Developing Vault Nanoparticles as a Delivery Platform for Burkholderia pseudomallei and Burkholderia mallei Antigens

摘要 类鼻疽伯克霍尔德菌（Bp）是热带地区的地方病，引起经常致命的类鼻疽病 在人类和动物中，对当地居民，游客和部署的美国军队构成重大威胁 人员的在东南亚的高度流行地区，类鼻疽预计将超过结核病， 是仅次于艾滋病毒/艾滋病的第二大传染病死亡原因。目前还没有有效 提供免疫力的方法，以及被内在抗生素耐药性混淆的治疗。的能力 在细胞内存活并利用膜融合作为在细胞间传播的手段 从体液免疫系统。先前提供针对Bp的保护性免疫的努力依赖于减毒活疫苗。 菌株或细菌亚单位，只有部分保护。因此，一种抗原递送策略， B需要抗体和细胞应答。假鼻疽，以及促进体内生存的因素 哺乳动物细胞将可能构成有效的抗原。这一点得到了动物模型中现有证据的支持 和恢复的类鼻疽患者，其中细胞毒性T淋巴细胞（CTL）通过识别和 杀死BP感染的细胞，抗体起辅助作用。我们的目标是利用内源性的人类穹窿 纳米颗粒作为一种新的，安全的平台，用于提供伯克霍尔德氏菌抗原，可以引发强的细胞介导的， 抗Bp的体液反应。穹窿有效地递送抗原并提供显著的免疫应答 而不需要额外的佐剂。穹窿是大的，自然发生的，细胞质核糖蛋白颗粒， 哺乳动物细胞。最近的观察表明，穹窿在免疫监视中起作用， 从死亡细胞中提取抗原以促进适应性免疫。重组库有效地将抗原递送到两个细胞， MHC I类和II类途径，并在APC中具有趋化样作用，诱导保护性CD 4和CD 8 T细胞 反应，同时也产生抗体反应。与此相反，本机或“空”保管库不会激活 本发明的化合物是炎性体或TLR依赖性炎性反应的化合物，并且是非免疫原性的。我们利用了这些 在几个系统中引发强大免疫应答的穹窿的性质，包括粘膜炎的小鼠模型， 细胞内衣原体感染，与Bp一样，是一种细胞内病原体。我们将联合收割机 致病机制的知识和制造重组库的实践生产方法 含有伯克霍尔德氏菌抗原（BurkAgs）。BurkAgs已经基于以下特性被识别： 假设与强大的免疫原性和潜在的广泛反应， 菌株Bp.重组BurkVaults诱导抗原特异性抗体、CD 4和CD 8 T细胞的能力 在i.n.或s.c.免疫将在建立的定量测定中确定。重组 显示最佳结构、组成和免疫原性特征的库将被用于研究 Bp致死性攻击BALB/c小鼠。

ABSTRACT Burkholderia pseudomallei (Bp) is endemic to tropical regions and causes the often-fatal disease melioidosis in humans and animals, posing a significant threat to native residents, tourists, and deployed U.S. military personnel. In highly-endemic areas of southeast Asia, melioidosis is projected to surpass tuberculosis as the second most common cause of infectious disease mortality after HIV/AIDS. There are currently no effective approaches for providing immunity, and treatment confounded by intrinsic antibiotic resistance. The ability to survive intracellularly and utilize membrane fusion as a means for spreading from cell to cell shelters the bacterium from the humoral immune system. Prior efforts to provide protective immunity against Bp rely on live-attenuated strains or bacterial subunits that are only partially protective. Therefore, an antigen-delivery strategy to elicit both antibody and cellular responses is needed for B. pseudomallei, and the factors that promote survival inside mammalian cells will likely constitute effective antigens. This is supported by current evidence in animal models and recovered melioidosis patients, where cytotoxic T lymphocytes (CTLs) play a central role by recognizing and killing Bp-infected cells, with antibody playing an ancillary role. Our goal is to utilize the endogenous human vault nanoparticle as a novel, safe platform for delivering Burkholderia antigens that can elicit strong cell-mediated and humoral responses against Bp. Vaults efficiently deliver antigens and provide remarkable immune responses without additional adjuvants. Vaults are large, naturally-occurring, cytoplasmic riboprotein particles found in all nucleated mammalian cells. Recent observations suggest that vaults function in immune surveillance, delivering antigens from dying cells to facilitate adaptive immunity. Recombinant vaults efficiently deliver antigens to both the MHC class I and II pathways, and have adjuvant-like effects in APCs, eliciting protective CD4 and CD8 T cell responses, while also generating antibody responses. In contrast, native or "empty" vaults do not activate inflammasome or TLR-dependent inflammatory reactions and are non-immunogenic. We have exploited these properties of vaults to elicit robust immune responses in several systems, including a mouse model of mucosal infection by intracellular Chlamydia, which like Bp, is an intracellular pathogen. We will combine our considerable knowledge of pathogenic mechanisms and practiced production approaches for making recombinant vaults containing Burkholderia antigens (BurkAgs). BurkAgs have been identified based on properties that are hypothesized to correlate with robust immunogenicity and the potential to elicit broad responses against diverse strains of Bp. The ability of recombinant BurkVaults to induce antigen-specific antibody, CD4 and CD8 T cell responses following i.n. or s.c. immunization will be determined in established, quantitative assays. Recombinant vaults that display optimal structural, compositional, and immunogenic profiles will be advanced for studies against lethal challenge with Bp in BALB/c mice.