Improved Therapeutics and Diagnostics for Pneumocystis Pneumonia

肺孢子虫肺炎仍然是艾滋病毒感染和其他免疫功能低下的严重并发症 states.最近的ICD 9/10数据显示，自2008年以来，每年有14- 15，000例住院治疗 仅在美国，每年的平均成本就接近10亿美元。此外，五氯苯酚的治疗方法没有改变 在25年中，人们担心抗微生物耐药性和药物：与TMP-SMX的药物相互作用（ PCP的一线治疗）。众所周知的CD 4+淋巴细胞计数与风险之间的负相关关系 PC感染的研究并不能解释宿主防御这种感染的机制的所有答案。现有 在资助期间，我们在确定肺孢子虫的转录组方面取得了重大进展， 滋养体形式以及两种形式的表面蛋白质组，通过使用 为念珠菌开发的技术。我们鉴定了营养蛋白，如Meu 10，一种GPI锚定的 蛋白质，以及葡聚糖合成酶1（GSC 1）等葡聚糖特异性蛋白质。我们能够克隆， 在酵母中表达GSC 1胞外域，用GSC 1免疫小鼠产生抗体， 的表面，并防止肺孢子虫的传播。我们也做 在区分定植和感染的诊断测定方面取得了重大进展。现在有几个团体 使用具有拷贝数阈值的定量PCR，这可能是一种有效的方法。上一个供资期间 开发并验证了一种选择性根除细菌的troph特异性测定法。此外，RNAseq 分析表明，营养体比β-葡聚糖具有更高的代谢活性。因此，我们创造了生命- 形成将在临床标本中检测的特定检测试剂盒。我们假设GSC 1免疫可以 预防肺孢子虫传播，粘膜免疫上级皮下免疫。 我们还预测，GSC 1抗血清或针对GSC 1胞外域的单克隆抗体可 减轻肺孢子虫IRIS。此外，由于我们的RNAseq分析也揭示了营养体显然是 由于肺中微生物的复制形式，我们假设检测营养体特异性基因的测定将是可行的。 PJP的诊断由于检测到生物体的复制形式。我们还将确定 这些靶基因在来自4大洲的样品中的异质性。我们将通过以下内容来检验这一假设 具体目标：目标1。确定全身（皮下或IM）与 粘膜GSC 1免疫预防PCP。目标2.确定抗细菌或抗营养素的功效 抗体治疗已建立的PCP和免疫重建综合征（IRIS）。目标3：确定并验证 在小鼠和人类样本中进行生命形式特异性PCR/CRISPR测定，并评估这些基因的遗传异质性。 在来自北美、非洲、西亚和东亚的样本中，

Pneumocystis (PC) pneumonia remains a serious complication of HIV infection and other immunocompromised states. Recent ICD9/10 data show that since 2008 there are consistently 14-15,000 hospitalizations per year with an average cost of close to $1B per annum in the US alone. Moreover treatment for PCP has not changed in 25 years and there is concern of anti-microbial resistance and drug:drug interactions with TMP-SMX (the frontline therapy for PCP). The well-known inverse relationship between CD4+ lymphocyte count and the risk of PC infection does not hold all of the answers to mechanisms of host defense against this infection. In the prior funding period, we made significant progress on defining the Pneumocystis transcriptome in both ascus and trophozoite forms as well as the surface proteome of both forms through surface labeling of the fungus using techniques that were developed for Candida. We identified troph proteins such as Meu10, a GPI-anchored protein, as well as ascus specified proteins such as glucan synthetase 1 (GSC1). We were able to clone and express the GSC1 ectodomain in yeast, and immunization of mice with GSC1 resulted in antibodies that stain the surface of the ascus and prevent transmission of Pneumocystis in cohousing experiments. We also made significant progress on diagnostic assays to discriminate colonization from infection. Several groups ae now using quantitative PCR with copy number thresholds, which may be a valid approach. In the prior funding period developed and validated a troph specific assay that selectively eradicates the ascus. Furthermore, RNAseq analysis revealed the troph to be much more metabolically active than the ascus. Thus, we have developed life- form specific assays that will be tested in clinical specimens. We hypothesize that GSC1 immunization can prevent pneumocystis transmission and that mucosal immunization is superior to subcutaneous immunization. We also predict that GSC1 antisera or monoclonal antibodies directed against the GSC1 ectodomain can mitigate Pneumocystis IRIS. Moreover, as our RNAseq analysis has also revealed that the troph is clearly the replicative form of the organism in the lung, we hypothesize that an assay to detect troph specific genes will be diagnostic of PJP due to the detection of the replicative form of the organism. We will also determine the genetic heterogeneity of these target genes in samples from 4 continents. We will test this hypothesis with the following specific aims: Aim 1. Determine the immunogenicity and efficacy of systemic (subcutaneous or IM) versus mucosal GSC1 immunization to prevent PCP. Aim 2. Determine the efficacy of anti-ascus or anti-troph antibodies to treat established PCP and immune reconstitution syndrome (IRIS). Aim 3. Determine and validate life-form specific PCR/CRISPR assays in murine and human samples and assess genetic heterogeneity of these targets in samples from North America, Africa, and western and eastern Asia.