Antiviral lectins as microbicides

说明(由申请人提供)：对于实际应用，杀菌剂必须大量可用，并且必须以成本效益的方式生产。这些要求可能会使重组蛋白不再被考虑，因为基于细胞的发酵系统的生产成本高得令人望而却步。我们开发了一种实用的解决方案，通过从烟草叶组织中表达和纯化重组形式的藻凝集素Griffithsin(GRFT)，GRFT是迄今描述的最有效的HIV-1进入抑制剂。GRFT在脊椎动物中没有已知的同系物，因此具有免疫原性，因此可能诱导与其作为杀微生物剂使用不相容的炎症反应。这是一个理论上的问题，适用于大多数，如果不是所有基于多肽的杀微生物剂，这一领域至关重要的是，更彻底地研究这一现象。我们将产生一个缺乏凝集素活性的GRFT突变体，以使我们能够回答凝集素活性是否在诱导粘膜炎症反应中发挥作用的问题。我们将使用我们的顾问和合作者Betsy Herold博士开发的一个全面的小鼠阴道给药模型来评估羟乙基纤维素配方GRFT的粘膜毒性。毒理学终点将包括组织学、炎症分子标志物的测量以及阴道组织中白细胞的表型。由于蛋白质杀菌剂的免疫原性是一个令人担忧的问题，我们还将在故意引发抗GRFT免疫反应的动物中测量对阴道杀菌剂的免疫反应。我们将使用Herold博士优雅的小鼠生殖器单纯疱疹病毒(HSV)传染性测试，作为杀菌剂安全性的替代测量。GRFT治疗的动物对HSV-2攻击的敏感性将告诉我们，GRFT治疗的复杂生物反应是否可以增加对HSV-2感染的易感性，从而可能增加人类对HIV-1感染的易感性。我们还将研究我们在选择实验中分离到的HIV-1分支C病毒突变株对GRFT的抗性机制。总而言之，我们在这项研究过程中产生的数据将全面评估GRFT作为阴道杀微生物剂的一种成分的效用，以防止艾滋病毒-1传播。 与公共卫生的相关性：在缺乏有效的艾滋病毒疫苗的情况下，迫切需要其他战略来预防艾滋病毒的性传播，例如经阴道使用的杀菌凝胶。我们的目标是回答这样一个问题，一种名为凝集素的蛋白质，它结合了艾滋病病毒表面发现的糖结构，当给药到小鼠的阴道时，是否会产生炎症反应。这些研究将提供重要的信息，有助于确定凝集素是否具有可接受的毒性特征，以证明进一步开发为杀微生物剂是合理的。

DESCRIPTION (provided by applicant): For practical application, microbicides must be available in massive amounts, and must be produced cost- effectively. These requirements might eliminate recombinant proteins from consideration as costs of production in cell-based fermentation systems are prohibitively high. We developed a practical solution by expressing and purifying recombinant forms of the algal lectin Griffithsin (GRFT), the most potent HIV-1 entry inhibitor yet described, from tobacco leaf tissues. GRFT has no known homologs in vertebrates, and hence has the potential to be immunogenic and thus possibly to induce an inflammatory response incompatible with its use as a microbicide. This is a theoretical concern that applies to most, if not all, peptide-based microbicides, and it is of vital importance to the field that this phenomenon be investigated more thoroughly. We will generate a mutant of GRFT that lacks lectin activity to allow us to answer the question whether lectin activity plays a role in induction of mucosal inflammatory responses. We will use a comprehensive mouse vaginal administration model, developed by our consultant and collaborator Dr. Betsy Herold, to evaluate the mucosal toxicity of hydroxyethylcellulose-formulated GRFT. Toxicology endpoints will include histology, measurement of molecular markers of inflammation, and phenotypes of leukocyte infiltrate in vaginal tissues. Since immunogenicity of protein microbicides is a concern, we will also measure immune responses to the vaginal microbicide in animals in which we have intentionally raised an anti-GRFT immune response. We will use Dr. Herold's elegant mouse genital herpes simplex virus (HSV) infectivity assay that functions as a surrogate measurement of microbicide safety. Susceptibility to challenge with HSV-2 in animals treated with GRFT will inform us whether the complex of biological responses to treatment with GRFT can increase susceptibility to infection with HSV-2, and hence to possible enhanced susceptibility to HIV-1 infection in humans. We will also study the mechanism of resistance to GRFT in a mutant HIV-1 Clade C virus that we isolated in a selection experiment. Collectively, the data that we generate in the course of this research will provide a comprehensive assessment of the utility of GRFT as a component of a vaginal microbicide to prevent HIV-1 transmission. PUBLIC HEALTH RELEVANCE: In the absence of an effective vaccine against HIV, there is an urgent need for alternative strategies to prevent sexual transmission of HIV, such as vaginally-applied microbicidal gels. We aim to answer the question whether proteins called lectins, which bind the sugar structures found on the surface of the AIDS virus, generate an inflammatory response when administered in the vagina of mice. These studies will provide important information that will help determine whether lectins have acceptable toxicity profiles to justify further development as microbicides.