New Candidate Biomarkers in the Female Genital Tract to Evaluate Microbicide Toxicity

Vaginal microbicides hold great promise for the prevention of viral diseases like HIV, but the failure of several microbicide candidates in clinical trials has raised important questions regarding the parameters to be evaluated to determine in vivo efficacy in humans. Clinical trials of the candidate microbicides nonoxynol-9 (N9) and cellulose sulfate revealed an increase in HIV infection, vaginal inflammation, and recruitment of HIV susceptible lymphocytes, highlighting the need to identify biomarkers that can accurately predict microbicide toxicity early in preclinical development and in human trials. We used quantitative proteomics and RT-PCR approaches in mice and rabbits to identify protein changes in vaginal fluid and tissue in response to treatment with N9 or benzalkonium chloride (BZK). We compared changes generated with N9 and BZK treatment to the changes generated in response to tenofovir gel, a candidate microbicide that holds promise as a safe and effective microbicide. Both compounds down regulated mucin 5 subtype B, and peptidoglycan recognition protein 1 in vaginal tissue; however, mucosal brush samples also showed upregulation of plasma proteins fibrinogen, plasminogen, apolipoprotein A-1, and apolipoprotein C-1, which may be a response to the erosive nature of N9 and BZK. Additional proteins down-regulated in vaginal tissue by N9 or BZK treatment include CD166 antigen, olfactomedin-4, and anterior gradient protein 2 homolog. We also observed increases in the expression of C-C chemokines CCL3, CCL5, and CCL7 in response to treatment. There was concordance in expression level changes for several of these proteins using both the mouse and rabbit models. Using a human vaginal epithelial cell line, the expression of mucin 5 subtype B and olfactomedin-4 were down-regulated in response to N9, suggesting these markers could apply to humans. These data identifies new proteins that after further validation could become part of a panel of biomarkers to effectively evaluate microbicide toxicity.

阴道杀微生物剂（vaginal microbicides）在预防HIV等病毒性疾病方面展现出巨大应用潜力，但多款候选杀微生物剂在临床试验中遭遇失败，引发了关于需评估哪些参数以确定其在人体内活体功效的关键问题。针对候选杀微生物剂壬苯醇醚-9（nonoxynol-9, N9）与硫酸纤维素的临床试验结果显示，HIV感染风险、阴道炎症程度以及HIV易感淋巴细胞的招募情况均有所上升，这凸显出亟需识别可在临床前开发早期及人体试验中准确预测杀微生物剂毒性的生物标志物。本研究采用定量蛋白质组学与逆转录聚合酶链反应（RT-PCR）技术，在小鼠与家兔模型中检测阴道液及组织经壬苯醇醚-9（N9）或苯扎氯铵（benzalkonium chloride, BZK）处理后的蛋白表达变化。我们将N9与BZK处理引发的蛋白表达变化，与替诺福韦凝胶（tenofovir gel）——一款被证实具备安全有效潜力的候选杀微生物剂——处理引发的变化进行了对比。两种化合物均可下调阴道组织中黏蛋白5亚型B（mucin 5 subtype B）与肽聚糖识别蛋白1（peptidoglycan recognition protein 1）的表达；不过，黏膜刷拭样本还显示血浆蛋白纤维蛋白原、纤溶酶原、载脂蛋白A-1及载脂蛋白C-1的表达出现上调，这或许是机体对N9与BZK腐蚀性的应答反应。经N9或BZK处理后，阴道组织中另有多款蛋白表达下调，包括CD166抗原（CD166 antigen）、嗅素-4（olfactomedin-4）以及前部梯度蛋白2同源物（anterior gradient protein 2 homolog）。我们还观察到，经上述化合物处理后，C-C趋化因子（C-C chemokines）CCL3、CCL5与CCL7的表达水平有所上升。小鼠与家兔模型中，多款上述蛋白的表达变化趋势保持一致。利用人类阴道上皮细胞系开展的验证实验显示，N9处理可下调黏蛋白5亚型B与嗅素-4的表达，这表明这些标志物或许可推广应用于人类相关研究。本研究鉴定出了全新的蛋白标志物，经进一步验证后，它们可组成一组生物标志物组合，用于有效评估杀微生物剂的毒性。