Alpha2-Macroglobulin-PA Complexes: Novel Anthrax Vaccin*

Alpha2-巨球蛋白-PA 复合物：新型炭疽疫苗*

• 批准号: 6665114
• 负责人: Salvatore V Pizzo
• 金额: $ 23.1万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6665114
• 关键词: Bacillus anthracis; Escherichia coli; SDS polyacrylamide gel electrophoresis; active immunization; anthrax; anthrax vaccines; bioterrorism /chemical warfare; enzyme linked immunosorbent assay; glycoproteins; immune complex; immunogenetics; immunomodulators; laboratory rabbit; macroglobulins; neutralizing antibody; proteolysis; serum; vaccine development; western blottings

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.

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（包括炭疽、肉毒杆菌中毒和鼠疫）的多种制剂的更有效的疫苗。该申请的长期目标是开发基于完全天然、非反应性佐剂α2-巨球蛋白（α2M）的新一代生物制剂疫苗。 Alpha2M 已被证明可以极大地增强许多抗原的免疫原性。 α2M 佐剂疫苗的开发将显着影响美国和世界的医疗保健，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使用起来更安全。 PA被认为是当前炭疽疫苗（AVA，吸收炭疽疫苗）中的主要保护性抗原，将被用作拟议研究的原型亚基候选者。本研究的具体目的是（1）确定炭疽杆菌 PA 的最佳大小及其共价掺入兔 alpha2M 的最佳条件； (2)测定PA与a2M共价偶联的各种复合物产生炭疽毒素中和抗体的能力； (3)确定PA与α2M复合的免疫原性是否可以通过与现有佐剂组合来增强。具体而言，全长 PA（83 kDa，rPA83）将在大肠杆菌中表达并纯化至均质。 rPA83 的蛋白水解将用于生成“带切口的 PA”（63 kDa，rPA63），或包含受体结合结构域的羧基末端片段（47 kDa，rPA47）。 a2M 将从兔血浆中纯化至同质。研究将确定不同大小的 rPA 在不同条件下掺入兔 a2M 的效率。然后使用兔 a2M 和 rPA (α2M-rPA) 的复合物对兔进行免疫，并将与单独吸收到明矾上的 rPA 进行比较其免疫原性。将评估免疫兔血清的抗 rPA 滴度（基于 ELISA）、免疫球蛋白同种型以及阻断炭疽毒素介导的巨噬细胞细胞毒性（中和活性）的能力。产生中和滴度的 Alpha2M-rPA 复合物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候选炭疽疫苗，其免疫原性得到提高，反应原性降低。