Multivalent Plague, Anthrax Vaccines Using Bacteriophage T4 Display

描述（由申请人提供）：我们假设噬菌体 T4 上抗原（Ag）的体外组装可以开发为构建下一代多价生物防御疫苗的通用平台。该提案的主要目标是检验这一假设并开发三价鼠疫疫苗和多价鼠疫炭疽疫苗，这些疫苗可以提供针对炭疽杆菌和鼠疫耶尔森氏菌（两种 A 类生物防御病原体）的保护。我们开发了一种明确的体外系统，可以在噬菌体 T4 纳米颗粒 (120 x 86 nm) 上高密度排列大的外源 Ag。全长炭疽毒素大至 90kDa，与两种非必需噬菌体 T4 外衣壳蛋白 Soc（小外衣壳蛋白；870 拷贝）和 Hoc（高抗原性外衣壳蛋白；155 拷贝）融合，在大肠杆菌中过表达，纯化至均质，并通过体外结合展示在 hoc_soc_ 衣壳上。多个Ag和大型异源寡聚复合物可以显示至饱和并且可以控制拷贝数。空间暴露且对称排列的 T4-rPA 颗粒在无需任何佐剂的情况下具有高度免疫原性，可引发强 PA 特异性和致命毒素中和抗体 (Ab) 滴度，为兔子提供针对 100 LD50 炭疽芽孢杆菌孢子攻击的完全保护。最好的疫苗配方正在恒河猴的临床前试验中进行测试。结合我们的生化、分子遗传学、结构和气溶胶攻击专业知识，设计策略开发一种新型三价 T4 鼠疫疫苗，由三种耶尔森氏菌 Ag、荚膜 Ag、Caf1、低钙反应 V Ag、LcrV 和耶尔森氏菌分泌复合因子 YscF 组成。 YscF 是一种高度保守的 III 型分泌系统针状形成亚基，对所有强毒力耶尔森氏菌物种至关重要，预计将产生一种优质且广泛有效的三价鼠疫疫苗。衣壳结合鼠疫抗原的结构配置和拷贝数将得到优化。 T4鼠疫抗原、佐剂、递送途径（包括皮肤贴剂）的组合将被测试抗体和细胞免疫反应以及针对荚膜菌株鼠疫耶尔森氏菌CO92（最致命的鼠疫菌株之一）攻击的保护作用。将在新的挪威褐鼠气溶胶挑战模型中评估保护效果。最好的 T4 炭疽和 T4 鼠疫疫苗将被结合起来，创造出一种新型多价炭疽-鼠疫疫苗。该疫苗预防吸入性炭疽和肺鼠疫的功效将在恒河猴气溶胶攻击模型中进行测试。

DESCRIPTION (provided by applicant): We hypothesize that in vitro assembly of antigens (Ags) on bacteriophage T4 can be developed as a common platform for construction of next generation multivalent biodefense vaccines. The major goal of this proposal is to test this hypothesis and develop trivalent plague and multivalent plague-anthrax vaccines that can confer protection against Bacillus anthracis and Yersinia pestis, two Category A biodefense pathogens. We have developed a defined in vitro system to array large foreign Ags at high density on phage T4 nanoparticle (120 x 86 nm). Full-length anthrax toxins, as large as 90kDa, are fused to the two non-essential phage T4 outer capsid proteins, Soc (small outer capsid protein; 870 copies) and Hoc (highly antigenic outer capsid protein; 155 copies), over-expressed in E. coli, purified to homogeneity, and displayed on hoc_soc_ capsid through in vitro binding. Multiple Ags and large hetero-oligomeric complexes can be displayed to saturation and the copy number can be controlled. The spatially exposed and symmetrically arrayed T4-rPA particles are highly immunogenic without any adjuvant, eliciting strong PA-specific and lethal toxin neutralizing Antibody (Ab) titers, conferring complete protection to rabbits against 100 LD50 B. anthracis Ames spore challenge. The best vaccine formulations are being tested in a preclinical trial using rhesus macaques. Combining our biochemical, molecular genetic, structural, and aerosol challenge expertise, strategies are designed to develop a novel trivalent T4-plague vaccine comprised of three Yersinia Ags, the capsular Ag, Caf1, the low calcium response V Ag, LcrV, and the Yersinia secretory complex factor, YscF. Inclusion of YscF, a highly conserved needle-forming subunit of type III secretion system that is essential to all virulent Yersinia species, is expected to generate a superior and broadly effective trivalent plague vaccine. The structural disposition and copy number of the capsid-bound plague Ags will be optimized. Combinations of T4-plague Ags, adjuvants, route of delivery (including skin patch), will be tested for Ab and cellular immune responses and protection against challenge with the capsular strain Y. pestis CO92, one of the most lethal plague strains. Efficacy of protection will be assessed in a new Brown Norway rat aerosol challenge model. The best T4-anthrax and T4-plague vaccines will be combined to create a novel multivalent anthrax-plague vaccine. The efficacy of this vaccine for protection against both inhalation anthrax and pneumonic plague will be tested in rhesus macaque aerosol challenge in model.