Preclinical studies of a Cryptococcus vaccine for AIDS patients

项目摘要/摘要 据估计，与艾滋病相关的死亡有15%是由隐球菌病引起的。我们已经通过基因工程制造了一种 新生隐球菌，命名为cda1∆2∆3∆，缺乏编码甲壳素的三个基因。 脱乙酰酶(CDA)。值得注意的是，给了一次肺内疫苗的小鼠，活的或热死的 CDA1∆2∆3∆对致命性新生葡萄球菌的挑战产生长期保护作用，即使CD4+T细胞 在真菌攻击时，细胞被耗尽。在壳聚糖生产中突变的其他隐球菌菌株，或 生长在不同介质中的野生型菌株也具有保护作用，尽管有些菌株会引发有害的促炎作用。 回应。这三个具体目标的重点是发展对 免疫学和疫苗保护性决定因素。长期目标是开发一种隐球菌 疫苗，以保护高危个人，特别是艾滋病毒携带者。目标1是确定以下各项的相关性 CDA1-∆-2-∆-3∆疫苗对CD4+T细胞充足小鼠的保护作用我们假设接种疫苗时 CD1-∆-2-∆-3-∆可产生和扩增Th1偏斜的抗原特异的CD+T细胞 通过产生细胞因子来招募和/或激活抗真菌效应细胞来协调疫苗免疫。我们 将剖析接种疫苗和感染后的细胞和细胞因子反应，询问 巨噬细胞和树突状细胞倾斜，并定义保护所需的细胞和细胞因子。目标2是 确定负责疫苗介导的保护的效应机制当CD4+T细胞 在挑战阶段耗尽。我们的初步数据表明，小鼠需要CD4+T细胞 接种CDA1∆2∆3∆以产生保护性免疫，但当小鼠收到 新生假单胞菌的致命挑战。这种可塑性表明了一种策略，即艾滋病毒携带者可以 当他们的CD4+T细胞升高时接种疫苗，当他们的CD4+T细胞升高时仍可免受隐球菌感染 细胞数量下降。我们将进一步定义对CD4+T细胞的要求，并确定其效应机制 这可以弥补CD4+T细胞的损失。目标3是确定新生隐孢子菌的成分 推动不同的宿主反应，重点关注高度炎症反应与保护性反应。 这一目标是继我们的发现之后发现的，全细胞隐球菌疫苗可以在 它们引起的肺部炎症的量。我们将描述保护性和炎症性的性质 并确定驱动这些反应的真菌配体。我们预计，在今年年底 资助期内，我们将对寄主和真菌因素有一个机械性的了解 CDA1-∆-2-∆-3∆疫苗株对CD_4+T细胞充足和缺陷小鼠的保护作用这项建议 解决了全球对开发隐球菌疫苗的主要健康需求，并可以建立证据 适用于其他艾滋病相关机会性感染和粘膜疫苗的原则。

Project Summary/Abstract An estimated 15% of AIDS-related deaths are due to cryptococcosis. We have genetically engineered a Cryptococcus neoformans strain, designated cda1∆2∆3∆, that it is deficient in three genes encoding for chitin deacetylases (CDA). Remarkably, mice given a single intrapulmonary vaccination with live or heat-killed cda1∆2∆3∆ develop long-term protection against an otherwise lethal C. neoformans challenge, even if CD4+ T cells are depleted at the time of fungal challenge. Other cryptococcal strains mutant in chitosan production, or wild type strains grown in different media, also are protective, although some elicit deleterious proinflammatory responses. The three specific aims are focused on developing a mechanistic understanding of the immunological and vaccine determinants of protection. The long-term objective is to develop a cryptococcal vaccine to protect at risk individuals, particularly persons living with HIV. Aim 1 is to determine correlates of cda1∆2∆3∆ vaccine-mediated protection in CD4+ T cell-sufficient mice. We hypothesize that vaccination with cda1∆2∆3∆ results in the generation and expansion of Th1-skewed antigen-specific CD4+ T cells which orchestrate vaccine immunity by producing cytokines which recruit and/or activate antifungal effector cells. We will dissect the cellular and cytokine response following vaccination and infection, interrogate the role of macrophage and dendritic cells skewing, and define the cells and cytokines required for protection. Aim 2 is to determine the effector mechanisms responsible for vaccine-mediated protection when CD4+ T cells are depleted during the challenge phase. Our preliminary data demonstrate that CD4+ T cells are required for mice vaccinated with cda1∆2∆3∆ to develop protective immunity, but then become dispensable when mice receive a lethal challenge of C. neoformans. This plasticity suggests a strategy whereby persons with HIV can be vaccinated when their CD4+ T cells are elevated and still be protected from cryptococcosis when their CD4+ T cells counts fall. We will further define the requirement for CD4+ T cells and identify the effector mechanisms that compensate for the loss of CD4+ T cells. Aim 3 is to determine the components of C. neoformans which drive disparate host responses, focusing on the highly inflammatory response versus the protective response. This aim follows up our discovery that whole cell cryptococcal vaccines can exhibit marked variations in the amount of lung inflammation they induce. We will characterize the nature of the protective and inflammatory response and determine the fungal ligands that drive these responses. We anticipate that at the end of the funding period, we will have a mechanistic understanding of the host and fungal factors responsible for protection of CD4+ T cell-sufficient and -deficient mice by the cda1∆2∆3∆ vaccine strain. The proposal addresses a major global health need for the development of cryptococcal vaccines and could establish proofs of principle applicable to other AIDS-related opportunistic infections and mucosal vaccines.