Figure 1.
Cellulose sulfate and PRO 2000 gels protect mice from genital herpes, but the response is modulated when virus is introduced in seminal plasma.
(A). Survival curves for mice pretreated with a single dose of each blinded gel or no gel (untreated) and challenged with an LD90 of HSV-29G) diluted in PBS. Survival curves for mice pretreated with a single dose of DE-021 (HEC), DE-018 (CS) or DE-020 (PRO 2000) and challenged with an LD90 of HSV-2(4674) diluted in PBS (B) or pooled human seminal plasma (C). Data were obtained from 2 independent experiments (10 mice per group). The asterisk denotes significant protection relative to HEC-treated mice (p<0.001 for virus introduced in PBS and p = 0.015 for virus introduced in seminal plasma).
Figure 2.
Vaginal washes from mice treated with cellulose sulfate 6% gel inhibit HSV-2 plaque formation.
To remove any residual drug within the vaginal lumen prior to HSV-2 challenge in the safety studies, vaginal washes were obtained with 100 µl of saline. The vaginal washes were tested in a viral plaque assay. Results are means (SEM) from 2 independent experiments with pooled washes from 5 mice each; the asterisk denotes p<0.05.
Figure 3.
Increased susceptibility to HSV-2 following seven daily gel applications.
Mice were challenged with ∼103, 104 or 105 pfu/mouse of HSV-2(4674) (upper, middle and bottom panels, respectively), representing an LD10, LD40, and LD90 12 h after receiving the seventh daily dose of N-9 or coded gel products and after vaginal washing. Animals were observed daily for signs and symptoms of disease. Mice developing severe genital or neurological disease were euthanized. Results show percent survival pooled from at least 2 independent experiments (n = 20 mice/group). The asterisks denote p<0.05.
Figure 4.
N-9 and cellulose sulfate causes disruption of the epithelium.
Mice were treated daily for seven days with microbicides and twelve hours after the final application, the mice were sacrificed and the entire vaginal canal up to the uterine bifurcation was excised. The tissue was then fixed and stained with EZ-Link Sulfo-NHS-Biotin to detect the apical surface (magenta) and DAPI to detect nuclei (blue), ZO-1 (green) and desmoglein (DSG, red) and viewed by confocal microscopy. Representative 3-dimensionsal (upper panel) and xz (lower panels) images are shown (A). Images were taken from at least 3 animals per treatment group and at least 6 independent randomly selected images were acquired per animal. To assess whether disruption of the epithelial barrier promoted HSV migration through the tissue, mice were inoculated with HSV-2 12 hours after the seventh daily gel application and were then sacrificed four hours after infection. Tissues were stained for viral capsids with a mAb to VP16; phalloidin, which stains actin and was used to delineate the cytoplasm; and DAPI to identify nuclei. Individual Z slices were analyzed and intensity density of the fluorescence quantified. The results are means obtained from 8 independent randomly selected images (2 images per genital tract sample) (B) and the cumulative area under the curve is shown in C; asterisks indicate a significant increase in the area under the curve (p<0.001, ANOVA).
Figure 5.
Changes in cytokines and chemokines in response to microbicides.
The levels of chemokines and cytokines were measured in vaginal washes pooled from 5 mice by BioLuminex. Results are presented as mean ± SE values from six different pools (5 mice per pool) for washes obtained after three daily doses and from at least three pools for washes obtained after seven daily doses. The asterisks denote p<0.05 relative to HEC-treated mice.