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)是最常见的两种感染 女性生殖道。这两种感染都与艾滋病毒感染风险增加有关。而单纯疱疹病毒2型和 BV在许多研究中都有流行病学联系，但它们相互作用的方向和机制尚不清楚 为人所知。我们小组首创了每天收集参与者的样本，以表明HSV-2已被检测到。 通常，在10%-28%的时间里，生殖器表面出现HSV-2脱落，大多数人没有症状 时间到了。最近的数据表明，HSV-2生殖器损害和无症状脱皮发作 与持续的炎症细胞反应有关，干扰素-γ产生单纯疱疹病毒特异性的cd4+和 组织驻留的CD8+T细胞。这些数据表明，单纯疱疹病毒2型感染会从根本上改变 生殖道发炎。Marrazzo博士和Fredricks博士同样使用了频繁的阴道采样和 用分子方法记录阴道微生物组的快速变化，并发现与BV相关的新病毒 细菌(BVAB)。这项建议旨在阐明HSV-2/BV的时间性和机械性 并确定对HSV-2的炎症反应中可能增加风险的成分 BV的。我们假设频繁的HSV-2生殖器脱落会导致炎症水平的增加 细胞因子，如IL-1ç和干扰素-γ，以及抗病毒天然免疫分子水平的降低(即分泌性 白细胞蛋白酶抑制剂)，这些变化导致阴道微生物群的波动 这使女性更容易患上BV。为了解决这些假设，我们将招募40名HSV-2血清阳性的患者， 过去一年有BV病史的HIV血清阴性妇女将自我收集生殖器和阴道 连续30天每天采集样本。我们还将招募20名无BV临床病史的HSV-2血清阳性女性。 我们将使用经过验证的聚合酶链式反应方法对HSV进行量化，并使用 革兰氏染色(纽金特评分)、定量聚合酶链式反应(QPCR)和广谱16S rRNA基因聚合酶链式反应 单纯疱疹病毒脱毒过程中的吞吐量测序。每日阴道天然免疫蛋白水平和 将测量与HSV-2脱落相关的细胞因子。单纯疱疹病毒2型脱落与病毒感染的关系 Nugent评分，BVAB的数量和存在，以及细胞因子水平将被确定。然后这些女人就会 加用抑制性阿昔洛韦，可使HSV-2的脱落减少70%-80%。我们将确定是否 抑制HSV-2的脱落导致阴道菌群的改善，通过Nugent评分的下降来衡量 和乳酸菌浓度的增加。这一综合提案将决定 HSV-2脱落与阴道微生物群的关系，并将深入了解这种联系的机制基础 为评估这些感染如何影响生殖道内的炎症提供了一个框架。 最终，这项建议将为设计干预措施提供重要和新颖的信息，这些干预措施可以 调节BV，将为我们提供更好地促进妇女生殖健康的知识。

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.