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

描述(由申请人提供)：我们提出了一种新的方法，利用噬菌体T4纳米颗粒递送平台来创造单剂、多价、生物防御疫苗。在两项非常成功的U01生物防御基金(17篇论文和5项专利)中，我们开发了T4保护性抗原(PA)炭疽疫苗和T4-F1mut-V(突变型囊膜抗原F1和低钙反应V抗原)鼠疫疫苗，T4-F1mut-V疫苗对鼠疫耶尔森氏菌CO92的5,000 LD50毒力提供了鼻腔或气溶胶攻击，对兔和猕猴提供了完全保护。在这些成功的基础上，这项提议将实现两个主要目标：i)开发一种单剂炭疽-鼠疫双重疫苗，既可以预防吸入性炭疽病，也可以预防肺炎鼠疫；ii)建立一个可以适应任何生物防御或新兴病原体的“即插即用”T4疫苗交付平台。炭疽杆菌PA和鼠疫F1-MUT-V抗原将展示在120 nm×86 nm噬菌体T4衣壳上，作为小外壳蛋白Soc的融合蛋白(870个拷贝)。与这些抗原对应的基因将在CMV强启动子的控制下克隆，并使用噬菌体T4DNA包装机包装在衣壳内。DNA分子将在宿主体内诱导数周至数月的抗原产生。将制备一系列含有PA和F1mut-V抗原、基因或两者兼有的纳米颗粒制剂(目标1)。使用我们新开发的炭疽-鼠疫双重挑战模型，将用这些配方的单剂免疫小鼠，而不使用佐剂，并对炭疽和鼠疫进行保护。特别令人感兴趣的是外层含有抗原、内层含有DNA的“优质强化”疫苗。通过不断表达抗原，蛋白质起到“启动”的作用，而DNA起到“助推”的作用，进而刺激强大的免疫反应。免疫学分析将量化结合抗体滴度、致死毒素中和滴度和细胞反应。将进一步评估最好的疫苗配方的反应持久性以及通过口服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个动物模型中进行测试，新西兰大白兔用于吸入炭疽病，布朗挪威鼠用于肺鼠疫(目标3)。最后，在食蟹猴模型中，用鼠疫耶尔森氏菌CO92和炭疽杆菌的Ames孢子进行双重气雾剂攻击，以评价它们的免疫原性和保护效果(目标4)。通过开发强大的疫苗交付平台、最先进的设施、与成熟的行业合作伙伴的合作伙伴关系以及一支具有互补优势的高度成功的研究团队的协同作用，这项提议将导致创建一种有效的炭疽-鼠疫双重疫苗，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.