Vaccines and Therapeutics for Anthrax

炭疽疫苗和治疗方法

• 批准号: 8156950
• 负责人: Stephen Leppla
• 金额: $ 150.63万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156950
• 关键词: Adenylate Cyclase; Alleles; Anabolism; Anthrax disease; Area; BAY 54-9085; Binding; Biochemical; Cancer Model; Chimeric Proteins; Clinical Management; Collaborations; Culture Media; Cyclic AMP; Cytosol; Data; Detection; Endothelial Cells; Engineering; Fibrin; Fibrinolysis; Gene Targeting; Genes; Growth; Growth Factor; Hospitals; Human; Infection; Inflammation; Laboratories; Leukocytes; Ligands; MAP Kinase Gene; MAPK14 gene; Measurement; Methods; Modification; Molecular Target; Mouse Strains; Mus; Mutate; Mutation; National Institute of Allergy and Infectious Disease; National Institute of Dental and Craniofacial Research; Organism; Pathway interactions; Physiological; Secondary to; Solid Neoplasm; Somatic Cell Genetics; Stromal Cells; Tumor-Derived; Variant; Virulence Factors; anthrax lethal factor; anthrax toxin receptors; antigen binding; bacterial resistance; base; improved; in vivo; killings; macrophage; prevent; therapeutic vaccine; tissue regeneration

Anthrax toxin, a major virulence factor of Bacillus anthracis, consists of the cellular binding moiety protective antigen (PA) and the enzymatic moieties lethal factor (LF) and edema factor (EF). To intoxicate host organisms, PA binds to its cellular receptors TEM8 and CMG2 and is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA heptamer, which in turn translocates LF and EF into the cytosol of cells. LF cleaves several MEKs, thereby inactivating the ERK, p38, and JUNK MAPK pathways. EF is an adenylate cyclase that generates abnormally high concentrations of cAMP. PA is a key virulence determinant of anthrax disease, and antibodies to PA protect against infection. Thus, PA is the focus of existing and new vaccines. Our group is continuing its long-term effort to improve methods for producing PA and PA-based vaccines. Avirulent strains of B. anthracis are being engineered to be sporulation deficient and to lack extracellular proteases. Proteins secreted from such strains are more easily purified with higher yields and with increased stability. During 2010, we evaluated additional B. anthracis strains having multiple extracellular proteases deleted. More details about these strains are provided in the report for project AI001030-03. In addition, we recognized the value of adding additional calcium ions to the growth medium. PA contains two tightly bound calcium ions, and it appears that adding calcium improves the folding of PA as it is secreted from the bacteria. The added calcium causes yields to increase 2-fold over previous levels, reaching nearly 100 mg per liter. Another approach to immunological defense against anthrax uses passive administration of monoclonal antibodies. We are continuing collaborative work with Dr. Purcell (LID, NIAID) on chimpanzee/human monoclonal antibodies (mAbs) to various anthrax antigens. In parallel work, we are developing new mouse mAbs against the anthrax toxin enzymatic moieties, EF and LF, to better understand immune responses to the toxins, and to identify mechanisms by which antibodies neutralize the toxins. Thus, during 2010 we prepared a new set of mAbs to EF and showed that they react with different epitopes and have several mechanisms of action. These mAbs were shown to be useful in detection and measurement of EF in the blood of infected animals. During 2010 we also arranged to obtain a set of incompletely characterized mAbs to LF. Full characterization of these mAbs is expected to identify reagents of value in diagnostics and in the characterization of toxin action. One area of work in this project seeks to use modified anthrax toxins to target cancer. We previously found that the toxicity of anthrax toxin can be redirected to cancers by changing PAs furin specificity to cancer-selective protease specificities. Therefore, we constructed uPA (urokinase type plasminogen activator) and MMP (matrix metalloproteinase) activated anthrax lethal toxins. uPA and MMPs are proteases that are overproduced by cancer tissues and required for invasive growth, serving as potential molecular targets for cancer treatments. We previously found that the MMP-activated LeTx has a potent anti-cancer activity due to its targeting of tumor vasculature. In 2010, collaborating with Dr. Arthur Frankel (Cancer Research Institute, Scott and White Memorial Hospital, Temple, Texas), we found that the MMP-activated LeTx is also very potent in treating human anaplastic thyroid carcinoma (ATC) in an orthotopic mouse model. We showed that the MMP-activated LeTx inhibits orthotopic ATC xenograft progression via reduced endothelial cell recruitment and subsequent tumor vascularization. This in turn translates to an improved long-term survival in mouse models that is comparable with that produced by the multikinase inhibitor sorafenib. The results also indicate that therapy with the MMP-activated LeTx is extremely effective against advanced cancers with well-established vascular networks. The results demonstrate that MMP-activated LeTx-mediated endothelial cell targeting is the primary in vivo antitumor mechanism of this novel toxin, suggesting its potential usefulness in the clinical management of many different types of solid tumors. In the year of 2010, we also developed a "humanized uPA" mouse strain. In this strain, termed Plau(GFDhu/GFDhu), we introduced four amino acid substitutions into the growth factor domain of uPA, resulting in a mutated uPA that can bind only to human uPAR but not to the endogenous mouse uPAR. In this strain, the interaction between endogenous uPA and uPAR is selectively abrogated, while other functions of both the uPA protease and its receptor are retained. Elucidation of the specific functions of the uPA-uPAR interaction in vivo has been difficult because uPA has important physiological functions that are independent of binding to uPAR and because uPAR engages multiple ligands. It was in part to resolve this problem that we, in collaboration with Dr. Thomas Bugge (NIDCR), generated this humanized uPA mouse strain using a gene targeting approach. Analysis of the Plau(GFDhu/GFDhu) mice revealed an unanticipated role of the uPA-uPAR interaction in suppressing inflammation secondary to fibrin deposition. In contrast, leukocyte recruitment and tissue regeneration were unaffected by the loss of uPA binding to uPAR. This study identifies a principal in vivo role of the uPA-uPAR interaction as being the promotion of the cell-associated fibrinolysis critical for suppression of fibrin accumulation and fibrin-associated inflammation. This mouse provides a valuable model for further exploration of the multifunctional uPAR receptor. In addition, this mouse will have value in evaluating the anti-cancer efficacy of uPA-activated anthrax toxins. We previously showed that the uPA-activated anthrax toxin has a potent anti-cancer efficacy in two syngeneic mouse cancer models. In these models uPA, which often is secreted by host-derived tumor stromal cells, binds to uPAR on cancer cells and activates the cell-associated uPA-activated anthrax toxin, resulting in killing of the cancer cells. However, because mouse uPA cannot bind human uPAR, this toxin is not expected to be active against human tumor xenografts in mouse models. The new mouse described here will allow such efficacy tests. This laboratory has for many years done mutagenic and somatic cell genetic analyses to identify host genes involved in the actions of bacterial protein toxins. We have previously used retroviral insertional mutagenesis to isolate anthrax toxin receptor-deficient CHO cell mutants and mutants defective in diphthamide modification on eukaryotic elongation factor-2 (eEF-2). Because diphthamide is the target of bacterial ADP-ribosylating toxins (such as Pseudomonas exotoxin A), the diphthamide-deficient CHO cell mutants are completely resistant to these bacterial toxins. In 2010, we completed the analysis of an additional CHO cell mutant obtained in the earlier selections. CHO PR3228 belongs to another class of CHO cell mutants that are resistant to the bacterial ADP-ribosylating toxins. We have now shown that these cells have a mutation in one allele of the eEF-2 gene. This mutation, Gly717Arg, is close to His715, the residue that is modified to become diphthamide. This Arg substitution prevents diphthamide biosynthesis at His715, rendering the mutated eEF-2 non-responsive to ADP-ribosylating toxins, while having no apparent effect on its function in protein synthesis. CHO PR328 cells are heterozygous, having one mutant eEF-2 allele which allows the cells to survive even in the presence of ADP-ribosylating toxins, indicating that 50% of active eEF-2 is sufficient to support long term cell survival.

炭疽毒素是炭疽芽孢杆菌的主要毒力因子，由细胞结合部分保护性抗原（PA）和酶促部分致死因子（LF）和水肿因子（EF）组成。为了吸入X型宿主生物，PA与其细胞受体TEM8和CMG2结合，并通过普遍表达的细胞表面脂肪蛋白蛋白酶蛋白水解激活，从而形成了活性PA含量，从而形成了活性PA含量，从而将LF转移到细胞的细胞质中。 LF切割了几种Mek，从而使ERK，p38和垃圾MAPK途径失活。 EF是一种腺苷酸环化酶，产生异常高浓度的cAMP。 PA是炭疽疾病的关键毒力决定因素，也是PA预防感染的抗体。因此，PA是现有疫苗和新疫苗的重点。我们的小组正在继续进行长期努力，以改善生产基于PA和PA的疫苗的方法。炭疽芽孢杆菌的无毒性菌株正在设计为孢子形成缺乏且缺乏细胞外蛋白酶。从这种菌株分泌的蛋白质更容易纯化，并具有较高的稳定性。在2010年期间，我们评估了具有多个细胞外蛋白酶的其他嗜血芽孢杆菌菌株。有关这些菌株的更多详细信息在项目AI001030-03的报告中提供。此外，我们认识到在生长培养基中添加其他钙离子的价值。 PA包含两个紧密结合的钙离子，似乎添加钙可以改善PA的折叠，因为它是从细菌中分泌的。增加的钙导致产量比以前的水平增加2倍，达到每升近100毫克。 针对炭疽的免疫防御的另一种方法使用了单克隆抗体的被动施用。我们将继续与Purcell博士（LID，NIAID）在黑猩猩/人类单克隆抗体（MABS）上与各种炭疽抗原进行合作。在同步工作中，我们正在开发针对炭疽毒素酶促的EF和LF的新小鼠mAb，以更好地理解对毒素的免疫反应，并鉴定抗体中和毒素的机制。因此，在2010年，我们为EF准备了一组新的单元单元，并表明它们与不同的表位反应，并具有多种作用机理。这些mAB被证明可用于在感染动物的血液中检测和测量EF。在2010年期间，我们还安排了一组未完全表征的mAb对LF。这些mAb的全面表征有望识别诊断和毒素作用表征中的价值试剂。 该项目的一个工作领域试图使用经过修改的炭疽毒素来靶向癌症。我们先前发现，通过将PAS Furin特异性转换为癌症选择性蛋白酶特异性，可以将炭疽毒素的毒性重定向到癌症。因此，我们构建了UPA（尿激酶型纤溶酶原激活剂）和MMP（基质金属蛋白酶）激活了炭疽致死毒素。 UPA和MMP是蛋白酶，由癌组织过多生产并侵入性生长所必需，作为癌症治疗的潜在分子靶标。我们先前发现，由于肿瘤脉管系统的靶向，MMP激活的LETX具有有效的抗癌活性。 2010年，我们与德克萨斯州坦普尔的Scott and White Memorial Hospital的Arthur Frankel博士合作，我们发现在原位小鼠模型中，MMP激活的LETX在治疗人体变性甲状腺癌（ATC）方面也非常有效。我们表明，通过减少内皮细胞募集和随后的肿瘤血管化，MMP激活的LETX抑制了原位ATC异种移植进展。这反过来又转化为在小鼠模型中的长期生存的改善，与多次激酶抑制剂索非尼产生的长期生存相当。结果还表明，用MMP激活的LETX治疗对具有良好的血管网络的高级癌症非常有效。结果表明，MMP激活的LETX介导的内皮细胞靶向是这种新型毒素的主要体内抗肿瘤机制，这表明其在许多不同类型的实体瘤的临床管理中的潜在有用性。 在2010年，我们还开发了一种“人性化的UPA”小鼠菌株。在这种称为PLAU（GFDHU/GFDHU）的菌株中，我们将四个氨基酸取代引入了UPA的生长因子结构域，从而导致突变的UPA可以仅与人UPAR结合，但不与内源性小鼠UPAR结合。在这种菌株中，内源性UPA和UPAR之间的相互作用被选择性地消除，而UPA蛋白酶及其受体的其他功能都被保留。很难阐明体内UPA-UPAR相互作用的特定功能，因为UPA具有重要的生理功能，这些功能独立于与UPAR结合，并且UPAR与多种配体接合。我们与Thomas Bugge博士（NIDCR）合作解决了这一问题，它使用基因靶向方法生成了这种人源化的UPA小鼠菌株。 PLAU（GFDHU/GFDHU）小鼠的分析表明，UPA-UPAR相互作用在抑制纤维蛋白沉积继发的炎症中的意外作用。相反，白细胞募集和组织再生不受UPA与UPAR结合的损失的影响。这项研究确定了UPA-UPAR相互作用的主要体内作用是促进与细胞相关的纤维蛋白溶解对抑制纤维蛋白积累和与纤维蛋白相关的炎症至关重要的。该小鼠为进一步探索多功能UPAR受体提供了宝贵的模型。 此外，该小鼠将具有评估UPA激活炭疽毒素的抗癌能力的价值。我们先前表明，在两个合成小鼠癌模型中，UPA激活的炭疽毒素具有有效的抗癌功效。在这些模型中，通常由宿主衍生的肿瘤基质细胞分泌的UPA与癌细胞上的UPAR结合并激活与细胞相关的UPA激活的炭疽毒素，从而杀死癌细胞。但是，由于小鼠UPA无法结合人UPAR，因此在小鼠模型中，这种毒素不会对人类肿瘤异种移植物具有活性。此处描述的新鼠标将允许这种功效测试。 该实验室多年来进行了诱变和体细胞细胞遗传分析，以鉴定与细菌蛋白毒素作用有关的宿主基因。我们以前已经使用逆转录病毒插入诱变来分离炭疽毒素受体缺陷型CHO细胞突变体和突变体在真核延伸因子2（EEF-2）上的双智酰胺修饰中有缺陷（EEF-2）。由于双乙酰胺是细菌ADP-核糖基毒素的靶标（例如假单胞菌Exotoxin A），因此缺乏双智胺的CHO细胞突变体对这些细菌毒素完全抗性。 在2010年，我们完成了对早期选择中获得的其他CHO细胞突变体的分析。 CHO PR3228属于对细菌ADP-核糖基化毒素具有抗性的另一种CHO细胞突变体。我们现在表明，这些细胞在EEF-2基因的一个等位基因中具有突变。该突变，Gly717arg，接近HIS715，该残留物被修饰为Diphthamide。这种ARG替换可阻止HIS715上的Diphthamide生物合成，从而使突变的EEF-2无反应性对ADP-核糖基化毒素，同时对其在蛋白质合成中的功能没有明显的影响。 CHO PR328细胞是杂合的，具有一个突变的EEF-2等位基因，即使存在ADP-核糖基毒素，也可以使细胞生存，这表明50％的活性EEF-2足以支持长期细胞存活。