Interaction between HSV-2 and the vaginal microbiome in the female genital tract

描述（由申请人提供）：2型单纯疱疹病毒（HSV-2）和细菌性阴道病（BV）是女性生殖道最常见的两种感染。这两种感染都增加了感染艾滋病毒的风险。虽然在许多研究中HSV-2和BV在流行病学上有联系，但它们相互作用的方向和机制尚不清楚。我们的研究小组率先每天对参与者进行采样，以显示在10-28%的天数内经常从生殖器表面检测到HSV-2（“脱落”），并且HSV-2脱落在大多数时间是无症状的。最近的数据表明，HSV-2生殖器病变和无症状性散毒发作与持续性炎症细胞应答相关，HSV特异性CD 4+和CD 8 + T细胞产生IFN-？。这些数据表明，HSV-2感染慢性和根本性地改变生殖道。Marrazzo和Fredricks博士同样使用频繁的阴道采样和分子方法来记录阴道微生物组的快速变化，并发现新的BV相关细菌（BVAB）。该提案旨在阐明HSV-2/BV关联的时间和机制性质，并确定可能调节该关联的对HSV-2和BV的炎症反应的组分。我们假设，频繁的HSV-2生殖器脱落导致炎症细胞因子水平升高，如IL-1？和IFN-？，以及抗病毒先天免疫分子（即分泌性白细胞蛋白酶抑制剂，SLPI）的减少，这些变化导致阴道微生物组的波动，使女性易患BV。为了解决这些假设，我们将招募100名HSV-2血清阳性、HIV血清阴性、有复发性BV和HSV-2病史的女性，她们将每天自我采集生殖器和阴道标本，持续28天。我们将使用经过充分验证的PCR测定法定量HSV，并将使用革兰氏染色（Nugent评分）、定量PCR（qPCR）和宽范围16 S rRNA基因PCR结合焦磷酸测序在HSV脱落发作期间测量微生物组的变化。将测量与HSV-2和BV相关的先天免疫蛋白和细胞因子的每日阴道水平。将确定HSV-2散毒与Nugent评分、BV相关细菌（BVAB）的数量和存在以及细胞因子水平之间的关系。在一部分女性中，我们将确定使用阿昔洛韦抑制HSV-2脱落是否会导致阴道植物群的改善，通过Nugent评分的降低来衡量。我们还将确定每周两次阴道内甲硝唑凝胶是否与HSV脱落率降低相关。这项综合提案将确定HSV-2脱落与阴道微生物组之间的关系，并将深入了解这种关联的机制基础。了解HSV-2/BV相关性将为了解这些感染如何影响生殖道内的炎症提供一个框架。最终，这项建议将为我们提供知识，以更好地促进妇女的生殖健康。

DESCRIPTION (provided by applicant): Herpes simplex virus type 2 (HSV-2) and bacterial vaginosis (BV) are two of the most prevalent infections of the female genital tract. Both infections are associated increased risk for HIV acquisition. While HSV-2 and BV have been epidemiologically linked in many studies, the direction and mechanism of their interaction is not known. Our group has pioneered daily participant-collected sampling to show that HSV-2 is detected ("shed") frequently, on 10-28% of days, from genital surfaces, and that HSV-2 shedding is asymptomatic the majority of the time. More recent data has demonstrated that HSV-2 genital lesions and asymptomatic shedding episodes are associated with a persistent inflammatory cellular response, with HSV-specific CD4+ and CD8+ T-cells producing IFN-?. These data suggest that HSV-2 infection chronically and fundamentally alters the genital tract. Drs. Marrazzo and Fredricks have similarly used frequent vaginal sampling and molecular methods to document rapid shifts in the vaginal microbiome, and to discover novel BV-associated bacteria (BVAB). This proposal seeks to elucidate the temporal and mechanistic nature of the HSV-2/BV association and to identify the components of the inflammatory response to HSV-2 and BV that may modulate the association. We hypothesize that frequent HSV-2 genital shedding causes increased levels of inflammatory cytokines, such as IL-1¿ and IFN-?, and decreases in antiviral innate immune molecules (i.e.secretory leukocyte protease inhibitor, SLPI) and that these changes result in fluctuations in the vaginal microbiome which predispose women to develop BV. To address these hypotheses, we will enroll 100 HSV-2 seropositive, HIV seronegative women with a history of recurrent BV and HSV-2, who will self-collect genital and vaginal specimens daily for 28 days. We will quantify HSV using a well-validated PCR assay, and we will measure changes in the microbiome using gram stain (Nugent score), quantitative PCR (qPCR) and broad range 16S rRNA gene PCR with pyrosequencing during HSV shedding episodes. Daily vaginal levels of innate immune proteins and cytokines associated with both HSV-2 and BV will be measured. Relationships between HSV-2 shedding and Nugent score, quantity and presence of BV-associated bacteria (BVAB), and cytokine levels will be determined. In a subset of women, we will determine whether suppression of HSV-2 shedding using acyclovir will result in improved vaginal flora as measured by a decrease in Nugent score. We will also determine whether twice weekly intravaginal metronidazole gel is associated with a decrease in HSV shedding rates. This integrated proposal will determine the relationship between HSV-2 shedding and the vaginal microbiome, and will give insight into the mechanistic basis of the association. Understanding the HSV-2/BV association will provide a framework for appreciating how these infections influence inflammation within the genital tract. Ultimately, this proposal will provide us with knowledge to better promote women's reproductive health.