Stability of MucoCept VDTs.

<p>Two batches of tablets were prepared and placed in storage at 4°C, 25°C, or 37°C. Potency was measured as CFU/g at time zero, one, two, three, six, and 12 months to assess stability. The mean initial potency of the tablets was 5.7x10¹¹ CFU/g. Error bars represent the standard error of the mean (SEM). The potency of tablets stored at 4°C and 25°C for one year was 4.9x10¹¹ CFU/g and 1.2x10¹¹ CFU/g, respectively. Tablets stored at 37°C lost 1.8 log₁₀ CFU/g of their initial potency after two months (to 9.4x10⁹ CFU/g). Stability at 37°C was discontinued after two months.</p

Vaginal colonization of macaques following administration of MucoCept VDTs.

<p>Macaques were vaginally administered one (closed symbols) or five tablets (open symbols) and colonization of <i>L</i>. <i>jensenii</i> 1153–1666 determined at days 14 and 21 post-dosing. In addition, macaques dosed with one tablet were swabbed after three days to determine the initial delivery of viable bacteria to the vagina. Colonization was expressed as CFU/swab following growth of vaginal bacterial dilutions on Rogosa agar and colony counting. Menses (M) is noted in three macaques on the day of sampling. Animals in follicular phase or luteal phase at the time of tablet administration are noted when accurate determination was possible. No significant difference was found between one tablet and five tablet administration at day 14 p = 0.88 or day 21 p = 0.99 based on a one sided Wilcoxon Rank Sum test.</p

Identification of <i>L</i>. <i>jensenii</i> 1153–1666 confirmed by strain-specific PCR.

<p>Genomic DNA was extracted from vaginal swabs and PCR performed using <i>L</i>. <i>jensenii</i> 1153-1666-cyanovirin cassette specific primers. All the macaques were PCR-positive at 21 days post-inoculation as indicated by the presence of 247 base pair PCR fragment that uniquely identifies the recombinant strain. The solid grey lines indicate macaques that received one tablet; solid white lines represent macaques that received five tablets.</p

Western blot to confirm mCV-N protein expression.

<p>Proteins in stationary phase conditioned media from <i>L</i>. <i>jensenii</i> 1153–1666 isolated from macaques that received one-tablet or five-tablets were separated on a 12% Bis-Tris gel. A representative gel is shown from the day 14-post dosing time point. The parental strain, <i>L</i>. <i>jensenii</i> 1153, is shown as the negative control, and <i>L</i>. <i>jensenii</i> 1153–1666 as the positive control. Three macaques from the five-tablet group and two macaques from the one-tablet group are shown, confirming that colonies of <i>L</i>. <i>jensenii</i> 1153–1666 recovered from the macaques continued to produce mCV-N protein.</p

MucoCept prototype VDT before and after disintegration.

<p>A) Mylar foil pouch opened to reveal tablet and desiccant. B) Tablet disintegration is shown at three minutes. The tablet is placed on a paper towel wetted with vaginal fluid simulant, to simulate the mucosal surface. The time required for the solution to completely wet the tablet surface was measured as the disintegration time. Tablets disintegrated within two minutes.</p