Single Dose, Multivalent, Anthrax Plague Vaccines using Bacteriophage T4 Nanopart

描述（由申请人提供）：我们提出了一种利用T4噬菌体纳米颗粒递送平台制造单剂量、多价、生物防御疫苗的新方法。在两次非常成功的U01生物防御拨款（17篇出版物和5项专利）中，我们开发了一种t4保护抗原（PA）炭疽疫苗，该疫苗为兔子和恒河猴提供了完全保护，免受100 LD50的炭疽杆菌Ames孢子的气溶胶攻击。T4-F1mut-V（突变荚膜抗原F1和低钙反应V抗原）鼠疫疫苗对小鼠和褐挪威大鼠提供完全保护，使其免受5000 LD50鼠疫耶尔森氏菌CO92的鼻内或气溶胶攻击。在这些成功的基础上，该提案将实现两个主要目标：1)开发一种可预防吸入性炭疽和肺鼠疫的单剂量炭疽-鼠疫双重疫苗，2)建立一种“即插即用”T4疫苗递送平台，可适用于任何生物防御或新出现的病原体。炭疽PA和鼠疫F1mut-V抗原将作为小外衣壳蛋白Soc（870拷贝）的融合蛋白显示在120nm × 86nm的噬菌体T4衣壳上。这些抗原对应的基因将在强CMV启动子的控制下克隆，并使用噬菌体T4 DNA包装机包装在衣壳内。DNA分子会在宿主体内诱导抗原的产生，持续数周到数月。将制备一系列含有PA和F1mut-V抗原、基因或两者的纳米颗粒制剂（目的1）。小鼠将使用我们新开发的炭疽-鼠疫双攻模型，单剂量接种这些制剂，不加佐剂，并对炭疽和鼠疫进行免疫筛选。特别令人感兴趣的是外部含有抗原而内部含有DNA的“初级强化”疫苗。这种蛋白质将充当“引物”，而DNA则充当“助推器”，通过不断表达抗原，进而刺激强有力的免疫反应。免疫学分析将量化结合抗体滴度，致命毒素中和滴度和细胞反应。将进一步评估最佳疫苗配方的反应持久性以及通过口服给药诱导粘膜免疫的能力（目标2）。来自小鼠研究的两种最佳配方将在第二种动物模型中进行测试，新西兰白兔用于吸入性炭疽，棕色挪威大鼠用于肺鼠疫（Aim 3）。最后，在食蟹猴模型中，采用鼠疫杆菌CO92和炭疽芽孢杆菌Ames孢子的双重气溶胶攻击来评估它们的免疫原性和保护效果（Aim 4）。通过开发强大的疫苗交付平台、先进的设施、与成熟的行业合作伙伴的伙伴关系以及具有互补优势的高度成就的研究人员团队的协同作用，该提案将导致创造一种有效的炭疽-鼠疫双重疫苗，这将对生物防御疫苗计划产生巨大影响。

DESCRIPTION (provided by applicant): We propose a novel approach to create single dose, multivalent, biodefense vaccines, using bacteriophage T4 nanoparticle delivery platform. In the two highly successful U01 biodefense grants (17 publications and 5 patents), we have developed a T4-protective antigen (PA) anthrax vaccine that provided complete protection to rabbits and rhesus macaques against 100 LD50 aerosol challenge of B. anthracis Ames spores, and a T4-F1mut-V (mutated capsular antigen F1 and low calcium response V antigen) plague vaccine that provided complete protection to mice and Brown Norway rats against intranasal or aerosol challenge with 5,000 LD50 of Yersinia pestis CO92. Building on these successes, this proposal will achieve two major goals: i) develop a single dose anthrax-plague dual vaccine that can protect against both inhalation anthrax and pneumonic plague, and ii) establish a "plug and play" T4 vaccine delivery platform that can be adapted to any biodefense or emerging pathogen. The anthrax PA and plague F1mut-V antigens will be displayed on the 120nm x 86nm phage T4 capsid shell, as fusion proteins of the small outer capsid protein, Soc (870 copies). The genes corresponding to these antigens will be cloned under the control of a strong CMV promoter and packaged inside the capsid, using the phage T4 DNA packaging machine. The DNA molecules will induce production of antigens in the host for weeks to months. A series of nanoparticle formulations will be prepared containing PA and F1mut-V antigens, genes, or both (Aim 1). Mice will be immunized with a single dose of these formulations, without an adjuvant, and screened for protection against both anthrax and plague, using our newly developed anthrax- plague double challenged model. Of particular interest are the "prime-boost" vaccines containing antigen outside and DNA inside. The protein would act as "prime" and the DNA as "boost," by continually expressing the antigens, and, in turn, stimulating potent immune responses. Immunological analyses will quantify binding antibody titers, lethal toxin neutralization titers, and cellular responses. The best vaccine formulations will be further evaluated for durability of the responses as well as the ability to induce mucosal immunity by oral delivery (Aim 2). The two best formulations from the mouse studies will be tested in a second animal model, New Zealand white rabbit for inhalation anthrax and Brown Norway rat for pneumonic plague (Aim 3). Finally, their immunogenicity and protective efficacy will be evaluated in the cynomolgus macaque model by double aerosol challenge with Y. pestis CO92 followed by Ames spores of B. anthracis (Aim 4). Synergized by the development of a robust vaccine delivery platform, state of the art facilities, partnership with an established industry partner, and a team of highly accomplished investigators with complementary strengths, this proposal would lead to the creation of an efficacious anthrax-plague dual vaccine that will have tremendous impact on the biodefense vaccine program.