Alpha2-Macroglobulin-PA Complexes: Novel Anthrax Vaccin*

说明(申请人提供)：迫切需要确定和开发新的方法，以提供保护性抗原，作为更有效的疫苗，预防可能被用作生物武器的各种制剂，包括炭疽病、肉毒杆菌中毒和鼠疫。拟议应用的长期目标是开发基于一种完全天然的、非反应性佐剂--α2-巨球蛋白(α2M)的新一代生物制剂疫苗。Alpha2M已被证明能极大地增强一些抗原的免疫原性。Alpha2M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单位的新一代疫苗，包括预防和治疗性疫苗，这种疫苗生产成本低，使用本质上更安全。PA被公认为当前炭疽疫苗(AVA，炭疽疫苗吸收)中的主要保护性抗原，将被用作拟议研究的原型亚单位候选。拟议研究的具体目的是(1)确定炭疽芽孢杆菌PA的最佳大小及其共价结合到兔Alpha2M中的最佳条件；(2)确定PA与a2M共价偶联的各种络合物产生中和炭疽毒素抗体的能力；以及(3)确定PA与Alpha2M络合物的免疫原性是否可以通过与现有佐剂的组合而增强。具体地说，全长PA(83 kDa，rPA83)将在大肠杆菌中表达并纯化至均一。RPA83的蛋白分解将被用来产生“镍PA”(63 kDa，rPA63)，或含有受体结合域(47 kDa，rPA47)的羧基末端片段。A2M将从兔血浆中纯化至均一。研究将确定在不同条件下将不同大小的RPA掺入兔a2M的效率。然后将兔a2M和RPA的复合物(α2M-RPA)用于免疫兔子，并将其与单独吸附在明矾上的RPA进行免疫原性比较。免疫兔子的血清将被评估抗RPA效价(基于ELISA法)、免疫球蛋白亚型和阻断炭疽毒素介导的巨噬细胞毒性的能力(中和活性)。产生中和效价的Alpha2M-RPA复合体将与其他佐剂结合进行进一步评估。这些研究将提供一种新的炭疽疫苗候选疫苗，既提高了免疫原性，又降低了反应性。

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.