Yvonne T. Cosgrove Sweeney

The vaginal microflora of pig-tailed macaques and the effects of chlorhexidine and benzalkonium on this ecosystem.

Background and Objectives: To characterize normal vaginal microflora of pig‐tailed macaques and to evaluate two commonly used intravaginal compounds, chlorhexidine (CHG), a vaginal antiseptic (Surgilube, E. Fougera, Melville, NY), and benzalkonium chloride (BZK) (spermicidal contraceptive) in this monkey model to assess effects on the vaginal microflora. Study Design: Vaginal swabs were collected for microbiologic analysis to characterize normal flora. Subsequently, the vagina was exposed to either CHG or BZK twice at 24‐hour intervals. Results: The vaginal microflora of 26 pig‐tailed macaques was found to be remarkably similar to the vaginal flora of the human with respect to frequency of vaginal colonization by H2O2‐producing lactobacilli, Prevotella species, and several other microorganisms. After two vaginal applications at 24‐hour intervals, CHG had only small effects on the vaginal microflora of five animals. By contrast, BZK applied by the same protocol had profound adverse effects on the lactobacilli and Peptostreptococcus and more transient effects on vaginal Prevotella and viridans streptococci of six animals. Conclusions: These observations demonstrate that the vaginal microflora of the pig‐tailed macaque is a useful model in which to further evaluate newly developed intravaginal contraceptives that may be microbicidal and/or virucidal before widespread intravaginal use in women.

Rectal applications of nonoxynol-9 cause tissue disruption in a monkey model

Background Efforts to develop topical microbicide products have all but ignored evaluation for rectal use. Goal The goal of this study was to assess the effects of multiple rectal applications of Conceptrol (containing 4% nonoxynol-9) on flora and mucosal tissues in the pig-tailed macaque model. Study Design Monkeys (8 per group) received daily rectal applications of Conceptrol, placebo gel, or no product, for 3 days. At each visit, a preapplication rectal lavage specimen and swab specimen for microbiology and pH determination were collected. Conceptrol or placebo gel (2.5 ml) was then administered intrarectally. Fifteen minutes after application, samples were again collected. Results Gross observation of rectal lavage indicated sheets of epithelium 15 minutes after application of the nonoxynol-9 product. Histopathology of these samples revealed epithelial sheets with stroma attached. The presence of H2O2-producing lactobacilli remained relatively constant, whereas that of H2O2-producing viridans streptococci diminished in all nonoxynol-9-exposed animals in which these organisms were detected at baseline. Conclusions Repeated applications of nonoxynol-9 disrupts the rectal mucosa of the pig-tailed macaque. The disruption of these tissues could have serious implications for an increase in likelihood of acquisition of sexually transmitted infection/HIV in humans.

Safety and Efficacy Evaluations for Vaginal and Rectal Use of BufferGel in the Macaque Model

Background: The nonhuman primate model allows for safety and efficacy testing of topical microbicide products. Goal: The goal of this study was to evaluate the safety and efficacy of vaginal and rectal applications of BufferGel (ReProtect, Inc.). Study Design: The safety of repeated product applications was evaluated by microflora, pH, vaginal colposcopy, and rectal lavage. To test efficacy in preventing chlamydia, infection was documented by culture and nucleic acid amplification tests. Results: Repeated vaginal or rectal applications of BufferGel were not associated with significant changes in microflora. BufferGel use had a transient acidifying effect on vaginal and rectal pH. Colposcopic observations remained relatively normal in all test animals. A slightly increased incidence of epithelial desquamation was noted after rectal product use compared with the control group. BufferGel did not prevent cervical or rectal chlamydial infection. Conclusion: BufferGel has an acceptable safety profile after repeated vaginal and rectal use, but does not prevent chlamydial infection in the macaque models.

Significant Reduction in Inflammatory Response in the Macaque Model of Chlamydial Pelvic Inflammatory Disease with Azithromycin Treatment

We inoculated 45 female macaques in the cervix with Chlamydia trachomatis once weekly for 5 weeks and randomly assigned them to treatment with doxycycline (n=12), azithromycin (n=12), or placebo (n=21). At hysterectomy, cervical cultures remained positive in 12 of 21 placebo-treated monkeys, versus 0 of 12 doxycycline- or azithromycin-treated monkeys (P<.01); cervical ligase chain reaction remained positive in 15 placebo-, 1 doxycycline-, and 0 azithromycin-treated monkeys. Tubal swabs remained positive in 3 placebo-, 1 doxycycline-, and 0 azithromycin-treated monkeys. Immunopathologic damage was moderate to widespread in upper and lower reproductive-tract tissues from placebo- and doxycycline-treated monkeys but were significantly reduced in azithromycin-treated monkeys. Transforming growth factor- beta was also significantly less prevalent in azithromycin-treated monkeys. Azithromycin treatment dramatically reduced the inflammatory response and was highly effective in eradicating C. trachomatis from the lower and upper reproductive tract (12/12), compared with doxycycline (7/12) and placebo (3/21).

Antibody Response to the Chlamydial Heat-Shock Protein 60 in an Experimental Model of Chronic Pelvic Inflammatory Disease in Monkeys (Macaca nemestrina)

A primate model of chlamydial pelvic inflammatory disease was used to characterize serum antibody responses to the 60 kDa chlamydial heat shock protein (CHSP60). Forty monkeys were infected in the fallopian tubes with Chlamydia trachomatis and then were treated. Twenty-three (58%) monkeys developed antibodies against CHSP60, of whom 6 (15%) had CHSP60 responses that persisted throughout the study and 17 (42.5%) had a transient response. A persistent CHSP60 antibody response was correlated with being culture- or ligase chain reaction-positive in the fallopian tubes (P=.004), but not in the cervix pretreatment, and with being tubal-positive posttreatment (P=. 02). Compared with tubal-negative monkeys, tubal-positive monkeys had more intense CHSP60 responses (P=.006) that lasted longer (P=. 002). Among CHSP60 responders, an OD>0.5 was correlated with more severe salpingeal pathology before treatment (P=.04). CHSP60 antibody response may be useful as a marker of persistent chlamydial infection in the fallopian tubes.

Effects of nonoxynol-9 on vaginal microflora and chlamydial infection in a monkey model.

Background and Objectives: Nonoxynol‐9, an intravaginal microbicide, is chlamydiacidal in vitro but also cytotoxic. This study examines the effects of nonoxynol‐9 in vivo, using a pigtail macaque model of chlamydial cervicitis. Goals: To establish a minimum infectious dose of Chlamydia trachomatis in the macaque, and to observe the effects of a single dose of nonoxynol‐9 on efficiency of chlamydial infection, vaginal microflora, and cervicovaginal irritation. Study Design: The effects of 4% nonoxynol‐9, C. trachomatis (5,000 or 10,000 IFU) or both nonoxynol‐9 application and chlamydial infection were studied in 17 macaques. Results: Following a single application of nonoxynol‐9, chlamydial infection was prevented in 4 of 6 monkeys infected with 10,000 IFU; there was a transient decrease in anaerobic gram‐negative rods (P < 0.05) and Peptostreptococci (P > 0.05), but no change in Lactobacillus. Mild cervicovaginal irritation was observed in the monkeys. Conclusions: A single dose of nonoxynol‐9 causes minimal vaginal flora and epithelial irritation, and may be useful for prevention of chlamydial infection.

Macaca fascicularis vs. Macaca nemestrina as a model for topical microbicide safety studies

Abstract:  Preclinical studies of topical microbicide products, using appropriate animal models for assessing the safety of repeated use are essential. The pig‐tailed macaque (Macaca nemestrina) model has been used to assess the safety of vaginally and rectally applied topical microbicide products. The availability of sexually mature female pig‐tailed macaques has become extremely restricted. Currently, M. fascicularis is more readily available, and was therefore evaluated as an alternative model for topical microbicide pre‐clinical evaluation. Twenty sexually mature M. fascicularis were assessed for feasibility to mimic the established models. The rectal and cervicovaginal microenvironments of the M. fascicularis were determined to be similar to those of M. nemestrina and humans. The gross anatomy was significantly smaller than that of the pig‐tailed macaque, such that colposcopic examinations and multiple biopsies would not be possible. Thus, the M. fascicularis may not be useful for vaginally applied topical microbicide safety studies yet adequate for assessing safety of rectally applied topical microbicide products.

A Summary of Preclinical Topical Microbicide Rectal Safety and Efficacy Evaluations in a Pigtailed Macaque Model

Background: There is widespread recognition of the potential promise of vaginal microbicides as a tool to combat global human immunodeficiency virus/acquired immunodeficiency syndrome and sexually transmitted infections epidemics, and candidate product development has maintained a rapid pace in recent years; however, rectal microbicide development has received less attention. As it is likely that commercial products developed for vaginal use will also be used rectally, there is a clear need to assess the safety and efficacy of candidate microbicide products specifically in the rectal compartment. Methods: We have developed a standardized protocol for preclinical rectal safety and (chlamydial) efficacy assessment of topical microbicide candidates in a nonhuman primate model. We evaluated a total of 12 test compounds for rectal safety (via rectal pH, microflora, and rectal lavage) and 1 compound for efficacy against rectal chlamydial infection. Results: In this article, we describe our methods in detail and summarize our results, particularly noting the ability of our model to distinguish products with deleterious effects on the rectal environment. We also outline the specific criteria used to recommend products move into preclinical rectal efficacy trials or be recommended for reformulation to the product developer. In summary, we observed significant adverse effects in 2 products. The single product that underwent efficacy evaluation was not observed to be protective against rectal chlamydial infection. Conclusions: A preclinical safety and efficacy model is critical to promoting rectal microbicide development, which will ultimately offer a significant opportunity for intervention in the global HIV/AIDS epidemic.

Vaginal and Rectal Topical Microbicide Development: Safety and Efficacy of 1.0% Savvy (c31g) in the Pigtailed Macaque

Background: A 1.0% gel formulation of C31G, a surfactant, has been shown to have in vitro antiviral and antibacterial activity. Goal: The goal of this study was to evaluate the safety and efficacy of vaginal and rectal applications of 1.0% Savvy (C31G) in the nonhuman primate model. Study Design: The safety of repeated 1.0% C31G application was evaluated by microflora, pH, vaginal biopsy, colposcopy, and rectal lavage. Efficacy in preventing chlamydial infection was documented by culture, nucleic acid amplification tests, and serology. Results: Repeated applications of Savvy (1.0% C31G) were not associated with significant changes in pH, microflora, or inflammatory infiltrates on tissues. No significant differences in epithelial desquamation were noted after rectal product use compared with placebo. Four of 6 animals were protected from chlamydial infection after pretreatment with Savvy. C31G was shown to be safe to both vaginal and rectal mucosal tissues and to the microflora with repeated daily use. Conclusion: Savvy has an acceptable safety profile after repeated vaginal and rectal use. A single intravaginal application of 1.0% C31G provided partial protection from acquiring cervical chlamydial infection.

Heat Shock Protein 60 Is the Major Antigen Which Stimulates Delayed-Type Hypersensitivity Reaction in the Macaque Model of Chlamydia trachomatis Salpingitis

ABSTRACT Chlamydial delayed-type hypersensitivity antigens were analyzed by using the subcutaneous salpingeal autotransplant model of Macaca nemestrina infected with Chlamydia trachomatis serovar E. Heat shock protein 60 was the only antigen shown to induce delayed-type hypersensitivity among other antigens tested, including UV-inactivated organisms, recombinant major outer membrane protein, purified outer membrane proteins, and heat shock protein 10.