CD134-based fusion polypeptides as novel FIV immuno-therapeutics

描述（由申请人提供）：猫免疫缺陷病毒（FIV）与灵长类慢病毒具有许多共同特征，因此被认为是研究艾滋病干预治疗的有价值的实验模型。与HIV-1一样，FIV进入是一个多步骤的过程。FIV包膜（Env）必须依次接合CD 134和CXCR 4，触发Env的构象变化，最终导致病毒和宿主细胞膜之间的融合。除了其在病毒进入中的关键作用外，FIV Env也是抗体介导的中和的主要靶标。然而，天然（或实验）引发的针对FIV Env的抗体失败或微弱地中和病毒感染。我们最近对FIV中和机制的研究表明，针对FIV Env的主要免疫显性结构域V3区的单克隆抗体（MAb）是CD 134诱导的（CD 134 i）抗体。它们阻断Env-CXCR 4相互作用，但弱中和感染。然而，在可溶性CD 134存在下，中和作用大大增强。我们假设CD 134 i表位仅在Env结合CD 134之后但在CXCR 4相互作用之前短暂暴露。然后，动力学和/或空间因素限制抗体接近V3区，并因此限制其中和效力。拟议工作的中心目标是开发基于CD 134的分子，能够使V3区域上的决定簇可被中和剂和阻断剂所接近。具体目标是：1.开发和测试体外嵌合蛋白，其含有通过柔性多肽接头连接到由我们最有效的抗V3 MAb的单链可变区（scFv）组成的第二部分的CD 134部分。概念是CD 134部分与FIV Env的结合将诱导抗V3 MAb表位的暴露;然后scFv部分将结合，从而阻断Env-CXCR 4相互作用。2.通过用CXCR 4的细胞外环替换scFv部分来扩展目标1中提出的模型。尽管第二个ECL对于FIV感染至关重要，但将检测所有ECL，包括N-末端尾部。

DESCRIPTION (provided by applicant): Feline immunodeficiency virus (FIV) shares many features with the primate lentiviruses and is thus considered a valuable experimental model to study AIDS intervention therapies. As with HIV-1, FIV entry is a multistep process. FIV envelope (Env) must sequentially engage CD134 and CXCR4, triggering conformational changes in Env that ultimately lead to fusion between the viral and host cell membranes. Beside its critical role in viral entry, FIV Env is also the primary target for antibody-mediated neutralization. However, antibodies elicited naturally (or experimentally) against FIV Env either failed or weakly neutralize virus infection. Our recent studies on the mechanism of FIV neutralization indicate that monoclonal antibodies (MAb) elicited against the V3 region, the major immunodominant domain of FIV Env, are CD134 induced (CD134i) antibodies. They blocked Env-CXCR4 interaction, but weakly neutralized infection. However, neutralization was greatly enhanced in the presence of soluble CD134. We hypothezise that the CD134i epitopes are only transiently exposed after Env binds CD134 but before CXCR4 interaction. Kinetic and/or steric factors then restrict the access of the antibodies to the V3 region, and hence their efficacy at neutralization. The central goal of the proposed work is to develop CD134-based molecules capable to render determinants on the V3 region accessible to neutralizing and blocking agents. The specific aims are to: 1. develop and test in vitro chimeric proteins containing a CD134 moiety attached via a flexible polypeptide linker to a second moiety consisting of the single-chain variable region (scFv) of our most potent anti-V3 MAbs. The concept is that binding of the CD134 moiety to FIV Env will induce the exposure of the anti-V3 MAb epitope; the scFv moiety would then bind, thereby blocking Env-CXCR4 interaction. 2. extend the model proposed in aim 1 by replacing the scFv moiety by the extracellular loops of CXCR4. Although the second ECL is critical for FIV infection, all ECL including the N-terminal tail will be tested.