CD134-based fusion polypeptides as novel FIV immuno-therapeutics

描述(申请人提供)：猫免疫缺陷病毒(FIV)与灵长类慢病毒有许多相似之处，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，进入FIV是一个多步骤的过程。FIV包膜(Env)必须顺序地与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。除了在病毒进入中的关键作用外，FIV Env也是抗体介导的中和的主要靶点。然而，自然(或实验)产生的针对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主要免疫优势区V3区的单抗是CD134诱导的(CD134i)抗体。它们阻断了Env-CXCR4的相互作用，但弱中和了感染。然而，在可溶性CD134的存在下，中和作用大大增强。我们推测，CD134i表位仅在Env与CD134结合后但在CXCR4相互作用之前短暂暴露。然后，动力和/或空间因素限制了抗体对V3区域的访问，从而限制了它们在中和时的效力。这项拟议工作的中心目标是开发基于CD134的分子，使V3区域的决定簇能够被中和剂和封闭剂访问。其具体目的是：1.开发和测试包含CD134部分的嵌合蛋白，该部分通过灵活的多肽连接物连接到第二部分，该部分由我们最有效的抗V3单抗的单链可变区(ScFv)组成。其概念是CD134部分与FIV Env结合将导致抗V3单抗表位暴露；ScFv部分随后将结合，从而阻止Env-CXCR4相互作用。2.通过用CXCR4的胞外环取代scFv部分来扩展目标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末端尾巴在内的所有ECL都将进行测试。

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.