HIV-envelope-specific DARPin-based microbicide strategies

描述(申请人提供)：HIV包膜特异性DARPin杀微生物剂策略为了成为可行的候选杀微生物剂，抗HIV策略必须是高效的，在相关的体外模型系统中阻断无细胞和细胞相关的感染，防止体内感染而不扰乱免疫功能或引起任何毒性反应，并且能够以低成本生产。通过最近建立的一项合作，我们确定了一种使用设计的Ankyrin重复蛋白(DARPin)技术来抑制HIV感染的新的、高度特异的方法。最初的概念验证研究证明，选择了高度特异和有效地防止多种艾滋病毒分离株以及纳摩尔水平的SIV体外感染的CD4特异性DARPins，而不干扰基本的细胞功能。DARPins具有独特的特性，表现出极高的稳定性和特异性，而且生产成本很低。DARPins将这些特点与预防艾滋病毒感染的能力相结合，是探索预防艾滋病毒传播的一种有前途的新战略。在这项新的合作研究的基础上，我们建议选择高活性的HIV Env特异性DARPins，这些DARPins将能够预防粘膜HIV感染。我们推测，识别gp120和gp41的环境特异性DARPins的鸡尾酒将(I)在体外有效地防止许多功能在低纳摩尔范围内的HIV和SIV分离株的无细胞和细胞相关感染，(Ii)不会损害固有或获得性免疫功能，(Iii)防止猕猴感染SHIV162P3，对当地免疫功能、菌群和pH没有不利影响。这项创新的抗艾滋病毒技术正在Trkola博士的实验室(与Plück thun博士的团队合作)开发，一旦通过严格的筛选测试被选中，有希望的候选人将使用Robbiani博士和Trkola博士的实验室建立的相关细胞和组织测试，在各种体外病毒和免疫系统中进行彻底测试。领先的候选新药(S)随后将通过罗比亚尼博士的实验室(与布兰查德博士合作)在猕猴身上进行体内活动测试。利用一种新的技术来开发抗艾滋病毒环境的特定战略，这种高风险的DARPins方法应该在预防艾滋病毒感染和传播方面取得关键进展。 项目摘要-相关性：这些研究将提供关于这种新型局部杀微生物剂在体内预防阴道感染的有效性的第一批数据。随着概念的证明，这是一个有希望的战略，以解决艾滋病毒在粘膜，我们将能够探索应用这种方法的性交独立的模式，以及潜在的应用，以防止直肠传播。这些数据将推动这项新技术的设计和未来的测试，以改进在女性和男性身上测试的杀微生物剂策略。

DESCRIPTION (provided by applicant): HIV-envelope-specific DARPin-based microbicide strategies In order to be a viable candidate microbicide, an anti-HIV strategy must be highly effective, block cell-free and cell-associated infections in relevant in vitro model systems, prevent in vivo infection without perturbing immune function or causing any toxic reactions, and be able to be produced at low costs. Through a recently established collaboration, we identified a novel, highly specific approach to inhibit HIV infection using the Designed Ankyrin Repeat Protein (DARPin) technology. The initial proof of concept studies documented the selection of CD4- specific DARPins that are highly specific and efficiently prevent in vitro infection with a wide range of HIV isolates as well as SIV at nanomolar levels, without interfering with basic cellular functions. DARPins possess unique characteristics exhibiting exceptional stability and specificity, and cost little to produce. Combining these features with the ability to prevent HIV infection, DARPins represent a promising novel strategy to explore for the prevention of HIV spread. Expanding on this new collaborative research, we propose to select highly active HIV Env-specific DARPins that will be able to prevent mucosal HIV infection. We hypothesize that cocktails of Env-specific DARPins, which recognize gp120 and gp41, will (i) efficiently prevent in vitro cell-free and cell-associated infection with numerous HIV and SIV isolates, functioning in the low nanomolar range, (ii) not impair innate or adaptive immune functions, and (iii) prevent mucosal infection with SHIV162P3 in macaques, with no adverse impact on local immune function, flora, and pH. This innovative anti-HIV technology is being developed in Dr. Trkola's laboratory (in collaboration with Dr. Pl¿ckthun's group) and, once selected through rigorous screening assays, promising candidates will be thoroughly tested in a variety of in vitro viral and immune systems using relevant cell and tissue-based assays established in both Drs. Robbiani's and Trkola's laboratories. The lead novel candidate(s) will then be tested for in vivo activity in macaques through Dr. Robbiani's laboratory (in collaboration with Dr. Blanchard). Utilizing a novel technology to develop an anti-HIV Env specific strategy, this high-risk DARPins approach should yield critical advances towards preventing HIV infection and spread. Project Summary-Relevance: These studies will provide the first data on the efficacy of this novel topical microbicide to prevent vaginal infection in vivo. With proof of concept that this represents a promising strategy to tackle HIV at the mucosa, we will be able to explore application of this approach to coitally independent modalities, as well as for the potential application against rectal transmission. These data will advance the design and future testing of this novel technology to improve microbicide strategies to test in women and men.