Development of a novel mouse model with progesterone-independent susceptibility to genital HSV infection

生殖器疱疹是由单纯疱疹病毒（HSV）引起的一种无法治愈的性传播感染（STI），对女性的影响不成比例，并可能导致性别特异性不良事件，如获得其他STI的风险增加以及分娩期间的垂直传播。小鼠常用作研究阴道HSV感染的免疫和发病机制的临床前模型。然而，小鼠模型的一个主要缺点是，与人类不同，小鼠仅在发情周期的某些阶段易受感染。因此，雌性小鼠通常用长效醋酸甲羟孕酮（DMPA，Depo-Provera）（一种基于孕酮的激素避孕药）预处理，以使小鼠保持在动情间期并确保对HSV感染的均匀易感性。然而，DMPA的使用限制了对发情周期的单个阶段的宿主-病原体相互作用的研究，尽管有大量证据表明雌性性激素对雌性生殖道的抗病毒防御和免疫监视具有重大影响。此外，DMPA的使用排除了与阴道细菌或真菌的共接种研究，因为这些微生物通常需要高雌激素的环境来稳固地定殖小鼠阴道。小鼠和人类阴道之间的一个主要差异是，上皮细胞上的病毒进入受体nectin-1的表达在小鼠中是激素依赖性的，但在人类中是组成性表达的。为了克服这一挑战，该提议将开发一种新的小鼠模型，其中nectin-1通过解决转录和翻译后调控机制在小鼠阴道上皮中组成型表达。连接蛋白-1表达将置于两个不同的组成型启动子的控制下，并且将引入两个突变以防止胞外域切割。上皮特异性表达的nectin-1将通过包含stop-flox盒和繁殖到Cre重组酶表达受人角蛋白14启动子控制的小鼠来实现。最后，将在发情周期的不同阶段测试这些新型小鼠的HSV易感性。成功开发一种新型的、更具转化性的HSV感染小鼠模型将有助于实现我们的长期目标，即探索性激素在调节阴道屏障处的宿主-病原体相互作用中的作用，以及研究阴道微生物生态失调或STI共感染对HSV发病机制的分子影响。我们希望未来的研究能够揭示生殖器疱疹保护性免疫的未知方面，包括疫苗诱导的免疫，并可能导致发现新的治疗靶点，旨在降低对HSV感染的易感性。

Genital herpes, an incurable sexually transmitted infection (STI) caused by herpes simplex virus (HSV), disproportionately affects women and can cause sex-specific adverse events such as increased risk of acquiring other STIs as well as vertical transmission during birth. The mouse is commonly used as a preclinical model to study immunity and pathogenesis of vaginal HSV infection. However, one major drawback of the murine model is that unlike humans, mice are susceptible to infection only at certain stages of the estrus cycle. Thus, female mice are typically pre-treated with depot medroxyprogesterone acetate (DMPA, Depo-Provera), a progesterone-based hormonal contraceptive, to hold the mice in diestrus and ensure uniform susceptibility to HSV infection. However, the use of DMPA limits the study of host-pathogen interactions a single stage of the estrus cycle, despite abundant evidence that female sex hormones have a major impact on antiviral defense and immunosurveillance of the female reproductive tract. Furthermore, use of DMPA precludes co-inoculation studies with commensal bacteria or fungi, as these microbes often require an estrogen-high environment to robustly colonize the murine vagina. One major difference between the mouse and human vagina is that expression of the viral entry receptor nectin-1 on epithelial cells is hormone dependent in mice, but constitutively expressed in humans. To overcome this challenge, this proposal will develop a novel mouse model in which nectin-1 is constitutively expressed in the murine vaginal epithelium by addressing transcriptional and post-translational regulatory mechanisms. Nectin-1 expression will be placed under the control two different constitutive promoters, and two mutations will be introduced to prevent ectodomain cleavage. Epithelium-specific expression of nectin-1 will be achieved through the inclusion of a stop-flox cassette and breeding to mice in which Cre recombinase expression is controlled by the human keratin 14 promoter. Finally, the HSV susceptibility of these novel mice will be tested at different stages of the estrus cycle. The successful development of a novel, more translational mouse model of HSV infection would help achieve our long-term goal of exploring the role of sex hormones in modulating host-pathogen interactions at the vaginal barrier as well as investigating the molecular impact of vaginal microbial dysbiosis or STI co- infection on HSV pathogenesis. We expect future studies to reveal unknown aspects of protective immunity against genital herpes, including that induced by vaccines, and potentially lead to the discovery of novel targets for therapeutics aimed at reducing susceptibility against HSV infection.