CD134-based fusion polypeptides as novel FIV immuno-therapeutics

描述（由申请人提供）：猫科免疫缺陷病毒（FIV）与灵长类慢病毒有许多共同的特征，因此被认为是研究艾滋病干预疗法的有价值的实验模型。与HIV-1一样，FIV的进入是一个多步骤的过程。FIV包膜（Env）必须依次与CD134和CXCR4结合，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。除了在病毒进入中起关键作用外，FIV Env也是抗体介导中和的主要靶点。然而，自然（或实验）诱导的针对FIV Env的抗体要么失败，要么弱中和病毒感染。我们最近对FIV中和机制的研究表明，针对FIV Env主要免疫优势结构域V3区诱导的单克隆抗体（MAb）是CD134诱导的（CD134i）抗体。它们阻断了Env-CXCR4的相互作用，但能微弱地中和感染。然而，在可溶性CD134的存在下，中和作用大大增强。我们假设CD134i表位仅在Env与CD134结合后、CXCR4相互作用前短暂暴露。动力学和/或位阻因素限制了抗体进入V3区，从而限制了它们的中和作用。提出的工作的中心目标是开发基于cd134的分子，能够使中和剂和阻断剂可以访问V3区域的决定因子。具体目标是：1。开发和测试含有CD134片段的体外嵌合蛋白，该嵌合蛋白通过柔性多肽连接物连接到由单链可变区（scFv）组成的第二个片段，该片段是我们最有效的抗v3单克隆抗体。概念是CD134片段与FIV Env结合将诱导抗v3 MAb表位的暴露；然后scFv片段结合，从而阻断Env-CXCR4的相互作用。2. 通过用CXCR4的细胞外环取代scFv片段来扩展目标1中提出的模型。虽然第二个ECL对FIV感染至关重要，但包括n端尾部在内的所有ECL都将被检测。

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.