Determination of the Neutralization Specificity in Broadly Neutralizing HIV Sera

罕见的HIV-1感染者表现出大量的广谱中和抗体，反映了中和模式(即广度)和效力(即相对较高的滴度)。我们假设，广泛中和患者血清中存在的中和特异性的定义将告诉我们，免疫原设计努力集中在哪些最佳靶点上，以引起广泛的中和反应。由于目前尚不清楚Env的哪些区域是血清展示广度的靶点，这是指导免疫原设计的重要信息。与已知的4种广为人知的中和抗体一样，这类血清表明，如果给予适当的免疫原性刺激，就有可能产生广泛的中和艾滋病毒抗体。我们打算采用多管齐下的方法，通过选择性蛋白质-多肽吸附、嵌合病毒和直接分离新的B细胞克隆来鉴定血清中和的特异性。同时，我们将在小动物中分析候选的基于Env的免疫原，并绘制诱导的中和活性的特异性图。 A)通过选择性蛋白质吸附定位血清中和特异性 我们确定了HIV-1患者的一部分，他们的血清中显示出广泛的中和活性。我们正在积极进行血清中和特异性的分析，以阐明广度是如何通过自然感染过程实现的。我们已经证明，对于两个最有效的血清，V3和V1/2肽不影响血清的中和能力，而全长gp120偶联到固相小球上可以吸附大部分HIV中和活性。我们将添加精选的点突变gp120糖蛋白来创建具有不同抗原性的蛋白质小组，以选择性地去除中和活性。我们已经将Bal CD4结合位点突变的gp120蛋白偶联到珠子(368D/R)上，并观察到对于两种最有效的血清，突变的蛋白不能消除针对4种不同病毒株的中和活性。突变蛋白可以去除具有CD4结合区远端特异性的单克隆的中和和结合活性。这些数据表明，在这两个有效的中和血清中，大部分广泛的中和活性是针对CD4结合部位的。我们将增加额外的对照，例如变性的gp120小球和牛血清白蛋白偶联小球，并将在一系列血清稀释度上执行中和/吸附。我们将从血清中去除或纯化免疫球蛋白，以确认中和是抗体所致。为了检测gp41膜近端定向中和(MPR)，我们将产生MPR微小蛋白小球和丙氨酸突变版本作为阴性对照。我们将继续用Antonio Lanzavecchia开发的新方法分析EBV诱导永生化的患者B细胞谱系，并与Mark Connors合作，用永生化B细胞的培养上清直接中和伪型病毒。我们计划与丹尼斯·伯顿合作，从选定患者的骨髓中产生抗体噬菌体展示文库。我们将继续开发一组相互作用的HIV/SIV嵌合病毒，作为确认患者血清中和特异性的另一种手段。在未来，通过适当的验证，这些工具将非常有价值地映射疫苗诱导的中和血清中的特异性。

Rare HIV-1 infected individuals display a significant level of broadly neutralizing antibodies that reflect the pattern of neutralization (i.e. breadth) and potency (i.e. relatively high titer). We hypothesize that the definition of the neutralization specificities present in broadly neutralizing patient sera will tell us the best targets upon which to focus immunogen design efforts to elicit broadly neutralizing responses. Because it is not known the regions of Env that are the targets for sera displaying breadth, this is important information to guide immunogen design. Like the 4 known broadly neutralizing antibodies, such sera demonstrate that given the proper immunogenic stimulus, it is possible to elicit broadly neutralizing HIV antibodies. Using a multi prong approach we intend to identify the neutralization specificity in the sera by a combination of selective protein-peptide adsorptions, chimeric viruses and by the direct isolation of novel B cell clones. In parallel, we will analyze candidate Env-based immunogens in small animals and will map the specificity of the elicited neutralizing activity. a) Mapping the Neutralizing Specificity in Sera by Selective Protein Adsorption We identified a subset of HIV-1 patients that display broad neutralizing activity in their sera. We are actively pursuing the analysis of the neutralizing specificity in the sera to elucidate how breadth is accomplished by the natural infection process. We have demonstrated that for the two most potent sera, V3 and V1/2 peptides do not affect the neutralizing capacity of the sera, whereas full-length gp120 coupled to solid phase beads can adsorb most of the HIV neutralizing activity. We will add selected point mutant gp120 glycoproteins to create a protein panel with different antigenic properties to selectively remove neutralizing activity. We have already coupled BaL CD4 binding site point mutant gp120 proteins to the beads (368D/R) and have observed that for the two most potent sera, the mutant proteins do not remove the neutralizing activity directed against 4 diverse viral strains. The mutant proteins can remove the neutralizing and binding activity of monoclonals of with specificities distal to the CD4 binding region. These data indicate that much of the broad neutralizing activity in these two potent neutralizing sera is directed against the CD4 binding site. We will add additional controls, for example, a denatured gp120 bead and a BSA coupled bead , and will perform the neutralization/adsorptions over a range of serum dilutions. We will remove or purify IgG from the sera to confirm that the neutralization is antibody meditated. For detection of gp41 membrane-proximal-directed neutralization (MPR), we will generate MPR miniprotein beads and an alanine-mutated version as a negative control. We will continue the analysis of the patient B cell repertoire by EBV-induced immortalization by new methods developed by Antonio Lanzavecchia and perform direct neutralization of pseudo-typed virus with the supernatants from the immortalized B cells in collaboration with Mark Connors. We plan to collaborate with Dennis Burton to generate antibody phage display libraries from the bone marrow of selected patients. And we will continue to develop a panel of reciprocal HIV/SIV chimeric viruses as another means to confirm the neutralization specificity in the patient sera. In the future, with the appropriate validation, these tools will be extremely valuable to map the specificity in vaccine-elicited neutralizing sera.