Antigen discovery and validation in Pneumocystis pneumonia

描述(申请人提供)：肺孢子虫(PC)是一种二相性真菌，在免疫功能低下的人中会导致肺炎，而肺孢子虫肺炎(PCP)是诊断为艾滋病毒的人中最常见的严重机会性感染。此外，PCP正重新出现在非HIV感染的免疫受损患者的临床环境中，这些患者正在接受挽救生命的免疫抑制治疗，如血液系统恶性肿瘤、移植和自身免疫疾病。随着高危人群的持续增加，在PC上寻找新的、保守的细胞外抗原是设计更有效的诊断测试、创造基于单抗的治疗方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分析。具体地说，表面生物素化的总PC，既包含了蚂蚁的胞囊寿命，也包含了蚂蚁的寿命 结果表明，组蛋白2B(H_2B)是PC上丰富的胞外蛋白。还对分离的PC包囊和trOphs进行了RNA测序，以确定在这两种生命形式中差异表达的靶点。与特罗夫型相比，H_2B在囊型中的表达增加了3倍，而其他蛋白质，如Meu10，被发现在特隆型中有更高的表达。为了确定H_2B的免疫原性，用H_2B DNA疫苗免疫小鼠。与未接种疫苗的对照组相比，接受H2B疫苗的小鼠血清中抗PC抗体水平显著升高，肺PC负荷显著降低。这些实验证明，H_2B是一种富含包囊的抗原，能够提供保护，防止DNA疫苗接种后出现PCP。这一拟议项目的目标是：1.2.确定接种H_2B DNA疫苗后产生的体液免疫反应。确定Meu10DNA疫苗的免疫原性，并评价基于生命周期的多价DNA疫苗所提供的保护作用。为了实现这些目标，拟议的研究将探索H2B DNA疫苗接种所产生的记忆B细胞反应，并检查疫苗提供保护的免疫学机制。此外，将使用Meu10DNA疫苗和针对富含胞囊的H2B和富含Ttroph10的Meu10的多价疫苗进行疫苗接种研究，以检验基于生命周期的靶向多价疫苗的效力。拟议的研究将确定先前设计的H_2B疫苗背后的基本免疫学机制，并将根据NIAID的使命声明探索针对新出现的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.