Plant-produced Actinohivin as a Candidate HIV Microbicide

植物产生的放线菌素作为候选 HIV 杀菌剂

• 批准号: 8685097
• 负责人: Nobuyuki Matoba
• 金额: $ 43.21万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-10 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8685097
• 关键词: Affinity; Animal Model; Antiviral Agents; Binding; Biochemical; Biological; Biological Assay; Cell Line; Cells; Clinical Research; Data Set; Dendritic Cells; Development; Dose; Drug Formulations; Drug Kinetics; Epidemic; Epithelial Cells; Evaluation; Gene Expression; Giant Cells; Goals; HIV; HIV-1; Human; Immune; In Vitro; Infection; Inflammatory; Inhibitory Concentration 50; Integration Host Factors; Lactobacillus; Lectin; Local Microbicides; Macaca; Mannose; Maximum Tolerated Dose; Methods; Modeling; Molecular; Mus; Oligosaccharides; Oryctolagus cuniculus; Peripheral Blood Mononuclear Cell; Pharmacodynamics; Pharmacologic Substance; Phase; Pilot Projects; Plant Viruses; Plants; Play; Polysaccharides; Preclinical Testing; Preparation; Procedures; Production; Proteins; Rage; Recombinant Fusion Proteins; Recombinants; Reporter Genes; Role; Safety; Seminal Plasma; Solutions; Specificity; Staging; System; T-Lymphocyte; Testing; Time; Toxic effect; Vagina; Viral; Virus; base; cervicovaginal; commensal microbes; cytotoxic; cytotoxicity; dimer; immunogenicity; improved; in vitro Assay; inhibitor/antagonist; irritation; meetings; microbicide; monomer; mouse model; novel; preclinical study; safety study; success; sugar; transmission process; vaginal microbicide

DESCRIPTION (provided by applicant): Safe, effective, and inexpensive topical microbicides are urgently needed to curb the global human immunodeficiency virus type-1 (HIV-1) epidemic. Actinohivin (AH) is an actinomycete-derived lectin. This lectin specifically binds to high-mannose clusters uniquely found on the HIV-1 envelope (Env), thereby eliciting nanomolar antiviral activity against multiple HIV strains. Preliminary analyses revealed that AH has a high safety profile in human peripheral blood mononuclear cells (PBMCs) and in the rabbit vaginal irritation assay. Meanwhile, a translational AH-AH fusion protein (recombinant dimer [rd] AH) was suggested to have stronger and broader anti-HIV-1 activity than the original monomer. Given these high potentials, we hypothesize that rAH and/or rdAH (r/rdAH) are excellent HIV-1 microbicide candidates. This project's goal is to reveal the feasibilities of r/rdAH in terms of manufacture, antiviral efficacy, and safety upon use as a vaginal microbicide. In the R21 phase, we will initially focus on developing a highly efficient, scalable production system for r/rdAH that allows for extensive efficacy and safety studies and possible global use. We will utilize recombinant plant virus-based expression systems and various molecular biological approaches for rapid and high-level expression of high-quality r/rdAH. Upon obtaining bulk r/rdAH active pharmaceutical ingredients with high purity standards, we will analyze HIV-1 neutralization effects against selected R5-type viruses in two in vitro HIV neutralization assays based on Env-pseudotyped virus-reporter gene expression and primary isolate- PBMC infection systems. Next, r/rdAH' cytotoxic, mitogenic, and inflammatory potentials will be tested in PBMCs and/or human cervicovaginal (CV) epithelial cell lines to establish the minimal safety profile. Our success criteria in the R21 phase are: (1) establishing the bulk preparation procedure; (2) demonstrating cross- clade antiviral effects to R5 viruses; and (3) demonstrating no apparent in vitro cytotoxicity, mitogenic activity, or inflammatory potential at >100 times above an average anti-HIV IC50, for plant-made r/rdAH. Upon approval of our transition to the R33 phase, we will comprehensively analyze anti-HIV-1 efficacy of r/rdAH for various modes of HIV-1 infection and transmission, using various in vitro assay systems. In addition, we will investigate potential overlap, complementation, synergy, and antagonism of anti-HIV activities between r/rdAH and other inhibitors toward potential microbicide combination strategies. Finally, we will perform extensive evaluations of r/rdAH upon vaginal application in rabbit and mouse models. We will thoroughly evaluate r/rdAH' vaginal toxicity, inflammatory potential, and stability. Upon determining the maximal tolerated dose of r/rdAH, we will examine their potential immunogenicity and toxicity after a long-term exposure. Potential toxicity to the symbiotic vaginal commensal bacteria, the Lactobacillus species, will be examined. In summary, the proposed studies should answer the question of whether r/rdAH is justified for advanced next-stage preclinical studies. . The proposed studies will analyze the feasibilities of the novel HIV-1-binding lectin Actinohivin and its derivative recombinant dimer, as a candidate vaginal HIV-1 microbicide. The proposed studies should generate a comprehensive data set that will reveal their large-scale producibility, anti-HIV-1 efficacy, and broad toxicity profile upon vaginal application, thereby providing criteria of whether Actinohivin and its derivative are justified for further extensive preclinical and clinical studies.

描述（由申请人提供）：迫切需要安全，有效和廉价的局部杀微生物剂来遏制全球人类免疫缺陷病毒1型（HIV-1）流行。放线菌素（Actinohivin，AH）是一种放线菌凝集素。这种凝集素特异性结合HIV-1包膜（Env）上独特的高甘露糖簇，从而引发针对多种HIV毒株的纳摩尔级抗病毒活性。初步分析显示，AH在人外周血单核细胞（PBMC）和家兔阴道刺激试验中具有较高的安全性。同时，一个翻译AH-AH融合蛋白（重组二聚体[rd] AH）被认为比原始单体具有更强和更广泛的抗HIV-1活性。鉴于这些高潜力，我们假设rAH和/或rdAH（r/rdAH）是优秀的HIV-1杀微生物剂候选物。该项目的目标是揭示r/rdAH作为阴道杀微生物剂在生产、抗病毒功效和安全性方面的特性。在R21阶段，我们将首先专注于开发一个高效、可扩展的r/rdAH生产系统，以便进行广泛的有效性和安全性研究，并可能在全球范围内使用。我们将利用重组植物病毒为基础的表达系统和各种分子生物学方法的快速和高水平的表达高品质的r/rdAH。在获得具有高纯度标准品的散装r/rdAH活性药物成分后，我们将在基于Env-假型病毒-报告基因表达和原代分离株- PBMC感染系统的两种体外HIV中和试验中分析HIV-1对选定R5型病毒的中和作用。接下来，将在PBMC和/或人宫颈阴道（CV）上皮细胞系中测试rrdAH的细胞毒性、促有丝分裂和炎性潜力，以建立最低安全性特征。我们在R21阶段的成功标准是：（1）建立批量制备程序;（2）证明对R5病毒的交叉进化枝抗病毒作用;和（3）证明在植物制备的r/rdAH的平均抗HIV IC 50以上>100倍时，没有明显的体外细胞毒性、促有丝分裂活性或炎症潜力。在批准我们过渡到R33阶段后，我们将使用各种体外试验系统全面分析r/rdAH对各种HIV-1感染和传播模式的抗HIV-1疗效。此外，我们还将研究r/rdAH和其他抑制剂之间抗HIV活性的潜在重叠、互补、协同和拮抗作用，以实现潜在的杀微生物剂组合策略。最后，我们将在兔和小鼠模型中对阴道应用r/rdAH进行广泛的评价。我们将彻底评估r/rdAH的阴道毒性、炎症潜力和稳定性。在确定r/rdAH的最大耐受剂量后，我们将检查其长期暴露后的潜在免疫原性和毒性。将检查对共生的阴道微囊细菌（乳杆菌属）的潜在毒性。总之，拟议的研究应该回答r/rdAH是否适合进行下一阶段的高级临床前研究的问题。.拟开展的研究将分析新型HIV-1结合凝集素Actinohivin及其衍生物重组二聚体作为HIV-1阴道杀微生物剂候选物的特性。拟议的研究应产生一个全面的数据集，将揭示其大规模生产，抗HIV-1的疗效，以及广泛的毒性特征后，阴道应用，从而提供标准是否放线菌素及其衍生物是合理的进一步广泛的临床前和临床研究。