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

<div><p>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 (<b>M</b>), zinc acetate (<b>Z</b>A), carrageenan (<b>C</b>G) and levonorgestrel (<b>L</b>NG) (<b>MZCL IVR</b>) 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 <i>gag</i> 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 <i>in vitro</i>. 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 <i>ex vivo</i> 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.</p></div

MIV-150 and LNG concentrations in different compartments 24h post IVR insertion.

<p>MIV-150 and LNG concentrations in different compartments 24h post IVR insertion.</p

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

<p>(<b>A</b>) 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>B</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 <i>gag</i> qRT-PCR in tissue supernatants. Summaries of Log₁₀ CUM analyses (d3-14 of culture) of 7 experiments (Mean±SEM) are shown. Input SIV <i>gag</i> copy numbers (Mean; post washout after challenge) are shown as dotted lines. Significant <i>p</i>-values of <0.0001(***) and <0.001(**) are indicated.</p

MIV-150 containing gels inhibit SHIV-RT infection in macaque vaginal explants up to 4d post gel exposure.

<p>Explants were challenged with SHIV-RT 24 h or 4d after exposure to diluted modified gels and cultured for 14d as described in Fig. 3. Shown are log₁₀-transformed p27 SOFT and CUM analyses (Mean±SEM) (d3-14 of culture). Summaries of n = 6–16 (24 h post gel exposure) and n = 3–6 (4d post gel exposure) experiments are shown. The LLOQ of the assay are denoted for both SOFT (solid line) and CUM (dotted line).</p