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: 8509580
• 负责人: Nobuyuki Matoba
• 金额: $ 40.89万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8509580
• 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菌剂来遏制全球人类免疫缺陷病毒1型（HIV-1）的流行。放线菌素（AH）是一种来源于放线菌的凝集素。这种凝集素特异性结合在HIV-1包膜（Env）上独特发现的高甘露糖簇，从而激发针对多种HIV毒株的纳摩尔抗病毒活性。初步分析表明，AH在人外周血单个核细胞（PBMCs）和兔阴道刺激试验中具有很高的安全性。同时，一种翻译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。鉴于这些高电位，我们假设rAH和/或rdAH （r/rdAH）是极好的HIV-1杀菌剂候选者。该项目的目标是揭示r/rdAH在生产、抗病毒功效和作为阴道杀菌剂使用的安全性方面的可行性。在R21阶段，我们将首先专注于开发一种高效、可扩展的r/rdAH生产系统，允许进行广泛的疗效和安全性研究，并可能在全球范围内使用。我们将利用重组植物病毒表达系统和各种分子生物学方法快速、高质量地表达r/rdAH。在获得具有高纯度标准的散装r/rdAH活性药物成分后，我们将基于env假型病毒报告基因表达和原代分离物- PBMC感染系统，在两种体外HIV中和试验中分析对选定r5型病毒的HIV-1中和效果。接下来，将在pbmc和/或人宫颈阴道（CV）上皮细胞系中测试r/rdAH的细胞毒性、有丝分裂性和炎症潜力，以确定其最低安全性。我们在R21阶段的成功标准是：(1)建立原料药制备程序；(2)证明了对R5病毒的跨进化抗病毒作用；(3)对植物合成的r/rdAH没有明显的体外细胞毒性、有丝分裂活性或炎症潜力，其抗hiv IC50值为平均抗hiv IC50的100倍。一旦我们被批准进入R33期，我们将使用各种体外检测系统，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，我们将研究r/rdAH和其他抑制剂之间潜在的重叠、互补、协同和抗hiv活性的拮抗作用，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r/rdAH阴道应用进行广泛的评估。我们将全面评估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。对共生阴道共生细菌（乳酸菌种）的潜在毒性将进行检查。总之，拟议的研究应该回答r/rdAH是否有理由进行下一阶段的临床前研究。本研究将分析新型HIV-1结合凝集素放线素及其衍生物重组二聚体作为阴道HIV-1杀菌剂的可行性。拟议的研究应该产生一个全面的数据集，以揭示它们的大规模生产、抗hiv -1功效和阴道应用后的广泛毒性特征，从而为放线菌素及其衍生物是否有理由进行进一步广泛的临床前和临床研究提供标准。