Immunity to Chlamydial Infection

本工作的目的是利用沙眼衣原体持续感染的体外模型，(I)了解导致沙眼衣原体持续感染和慢性疾病的衣原体免疫逃避的分子基础，(Ii)定义保护性细胞毒性T细胞识别的新抗原，以代表新的疫苗靶点。我们对所有15个衣原体参考菌株和500多个眼球生殖器临床分离株进行了功能基因组研究，重点放在病原体的可塑性区域(PZ)内的基因，即位于基因组复制终止区的一个小的多态区域。PZ含有细胞毒素基因、磷脂酶D基因家族和色氨酸操纵子(TrpRBA)。TrpRBA编码Trp抑制物和TrpB和TrPA多肽，形成异源二聚体通道色氨酸合成酶(TS)。我们发现，感染和疾病器官趋向性不同的沙眼衣原体菌株与病原体TRPA多肽的错义或无义失活突变有关。这些突变导致了所有生殖器分离株的功能全长TS和所有眼部菌株的截短的非功能性TS。这种功能基因组分析已经建立了仅基于TS功能的眼部和生殖器组织嗜性菌株之间的独特范例。这种本地化和高度特异性的遗传多样性是这些在遗传上非常相似的菌株在基因组组织上的主要差异，这强烈地暗示了TS是嗜生殖器分离株的主要毒力因素。此外，功能性TS活性通过吲哚拯救机制与人上皮细胞对干扰素-γ的抵抗直接相关。因此，生殖器菌株存在很大的选择压力，以维持能够利用吲哚进行色氨酸生物合成的功能TS。有趣的是，吲哚不是由哺乳动物细胞或衣原体合成的，这增加了吲哚是通过共同感染产生吲哚的女性泌尿生殖道微生物群获得的可能性。利用吲哚合成色氨酸可以使衣原体通过IDO色氨酸剥夺途径逃避干扰素-γ介导的免疫清除，从而建立一种在雌性宿主中持续感染和持续传播的机制。这是细菌疾病发病机制中这种独特的宿主-寄生虫-微生物区系关系的第一个例子。未来的研究将集中在开发人源化的小鼠动物模型，以实验定义这种独特的宿主-病原体-微生物相互作用。 我们也正在使用干扰素-γ介导的持续感染的体外模型作为一种新的方法来发现由细胞毒T细胞识别的新的衣原体抗原(S)。细胞毒性CD8和CD4T细胞是保护性免疫中重要的效应淋巴细胞亚群，但这些细胞毒性T细胞识别的抗原在很大程度上仍不清楚。有趣的是，在衣原体感染的人宫颈上皮细胞中，人类白细胞抗原II类是由干扰素-γ诱导的，而在持续感染的宫颈上皮细胞中，衣原体抗原是由人类白细胞抗原II类来加工和呈递的。因此，这种独特的感染靶点代表了细胞毒性T细胞的新靶点，由这些细胞提出的人类白细胞抗原(S)是潜在的新的和重要的衣原体疫苗靶点。我们正在从经干扰素-γ处理的衣原体感染的宫颈上皮细胞中提纯人类白细胞抗原I类和II类分子，洗脱与人类白细胞抗原结合的多肽，并用高分辨质谱仪对其进行鉴定。根据衣原体基因组数据库对多肽序列进行炮轰，以确定多肽的来源基因。到目前为止，我们已经用这种方法鉴定了21个新的HLAI类抗原和12个新的HLAII类肽。未来的工作将集中在使用从自然感染的人中分离的T细胞来描述这些蛋白的抗原性，产生特定的T细胞克隆，并鉴定具有衣原体特异性细胞毒功能的克隆。编码这些蛋白的基因将被克隆并在减毒牛痘和流感病毒中表达，并作为新的生殖器或鼻腔注射疫苗在临床前进行测试。

The purpose of this work is to utilize in vitro models of C. trachomatis persistent infection to (i) understand the molecular basis of chlamydial immune evasion that leads to persistence and chronic disease and, (ii) define new antigens recognized by protective cytotoxic T cells that represent novel vaccine targets. We have performed functional genomic studies on all 15 chlamydial reference strains and over 500 oculogenital clinical isolates focusing on the genes that reside within the pathogen's plasticity zone (PZ); a small polymorphic region located at the replication termination region of the genome. The PZ contains the cytotoxin gene, the phospholipase D gene family, and the tryptophan operon (trpRBA). The trpRBA encodes the Trp repressor and TrpB and TrpA polypeptides that form the heterodimeric channeling tryptophan synthase (TS) enzyme. We found that C. trachomatis strains differing in infection and disease organotropism correlated with either missense or nonsense inactivating mutations in the pathogen's TrpA polypeptide. These mutations resulted in a functional full length TS for all genital isolates and a truncated non-functional TS in all ocular strains. This functional genomic analysis has established the unique paradigm between ocular and genital tissue tropic strains based solely on TS functionality. This localized and highly specific genetic diversity is the primary difference in the genomic organization among these otherwise genetically very similar strains strongly implicating the TS a major virulence factor for genital tropic isolates. Moreover, functional TS activity was directly correlated to IFN-gamma resistance in human epithelial cells through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain functional TS capable of using indole for tryptophan biosynthesis. Interestingly, indole is not synthesized by mammalian cells or chlamydiae raising the possibility that indole is acquired from co-infecting indole producing microflora of the female urogenital tract. Utilizing indole to synthesize tryptophan would allow chlamydiae to escape IFN-gamma mediated immune eradication through the IDO tryptophan deprivation pathway, thereby establishing a mechanism of persistent infection and sustained transmission in the female host. This is the first example of such a unique host-parasite-microflora relationship in the pathogenesis of bacterial disease. Future investigations will focus on the development of a humanized murine animal model to experimentally define this unique host-pathogen-microbial interaction. We are also using the in vitro model of IFN-gamma mediated persistent infection as a novel approach to discover new chlamydial antigen(s) recognized by cytotoxic T cells. Cytotoxic CD8 and CD4 T cells are important effector lymphocyte subsets in protective immunity yet the antigens recognized by these cytotoxic T cells remain largely uncharacterized. Interestingly, HLA class II is induced by IFN-gamma in chlamydial infected human cervical epithelial cells and chlamydial antigens are processed and presented by in the context HLA class II in persistently infected epithelial cells. Thus this unique target of infection represents a novel target for cytotoxic T cells and the HLA antigen(s) presented by these cells are potentially new and important chlamydial vaccine targets. We are purifying HLA class I and II molecules from IFN-gamma treated chlamydial infected cervical epithelial cells, eluting the HLA bound peptides, and identifying the peptides by high resolution mass spectrometry. Peptide sequences are blasted against the chlamydial genome databases to identify the genes from which the peptides are derived. To date, we have identified 21 novel HLA class I antigens, and 12 novel HLA class II peptides using this approach. Future work will focus on describing the antigenicity of the proteins using isolated T cells obtained from naturally infected humans, generating specific T cells clones, and identifying clones that exhibit chlamydial specific cytotoxic function. The genes encoding these proteins will be cloned and expressed in attenuated vaccinia and influenza viruses and tested as novel genital or intranasal administered vaccines in a pre-clinic