Immunity To Chlamydial Infection

这项工作的目的是确定介导对女性生殖道沙眼衣原体感染的保护性免疫的免疫学基础。长期目标是利用这些信息研制一种安全有效的预防衣原体引起的性传播疾病的疫苗，该项目包括研究女性生殖道衣原体感染小鼠模型的免疫力。这项工作的目标是确定保护性免疫和保护性抗原的机制，这些信息可以用来制定新的疫苗，在临床前模型中进行测试。长期目标是将这些最有希望的疫苗转移到人体临床试验，以评估其在预防衣原体性传播疾病方面的安全性和有效性。该实验室过去使用基因敲除小鼠、免疫T细胞过继转移和体内T细胞亚群耗损的研究强烈暗示CD4+ Th1细胞介导的免疫是针对衣原体生殖器感染的免疫反应的主要保护臂。相反，CD8+ T细胞、γ / δ T细胞和抗体在介导保护性免疫中只发挥有限的作用。我们沿着这条路线继续研究，以进一步确定CD4+ Th1介导的免疫和淋巴细胞归巢到生殖器粘膜的效应功能。我们的研究结果支持ifn - γ而非tnf - α、iNOS或Fas介导的凋亡杀伤在抗衣原体介导的保护性T细胞免疫中的作用。ifn - γ的作用机制是通过诱导吲哚胺2,3-二氧合酶（IDO）和随后被剥夺宿主色氨酸的衣原体饥饿。因此，能够在生殖器粘膜产生衣原体特异性1型CD4+免疫应答的疫苗可能引发保护性免疫。淋巴细胞归巢研究暗示了系统和粘膜整合素及其同源受体在淋巴细胞归巢生殖器粘膜中的作用。迄今为止，使用传统方法进行疫苗接种，如靶向衣原体重组蛋白或DNA编码蛋白（有或没有Th1促进佐剂）全身性或局部递送，均未能在生殖器粘膜引发保护性Th1免疫。体外脉冲树突状细胞(DC？是对衣原体生殖器再次挑战产生保护性免疫的唯一高效方法。我们的结论是，衣原体的抗原复杂性、其复杂的生命周期和对粘膜上皮细胞的趋向性对传统疫苗的产生构成了巨大的挑战。因此，我们目前的重点是研制减毒活疫苗株。这是通过克隆选择无法合成色氨酸合酶的变异来实现的，色氨酸合酶是病原体在宿主防御下持续存在于上皮细胞中的关键酶。我们通过在补充了5-氟吲哚（5- fi）的trp缺失培养基中生长来选择trp-/-减毒菌株。具有色氨酸合酶基因（trpRBA）突变的生物体不会吸收5-FI，并在其致命作用下存活下来。5-FI抗性克隆的生物学特征包括体外对ifn - γ的敏感性、吲哚拯救性和trpRBA序列分析。体外生长对ifn - γ高度敏感和trpRBA基因突变的克隆将在体内感染模型中进行测试，以观察其生长减弱和病理特性。在小鼠雌性生殖道中表现出体外生长减弱的菌株，随后将测试其诱导保护性免疫抵御致病性衣原体攻击感染的能力。这些研究应提供重要信息，以开发能够预防或控制衣原体引起的人类性传播疾病的高效和安全疫苗。

The purpose of this work is to define the immunological basis that mediates protective immunity against Chlamydia trachomatis infection of the female genital tract. The long-term goal is to then use this information to develop a safe and efficacious vaccine against chlamydial caused sexually transmitted diseases (STD. The project involves the study of immunity in a murine model of chlamydial infection of the female genital tract. The goal of the work is to identify mechanisms of protective immunity and protective antigens, information that then can used to formulate novel vaccines to test in this pre-clinical model. The long-range goal is to move the most promising of these vaccines to human clinical trials to assess their safety and efficacy in preventing chlamydial STDs. Past studies from this laboratory using gene knock out mice, adoptive transfer of immune T cells, and in vivo depletion of T cell subsets strongly implicate CD4+ Th1 cell mediated immunity as the major protective arm of the immune response against chlamydial genital infection. Conversely, CD8+ T cells, gamma/delta T cells, and antibodies play only a limited role in mediating protective immunity. We have continued studies along these lines to further define effector function(s) of CD4+ Th1 mediated immunity and lymphocyte homing to the genital mucosal. Our findings support a role for IFN-gamma but not TNF-alpha, iNOS, or Fas mediated apoptopic killing in protective anti-chlamydial mediated T cell immunity. The effector mechanims by which IFN-gamma functions is through the induction of indoleamine 2,3-dioxygnease (IDO) and subsequent starvation of chlamydiae by the deprivation host tryptophan. Thus, vaccines capable of generating a chlamydial specific type 1 CD4+ immune response at the genital mucosae are likely to elicit protective immunity. Lymphoctye homing studies implicate both systemic and mucosal integrins and their cognate receptors in lymphocyte homing to the genital mucosa. To date, vaccination using conventional approaches such as targeted chlamydial recombinant proteins or DNA encoding proteins delivered either systemically or locally with and without Th1 promoting adjuvants have failed in eliciting a protective Th1 immunity at the genital mucosae. Infection of the genital tract or immunization with ex vivo pulsed dendritic cells (DC? are the only highly efficacious ways of generating protective immunity against chlamydial genital re-challenge. Our conclusions are that the antigenic complexity of the chlamydiae, its complex life cycle, and tropism for mucosal epithelial cells constitute overwhelming challenges for the generation of a conventional vaccine. Consequently, we are currently focusing on the generation of live-attenuated vaccine strains. This is being accomplished by the clonal selection of variants that are incapable of synthesizing tryptophan synthase, a key enzyme in the ability of the pathogen to persist in epithelial cells in the presence host defense. We are selecting attenuated trp-/- strains by growth in trp deficient medium supplemented with 5 fluoroindole (5-FI). Organisms with mutations in the tryptophane synthase gene (trpRBA) will not incorporate 5-FI and will survive its lethal effect. 5-FI resistant clones will be characterized biologically for sensitivity to IFN-gamma in vitro, indole rescuablility, and trpRBA sequence analysis. Clones whose growth is highly sensitive to IFN-gamma in vitro and that have mutations in the trpRBA genes will be tested in in vivo models of infection for attenuated growth and pathological properties. Strains demonstrating attenuated in vitro growth for the murine female genital tract will then be tested for their ability to induce protective immunity against challenge infections with virulent chlamydiae. These studies should yield information important to the development of a highly efficacious and safe vaccine capable of preventing or controlling chlamydial caused STDs in humans.