Preclinical evaluation of synthetic -2 RANTES as a candidate vaginal microbicide to target CCR5

Abstract

A potential strategy that can be used to combat the worldwide AIDS epidemic is the development of a vaginal microbicide that prevents the sexual transmission of human immunodeficiency virus type 1 (HIV-1). Certain CC chemokines, including RANTES, MIP-1alpha, and MIP-1beta, might facilitate the development of such microbicides since they potently suppress HIV-1 infection by binding to CCR5, the viral coreceptor used by most sexually transmitted strains of HIV-1 to enter host cells. In this study, we evaluated whether a CCR5-specific fragment of RANTES that lacks two N-terminal residues (-2 RANTES) and possesses especially potent HIV-1 suppressive activity has toxicity profiles conducive to the advancement of testing in candidate microbicide formulations. Analyses were carried out with a synthetic version of the chemokine, which was formulated with either Novasomes 7474, a nonphospholipid liposome, or methylcellulose gel. Dialysis studies demonstrated that the formulated -2 RANTES was released from both vehicles and retained anti-HIV-1 activity. Preclinical toxicity studies carried out with Swiss Webster mouse and New Zealand White rabbit vaginal irritation models demonstrated minimal inflammation and minimal adverse changes in cervicovaginal tissue integrity after short-term (10 min) and long-term (24 h) exposure to formulations containing up to 1 mg/ml of -2 RANTES. Similarly, no toxicity was observed with formulations of bioactive murine RANTES in the Swiss Webster mouse vaginal irritation model. Overall, these preclinical studies suggest that -2 RANTES is suitable for further testing as a candidate anti-HIV-1 microbicide.

FIG. 1.

Analysis of synthetic human −2 RANTES toxicity in vitro. HeLa cells were exposed to increasing concentrations of −2 RANTES or N-9 (positive control) for 30 min (A), 2 h (B), 4 h (C), and 24 h (D). Microbicide concentrations up to 1 mg/ml of −2 RANTES and N-9 were evaluated at the indicated time points. Cellular viability was measured immediately following microbicide exposure by using an MTS cellular viability assay. The results are expressed relative to those for mock-exposed cells. The data in each graph represent the average of three experiments, in which each concentration was tested in triplicate. Error bars indicate the standard deviations of the calculated mean values.

FIG. 2.

Effects of synthetic human −2 RANTES on the vaginal mucosa in the Swiss Webster mouse model. Swiss Webster mice were inoculated intravaginally with 60 μl of −2 RANTES (1 mg/ml). The genital tracts were harvested from the mice at 30 min, 2 h (C and D), 4 h, 8 h, and 24 h (E and F) following application. Formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin and eosin (H & E) for gross morphological analyses of the surface epithelium (A, C, and E). Immunohistochemical analyses were also performed for the detection of CD45-positive stained cells, as indicated by arrows (B, D, and F). The inset in panel B magnifies cells staining positive for CD45. Two independent experiments were performed with three mice per time point, and representative tissue sections are presented. PBS-treated mice (2 h) (A and B) and 1% N-9-treated (data not shown) mice were included in each experiment as reference controls.

FIG. 3.

Effects of synthetic human −2 RANTES on the cervical mucosa in the Swiss Webster mouse model. Swiss Webster mice were inoculated intravaginally with 60 μl of −2 RANTES (1 mg/ml). The genital tracts were harvested from the mice at 30 min, 2 h (C and D) 4 h, 8 h, and 24 h (E and F) following application. Formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin and eosin (H & E) for gross morphological analyses of the surface epithelium (A, C, and E). Immunohistochemical analyses were also performed for the detection of CD45-positive stained cells, as indicated by arrows (B, D, and F). The inset in panel B magnifies cells staining positive for CD45. Two independent experiments were performed with three mice per time point, and representative tissue sections are presented. PBS-treated mice (2 h) (A and B) and 1% N-9-treated (data not shown) mice were included in each experiment as reference controls.

FIG. 4.

Effects of recombinant murine RANTES on cervicovaginal mucosa in the Swiss Webster mouse model. Murine cervicovaginal epithelium was treated with 2 μg/ml of murine RANTES, and tissues were assessed by hematoxylin and eosin staining for evidence of gross morphological damage or evidence of inflammation. Representative vaginal and cervical tissues collected at 30 min, 2 h, 4 h, and 24 h are displayed in the columns labeled vagina and cervix, respectively. Arrows indicate the initial points when increased cell infiltrate was observed below the epithelium. Three independent experiments were performed with at least three mice per time point.

FIG. 5.

Release of −2 RANTES from carrier vehicles. Dialysis experiments (see Materials and Methods) were used to evaluate the release of chemokine from methylcellulose (MC) and Novasomes 7474 carrier vehicles. (A) PBS formulations containing the indicated concentrations of −2 RANTES were placed in dialysis bags and dialyzed against PBS. Samples of the dialysis buffer were collected at the indicated times and assayed for −2 RANTES by ELISA. (B) Various concentrations of −2 RANTES were formulated in Novasomes 7474 or methylcellulose vehicles and placed in dialysis bags. The amount of chemokine detected in the buffer after 360 min of dialysis is presented as the percentage of that released from the corresponding −2 RANTES formulations in PBS. The data shown represent the average of values obtained from at least two independent experiments. Error bars indicate standard deviations of the calculated mean values.

FIG. 6.

Anti-HIV activity of −2 RANTES released from vehicle formulations. Samples collected after 120 min of dialysis were tested in infectivity assays with HIV-1_BaL (see Materials and Methods). After 6 days, infection was determined as a function of HIV p24 levels in the culture supernatants. Replicate values were averaged and were used to generate dose-effect curves. The curves were used to calculate the IC₅₀ of each test formulation. An average value from two independent experiments is presented.

FIG. 7.

−2 RANTES formulations are nontoxic to the murine cervicovaginal mucosa. Swiss Webster mice were anesthetized and intravaginally inoculated with 60 μl of −2 RANTES (1 mg/ml) formulated in Novasomes 7474 and methylcellulose carrier vehicles. The results for controls treated with vehicle alone are also presented. The genital tracts were harvested from the mice at 30 min, 2 h, 4 h, 8 h, and 24 h following application. Formalin-fixed, paraffin-embedded tissue sections were stained with standard hematoxylin and eosin for gross morphological analyses of the surface epithelium. Representative tissue sections harvested at 2 h are presented here. Three independent experiments were performed with at least two mice per time point.