Antigen discovery and validation in Pneumocystis pneumonia

描述（由申请人提供）：肺孢子虫（PC）是一种双相真菌，可导致免疫功能低下个体的肺炎，而PC肺炎（PCP）是诊断为HIV感染者最常见的严重机会性感染。此外，PCP在非HIV感染的免疫功能低下个体的临床环境中重新出现，这些个体接受挽救生命的免疫抑制治疗，以治疗血液学恶性肿瘤、移植和自身免疫性疾病等疾病。随着高危人群的不断增加，在PC上发现新的、保守的细胞外抗原是设计更有效的诊断测试、创建基于单克隆抗体的疗法和开发疫苗的第一步。为了发现新的抗原，对PC进行了蛋白质组学和转录组学分析。具体地说，表面生物素化的总PC，包含两个囊蚂蚁营养生命 组蛋白2B（H2B）是PC上丰富的胞外蛋白。还对分离的PC孢囊和营养体进行RNA测序，以鉴定在两种生命形式中差异表达的靶标。与营养型相比，H2B在包囊型中的表达增加了3倍，而其他蛋白质如Meu10在营养型中的表达更高。为了确定H2B的免疫原性，用H2B DNA疫苗接种小鼠。与未接种疫苗的对照组相比，接受H2B疫苗的小鼠抗PC血清IgG显著增加，肺PC负荷显著降低。这些实验表明，H2B是一种富含包囊的抗原，能够在DNA疫苗接种后提供针对发展中PCP的保护。本项目的目标是：1）确定H2B DNA疫苗接种产生的体液免疫应答，以及2.）确定Meu10 DNA疫苗的免疫原性，并评价基于生命周期的多价DNA疫苗接种提供的保护作用。为了实现这些目标，拟议的研究将探讨记忆B细胞反应产生的H2 B DNA疫苗接种，并检查疫苗提供的免疫保护机制。此外，还将使用Meu10 DNA疫苗和多价疫苗进行疫苗接种研究，以检测基于生命周期的靶向多价疫苗的有效性。根据NIAID的使命声明，拟议的研究将确定先前设计的H2B疫苗背后的基本免疫机制，并将探索用于重新出现的PC传染病的疫苗开发的新策略。

DESCRIPTION (provided by applicant): Pneumocystis (PC) is a dimorphic fungus that causes pneumonia in immunocompromised individuals and PC pneumonia (PCP) is the most common serious opportunistic infection in individuals diagnosed with HIV. Furthermore, PCP is re-emerging in the clinical setting of non-HIV infected immunocompromised individuals receiving life-saving immunosuppressive therapies for conditions such as hematological malignancies, transplantation, and autoimmune conditions. As the at-risk population continues to increase, finding novel, conserved, extracellular antigens on PC is the first step towards designing more effective diagnostic tests, creating monoclonal antibody based therapies, and developing vaccines. To discover novel antigens, proteomic and transcriptome analysis of PC was conducted. Specifically, surface biotinylation of total PC, containing both the cyst ant troph life form, was performed and demonstrated that histone 2B (H2B) was an abundant extracellular protein on PC. RNA sequencing was also performed on separated PC cysts and trophs to identify targets differentially expressed in the two life forms. H2B had a 3-fold increase in expression in the cyst form when compared to the troph form, while other proteins, such as a Meu10, were found to have higher expression in the troph form. To determine the immunogenicity of H2B, mice were vaccinated with an H2B DNA vaccine. Mice receiving the H2B vaccine had a significant increase in anti-PC serum IgG and had a significant decrease in lung PC burden when compared to unvaccinated controls. These experiments demonstrate that H2B is a cyst-enriched antigen and is capable of providing protection against developing PCP following DNA vaccination. The aims of this proposed project are: 1.) To define the humoral immune response generated by H2B DNA vaccination, and 2.) To determine the immunogenicity of a Meu10 DNA vaccine and evaluate the protection provided by a life cycle based multivalent DNA vaccination. To accomplish these aims, the proposed studies will explore the memory B cell response generated by H2B DNA vaccination and examine the immunologic mechanisms of protection provided by the vaccine. Furthermore, vaccination studies will be conducted with a Meu10 DNA vaccine and a multivalent vaccine targeting both the cyst-enriched H2B and the troph-enriched Meu10 to examine the efficacy of a targeted, life cycle based multivalent vaccine. The proposed studies will define the fundamental immunological mechanisms behind a previously designed H2B vaccine and will explore a novel strategy for vaccine development for the re- emerging infectious disease of PC in accordance with the mission statement of the NIAID.