HIV-1 acidic epitope discovery from broadly neutralizing seroantibodies

 描述（由申请方提供）：背景/原理：有限数量的HIV-1感染者产生循环血浆抗体，可有效中和多种HIV-1分离株。这些抗体的特性和特异性可以指导HIV-1候选疫苗的开发。目前研究这些抗体的方法包括从记忆B细胞中分离抗体，这可能并不总是反映循环抗体。我们的研究主要集中在直接从患者血浆中鉴定和表征广谱中和抗体（无潜在的选择偏倚）。我们已经注意到特定的生物化学特征，这些特征指向一个共同的显性酸性表位，该表位靶向HIV的包膜。 目的：该建议的具体假设是，gp 120包膜上的共享酸性表位结合共享免疫球蛋白基因家族，从而引起血浆中广泛的HIV-1中和反应。该项目的具体目标是：1）从具有广泛中和作用的HIV-1感染者的血浆中直接分离负责广泛中和作用的抗体并对其进行测序，2）确定中和抗体与非中和抗体的基因家族用途，以检验共同的免疫球蛋白基因家族负责血浆中广泛的HIV-1中和反应的假设，3）绘制广谱中和抗体的相应表位，以检验共享酸性表位 gp 120的表达负责血浆中广泛的HIV-1中和反应。 方法：我们确定了10例广泛中和的患者，其中3例将进行详细研究。亲和纯化（抗原、亚类和轻链特异性）和分级分离（自由流等电聚焦）方案可直接将血浆中的目标抗体缩小至单个种属，并对其进行广泛中和试验。确认后，将使用LC-MS对单个抗体条带进行从头测序。将使用Elisa、诱变研究和X射线晶体学对新mAb进行表位作图。一旦鉴定了表位，将使用Elisa和诱变研究来测试其他7个个体的活性部分，以确定该表位是否负责广泛中和。我们还将确定20例患者中中和抗体与非中和抗体的基因家族使用情况。预期的结果将是分离新的单克隆抗体和鉴定一个共同的酸性表位，可以直接广泛的HIV-1中和。我们已经完成了本文所述技术（以及每个替代计划）的足够工作，以确保目标是可行的，并将完成。 影响：如果我们的假设被证明是正确的，那么其结果在方法上是新颖的，并且直接适用于HIV疫苗的研究。这项研究将提供对人类广泛的HIV-1中和反应的可能性的更深入的理解，并且它还将确定一个天然存在的表位，该表位可以成为针对HIV-1的有效交叉进化枝抗体的靶标以及负责的基因家族。

 DESCRIPTION (provided by applicant): Background/Rationale: A limited number of persons infected with HIV-1 develop circulating plasma antibodies that potently neutralize a wide variety of HIV-1 isolates. The characteristics and specificities of such antibodies can guide the development of HIV-1 vaccine candidates. Current methods for the study of these antibodies include isolation of antibodies from memory B cells, which may not always reflect the circulating antibodies. Our research has focused on the identification and characterization of broad neutralizing antibodies directly from patient plasma (without potential bias of selection). We have noted particular biochemical signatures that point to a common dominant, acidic epitope that is targeted on the envelope of HIV. Objectives: The specific hypothesis of this proposal is that a shared acidic epitope on the gp120 envelope binds a shared immunoglobulin gene family giving rise to the broad HIV-1 neutralizing response in plasma. The specific aims of the project are to 1) Directly isolate and sequence the antibodies responsible for the broad neutralization from the plasma of HIV-1 infected individuals with broad neutralization, 2) Define the gene family usage of neutralizing versus non-neutralizing antibodies to test the hypothesis that a shared immunoglobulin gene family is responsible for the broad HIV-1 neutralization response in plasma, 3) Map the corresponding epitope(s) of the broad neutralizing antibodies to test the hypothesis that a shared acidic epitope of gp120 is responsible for the broad HIV-1 neutralization response in plasma. Methods: We have identified 10 patients with broad neutralization, of which 3 will be studied in detail. The affinity purification (antigen, subclass, and light chain specific) and fractionation (free-flow isoelectric focusing) scheme can narrow the antibodies of interest, directly, from the plasma to individual species, which will be tested for broad neutralization. Upon confirmation, the individual antibody bands will be sequenced de novo using LC-MS. Epitope mapping of the new mAbs will be undertaken with Elisa, mutagenesis studies, and X-ray crystallography. Once the epitope is identified, Elisa and mutagenesis studies will be used to test the active fraction o the other 7 individuals to determine if this epitope is responsible for broad neutralization. We wil also define the gene family usage of neutralizing versus non-neutralizing antibodies in 20 patients. Anticipated results will be isolation of new mAbs and the identification of a common acidic epitope that can direct broad HIV-1 neutralization. We have completed enough work on the techniques described herein (as well as each alternative plan) to ensure that the aims are feasible and will be completed. Impact: If our hypothesis proves correct, then the results will be novel in its method, and directl applicable to the study of HIV vaccines. This study will provide a deeper understanding of possibilities of the broad HIV-1 neutralizing response in humans, and it will also have identified a naturally occurring epitope(s) that can be the target of potent cross-clade antibodies against HIV-1 as well as the gene family(ies) responsible.