A Helminth-Derived Immune Potentiator for Biodefense

描述（由申请人提供）： 最终目标是开发一种安全有效的人体免疫增强剂，对抗可能用作生物武器的病原体，我们将描述从人类寄生虫盘尾丝虫克隆的强免疫刺激蛋白在小鼠中启动的免疫机制。在我们的原理验证研究中，无LPS的重组O。肠扭转激活相关蛋白-1（rOv-ASP-1）在用以下疫苗接种的小鼠中充当抗体应答的强有力佐剂：1）卵白蛋白; 2）来自SARS-CoV的刺突区域的肽; 3）HIV-1多肽。由蛋白质增强的抗原的抗体滴度超过了由最佳剂量的明矾或Ribi佐剂佐剂辅助的抗体滴度，并且小鼠对蛋白质耐受良好。接受卵清蛋白与寄生虫蛋白的小鼠的细胞反应主要是Th 1型。该蛋白质还刺激高水平的促炎细胞因子（IFN-γ，TNF-α和GM-CSF）和IL-10分泌从幼稚的人白细胞在体外。rOv-ASP-1与> 94%的人B细胞和单核细胞、29%的NK细胞和14.5%的CD 8 + T细胞结合。为了使佐剂蛋白的可能的治疗应用最大化，现在重要的是首先详细建立它针对明确定义的共同施用的病原体抗原启动的体液和细胞应答。为此，我们提出了两个具体目标。首先，定义在用已知为保护性免疫的靶标的人病原体抗原免疫的小鼠中由rOv-ASP-1蛋白佐剂引发的体液和细胞应答的类型。具体地说，我们将使用重组SARS-CoV受体结合结构域，当与弗氏佐剂和土拉热弗朗西丝菌的重组TUL 4抗原组合时，该结构域在小鼠中刺激有效的中和抗体，该抗原是免疫小鼠中细胞介导免疫的经证实的靶标。其次，基于我们对蛋白质的分子结构（包括推定的趋化因子样结构域）的了解，对rOv-ASP-1的亚基进行结构-功能研究，目的是鉴定具有最佳佐剂性的可能活性位点。基于初步的数据，我们假设rOv-ASP-1将能够刺激抗体和Th 1/CD 8 + T细胞应答，这些应答与SARS-CoV和F.土拉热。此外，研究合理选择的rOv-ASP-1亚基的生物活性将有助于确定蛋白质的作用模式，并有助于设计具有最佳佐剂性和/或确定的引发特定效应途径的能力的分子。

DESCRIPTION (provided by applicant): With the ultimate goal of developing a safe and effective human immune potentiator against pathogens that may be used as bioweapons, we will characterize the immune mechanisms initiated in mice by a strongly immunostimulant protein cloned from the human parasite, Onchocerca volvulus. In our proof-of-principle studies, the LPS-free recombinant O. volvulus activation-associated protein-1 (rOv-ASP-1) acted as a powerful adjuvant for antibody responses in mice vaccinated with: 1) ovalbumin; 2) a peptide from the spike region of the SARS-CoV; 3) an HIV-1 polypeptide. Antibody titers to the antigens augmented by the protein exceeded those adjuvanted by optimal doses of alum or Ribi adjuvants and mice tolerated the protein well. Cellular responses in mice receiving ovalbumin with the parasite protein were predominantly Th1-type. The protein also stimulated high levels of pro-inflammatory cytokine (IFN-gamma, TNF-alpha and GM-CSF) and IL-10 secretion from naive human leukocytes in vitro. rOv-ASP-1 binds to > 94% of human of human B cells and monocytes, 29% of NK cells and 14.5% of CD8+ T cells. To maximize the possible therapeutic applications of the adjuvant protein, it is now important to first establish in detail the humoral and cellular responses it initiates against well-defined, coadministered pathogen antigens. To achieve this we propose two specific aims. Firstly, to define the types of humoral and cellular responses initiated by the rOv-ASP-1 protein adjuvant in mice immunized with human pathogen antigens known to be targets of protective immunity. Specifically, we will use recombinant SARS-CoV receptor binding domain which stimulates potent neutralizing antibodies in mice when combined with Freund's adjuvant and recombinant TUL4 antigen of Francisella tularensis, a proven target of cell-mediated immunity in immunized mice. Secondly, to perform structure-function studies on subunits of rOv- ASP-1, based on what we know about the molecular structure of the protein (including a putative chemokine-like domain), with the goal of identifying possible active sites with optimal adjuvanticity. Based on the preliminary data, we hypothesize that rOv-ASP-1 will be able to stimulate both antibody and Th1/CD8+ T cell responses relevant to protection against SARS-CoV and F. tularensis. Furthermore, studying the bioactivities of rationally-selected subunits of rOv-ASP-1 will help determine the mode of action of the protein and aid in the design of a molecule with optimal adjuvanticity and/or defined ability to elicit particular effector pathways.