Vaccinating at Mucosal Surfaces with Nanoparticle Conjugated Antigen and Adjuvant

近年来，由于全球负担，退伍军人结核病（TB）的发病率有所增加。 结核病是巨大的。这种负担随着耐多药和极端耐药的出现而升级。 耐药结核分枝杆菌（Mtb）菌株，因为目前的疫苗不引起持久的 对结核病的保护性免疫力，尤其是在成年人中。因此，新一代疫苗的开发， 将提供持久的结核病保护，将大大提高我们退伍军人的生活质量。我们的计划 提出了临床前研究，将确定保护性CD 8 + T细胞表位和开发粘膜疫苗 为设计下一代结核病疫苗提供平台。结核杆菌通过呼吸道进入宿主体内。 道。因此，最佳的保护将需要肺驻留的CD 4+和CD 8+记忆T细胞定位在 第一时间应对感染。传统的疫苗和批准的佐剂通常会引起 弱的、短暂的T细胞反应，并且肠胃外疫苗接种在建立保护性免疫方面无效 在组织粘膜内。此外，大多数病毒载体和亚单位TB疫苗使用Mtb的一个小子集， 抗原，导致最佳保护的表位多样性不足，部分原因是 在Mtb感染期间呈现并赋予保护性免疫力的基因尚未被探索。因此，我们的总体 目的是发现在感染过程中产生的免疫原性Mtb表位，并将它们整合到 创新的基于纳米颗粒（NP）的鼻内疫苗，旨在促进肺CD 4+平衡 和CD 8 + T细胞反应，这将防止结核病。在初步实验中，我们已经确定了41个新的 使用蛋白质组学方法从Mtb H37 Rv感染的原代巨噬细胞中提取肽。其中，17个是 假定的HLA-B*07;02结合表位，我们将表征其以推进抗TB疫苗设计。引发 补充CD 4 + T细胞应答的CD 8 + T细胞需要由HLA类呈递亚单位抗原 在适当的炎症线索的背景下，I分子用于CD 8 + T细胞交叉致敏， T细胞和CD 4分化。我们的团队最近开创了一种“病原体模仿”疫苗， pH敏感的核内体破裂的NP化学这样的NP促进抗原和核酸的递送 佐剂进入细胞质。初步研究表明， 与结构优化的5 'ppp-RNA发夹佐剂结合的负载有卵清蛋白的NP， 激活细胞质视黄酸诱导基因-I，激发一种稳健、持久和保护性的抗原特异性T细胞， 肺部的细胞反应。基于这些令人兴奋的新发现，我们假设鼻内免疫 用共装载有天然加工的I类限制性Mtb衍生表位和5 ′ ppp-RNA的NP疫苗 佐剂将显著增强气道中的组织驻留CD 8+记忆T细胞应答， 肺的实质我们检验这一假设的策略是：（a）评估免疫原性， 由HLA-B*07：02 I类分子呈递的天然加工Mt B表位的保护潜力;（B） 确定HLA-B *07：02和B*35：01-两个密切相关的HLA分子-是否呈现相似的表位; 和（c）开发NP疫苗，其激发持久的肺驻留CD 8+和CD 4+记忆T细胞应答， Mtb亚单位抗原赋予针对TB的保护。我们的多学科团队-由生物化学家， 免疫学家，微生物学家和生物工程师-是理想的位置，以追求三个特定的目标。我们 预计成功完成拟议的研究将产生非常重要的知识， 用于下一代抗结核疫苗的设计。我们期望发现新的保护性Mtb表位， 由多种HLA分子识别。我们还将开发一种新的粘膜给药平台， 疫苗接种将允许抗原和佐剂的共递送用于细胞质免疫监视。我们 创新性“发现和提供”疫苗设计方法将影响结核病的临床实践模式 和其他鼻内感染，因此，将更好地照顾我们的退伍军人。

The incidence of tuberculosis (TB) has increased among the Veterans in recent years because the global burden of TB is enormous. This burden has escalated with the emergence of multidrug-resistant and extremely drug resistant Mycobacterium tuberculosis (Mtb) strains and because current vaccines do not elicit long-lasting protective immunity against TB especially in adults. Hence, the development of new generations of vaccines that will confer durable protection against TB will significantly improve the quality of life of our Veterans. Our plan proposes pre-clinical studies that will identify protective CD8+ T cell epitopes and develop mucosal vaccine delivery platforms for the design of next generation TB vaccines. Mtb enters the host through the respiratory tract. Hence, optimal protection will require lung-resident CD4+ and CD8+ memory T cells to be positioned at the frontline to respond immediately to infection. Traditional vaccines and approved adjuvants typically elicit weak, short-lived T cell responses, and parenteral vaccination is ineffective at installing protective immunity within tissue mucosae. Moreover, most virus-vectored and subunit TB vaccines employ a small subset of Mtb antigens, resulting in insufficient epitope diversity for optimal protection, partly because the epitopes that are presented during Mtb infection and confer protective immunity have not been explored. Hence, our overall objective is to discover immunogenic Mtb epitopes generated during infection and to incorporate them in an innovative nanoparticle (NP)-based intranasal vaccine that is designed to promote a balanced pulmonary CD4+ and CD8+ T cell responses that will protect against TB. In preliminary experiments, we have identified 41 novel peptides from Mtb H37Rv-infected primary macrophages using a proteomics approach. Among these, 17 are putative HLA-B*07;02-binding epitopes, which we will characterize to advance anti-TB vaccine design. Eliciting CD8+ T cells that complement a CD4+ T cell response requires that subunit antigens be presented by HLA class I molecules for CD8+ T cell cross-priming in the context of appropriate inflammatory cues that drive both CD8+ T cell and CD4 differentiation. Our team has recently pioneered a “pathogen-mimicking” vaccine that is based on pH-responsive, endosome-rupturing NP chemistry. Such NPs promote delivery of antigens and nucleic acid adjuvant cargoes into the cytooplasm. Preliminary studies demonstrated that a single intranasal administration of NPs loaded with ovalbumin in conjunction with a structurally optimized 5'ppp-RNA hairpin adjuvant, that activates cytoplasmic retinoic acid-inducible gene-I, elicits a robust, durable and protective antigen-specific T cell response in the lungs. Based on these exciting new findings, we hypothesize that intranasal immunization with NP vaccines co-loaded with naturally processed class I-restricted Mtb-derived epitopes and 5'ppp-RNA adjuvant will significantly enhance tissue resident CD8+ memory T cell responses in the airways and parenchyma of the lungs. Our strategy to test this hypothesis is to, (a) evaluate the immunogenicity and protective potential of naturally processed Mtb epitopes presented by HLA-B*07:02 class I molecules; (b) determine whether HLA-B*07:02 and B*35:01—two closely-related HLA molecules—present similar epitopes; and (c) develop a NP vaccine that elicits a durable lung-resident CD8+ and CD4+ memory T cell responses to Mtb subunit antigen(s) that confers protection against TB. Our multidisciplinary team —consisting of biochemists, immunologists, microbiologists and bio-engineer— is ideally situated to pursuing the three Specific Aims. We anticipate that successful completion of the proposed research will yield highly significant knowledge essential for next generation anti-TB vaccine design. We expect to discover novel protective Mtb epitopes presented and recognized by multiple HLA molecules. We will also have developed a new delivery platform for mucosal vaccination that will allow co-delivery of antigens and adjuvants for cytoplasmic immune surveillance. Our innovative “discover and deliver” approach to vaccine design, will impact clinical practice paradigms against TB and other intranasal infections and, hence, will better the healthcare of our Veterans.