Preclinical testing of candidate topical microbicides for anti-human immunodeficiency virus type 1 activity and tissue toxicity in a human cervical explant culture

Abstract

A human cervical explant culture was utilized for the preclinical assessment of anti-human immunodeficiency virus type 1 (HIV-1) activity and tissue toxicity of formulated, candidate topical microbicides. Products tested included cellulose acetate 1,2-benzene dicarboxylate (CAP), a carrageenan-based product (PC-515), a naphthalene sulfonate polymer (PRO 2000), a lysine dendrimer (SPL7013), a nonnucleoside reverse transcriptase inhibitor (UC781), and an antimicrobial peptide (D2A21), along with their placebos. Cervical explants were cultured overnight with HIV-1 with or without product, washed, and monitored for signs of HIV-1 infection. HIV-1 infection was determined by p24gag levels in the basolateral medium and by immunohistochemical analysis of the explant. Product toxicity was measured by the MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] assay and histology. CAP, PRO 2000, SPL7013, and UC781 consistently prevented HIV-1 infection in all explants tested. PC-515 and D2A21 prevented HIV-1 infection in 50% or fewer of the explants tested. Placebos did not prevent infection in any of the explants tested. With the exception of PRO 2000 (4%), the MTT assay and histological analysis of the other products and placebos showed minimal toxicity to the epithelium and submucosa. Collectively, these data suggest that this culture system can be used for evaluating the safety and efficacy of topical microbicides designed for vaginal use.

FIG. 1.

Cervical explant culture. (A) Circular tissue explants were inserted through a hole in a transwell insert with the epithelium oriented upward in the apical chamber. The epithelial surface of the explant was surrounded with 2% agarose to maintain tissue orientation and minimize seepage of microbicides and/or virus around the tissue edges. The stroma was cultured in cDMEM in the basolateral chamber. HIV-1 alone in medium or mixed with microbicide was applied to the apical surface to simulate mucosal exposure in vivo. (B to D) Histology of a representative hematoxylin-and-eosin-stained normal human cervix at day 0 (day of surgery; B), day 3 (C), and day 7 (D) of culture. Original magnification, ×50.

FIG. 2.

(A) Immunohistochemical analysis of cervical tissue infected with HIV-1_BaL. Colocalization of HIV-1 p24 antigen (red) with cell markers (brown) for T cells (CD3, left), macrophages (CD68, middle), and dendritic cells (S100, right) in human cervical tissues. Arrows point to macrophages (CD68⁺ cells) with brown cytoplasmic staining that also stain red, denoting the presence of p24 antigen (original magnification, ×50). (B) The integrity of the cervical explant model system was evaluated by examining the transmission of fluorescent microspheres (0.026-μm diameter) across the tissues. On the first day of culture, 0.5 ml of the fluorescent microspheres (1 × 10¹⁴/ml) was added to the epithelial surface of three explants (1, 2, and 3; same tissue donor) and a well filled with 2% agarose. The basolateral medium was sampled at 1, 3, and 7 days, and the percent transmission was calculated by dividing the fluorescent signal in the basolateral supernatant from the explants and agarose control by that from the blank well.

FIG. 3.

Growth kinetics of HIV-1 in cervical tissues. After an overnight incubation with 10⁴ TCID₅₀ HIV-1_BaL or primary HIV-1 isolates (96USSN20/A CCR5/CXCR4-using, 97USSN54/A CCR5-using, and 92US660/B CCR5-using), the explants were washed and maintained in culture for 21 days. Basolateral supernatants were sampled every 3 to 4 days and assayed for p24gag protein.

FIG. 4.

The effects of microbicides and placebos on HIV-1_BaL infection in representative cervical explant cultures. (A) After overnight incubation with HIV-1_BaL alone (5 × 10⁴ TCID₅₀) in medium or a 1:10 dilution of microbicide or placebo (final concentrations are indicated on the figure) with virus, the explants were washed and maintained in culture. Basolateral supernatants were sampled twice a week for up to 17 days and assayed for p24gag protein. (B) Comparison of HIV-1-infected cervical explant cultures treated with a representative placebo or each of the tested microbicides. Products were diluted 1:10 in culture medium (final concentrations are indicated on the figure) containing HIV-1 (5 × 10⁴ TCID₅₀) and added to the apical surface of the tissue. After overnight incubation, the tissues were washed and maintained in culture. At the study endpoint, tissues were fixed and examined by immunohistochemical analysis for the presence of p24 antigen (red). Representative photographs from an explant treated with a placebo (polyacrylic acid) (top left) or each of the microbicides are shown (original magnification, ×50).

FIG. 5.

(A) Effects of diluted microbicides and placebos on cervical explant viability as determined using the MTT assay. After an overnight (18-h) incubation, the effect of each product on tissue viability was determined by comparing the viability of the treated explants to that of the untreated tissue control (same donor). Data are expressed as the average percent viabilities (± standard deviations) for each treatment tested in two donors (*, P < 0.05). (B) Histopathologic comparison of the effect of the different microbicides in cervical tissues. Microbicides or their placebos were diluted 1:10 in culture medium and added to the apical surface of the tissue. After overnight incubation, the tissues were washed and maintained in culture for 21 days. At the study endpoint, tissues were fixed and examined histologically for morphological changes. Representative photographs for one placebo (polyacrylic acid) and each of the microbicides are shown (hematoxylin and eosin stain; original magnification, ×50). The final concentrations of each product and placebo are indicated.