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

描述(申请人提供)：单纯疱疹病毒2型(HSV-2)和细菌性阴道病(BV)是女性生殖道最常见的两种感染。这两种感染都与艾滋病毒感染风险增加有关。虽然HSV-2和BV在许多研究中存在流行病学联系，但它们相互作用的方向和机制尚不清楚。我们小组率先每天收集参与者样本，以表明在10%-28%的日子里，生殖器表面频繁地检测到HSV-2，并且HSV-2的脱落在大部分时间是无症状的。最近的数据表明，HSV-2生殖器病变和无症状的脱皮发作与持续的炎症细胞反应有关，HSV特异性的CD4+和CD8+T细胞产生干扰素？这些数据表明，HSV-2感染会从根本上慢性改变生殖道。Marrazzo博士和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)。这一建议试图阐明HSV-2/BV相关性的时间和机制性质，并确定可能调节这种相关性的HSV-2和BV炎症反应的成分。我们推测，频繁的HSV-2生殖器脱落导致炎性细胞因子水平升高，如IL-1β和干扰素-β，而抗病毒天然免疫分子(即分泌性白细胞蛋白酶抑制物，SLPI)水平下降，这些变化导致阴道微生物群的波动，使女性更容易患上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、有反复BV和HSV-2病史的妇女，她们将在28天内每天自我收集生殖器和阴道样本。我们将使用一种有效的PCR方法对HSV进行定量，并将使用革兰氏染色(Nugent Score)、定量PCR(QPCR)和广谱16S rRNA基因聚合酶链式反应(16S RRNA)结合焦磷酸测序来测量HSV脱落过程中微生物组的变化。每天将测量与HSV-2和BV相关的阴道天然免疫蛋白和细胞因子的水平。将确定HSV-2脱落与Nugent评分、BV相关细菌(BVAB)的数量和存在以及细胞因子水平的关系。在一组妇女中，我们将通过Nugent评分的降低来确定使用阿昔洛韦抑制HSV-2脱落是否会导致阴道菌群的改善。我们还将确定每周两次的阴道内甲硝唑凝胶是否与HSV脱落率的降低有关。这一综合建议将确定HSV-2脱落和阴道微生物群之间的关系，并将深入了解这种联系的机制基础。了解HSV-2/BV的相关性将为评估这些感染如何影响生殖道内的炎症提供一个框架。最终，这项提议将为我们提供更好地促进妇女生殖健康的知识。

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.