Alpha2-Macroglobulin-PA Complexes: Novel Anthrax Vaccin*

描述（由申请人提供）：迫切需要鉴定和开发用于递送保护性抗原的新方法，作为针对可能用作生物武器的各种试剂（包括炭疽、肉毒杆菌中毒和鼠疫）的更有效的疫苗。该申请的长期目标是开发基于完全天然的非反应原性佐剂α 2-巨球蛋白（α 2 M）的新一代生物制剂疫苗。已显示Alpha 2 M极大地增强许多抗原的免疫原性。alpha 2 M佐剂疫苗的开发将对美国和世界的医疗保健产生重大影响，因为它将允许基于蛋白质亚基的新一代预防性和治疗性疫苗，这些疫苗可以廉价生产，并且本质上更安全。PA被认为是当前炭疽疫苗（AVA，吸附炭疽疫苗）中的主要保护性抗原，将用作拟定研究的原型候选亚单位。所提出的研究的具体目的是（1）鉴定炭疽芽孢杆菌PA的最佳大小和其共价掺入兔α 2 M的最佳条件：（2）确定与α 2 M共价偶联的PA的各种复合物产生针对炭疽毒素的中和抗体的能力;和（3）确定与现有佐剂组合是否可以增强与alpha 2 M复合的PA的免疫原性。具体而言，全长PA（83 kDa，rPA 83）将在E. coli中，并纯化至均一。rPA 83的蛋白水解将用于产生“切口PA”（63 kDa，rPA 63）或含有受体结合结构域的羧基末端片段（47 kDa，rPA 47）。将从兔血浆中纯化α 2 M至均一。研究将确定在各种条件下将不同大小的rPA掺入兔α 2 M中的效率。然后将兔α 2 M和rPA的复合物（α 2 M-rPA）用于免疫兔，并将针对吸收到明矾上的单独rPA的免疫原性进行比较。将评价来自免疫兔的血清的抗rPA滴度（基于ELISA）、免疫球蛋白同种型和阻断炭疽毒素介导的巨噬细胞细胞毒性的能力（中和活性）。产生中和滴度的α 2 M-rPA复合物将与其他佐剂组合进行进一步评价。这些研究将提供一种新的炭疽疫苗候选者，具有改善的免疫原性和降低的反应原性。

DESCRIPTION (provided by applicant): An urgent need exists for the identification and development of novel approaches for delivering protective antigens as more effective vaccines against a variety of agents which might be used as biological weapons, including anthrax, botulism and plague. The long-term objective of the proposed application is to develop a new generation of vaccines against biological agents based on a totally natural, non-reactogenic adjuvant, alpha2-Macroglobulin (alpha2M). Alpha2M has been shown to greatly enhance immunogenicity of a number of antigens. The development of alpha2M adjuvanted vaccines will significantly impact healthcare in both the U.S. and the world, as it will allow a new generation of vaccines, both prophylactic and therapeutic, based on protein subunits, which can be produced inexpensively and are intrinsically safer to use. PA, recognized as the major protective antigen in the current anthrax vaccine (AVA, Anthrax Vaccine Absorbed), will be used as a prototypical subunit candidate for the proposed studies. The specific aims of the proposed studies are to (1) identify the optimal size of Bacillus anthracis PA and the optimal conditions for its covalent incorporation into rabbit alpha2M; (2) determine the ability of various complexes of PA covalently coupled with a2M to generate neutralizing antibodies to anthrax toxin; and (3) determine if the immunogenicity of PA complexed with alpha2M can be enhanced by combination with existing adjuvants. Specifically, full-Iength PA (83 kDa, rPA83) will be expressed in E. coli and purified to homogeneity. Proteolysis of rPA83 will be used to generate "nicked PA" (63 kDa, rPA63), or a carboxy-terminal fragment containing the receptor-binding domain (47 kDa, rPA47). a2M will be purified to homogeneity from rabbit plasma. Studies will determine the efficiency of incorporation of rPA of varying size into rabbit a2M under various conditions. Complexes of rabbit a2M and rPA (alpha2M-rPA) will be then be used to immunize rabbits and will be compared for immunogenicity against rPA alone absorbed onto alum. Sera from immunized rabbits will be evaluated for anti-rPA titers (based on ELISA), immunoglobulin isotypes, and ability to block anthrax toxin mediated macrophage cytotoxicity (neutralizing activity). Alpha2M-rPA complexes, which generate neutralizing titers, will be further evaluated in combination with other adjuvants. These studies will provide a new anthrax vaccine candidate with both improved immunogenicity and decreased reactogenicity.