Impact of HSV-2 shedding on the vaginal microbiome in the female genital tract

项目摘要 单纯疱疹病毒2型（HSV-2）和细菌性阴道病（BV）是两种最常见的感染， 女性生殖道这两种感染都增加了感染艾滋病毒的风险。而HSV-2和 许多研究表明BV与流行病学存在联系，但其相互作用的方向和机制尚不清楚 知道的我们的团队率先每天收集参与者的样本，以显示HSV-2被检测到（“脱落”） 通常，在10-28%的天数，从生殖器表面，HSV-2脱落是无症状的， 当时最近的数据表明，HSV-2生殖器病变和无症状的脱落事件， 与持续的炎症细胞反应有关，IFN-γ产生HSV特异性CD 4+， 组织驻留的CD 8 + T细胞。这些数据表明，HSV-2感染慢性和根本性改变， 生殖道炎症。Marrazzo博士和Fredricks博士同样使用频繁的阴道采样， 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）。该建议旨在阐明HSV-2/BV的时间和机制性质 相关性，并确定可能增加风险的HSV-2炎症反应的成分 的BV。我们假设频繁的HSV-2生殖器脱落导致炎症水平的增加， 细胞因子，如IL-1 β和IFN-γ，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），这些变化导致阴道微生物组的波动 使妇女容易患上BV。为了解决这些假设，我们将招募40名HSV-2血清阳性， 在过去一年中有BV病史的HIV血清阴性女性，将自行收集生殖器和阴道 每天采集标本30天。我们还将招募20名HSV-2血清阳性且无BV临床病史的女性。 我们将使用经验证的PCR检测来量化HSV，我们将使用 革兰氏染色（Nugent评分）、定量PCR（qPCR）和宽范围16 S rRNA基因PCR， 在HSV脱落发作期间进行通量测序。先天免疫蛋白的每日阴道水平， 将测量与HSV-2脱落相关的细胞因子。HSV-2排毒与 将确定Nugent评分、BVAB的数量和存在以及细胞因子水平。这些女性将 可以放置在抑制性阿昔洛韦，减少HSV-2脱落70- 80%。我们将决定 抑制HSV-2脱落导致阴道植物群改善，如通过Nugent评分降低所测量的 和乳酸杆菌浓度增加。这一综合提案将确定 HSV-2脱落和阴道微生物组，并将深入了解这种关联的机制基础 为了解这些感染如何影响生殖道炎症提供一个框架。 最终，该提案将为干预措施的设计提供重要和新颖的信息， 调节BV，并将为我们提供知识，以更好地促进妇女的生殖健康。

Project Summary 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 IFN-γ producing HSV-specific CD4+ and tissue resident CD8+ T-cells. These data suggest that HSV-2 infection chronically and fundamentally alters inflammation 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 that may increase the risk of BV. We hypothesize that frequent HSV-2 genital shedding causes increased levels of inflammatory cytokines, such as IL-1ß and IFN-γ, and decreased levels of 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 40 HSV-2 seropositive, HIV seronegative women with a history of BV in the past year who will self-collect genital and vaginal specimens daily for 30 days. We will also enroll 20 HSV-2 seropositive women without a clinical history of BV. We will quantify HSV using a 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 high throughput sequencing during HSV shedding episodes. Daily vaginal levels of innate immune proteins and cytokines associated with HSV-2 shedding will be measured. Relationships between HSV-2 shedding and Nugent score, quantity and presence of BVAB, and cytokine levels will be determined. These women will then be placed on suppressive acyclovir, which decreases HSV-2 shedding by 70-80%. We will determine whether suppression of HSV-2 shedding results in improved vaginal flora as measured by a decrease in Nugent score and increased concentration of lactobacilli. 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 to provide a framework for appreciating how these infections influence inflammation within the genital tract. Ultimately, this proposal will contribute vital and novel information to the design of interventions that can modulate BV, and will provide us with knowledge to better promote women's reproductive health.