Vaccinating at Mucosal Surfaces with Nanoparticle Conjugated Antigen and Adjuvant

近年来，退伍军人中结核病(TB)的发病率有所增加，因为全球负担 结核病的数量是巨大的。这种负担随着多重耐药和极端毒品的出现而升级 耐药结核分枝杆菌(Mtb)菌株，因为目前的疫苗不能诱导长效 对结核病的保护性免疫，特别是在成年人中。因此，新一代疫苗的开发 将赋予对结核病的持久保护，将显著改善我们退伍军人的生活质量。我们的计划 建议进行临床前研究，以确定保护性CD8+T细胞表位并开发粘膜疫苗 用于设计下一代结核病疫苗的交付平台。结核分枝杆菌通过呼吸道进入宿主 一条小路。因此，最佳保护需要驻留在肺中的CD4+和CD8+记忆T细胞位于 前线对感染立即做出反应。传统疫苗和批准的佐剂通常会引发 微弱、短暂的T细胞反应和非肠道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结核病疫苗使用的是结核分枝杆菌的一小部分。 抗原，导致表位多样性不足，无法实现最佳保护，部分原因是 在结核分枝杆菌感染期间出现的和授予保护性免疫还没有被探索。因此，我们的整体 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 创新的纳米粒(NP)鼻腔疫苗，旨在促进平衡的肺CD4+ 和CD8+T细胞反应，可以预防结核病。在初步实验中，我们已经确定了41部小说 用蛋白质组学方法从结核分枝杆菌H37Rv感染的原代巨噬细胞中提取多肽。其中，17个是 推测的HLA-B*07；02结合表位，我们将对其进行表征，以推进抗结核疫苗的设计。诱人 CD8+T细胞作为对CD4+T细胞应答的补充，要求其亚单位抗原由人类白细胞抗原分类递呈 CD8+T细胞的I分子在驱动CD8+T细胞的适当炎症信号的背景下交叉启动 T细胞和CD4分化。我们的团队最近率先研发出一种“病原体模拟”疫苗，这种疫苗基于 关于pH反应、内体破裂的NP化学。这种核糖核酸促进抗原和核酸的传递。 佐剂货物进入细胞质。初步研究表明，单次鼻腔给药 与结构优化的5‘PPP-RNA发夹佐剂一起负载卵清蛋白的NPs， 激活细胞质维甲酸诱导基因-I，诱导出强大、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，我们假设鼻腔免疫 NP疫苗与自然处理的I类限制性Mtb衍生表位和5‘PPP-RNA共负载 佐剂将显著增强呼吸道和组织中驻留的CD8+记忆T细胞反应 肺的实质。我们检验这一假设的策略是：(A)评估免疫原性和 人类白细胞抗原-B*07：02 I类分子呈现的天然处理的MTB表位的保护潜力；(B) 确定人类白细胞抗原-B*07：02和B*35：01这两个密切相关的人类白细胞抗原分子是否存在相似的表位； 以及(C)开发一种NP疫苗，该疫苗可引起持久的驻肺CD8+和CD4+记忆T细胞对 结核分枝杆菌亚单位抗原(S)，提供对结核病的保护。我们的多学科团队-由生物化学家组成， 免疫学家、微生物学家和生物工程师是追求这三个具体目标的理想人选。我们 预计成功完成拟议研究将产生至关重要的重要知识 用于下一代抗结核疫苗的设计。我们希望发现新的保护性Mtb表位和 被多个人类白细胞抗原分子识别。我们还将开发一种新的粘膜递送平台 疫苗接种将允许抗原和佐剂的联合传递，以进行细胞质免疫监测。我们的 创新的“发现并交付”疫苗设计方法，将影响结核病的临床实践范例 和其他鼻腔感染，因此，将改善我们退伍军人的医疗保健。

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.