Ex Vivo 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+HLA-DR+, 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+α4ß7high+ 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+α4ß7high+ 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.

Results of a phase 1, randomized, placebo-controlled 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.

Mucosal Susceptibility to Human Immunodeficiency Virus Infection in the Proliferative and Secretory Phases of the Menstrual Cycle.

Factors underlying HIV acquisition in women remain incompletely understood. This study evaluated ex vivo mucosal HIV-1BaL infection (ectocervix, endocervix), T cell frequencies and phenotype (ectocervix, endocervix, peripheral blood), and HIV-1BaL-induced tissue immune responses (ectocervix) in the proliferative and secretory phases of the menstrual cycle using samples obtained from women undergoing hysterectomies. Tissue infectivity (number of productively infected explants) and infection level following 500 and/or fifty 50% tissue culture infectious dose (TCID50) HIV-1BaL challenge were similar in the proliferative and secretory phases. Although not associated with infection outcomes, higher frequencies of HIV target CD4+α4ß7+ T cells, and stronger HIV-1BaL-induced proinflammatory responses were detected in ectocervix in the secretory versus proliferative phase. Independently of the cycle phase, serum E2 concentrations were inversely associated with ectocervical and endocervical tissue infection levels following high-dose 500 TCID50 HIV-1BaL challenge, with frequencies of CD4+α4ß7+ T cells in endocervix, and with HIV-induced interleukin (IL)2R and IL4 in ectocervix. Although serum P4 concentrations and P4/E2 ratios were neither associated with tissue infection level nor infectivity, high P4 concentrations and/or P4/E2 ratios correlated with high frequencies of CD4+α4ß7+ T cells in ectocervix, low frequencies of CD4+CD103+ blood T cells, low CD4+LFA-1+ T cells in endocervix, and high proinflammatory (IL1ß, IL17, tumor necrosis factor α) ectocervical tissue responses to HIV-1BaL. The data suggest an inhibitory effect of E2 on mucosal HIV infection, provide insights into potential mechanisms of E2-mediated anti-HIV activity, and highlight P4-associated immune changes in the mucosa.

Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo.

Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) strategies with proven in vivo efficacy rely on antiretroviral drugs, creating the potential for drug resistance and complicated treatment options in individuals who become infected. Moreover, on-demand products are currently missing from the PrEP development portfolio. Griffithsin (GRFT) is a non-antiretroviral HIV entry inhibitor derived from red algae with an excellent safety profile and potent activity in vitro. When combined with carrageenan (CG), GRFT has strong activity against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) in vitro and in vivo. Here, we report that GRFT/CG in a freeze-dried fast dissolving insert (FDI) formulation for on-demand use protects rhesus macaques from a high dose vaginal SHIV SF162P3 challenge 4 h after FDI insertion. Furthermore, the GRFT/CG FDI also protects mice vaginally against HSV-2 and HPV pseudovirus. As a safe, potent, broad-spectrum, on-demand non-antiretroviral product, the GRFT/CG FDI warrants clinical development.

MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal Mucosa and SHIV-RT in Rectal Mucosa.

The Population Council's microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad-spectrum microbicide against HIV, herpes simplex virus (HSV), and human papillomavirus. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection, and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and human papillomavirus in murine models. Recently, we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 coinfection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC, and compared quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay readouts for monitoring SHIV-RT infection. MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosas and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by 1-step simian immunodeficiency virus gag quantitative RT-PCR and p27 enzyme-linked immunosorbent assay demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of quantitative RT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

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.

MIV-150/zinc acetate gel inhibits cell-associated simian-human immunodeficiency virus reverse transcriptase infection in a macaque vaginal explant model.

The transmission of both cell-free and cell-associated immunodeficiency viruses has been demonstrated directly in multiple animal species and possibly occurs in humans, as suggested by genotyping of the infecting human immunodeficiency virus (HIV) in acutely infected women and in semen from their partners. Therefore, a microbicide may need to block both mechanisms of HIV transmission to achieve maximum efficacy. To date, most of the preclinical evaluation of candidate microbicides has been performed using cell-free HIV. New models of mucosal transmission of cell-associated HIV are needed to evaluate candidate microbicide performance. The MIV-150/zinc acetate/carrageenan (MZC) gel protects Depo-Provera-treated macaques against cell-free simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection when applied vaginally up to 8 h before challenge. We recently demonstrated the potent activity of MZC gel against cell-free SHIV-RT in macaque vaginal explants. In the current study, we established a cell-associated SHIV-RT infection model of macaque vaginal tissues and tested the activity of MZC gel in this model. MZC gel protected tissues against cell-associated SHIV-RT infection when present at the time of viral exposure or when applied up to 4 days prior to viral challenge. These data support clinical testing of the MZC gel. Overall, our ex vivo model of cell-associated SHIV-RT infection in macaque vaginal mucosa complements the cell-free infection models, providing tools for prioritization of products that block both modes of HIV transmission.

A MIV-150/zinc acetate gel inhibits SHIV-RT infection in macaque vaginal explants.

To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC's activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51-62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65-74%) did not significantly differ from CG (32-45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.

MIV-150-containing intravaginal rings protect macaque vaginal explants against SHIV-RT infection.

Recent studies demonstrated that intravaginal rings (IVRs) containing 100 mg of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 significantly protect macaques against a chimeric simian-human immunodeficiency virus that expresses the HIV-1 HxB2 reverse transcriptase (SHIV-RT) when present before and after vaginal challenge. The objectives of this study were to (i) evaluate the pharmacodynamics (PD) of MIV-150 in vaginal fluids (VF) and in ectocervical and vaginal tissues following 100-mg MIV-150 IVR exposure and to (ii) gain more insight whether pharmacokinetics (PK) of MIV-150 can predict PD. MIV-150 in VF collected at 1 day and 14 days post-MIV-150 IVR insertion inhibited ex vivo SHIV-RT infection in vaginal biopsy specimens from untreated animals (not carrying IVRs) in a dose-dependent manner. Previous PK studies demonstrated a significant increase of ectocervical and vaginal tissue MIV-150 concentrations 14 days versus 1 day post-IVR insertion, with the highest increase in vaginal tissue. Therefore, we tested PD of MIV-150 in tissues 14 days post-MIV-150 IVR insertion. Ex vivo SHIV-RT infection of vaginal, but not ectocervical, tissues collected 14 days post-MIV-150 IVR insertion was significantly inhibited compared to infection at the baseline (prior to MIV-150 IVR exposure). No changes in vaginal and ectocervical tissue infection were observed after placebo IVR exposure. Overall, these data underscore the use of the ex vivo macaque explant challenge models to evaluate tissue and VF PK/PD of candidate microbicides before in vivo animal efficacy studies. The data support further development of MIV-150-containing IVRs.