A Novel Microbicide/Contraceptive Intravaginal Ring Protects Macaque Genital Mucosa against SHIV-RT Infection Ex Vivo

Abstract

Women need multipurpose prevention products (MPTs) that protect against sexually transmitted infections (STIs) and provide contraception. The Population Council has developed a prototype intravaginal ring (IVR) releasing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (M), zinc acetate (ZA), carrageenan (CG) and levonorgestrel (LNG) (MZCL IVR) to protect against HIV, HSV-2, HPV and unintended pregnancy. Our objective was to evaluate the anti-SHIV-RT activity of MZCL IVR in genital mucosa. First, macaque vaginal tissues were challenged with SHIV-RT in the presence of (i) MIV-150 ± LNG or (ii) vaginal fluids (VF); available from studies completed earlier) collected at various time points post insertion of MZCL and MZC IVRs. Then, (iii) MZCL IVRs (vs. LNG IVRs) were inserted in non-Depo Provera-treated macaques for 24h and VF, genital biopsies, and blood were collected and tissues were challenged with SHIV-RT. Infection was monitored with one step SIV gag qRT-PCR or p27 ELISA. MIV-150 (LCMS/MS, RIA), LNG (RIA) and CG (ELISA) were measured in different compartments. Log-normal generalized mixed linear models were used for analysis. LNG did not affect the anti-SHIV-RT activity of MIV-150 in vitro. MIV-150 in VF from MZC/MZCL IVR-treated macaques inhibited SHIV-RT in vaginal mucosa in a dose-dependent manner (p<0.05). MIV-150 in vaginal tissue from MZCL IVR-treated animals inhibited ex vivo infection relative to baseline (96%; p<0.0001) and post LNG IVR group (90%, p<0.001). No MIV-150 dose-dependent protection was observed, likely because of high MIV-150 concentrations in all vaginal tissue samples. In cervical tissue, MIV-150 inhibited infection vs. baseline (99%; p<0.05). No cervical tissue was available for MIV-150 measurement. Exposure to LNG IVR did not change tissue infection level. These observations support further development of MZCL IVR as a multipurpose prevention technology to improve women's sexual and reproductive health.

Fig 1. LNG does not affect the activity of MIV-150 (non-toxic concentrations) in macaque vaginal mucosa.

(A) Macaque vaginal explants were immersed in culture media containing LNG or MIV-150 (vs. 1:10 diluted gynol) for ~18h. Tissue viability was determined using MTT assay (OD₅₇₀ of the formazan product was measured in triplicates and normalized to the dry weight of the explants). Each symbol indicates an individual donor and the Mean±SEM of the Log₁₀ OD₅₇₀/g of tissue for each condition is shown. (B) PHA/IL2 stimulated explants were challenged with SHIV-RT (10⁴ TCID₅₀/explant; triplicates) in the presence of 1.5 or 0.15 μM MIV-150 and/or 6 or 0.6 μM LNG (vs. untreated control). 18h later, tissues were washed and cultured for 14d with IL2 and infection was monitored by SIV gag qRT-PCR in tissue supernatants. Summaries of Log₁₀ CUM analyses (d3-14 of culture) of 7 experiments (Mean±SEM) are shown. Input SIV gag copy numbers (Mean; post washout after challenge) are shown as dotted lines. Significant p-values of <0.0001(***) and <0.001(**) are indicated.

Fig 2. MIV-150 in VF inhibits SHIV-RT infection in vaginal mucosa in a dose-dependent manner.

PHA/IL2-stimulated explants from untreated animals were challenged with 10⁴ TCID₅₀ SHIV-RT/explant (duplicates/triplicates) in the presence of 1:5 diluted VF collected at the baseline and after MZC or MZCL IVR insertion at 4h, 48h, 72h and 9d (post IVR). The concentrations of MIV-150 correspond to 1:5 diluted VF. The explants were cultured for 14d in the presence of IL2. Infection was monitored by p27 ELISA (d0-14 of culture). The activity of each baseline IVR and post IVR VF pair was tested in explants from different donors represented by different symbols. Changes in CUM analyses (d3-14 of culture) in the presence of post IVR VF are shown relative to MIV-150 concentrations in diluted VF.

Fig 3. Tissue-associated MIV-150 inhibits ex vivo SHIV-RT infection of vaginal and cervical mucosa.

MZCL (n = 5) or LNG (n = 4) IVRs were inserted for 24h. Vaginal and ectocervical biopsies were collected immediately after IVR removal (Post) and at the baseline. Non-stimulated vaginal (n = 2–6) and cervical (n = 2–4) explants were challenged with SHIV-RT (10⁴ TCID₅₀/explant) for ~18h, washed and cultured for 14d with IL2. Infection was monitored and analyzed as in Fig 1. Shown are (A) SIV gag copies/ml of each animal (Mean of replicates ± SEM; symbols match those shown in panel B) and (B) SIV gag CUM analyses of Log₁₀ transformed data (Mean ± SEM) in vaginal tissues. Each symbol represents an individual animal (MZCL group: closed symbols; LNG group: open symbols). Shown are (C) SIV gag copies/ml of each animal (Mean of replicates ± SEM; symbols match those shown in panel D) and (D) SIV gag CUM analyses of Log₁₀ transformed data (Mean ± SEM) in cervical tissues. Each symbol represents an individual animal (MZCL group). Significant p-values of <0.0001(***) and <0.001(**) are indicated. Input SIV gag copy numbers (Mean; post washout after challenge) are shown as dotted lines.