 afforded full protection (21 of 21 animals) for up to 24 h after 2 weeks of daily application. Partial protection was achieved with the MIV-150 gel (56% of control at 8 h after last application, 11% at 24 h), while the zinc acetate gel afforded more pronounced protection (67% at 8-24 h). Marked protection persisted when the zinc acetate or MIV-150/zinc acetate gels were applied every other day for 4 weeks prior to challenge 24 h after the last gel was administered (11 of 14 protected). More MIV-150 was associated with cervical tissue 8 h after daily dosing of MIV-150/zinc acetate versus MIV-150, while comparable MIV-150 levels were associated with vaginal tissues and at 24 h.A combination MIV-150/zinc acetate gel and a zinc acetate gel provide significant protection against SHIV-RT infection for up to 24 h. This represents a novel advancement, identifying microbicides that do not contain anti-viral agents used to treat HIV infection and which can be used repeatedly and independently of coitus, and underscores the need for future clinical testing of their safety and ability to prevent HIV transmission in humans

Results of a phase 1, randomized, placebocontrolled first-in-human trial of griffithsin formulated in a carrageenan vaginal gel

HIV pre-exposure prophylaxis (PrEP) is dominated by clinical therapeutic antiretroviral (ARV) drugs. Griffithsin (GRFT) is a non-ARV lectin with potent anti-HIV activity. GRFT’s preclinical safety, lack of systemic absorption after vaginal administration in animal studies, and lack of cross-resistance with existing ARV drugs prompted its development for topical HIV PrEP. We investigated safety, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of PC-6500 (0.1% GRFT in a carrageenan (CG) gel) in healthy women after vaginal administration. This randomized, placebo-controlled, parallel group, double-blind first-in-human phase 1 study enrolled healthy, HIV-negative, non-pregnant women aged 24–45 years. In the open label period, all participants (n = 7) received single dose of PC- 6500. In the randomized period, participants (n = 13) were instructed to self-administer 14 doses of PC-6500 or its matching CG placebo (PC-535) once daily for 14 days. The primary outcomes were safety and PK after single dose, and then after 14 days of dosing. Exploratory outcomes were GRFT concentrations in cervicovaginal fluids, PD, inflammatory mediators and gene expression in ectocervical biopsies. This trial is registered with ClinicalTrials. gov, number NCT02875119. No significant adverse events were recorded in clinical or laboratory results or histopathological evaluations in cervicovaginal mucosa, and no anti-drug (GRFT) antibodies were detected in serum. No cervicovaginal proinflammatory responses and no changes in the ectocervical transcriptome were evident. Decreased levels of proinflammatory chemokines (CXCL8, CCL5 and CCL20) were observed. GRFT was not detected in plasma. GRFT and GRFT/CG in cervicovaginal lavage samples inhibited HIV and HPV, respectively, in vitro in a dose-dependent fashion. These data suggest GRFT formulated in a CG gel is a safe and promising on-demand multipurpose prevention technology product that warrants further investigation

\u3ci\u3eEx vivo\u3c/i\u3e colonic tissue susceptibility to HIV-1 in cisgender men and women

The biology of HIV-1 acquisition through unprotected receptive anal intercourse is understudied. Considering that sex hormones are implicated in intestinal physiology, pathology, and HIV acquisition and pathogenesis, we explored links between sex hormones, ex vivo HIV-1BaL infection of colonic mucosa, and candidate biomarkers of susceptibility to HIV-1 (CD4+ T cell frequencies and immune mediators) in cisgender women and men. No consistent significant associations between sex hormone concentrations and ex vivo tissue infection with HIV- 1BaL were detected. In men, serum estradiol (E2) concentrations were positively associated with tissue proinflammatory mediators (IL17A, GM-CSF, IFNγ, TNFα, and MIG/CXCL9) and serum testosterone concentrations were negatively associated with frequencies of activated CD4+ T cells (CD4+CCR5+, CD4+HLADR+, and CD4+CD38+HLA-DR+). In women, the only significant interactions were positive associations between progesterone (P4)/E2 ratios and tissue ILRA concentrations and between P4/E2 ratios and frequencies of tissue CD4+α4β7high+ T cells. The study did not reveal relationships between biological sex or phase of the menstrual cycle and ex vivo tissue HIV-1BaL infection and tissue immune mediators. A comparison of CD4+ T cell frequencies between study groups revealed a higher frequency of tissue CD4+α4β7high+ T cells in women versus men. In contrast, higher frequencies of tissue CD4+CD103+ T cells were detected in men versus women in the follicular phase of the menstrual cycle. Overall, the study identified associations between systemic sex hormone concentrations, biological sex, and tissue candidate biomarkers of susceptibility to HIV-1. The significance of these results for tissue susceptibility to HIV-1 and early HIV-1 pathogenesis warrants further investigation

Development of a vaginal fast-dissolving insert combining griffithsin and carrageenan for potential use against sexually transmitted infections

Precoital, on-demand topical microbicides to reduce a woman’s risk of sexually transmitted infections have been in development for nearly three decades, but no product has been approved due to acceptability issues and poor adherence in clinical trials. We set out to develop a self-administered vaginal fast-dissolving insert (FDI) produced by freeze-drying that would deliver safe and effective amounts of the antiviral agents griffithsin (GRFT) and carrageenan (CG) and would have properties women and their partners find acceptable. We evaluated FDI physical criteria, attributes of the gel produced upon dissolving, and GRFT stability. The lead formulation, FDI-024, was selected from 13 candidates and contains 4 mg of GRFT, 15 mg of CG, and excipients (the cryoprotectant sucrose and bulking agents dextran 40 and mannitol). The FDI exhibits good friability and hardness and is stable for at least 6 months at up to 40°C/75% relative humidity. It disintegrates in less than 60 seconds in a physiologically relevant volume (∼1 mL) of simulated vaginal fluid, forming a viscous semi-solid gel with favorable mucoadhesive and spreading properties. The formulation retains the antiviral activity of GRFT and CG against human immunodeficiency virus type 1 and human papillomavirus, respectively, in cell-based assays

A novel intravaginal ring (IVR) protects macaques against SHIV-RT infection and reduces HSV-2 shedding after repeated SHIV-RT/HSV-2 co-challenge

Background: MZC gel (MIV-150, zinc acetate [ZA], carrageenan [CG]) significantly reduces vaginal SHIV-RT and HSV-2 infection in macaques when given 8h before challenge. CG gels significantly reduce HPV infection in mice when dosed 24h before challenge. We aim to develop a 90d IVR releasing MZC and levonorgestrel (LNG) to prevent HIV, HSV-2, HPV, and conception. Methods: IVRs contained 3mg MIV-150±0.6mg LNG in the matrix and 30mg ZA/70mg CG in the core (open to fluids via a matrix pore). We measured CG, MIV-150, and LNG in macaque blood and/or vaginal swabs during and after 28d of IVR insertion. For efficacy, we exchanged IVRs every 21d, challenging macaques with 200 TCID_50 SHIV-RT/10^7 pfu HSV-2 on d7, 10, 14 and 17 post IVR insertion (20 challenges total); n = 4 placebo IVR, n = 4 LNG IVR, n=12 MZC IVR, n = 12 MZCL IVR. Results: MIV-150 was detected in swabs and plasma 1h post IVR insertion, peaked at d1 in swabs and d1-3 in plasma, declined from d7-d28, and was undetectable 24h after IVR removal. LNG was detected in serum within 4h, plateaued by d7, and was undetectable 24h after IVR removal. CG was detected in swabs of most animals from d3 until the IVRs were removed. Preliminary data show MZCL IVRs protected (92%) macaques against SHIV-RT (1/12 vs. 4/4 infected in LNG IVR controls; p=0.003); 2/12 MZC and 2/4 placebo IVR animals became SHIV-RT infected (67% protection). MZC and MZCL IVRs significantly protected compared to control IVRs (3/24 vs. 6/8 infected, p = 0.002). Initial data suggest that ∼30% fewer animals became infected with HSV-2 after treatment with MZC and MZCL IVRs (17/24 vs. 8/8 infected controls); the frequency (18/30 vs. 23/95 time points positive; p \u3c 0.0006) and levels (67/180 vs. 31/570 replicates positive; p \u3c 0.0001) of HSV-2 shedding were significantly lower than in the controls. Conclusions: We developed an IVR that releases APIs targeting HIV, HSV-2, HPV, and conception. The IVR significantly reduces SHIV-RT infection and HSV-2 shedding in macaques

Antiviral activity and mode of action of Griffithsin against HSV-2 and HPV: Preliminary studies of a potential non-ARV combination microbicide

Background: Griffithsin (GRFT) is a promising HIV microbicide candidate. Nixon et al. have shown that GRFT blocks herpes simplex 2 (HSV-2) infection in a mouse model, proposing inhibition of cell-to-cell spread as the mode of action (MOA). Using in vitro studies we further investigated the MOA of GRFT against HSV-2 and studied its antiviral activity against human papillomavirus (HPV). We also combined GRFT with zinc acetate (ZA) and/or carrageenan (CG) to render a more potent microbicide. Methods: We used XTT assay to define non-cytotoxic concentrations of GRFT, ZA, CG or their combinations. Assays for anti-HIV, anti-HPV and anti-HSV-2 activities were performed in TZM-bl cells or PBMCs using MAGI and p24 ELISA; in HeLa cells using a luciferase assay; and in Vero cells using plaque forming units (pfu) assay. We performed time-of-addition and temperature dependence experiments to differentiate inhibition of viral adsorption from entry. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins and immunohistochemistry was used to determine the specific glycoprotein involved. Antiviral activities of prototype GRFT/CG (GC) and GRFT/ZA/CG (GZC) gels in a vaginal HSV-2 mouse model were evaluated. Results: GRFT shows modest in vitro antiviral activity against HSV-2 G (IC_50 = 5.8μg/ml) and HPV 6, 16, 18, 45 PsVs (IC_50 = 10.8-26.3μg/ml), compared to potent anti-HIV activity (IC_50 = 0.7-1.4ng/ml). As with HIV, GRFT blocks the entry but not the adsorption of HSV-2 and HPV to target cells. The combined analyses of SPR and immunohistochemistry for HSV-2 gD, suggest that GRFT binds to HSV-2 gD. GC and GZ had synergistic in vitro antiviral activity against HIV and HPV (CI \u3c 1). GC and GZC gels significantly reduced (p \u3c 0.05) HSV-2 vaginal infection in vivo when administered up to 2h before challenge with 10^6pfu/mouse. Conclusions: GRFT blocks HSV-2 and HPV entry to target cells and combination with CG and/or ZA may result in a potent/broad-spectrum non-ARV microbicide

New approaches for developing biomarkers of hormonal contraceptive use

To identify biomarkers of hormonal contraceptive (HC) use in urine and saliva, we conducted a pilot study with 30 women initiating levonorgestrel (LNG) containing combined oral contraceptives (COCs) or depot medroxyprogesterone acetate (DMPA) (15/group). Based on established COC pharmacokinetics, we collected serum and urine samples before COC ingestion and during Days one and three of use, or before DMPA injection and on Days 21 and 60 post-injection. We used liquid chromatography-tandem mass spectrometry (LC–MS/MS) to measure serum/urine LNG and MPA. LNG was undetectable at baseline (specificity 100%); post ingestion, most urine samples had detectable LNG levels (sensitivity: 80% 6 h post Dose one, 93% 6 h post Dose three). We used a DetectX LNG immunoassay kit and showed 100% sensitivity measuring urine LNG. Urine MPA levels were undetectable in 14/15 women at baseline (specificity 91%); post-injection all urine samples had detectable MPA levels (sensitivity: 100% days 21 and 60). Results suggest urine sampling can be used to identify a biomarker of LNG and MPA use. Based on evidence from other steroidal hormonal studies showing changes affecting the transcriptome profile of saliva at 24 h, we used the same (COC, DMPA) timepoints to collect saliva. We performed transcriptome analysis and detected several differentially expressed genes in DMPA users’ saliva on Days 21 and 60 compared to baseline; none among COC users. We plan further research of differential gene expression in saliva as a HC biomarker of DMPA use, and will explore longer periods of COC use and saliva collection times, and application of microRNA sequencing to support using saliva as a COC biomarker

MZC and 1% TFV gel: Multipurpose prevention approaches

Background: Next generation microbicides may be broad-spectrum multipurpose prevention technologies with improved efficacy and safety. CAPRISA-004 showed that 1% TFV vaginal gel prevented HIV-1 and HSV-2. Here we compare the in vitro and in vivo safety and efficacy profile of 1% TFV gel versus a microbicide gel (MZC) containing 50 mM MIV-150 (M), 14 mM zinc acetate (Z) and 3% carrageenan (CG). Methods: Increased HSV-2 susceptibility in mice (n=20), TEER in Caco-2 cells, and macaque vaginal explant histology were used to estimate damage to epithelia. XTT and Cyquant were used to evaluate in vitro cytotoxicity in TZM-bls and PBMCs. Anti-HIV activity was tested against HIV-1 lab strains (n=5), primary isolates and multidrug resistant strains/clones (n=28) in TZM-bls or PBMCs. CC_50 and IC_50 values were used to estimate therapeutic indexes (TI=CC_50/IC_50). Anti-SHIV-RT activity (10^4 TCID_50/explant) of diluted gels was assessed in macaque vaginal explants. In vivo anti-HSV-2 activity (5x103 pfu/mouse, n=20/treatment) was evaluated in mice (gel applied 1 h before and after HSV-2 vaginal challenge). Levels of TFV and TFV-DP in mouse cervicovaginal tissues were quantified by LC-MS/MS. Fisher\u27s exact test (P \u3c 0.05) was used for statistical comparison in all HSV-2 murine models. Kruskal-Wallis and Dunn\u27s Multiple Comparison test (P \u3c 0.05) in the explant model. Results: Both gels were safe. However, diluted 1% TFV reduced TEER values in Caco-2 monolayers. MZC showed greater in vitro antiviral activity (2 to 80-fold) than 1% TFV gel against HIV-1 in TZM-bls. Both gels showed good TI ( \u3e 25-800) in PBMCs, except for one and two MDR HIVs with 1%TFV and MZC, respectively. MZC fully protected explants from SHIV-RT (p \u3c 0.009) with greater anti-SHIV-RT activity than 1% TFV gel. MZC fully protected mice from HSV-2 (p \u3c 0.0001), but the 1% TFV gel did not. The lack of protection could be associated with sub-therapeutic levels of TFV and TFV-DP in mouse tissues. Conclusions: MZC is a promising microbicide candidate for clinical use

Griffithsin and carrageenan combination to target herpes simplex virus 2 and human papillomavirus

Extensive preclinical evaluation of griffithsin (GRFT) has identified this lectin to be a promising broad-spectrum microbicide. We set out to explore the antiviral properties of a GRFT and carrageenan (CG) combination product against herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) as well as determine the mechanism of action (MOA) of GRFT against both viruses. We performed the experiments in different cell lines, using time-of-addition and temperature dependence experiments to differentiate inhibition of viral attachment from entry and viral receptor internalization. Surface plasmon resonance (SPR) was used to assess GRFT binding to viral glycoproteins, and immunoprecipitation and immunohistochemistry were used to identify the specific glycoprotein involved. We determined the antiviral activity of GRFT against HSV-2 to be a 50% effective concentration (EC_50) of 230 nM and provide the first evidence that GRFT has moderate anti-HPV activity (EC_50 = 0.429 to 1.39 μM). GRFT blocks the entry of HSV-2 and HPV into target cells but not the adsorption of HSV-2 and HPV onto target cells. The results of the SPR, immunoprecipitation, and immunohistochemistry analyses of HSV-2 combined suggest that GRFT may block viral entry by binding to HSV-2 glycoprotein D. Cell-based assays suggest anti-HPV activity through α6 integrin internalization. The GRFT-CG combination product but not GRFT or CG alone reduced HSV-2 vaginal infection in mice when given an hour before challenge (P = 0.0352). While GRFT significantly protected mice against vaginal HPV infection when dosed during and after HPV16 pseudovirus challenge (P \u3c 0.026), greater CG-mediated protection was afforded by the GRFT-CG combination for up to 8 h (P \u3c 0.0022). These findings support the development of the GRFT-CG combination as a broad-spectrum microbicide

Multipurpose prevention approaches with antiretroviral-based formulations

We compared the preclinical safety and efficacy of Tenofovir (TFV) 1% gel and MZC gel containing 50 μM MIV-150 (M), 14 mM Zn(O_2CCH_3)_2(H2O)2 (Z) and 3% carrageenan (C) through a series of in vitro, ex vivo and in vivo assays. Both gels showed good antiviral therapeutic indexes ( \u3e 25-800). MZC showed greater anti-SHIV-RT activity than TFV 1% gel in rhesus macaque vaginal explants. MZC protected mice from vaginal HSV-2 challenge (p \u3c 0.0001), but the TFV 1% gel did not