Mechanistic evaluation of mast cell agonists combined with TLR, NOD and STING agonists.

摘要 亚单位疫苗比其他疫苗更安全，可以更广泛地在人群中应用 制剂如减毒活的。然而，亚单位抗原通常是抗原性差的，并且需要 制剂与免疫刺激佐剂以获得保护。此外，一些疫苗需要更多 而不是一种佐剂，需要组合佐剂来刺激保护性应答。此外， 佐剂可以减少疫苗接种加强，并提供更长的保护。一种评估组合的途径 佐剂是与肥大细胞（MC）激动剂。MC遍布全身，并驻留在许多界面的 宿主和环境。当激活的MC将单核细胞和白细胞募集到局部区域并有助于 促进适应性反应。MC激动剂与toll样受体（TLR）联合，核苷酸结合 寡聚化结构域蛋白2（NOD-2）或干扰素基因刺激物（STING）激动剂应 不仅引发体液反应，而且引发细胞反应，以产生有效的疫苗。 本文中，我们将评估MC激动剂与TLR、NOD-2或STING激动剂的组合以鉴定协同对。 将在小鼠和人细胞以及来自协作交叉（CC）品系的细胞中评价细胞对。的 CC品系是一大组重组近交系小鼠品系，其遗传变异可以模拟 以及深入了解有助于佐剂机制遗传变量。确保 这些佐剂是共同递送的，并提供剂量调整、冷链外储存和控制 为了释放佐剂，我们将它们配制成缩醛化葡聚糖（Ac-DEX）微粒。醋酸地塞米松 制剂已经显示了STING、NOD-2、TLR和MC激动剂在体外和体内的增强递送， 高于其它载体如脂质体或PLGA颗粒。确定的最佳佐剂组合为 在具有亚单位抗原BR8的牛痘疫苗的小鼠模型中评估。我们将使用模式识别 受体（PRR）敲除小鼠以及细胞缺陷型小鼠，以阐明组合佐剂的 机制此外，我们将采用基因测序工具，以机械地确定组合 佐剂机制

ABSTRACT Subunit vaccines are safer and can more broadly be applied across the population then other vaccine formulations such as live-attenuated. However, subunit antigens are often poorly antigenic and require formulation with an immune stimulating adjuvant to garner protection. Additionally, some vaccines require more than one adjuvant, necessitating combined adjuvants to stimulate a protective response. Also, a combined adjuvant could decrease vaccination boosts and provide longer protection. One avenue to evaluate combined adjuvants is with mast cell (MC) agonists. MCs are throughout the body and reside at many interfaces of the host and the environment. When activated MCs recruit monocytes and leukocytes to the local area and help to promote an adaptive response. MC agonists combined with toll-like receptor (TLR), nucleotide-binding oligomerization domain-containing protein 2 (NOD-2), or stimulators of interferon genes (STING) agonists should elicit not only a humoral response, but also a cellular response to create an efficacious and effective vaccine. Herein we will evaluate combined MC agonists with TLR, NOD-2 or STING agonist to identify synergistic pairs. Pairs will be evaluated in mice and human cells as well as with cells from collaborative cross (CC) strains. The CC strains are a large panel of recombinant inbred mouse strains with genetic variation that can mimic the human population as well as give insight into genetic variables that contribute to adjuvant mechanism. To ensure that the adjuvants are co-delivered as well as offer dose sparring, storage outside the cold chain and controlled release of adjuvant, we will formulate them into acetalated dextran (Ac-DEX) microparticles. Ac-DEX formulations have illustrated enhanced delivery of STING, NOD-2, TLR and MC agonists in vitro and in vivo, above that of other carriers like liposomes or PLGA particles. The best identified adjuvant combination will be evaluated in a mouse model of a vaccinia vaccine with subunit antigen BR8. We will use pattern recognition receptor (PRR) knock-out mice as well as cell deficient mice to elucidate aspects of the combination adjuvant's mechanism. Additionally, we will employ genetic sequencing tools to mechanistically identify the combination adjuvants mechanism.