Plant-produced Actinohivin as a Candidate HIV Microbicide

描述(由申请人提供)：安全、有效和廉价的局部杀微生物剂是遏制全球人类免疫缺陷病毒1型(HIV-1)流行的迫切需要。放线菌素(Actinohivin，AH)是一种来源于放线菌的凝集素。这种凝集素特异性地结合在HIV-1包膜(Env)上唯一发现的高甘露糖簇，从而引发针对多种HIV毒株的纳分子抗病毒活性。初步分析表明，在人外周血单个核细胞(PBMCs)和兔阴道刺激试验中，AH具有很高的安全性。同时，翻译的AH-AH融合蛋白(重组二聚体[RD]AH)比原始单体具有更强、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杀微生物剂候选者。该项目的目标是揭示r/rdah作为阴道杀菌剂在制造、抗病毒效果和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，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。我们将利用基于植物病毒的重组表达系统和各种分子生物学方法快速、高效地表达高质量的r/rdAH。在获得高纯度标准的原装r/rdAH活性成分后，我们将基于伪型病毒报告基因表达和原代分离株-PBMC感染系统，在两种体外HIV中和试验中分析HIV-1对选定的R5型病毒的中和作用。下一步，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、有丝分裂和炎症潜能，以建立最低安全性。我们在R21阶段的成功标准是：(1)建立批量制备程序；(2)表现出对R5病毒的交叉抗病毒作用；(3)对于植物制成的r/rdAH，在体外没有表现出明显的细胞毒性、有丝分裂活性或炎症潜力，高于平均抗HIV IC50的100倍。在我们被批准过渡到R33阶段后，我们将使用各种体外检测系统，综合分析r/rdAH针对各种HIV-1感染和传播方式的抗HIV-1效果。此外，我们将研究r/rdAH和其他抑制剂之间潜在的重叠、互补、协同和拮抗抗HIV活性，以寻找潜在的杀微生物剂组合策略。最后，我们将对r/rdAH在兔和小鼠模型上的阴道应用进行广泛的评估。我们将彻底评估r/rdah的阴道毒性、炎症潜在性和稳定性。在确定r/rdAH的最大耐受量后，我们将检测其长期暴露后的潜在免疫原性和毒性。对共生的阴道共生细菌，乳杆菌物种的潜在毒性将被检测。总之，拟议的研究应该回答了r/rdAH是否适用于下一阶段的高级临床前研究的问题。。这些研究将分析新型HIV-1结合凝集素放线菌素及其衍生的重组二聚体作为候选阴道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，揭示它们的大规模可生产性、抗HIV-1有效性和阴道应用的广泛毒性，从而为进一步广泛的临床前和临床研究提供是否合理的标准。

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.