Dorothy L. Patton

The Chlamydia trachomatis plasmid and inclusion membrane protein CT135 are virulence factors in the pathogenesis of murine female genital tract infection. To determine if these virulence factors play a similar role in female nonhuman primates, we infected pig-tailed macaques with the same C. trachomatis strains shown to be important in the murine model. Wild-type C. trachomatis and its isogenic mutant strain deficient in both plasmid and CT135 were used to infect macaques. Macaques were given primary and repeated cervicovaginal challenges with the wild-type and mutant strains. The infection rate, infection duration, and antibody response were similar among macaques infected with both strains. Unexpectedly, colposcopy, laparoscopy, and histologic analysis revealed no substantial genital tract pathology following either primary or repeated cervicovaginal challenges. Cytokine analysis of cervicovaginal secretions from both challenged groups revealed low concentrations of interleukin 1β (IL-1β) and elevated levels of the interleukin 1 receptor agonist (IL-1RA). We propose that an imbalance of IL-1β and IL-1RA in macaques is the reason for the mild inflammatory responses observed in infected urogenital tissues. Thus, understanding the pathobiology of chlamydial infection requires a better understanding of host epigenetic and chlamydial genetic factors. Our findings also have implications for understanding the high frequency of asymptomatic infections in humans.

Background Inclusion of sexual activity in the macaque model for topical microbicide safety evaluation would more closely mimic human use of topical microbicides and provide a more rigorous safety assessment. Methods Initially, male–female partners were monitored in cohousing arrangements to determine whether macaques would copulate ad libitum. The logistics of performing vaginal examinations before and after coital visits were analyzed and optimized. Findings from cervicovaginal examinations conducted before and after sexual activity were compared. Results Coital activity was reliably observed in the majority of cohousing sessions, representing all phases of the menstrual cycle. Female macaques were trained to be restrained while fully alert for pre‐coital vaginal sampling. Post‐coital examinations occur under general sedation. Post‐coital examinations reveal alterations to tissues, microbiology, and pH compared with pre‐coital visits. Conclusions This work clearly demonstrates that it is feasible to incorporate sexual activity in the macaque model for topical microbicide safety assessment.

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.

The development of topical microbicides represents a new and exciting field in the prevention of sexually transmitted diseases, and it is especially important that candidate products undergo rigorous preclinical safety and efficacy testing before advancing to clinical trials. We have developed a standardized protocol for preclinical vaginal safety and efficacy assessment of topical microbicide candidates in a nonhuman primate model. Over 7 years of funding under an NIH contract, we evaluated a total of 28 test compounds for vaginal safety (via colposcopy, vaginal pH, and microflora) and 9 compounds for efficacy against cervical chlamydial infection. 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 cervicovaginal environment. We also outline the specific criteria used to determine which products should move into efficacy trials and which should be recommended for reformulation to the manufacturer. Overall, we noted acceptable safety profiles for 24 of 28 candidate products. Common findings included a transient decrease in vaginal pH, petechiae, and mild erythema. Four products were associated with significant adverse colposcopic findings including blisters, epithelial abrasions, and friability; all 4 products were successfully reformulated and showed acceptable safety profiles at lower concentrations. No products showed complete protection against cervical chlamydial infection. The macaque preclinical safety and efficacy model is critical to maintaining the pace of topical microbicide development, which could ultimately offer a significant opportunity for intervention in the global HIV/AIDS epidemic.

The nonnucleoside reverse transcriptase inhibitor UC781 is under development as a potential microbicide to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1). Two gel formulations of UC781 (0.1% and 1.0%) were evaluated in a range of preclinical safety assessments, including systemic absorption analysis following topical application in the pig-tailed macaque models for vaginally and rectally applied topical microbicides. High-sensitivity high-performance liquid chromatography analysis of serum samples showed that no systemic absorption of UC781 was detected after repeated vaginal or rectal application of either product. However, high levels of UC781 were detectable in the cervicovaginal lavage samples up to 6 h after product exposure. Both formulations were safe to the vaginal microenvironment, even with repeated daily use, as evidenced by colposcopy, cytokine analysis, and lack of impact on vaginal microflora. By contrast, rectal application of the 1.0% UC781 formulation caused an increased expression of numerous cytokines not observed after rectal application of the 0.1% UC781 formulation. These results provide additional support for the continued development of UC781 formulations as anti-HIV microbicides.

Problem: The purpose was to identify distinct inflammatory markers in endometrial tissues of women with abnormal uterine bleeding (AUB) and Chlamydia trachomatis infection.Method of study: Archived endometrial specimens from 92 randomly selected premenopausal women with AUB were examined for C. trachomatis using the species‐specific monoclonal antibody against major outer membrane protein (MOMP) and for histopathology associated with inflammation. Statistical analyses included single and multiple logistic regression. Diagnostic accuracy was summarized using receiver operating characteristic (ROC) curves.Results: Chlamydia trachomatis was detected in 44 (48%) of 92 AUB specimens. There were statistically significant correlations of positive MOMP with higher counts of plasma cells (P < 0.01), macrophages (P < 0.0001), and lymphocytic foci (P = 0.01). The ROC curve for macrophages was the strongest predictor (area under the curve = 0.82) for C. trachomatis.Conclusion: The prevalence of C. trachomatis in women with AUB is under‐estimated. Macrophages appear to be a strong marker for the presence of C. trachomatis in the endometrium.

Objective: Trichomoniasis, caused by Trichomonas vaginalis, is a prevalent sexually transmitted infection associated with increased risk of HIV infection. An animal model of T. vaginalis infection would enable scientists to further investigate trichomoniasis. Study Design: Seven macaques (4 test vs. 3 control) were enrolled in a 2-week pilot study. Eight additional animals participated in a 2-arm (T. vaginalis vs. sham inoculated) crossover study lasting 5 weeks before treatment. In all, 12 Macaca nemestrina monkeys were challenged with a single intravaginal inoculation of 6.6 to 7.1 × 10⁵ trichomonads (ATCC 50148). Vaginal culture (InPouch TV), colposcopy, microbiology, pH, and cervical cytokines were assessed at baseline, day 2, and weekly thereafter. Results: Ten of 12 challenged animals tested positive for trichomoniasis for 2 weeks or longer. One animal tested positive on days 2 and 7 but negative thereafter. Only one animal was not infected. Oral metronidazole treatment (35 mg/kg per day for 3 days) resolved infection in all animals. Trichomoniasis infection did not lead to shifts in vaginal microbiology or pH. Conclusions: A single T. vaginalis inoculation results in persistent infection 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.

Three gel formulations (1%, 3%, and 5% [wt/wt]) of SPL7013, a dendrimer known to have antiviral (anti-human immunodeficiency virus and anti-herpes simplex virus) activities, completed a range of preclinical tests in the pigtailed macaque models for vaginally and rectally applied topical microbicide safety assessments. The vaginal safety profile of the 3% SPL7013 gel formulation was equal to that of the 1% formulation but was superior to that of the 5% formulation. The 3% SPL7013 gel was further evaluated for rectal safety and for antichlamydial efficacy with cervical challenge with Chlamydia trachomatis . This first-generation dendrimer-based product was shown to be safe to the vaginal and rectal microenvironments with repeated daily use. However, a single intravaginal application of the 3% (wt/wt) SPL7013 gel did not provide protection from the acquisition of cervical chlamydial infection.