Thwarting HIV evasion of antibody avidity with novel antibody architectures

描述 摘要： 尽管经过了几十年的努力，目前还没有一种疫苗能在阻止艾滋病毒感染的浓度上产生中和抗体。除了HIV包膜尖峰的结构特征有助于抗体逃避外，我们认为HIV尖峰的低密度和有限的横向流动性阻碍了抗体的二价结合。由此产生的以单价为主的结合最小化了亲和力，从而使高亲和力结合和有效的中和，从而扩大了允许抗体逃避的艾滋病毒突变的范围。HIV尖峰三聚体的几何结构不允许自然产生的二价抗体在尖峰内交联，但我们可以构建能够高亲和力的三价结合到尖峰的蛋白质，用于基因治疗和/或被动免疫。我们将设计、表达和测试三聚体的尖峰内交联剂，这种试剂可以与每个尖峰单体的两个或三个非重叠的位点结合(每个三聚体6-9个位点)。选择HIV结合蛋白和如何连接它们将从15-30个HIV刺突结合蛋白与双链DNA识别标签连接的文库开始组合完成。这些将使用可变长度的DNA连接起来，形成相隔不同距离的双特异性试剂。混合的双特异性试剂将通过亲和层析分离，以分离最紧密的结合对，这些结合对将被PCR扩增/测序，以确定两个蛋白质组分和连接子长度。在确定连接体的最佳长度后，用长度相当的蛋白连接体替换DNA，并将双结合蛋白试剂连接到HIV结合文库中的第三结合蛋白。最佳的两组分或三组分的HIV结合蛋白将通过首先通过DNA连接物连接三聚基序来确定最佳长度，然后使用基于蛋白质的连接物来三聚化。这种三聚体试剂的尖峰内交联会降低灭菌免疫所需的浓度，使艾滋病毒的尖峰低

DESCRIPTION Abstract: Despite decades of effort, no current vaccine elicits neutralizing antibodies at concentrations blocking HIV infection. In addition to structural features of HIV's envelope spike that facilitate antibody evasion, we propose that the low density and limited lateral mobility of HIV spikes impedes bivalent binding by antibodies. The resulting predominantly monovalent binding minimizes avidity and thereby high affinity binding and potent neutralization, thus expanding the range of HIV mutations permitting antibody evasion. The HIV spike trimer geometry does not allow intra-spike cross-linking by naturally-occurring bivalent antibodies, but we can construct proteins capable of high-avidity trivalent binding to a spike for gene therapy and/or passive immunization. We will design, express, and test trimeric intra-spike cross-linking reagents that bind to two or three non-overlapping sites per spike monomer (6-9 sites per trimer). Choosing HIV-binding proteins and how to link them will be done combinatorially starting with a library of 15-30 HIV spike-binding proteins coupled to double-stranded DNA identifying tags. These will be linked using variable-length DNA to form bispecific reagents separated by different distances. Pooled bispecific reagents will separated by affinity chromatography to isolate the tightest binding pairs, which will be PCR amplified/sequenced to determine the two protein components and the linker length. Upon identifying the optimal length for the linker, the DNA is replaced with a comparable-length protein linker, and the two-binding-protein reagent is linked to a third binding protein from the HIV-binding library. Optimal two- or three-component HIV binding proteins will be trimerized by attaching a trimerization motif first via DNA linkers to determine the optimal length, then using protein-based linkers. The trimeric reagent's intra-spike cross-linking would reduce the concentration required for sterilizing immunity, making HIV's low spike