A Helminth-Derived Immune Potentiator for Biodefense

描述(由申请人提供)： 我们的最终目标是开发一种安全有效的人类免疫增强剂，以对抗可用作生物武器的病原体，我们将研究从人类寄生虫克隆的一种强大的免疫刺激蛋白在小鼠体内启动的免疫机制。在我们的原理验证研究中，无内毒素的重组卷弧菌激活相关蛋白-1(ROV-ASP-1)可作为强大的佐剂，用于免疫以下疫苗的小鼠的抗体反应：1)卵清蛋白；2)SARS冠状病毒棘突区的多肽；3)HIV-1多肽。这种蛋白质增强的抗原的抗体效价超过了最佳剂量的明矾或Ribi佐剂，小鼠对这种蛋白质的耐受性很好。在接受带有寄生虫蛋白的卵清蛋白的小鼠中，细胞反应主要是Th1型。该蛋白还能刺激体外培养的人类幼稚白细胞分泌高水平的促炎细胞因子(干扰素-γ、肿瘤坏死因子-α和GM-CSF)和IL-10。ROV-ASP-1能与人94%的B细胞和单核细胞、29%的NK细胞和14.5%的CD8+T细胞结合。为了最大化佐剂蛋白可能的治疗应用，现在重要的是首先详细建立它对明确定义的联合给药病原体抗原启动的体液和细胞反应。为了实现这一目标，我们提出了两个具体目标。首先，确定ROV-ASP-1蛋白佐剂在以已知为保护性免疫靶标的人类病原体抗原免疫的小鼠中引发的体液和细胞反应的类型。具体地说，我们将使用重组SARS-CoV受体结合域，当与弗氏佐剂和土拉方杆菌重组TUL4抗原相结合时，它能在小鼠中激发强大的中和抗体，图拉氏方济氏菌TUL4抗原已被证明是免疫小鼠细胞免疫的目标。其次，根据已知的蛋白质分子结构(包括可能的趋化素样结构域)，对ROV-ASP-1的亚基进行结构-功能研究，目的是确定具有最佳佐剂活性的可能活性部位。根据初步数据，我们推测ROV-ASP-1将能够刺激抗体和Th1/CD8+T细胞反应，与SARS冠状病毒和图拉氏丝虫的保护相关。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和/或特定能力的分子来引发特定的效应通路。

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.