Antiviral lectins as microbicides

描述（由申请人提供）：为了实际应用，杀菌剂必须大量可用，并且必须具有成本效益。这些要求可能会消除重组蛋白的考虑，因为在基于细胞的发酵系统中生产成本过高。我们开发了一种实用的解决方案，通过表达和纯化重组形式的藻凝集素Griffithsin (GRFT)，迄今为止描述的最有效的HIV-1进入抑制剂，来自烟叶组织。GRFT在脊椎动物中没有已知的同源物，因此有可能具有免疫原性，从而可能诱导炎症反应，与它作为杀微生物剂的使用不相容。这是一个适用于大多数（如果不是全部的话）基于肽的杀菌剂的理论问题，对这一现象进行更彻底的研究对该领域至关重要。我们将生成一个缺乏凝集素活性的GRFT突变体，以回答凝集素活性是否在诱导粘膜炎症反应中起作用的问题。我们将使用由我们的顾问和合作者Betsy Herold博士开发的综合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、炎症分子标记的测量和阴道组织中白细胞浸润的表型。由于蛋白质杀微生物剂的免疫原性是一个问题，我们也将测量动物对阴道杀微生物剂的免疫反应，我们有意提高了抗grft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）感染性试验，作为杀微生物剂安全性的替代测量。接受GRFT治疗的动物对HSV-2的易感性将告诉我们，对GRFT治疗的生物反应复合物是否会增加对HSV-2感染的易感性，从而可能增强人类对HIV-1感染的易感性。我们还将研究我们在选择实验中分离的突变HIV-1 Clade C病毒对GRFT的抗性机制。总的来说，我们在这项研究过程中产生的数据将对GRFT作为阴道杀微生物剂的组成部分预防HIV-1传播的效用进行全面评估。

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.