Vaccinating at Mucosal Surfaces with Nanoparticle Conjugated Antigen and Adjuvant

描述（由申请人提供）： 本修订版VA Merit提案的目的是设计针对感染性疾病的CD 8 + T细胞靶向无病原体（亚单位）疫苗，并设计一种将其递送至粘膜部位的有效方法，粘膜部位标志着病原体进入的主要端口。目前使用的大多数疫苗通过抗体介导的中和和/或调理作用起作用。尽管CD 8 + T细胞在保护免受由细胞内微生物引起的感染性疾病中被证明具有关键作用，但目前还不存在靶向该T细胞亚群的有效疫苗。这是因为 天然加工和呈递的CD 8 + T细胞表位和赋予保护性免疫的那些表位仍然未知。为了填补这一空白，使用最成功的天花疫苗作为模型，我们首先发现并表征了许多HLA-A *02;01和-B*07;01 I类限制性的、天然加工的CD 8 + T细胞表位，这些表位来源于牛痘病毒（VacV），在天花病毒（天花的病原体）中也是保守的。然后，我们发现，用含有这些表位的工程化VacV蛋白与NKT细胞靶向佐剂β-半乳糖神经酰胺（β GC）-快速激活NKT细胞并有效地反式激活树突状细胞以及自然杀伤细胞，T和B细胞-引发小鼠的初免-加强疫苗接种引发了功能性CD 8 + T细胞应答。这些CD 8 + T细胞应答保护小鼠免受致命的呼吸道痘病毒攻击。在这些发现的指导下，我们将测试中心假设， “用最佳佐剂配制的微生物蛋白亚单位疫苗的基于纳米颗粒的粘膜递送通过靶向天然加工的CD 8 + T细胞表位赋予针对致命呼吸道挑战的保护性免疫力”。我们测试中心假设的方法是：（a）制备与γ-GC（GC-np）或IFN γ诱导的GC变体氟苄基-酰基-γ-GC偶联的纳米颗粒（np），（FBzGC-np），并表征其作用机制;（B）确定肺CD 8 + T细胞对用纳米颗粒免疫的应答的质量和稳健性，所述纳米颗粒将共递送偶联的重组VacV亚基- np和GC-1。np或FBzGC-np;和（c）阐明这种疫苗策略对抗异型（VacV-WR株）和同型（鼠痘）痘病毒模型的致死性呼吸道攻击的保护潜力。本文提出的方法是可行的，因为我们的新数据显示，rVV-np+GC-np在鼻内接种后比腹膜内免疫在人源化小鼠中引起更好的保护性应答。因此，我们提出了创新的方法来开发含有天然加工抗原和NKT细胞靶向佐剂的无微生物粘膜疫苗，这些疫苗在纳米颗粒载体上递送时，通过CD 8 + T细胞介导保护作用。这样的粘膜疫苗制剂靶向先前未被利用但对保护免受细胞内传染性疾病病原体至关重要的免疫系统的非常有效的分支-即，先天性NKT细胞和适应性CD 8 + T细胞。由于该疫苗将在异型和同型病原体-宿主模型以及人源化小鼠模型中进行测试，我们期望获得与下一代无病原体疫苗设计相关的新见解。这些疫苗将有可能改变临床实践模式，特别是针对直接影响世界人民健康和福祉的传染病，因此也将使退伍军人受益。

DESCRIPTION (provided by applicant): The objective of this revised VA Merit Proposal is to design CD8+ T cell-targeted pathogen- free (subunit) vaccines against infectious diseases and to devise an efficacious method for their delivery to mucosal sites, which mark the major port of pathogen entry. Most currently used vaccines work by antibody-mediated neutralization and/or opsonization. Despite the proven critical role for CD8+ T cells in protection against infectious diseases caused by intracellular microbes, effective vaccines that target this T cell subset currently do not exist. This is because naturally processed and presented CD8+ T cell epitopes and those that confer protective immunity remain unknown. To fill this gap, using the most successful smallpox vaccine as a model, we first discovered and characterized numerous HLA- A*02;01 and -B*07;01 class I restricted, naturally processed CD8+ T cell-epitopes derived from vaccinia virus (VacV) that are also conserved in variola virus-the agent of smallpox. We then found that prime-boost vaccination of mice with engineered VacV proteins containing such epitopes in combination with an NKT cell-targeted adjuvant �- galactosylceramide (�GC)-which rapidly activates NKT cells and potently trans-activates dendritic cells as well as natural killer, T and B cells-elicited robut, functional CD8+ T cell responses. These CD8+ T cell responses protected mice from lethal respiratory poxvirus challenge. Guided by these findings, we will test the central hypothesis that, "Nanoparticle- based mucosal delivery of microbial protein subunit vaccines formulated with an optimal adjuvant confers protective immunity against lethal respiratory challenge by targeting naturally processed CD8+ T cell epitopes". Our approaches to test the central hypothesis are: (a) to prepare nanoparticles (np) coupled with �GC (GC-np) or the IFNy-inducing GC variant, flurobenzyl-acyl-�GC (FBzGC-np), and to characterize the mechanism of their action; (b) to determine the quality and robustness of pulmonary CD8+ T cell responses to immunization with nanoparticles that will co-deliver coupled recombinant VacV subunit(s)- np and GC-np or FBzGC-np; and (c) to elucidate the protective potential of such a vaccine strategy against lethal respiratory challenge with heterotypic (VacV-WR strain) and homotypic (Ectromelia) poxvirus models. The approaches proposed herein are feasible because our new data revealed that rVV-np+GC-np elicited better protective response in humanized mice upon intranasal vaccination than intra-peritoneal immunization. Hence, we have proposed innovative approaches to develop microbe-free, mucosal vaccines containing naturally processed antigens and NKT cell-targeted adjuvant, which when delivered on nanoparticle carriers, mediate protection through CD8+ T cells. Such mucosal vaccine formulation targets previously unemployed, yet greatly potent arms of the immune system critical for protection against intracellular infectious disease agents-viz., the innate-like NKT cells and the adaptive CD8+ T cells. Because the vaccine will be tested in both heterotypic and homotypic pathogen-host models and a humanized mouse model, we expect to gain novel insights relevant to the design of next generation pathogen-free vaccines. Such vaccines will have the potential to shift clinical practice paradigms, especially against infectious diseases that directly impact the health and well-being of this World's peoples, and, hence, will benefit Veterans as well.