The Characterization of Pneumocystis Surface Antigens

肺孢子菌表面抗原的表征

• 批准号: 8565266
• 负责人: JOSEPH A KOVACS
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8565266
• 关键词: Antibodies; Antibody Formation; Antigens; Disease; Enzyme-Linked Immunosorbent Assay; Epidemiology; Genes; Geographic Locations; Goals; HIV Infections; High Pressure Liquid Chromatography; Human; Immune response; Immunocompromised Host; Immunofluorescence Immunologic; Individual; Length; Mass Spectrum Analysis; Membrane Glycoproteins; Methods; Molecular; Monoclonal Antibodies; Mus; Organism; Pathogenesis; Patients; Plasmids; Pneumocystis; Pneumocystis Infections; Pneumocystis carinii Pneumonia; Proteins; Rattus; Reading; Recombinants; Recording of previous events; Serum; Site; Source; Subgroup; Surface; Surface Antigens; Tandem Repeat Sequences; Variant; base; cDNA Library; next generation; pathogen; prevent

We have an ongoing project to characterize the antigens of mouse, rat, and human Pneumocystis. We have previously purified the major surface glycoprotein (MSG) of both rat and human pneumocystis using HPLC. It is necessary to use Pneumocystis from both sources because the organisms are different species. Subsequently, we identified a number of clones from a cDNA library of rat Pneumocystis that contain genes encoding for the MSG. These clones were clearly related but not identical, demonstrating that multiple genes encode the MSG. We have continued studies to characterize potential antigens of Pneumocystis. We have cloned a number of human Pneumocystis MSG genes and have expressed a full-length MSG in two fragments. We developed an ELISA to examine antibody responses to these antigens, and have utilized it to examine sera from patients with or without HIV infection, and with or without a history of PCP, as well as sera from a variety of healthy controls. In about 15% of healthy patients followed serially we have been able to document changes in antibody titers, suggesting that these individuals have developed reinfection or reactivation of Pneumocystis infection. We will continue these studies to better understand the epidemiology of Pneumocystis infection in humans. We have also identified the unique expression site of MSG in human Pneumocystis, and can now identify the MSG variants that are expressed in a patient with PCP. Within this expression site we have identified a region of tandem repeats that varies among different Pneumocystis isolates, and thus provides a new method for typing human Pneumocystis. We have sequenced MSG variants from a single human pneumocystis isolate and have shown that the isolates can be divided, into 2 subgroups that are genetically distinct. We have cloned and sequenced MSG variants from additional patients from around the world to see if this will provide information about the distribution of different Pneumocystis strains. To date we have found substantial variability in MSG variants from different parts of the world, but no clustering by geographic location. To better examine the MSG repertoire and MSG variability, we have utilized next generation sequencing. By 454 sequencing, we were unable to reconstruct the original sequences using a mixture of plasmids, in large part because of the conserved regions in MSG variants. We are currently evaluating PacBio sequencing, since this allows potentially longer reads that may overcome the assembly problem. We are also trying to identify the Pneumocystis protein that is recognized by monoclonal antibody 4D7. This is a protein present on the surface of Pneumocystis, based on immunofluorescence, and is found in all species of Pneumocystis studied to date. We have partially purified the antigen and by mass spectrometry analysis have identified a number of Pneumocystis proteins that are potential candidates for the 4D7 protein. We are currently attempting to express these proteins and determine if any of them are recognized by 4D7. The goal of this study is to better understand the pathogenesis of Pneumocystis pneumonia with the hope that we can use this information to control or prevent this disease.

我们有一个持续的项目，以表征小鼠，大鼠和人肺炎雄细胞的抗原。我们先前使用HPLC纯化了大鼠和人肺炎的主要表面糖蛋白（MSG）。由于生物是不同的物种，因此有必要使用两个来源的气囊藻。随后，我们从大鼠气囊藻的cDNA库中鉴定了许多克隆，这些克隆包含编码味精的基因。这些克隆显然是相关的，但并不相同，表明多个基因编码了味精。我们继续研究以表征性肺炎的潜在抗原。我们已经克隆了许多人肺炎藻msg基因，并在两个片段中表达了全长的味精。我们开发了ELISA来检查对这些抗原的抗体反应，并利用它来检查有或没有HIV感染患者的血清，以及有或没有PCP史以及各种健康对照组的血清。在大约15％的健康患者中，我们已经能够记录抗体滴度的变化，这表明这些人已经重新感染或重新激活了肺类细胞胸膜感染。我们将继续这些研究，以更好地了解人类肺囊肿感染的流行病学。我们还确定了人肺囊肿中MSG的独特表达位点，现在可以识别在PCP患者中表达的MSG变体。在此表达位点中，我们确定了一个串联重复区域，该区域在不同的肺炎藻菌分离株之间有所不同，因此提供了一种新方法来键入人肺囊肿。我们已经测序了来自单个人肺结压藻分离株的味精变体，并表明可以将分离株分为两个遗传上不同的亚组。我们已经克隆并测序了来自世界各地其他患者的味精变体，以查看这是否提供有关不同肺炎菌株分布的信息。 迄今为止，我们发现来自世界各地的MSG变体的差异很大，但没有按地理位置进行聚类。为了更好地检查MSG曲目和MSG变异性，我们使用了下一代测序。通过454个测序，我们无法使用质粒的混合物重建原始序列，这在很大程度上是由于MSG变体中保守的区域。我们目前正在评估PACBIO测序，因为这允许可能要克服装配问题的可能更长的读取。 我们还试图鉴定单克隆抗体4D7识别的肺类囊蛋白。 这是基于免疫荧光的肺炎表面上存在的一种蛋白质，并且在迄今为止研究的所有肺类细胞中都可以发现。我们已经部分纯化了抗原，并通过质谱分析确定了许多肺炎壳蛋白，它们是4D7蛋白的潜在候选者。我们目前正在尝试表达这些蛋白质，并确定其中任何一个是否均被4D7识别。 这项研究的目的是更好地了解肺炎肺炎肺炎的发病机理，希望我们可以使用这些信息来控制或预防这种疾病。