Determination of the Neutralization Specificity in Broadly Neutralizing HIV Sera

罕见的HIV-1感染个体显示出显著水平的广泛中和抗体，其反映了中和模式（即宽度）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最好的靶点，重点是免疫原设计工作，以引起广泛中和反应。因为不知道Env的区域是显示宽度的血清的靶，所以这是指导免疫原设计的重要信息。与4种已知的广泛中和抗体一样，这些血清证明，在适当的免疫原性刺激下，有可能引发广泛中和的HIV抗体。使用多管齐下的方法，我们打算通过结合选择性蛋白-肽吸附、嵌合病毒和直接分离新型B细胞克隆来鉴定血清中的中和特异性。同时，我们将在小动物中分析候选的基于Env的免疫原，并将绘制引发的中和活性的特异性。 a）通过选择性蛋白质吸附绘制血清中的中和特异性 我们确定了一个子集的HIV-1患者显示广泛的中和活性在他们的血清中。我们正在积极进行血清中中和特异性的分析，以阐明自然感染过程是如何实现广度的。 我们已经证明，对于两种最有效的血清，V3和V1/2肽不影响血清的中和能力，而全长gp 120偶联到固相珠可以吸附大部分的HIV中和活性。我们将添加选定的点突变gp 120糖蛋白，以创建具有不同抗原特性的蛋白质组，从而选择性地去除中和活性。我们已经将BaL CD 4结合位点突变gp 120蛋白偶联到珠粒（368 D/R）上，并观察到对于两种最有效的血清，突变蛋白不会去除针对4种不同病毒株的中和活性。突变蛋白可以去除具有特异性的CD 4结合区远端的单克隆抗体的中和和结合活性。这些数据表明，这两种强效中和血清中的大部分广泛中和活性针对CD 4结合位点。我们将添加其他对照品，例如变性gp 120微珠和BSA偶联微珠，并将在一系列血清稀释液中进行中和/吸附。我们将从血清中去除或纯化IgG，以确认中和是抗体介导的。为了检测gp 41近膜定向中和（MPR），我们将生成MPR微蛋白珠和丙氨酸突变版本作为阴性对照。我们将继续通过Antonio Lanzavecchia开发的新方法通过EBV诱导的永生化分析患者B细胞库，并与Mark Connors合作用永生化B细胞的上清液直接中和假型病毒。我们计划与Dennis Burton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的HIV/SIV嵌合病毒，作为另一种手段来确认患者血清中的中和特异性。在未来，通过适当的验证，这些工具将非常有价值的疫苗引起的中和血清中的特异性映射。

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.