Vaccinating at Mucosal Surfaces with Nanoparticle Conjugated Antigen and Adjuvant

描述(由申请人提供)： 这项修订的VA Merit提案的目标是设计CD8+T细胞靶向无病原体(亚单位)疫苗，以对抗传染病，并设计一种有效的方法将其运送到标志着病原体进入的主要口岸的粘膜部位。目前使用的大多数疫苗都是通过抗体介导的中和和/或调理作用发挥作用的。尽管CD8+T细胞在预防由细胞内微生物引起的传染病方面发挥了关键作用，但目前还没有针对这一T细胞亚群的有效疫苗。这是因为 自然处理和呈递的CD8+T细胞表位以及那些赋予保护性免疫的表位仍不清楚。为了填补这一空白，以最成功的天花疫苗为模型，我们首先发现并鉴定了大量的人类白细胞抗原-A*02；01和-B*07；01-I类限制性的、自然处理的CD8+T细胞表位，这些表位来自痘苗病毒(VacV)，也在天花病原体天花病毒中保守。然后，我们发现，用含有这些表位的工程VacV蛋白与NKT细胞靶向佐剂�-半乳糖神经酰胺(�GC)相结合的初始加强免疫小鼠，可以快速激活NKT细胞，并有效反式激活树突状细胞以及自然杀伤细胞、T细胞和B细胞，从而引发正常的CD8T细胞反应。这些CD8+T细胞反应保护小鼠免受致死性呼吸道痘病毒攻击。在这些发现的指导下，我们将检验中心假设， 用最佳佐剂配制的微生物蛋白亚单位疫苗以纳米颗粒为基础的粘膜递送通过靶向自然处理的CD8+T细胞表位来提供针对致命呼吸道攻击的保护性免疫。我们检验这一中心假设的方法是：(A)制备与VacVGC(GC-NP)或IFNY诱导的GC变异体(FBZGC-NP)偶联的纳米粒(NP)，并表征它们的作用机制；(B)确定将共传递偶联重组�亚单位(S)-NP和GC-NP或FBzGC-NP的纳米粒免疫肺部CD8T细胞的质量和稳健性；以及(C)用异型(VacV-WR株)和同型(Etromelia)痘病毒模型阐明这种疫苗策略对致命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在人源化小鼠鼻内免疫时比腹膜内免疫产生了更好的保护性反应。因此，我们提出了创新的方法来开发无微生物的粘膜疫苗，该疫苗包含自然处理的抗原和NKT细胞靶向佐剂，当以纳米颗粒载体递送时，通过CD8+T细胞介导保护。这种粘膜疫苗配方针对的是先前失业但功能强大的免疫系统手臂，这些手臂对于抵御细胞内传染病病原体至关重要--即先天类似的NKT细胞和适应性CD8+T细胞。由于疫苗将在异型和同型病原体宿主模型和人源化小鼠模型中进行测试，我们预计将获得与下一代无病原体疫苗设计相关的新见解。这种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.