HIV-1 acidic epitope discovery from broadly neutralizing seroantibodies

 说明(申请人提供)：背景/原理：少数感染HIV-1的人产生循环血浆抗体，可有效中和多种HIV-1分离株。这些抗体的特征和特异性可以指导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。我们的研究集中于直接从患者血浆中鉴定和鉴定广泛的中和抗体(没有潜在的选择偏见)。我们注意到了特定的生化特征，这些特征指向HIV包膜上的一个共同的主导酸性表位。 目的：这一建议的具体假设是gp120包膜上的一个共同的酸性表位与一个共同的免疫球蛋白基因家族结合，从而在血浆中引起广泛的HIV-1中和反应。该项目的具体目标是：1)直接从具有广泛中和能力的HIV-1感染者的血浆中分离负责广泛中和反应的抗体并对其进行测序；2)定义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对血浆中广泛的HIV-1中和反应负责的假设；3)绘制广泛中和抗体的相应表位(S)，以验证共享酸性表位的假设 Gp120的表达与血浆中广泛的HIV-1中和反应有关。 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(自由流动等电聚焦)方案可以直接将感兴趣的抗体从血浆缩小到单个物种，这将进行广泛的中和测试。确认后，将使用LC-MS重新对单个抗体条带进行测序。新单抗的表位定位将通过酶联免疫吸附试验、诱变研究和X射线结晶学进行。一旦确定了表位，将使用ELISA和诱变研究来检测其他7个个体的活性部分，以确定该表位是否对广泛的中和负责。我们还将确定20名患者中和抗体与非中和抗体的基因家族用途。预期的结果将是分离出新的单抗，并鉴定出可以指导广泛的HIV-1中和的共同酸性表位。我们已经完成了这里描述的技术(以及每个替代计划)的足够工作，以确保这些目标是可行的，并将完成。 影响：如果我们的假设被证明是正确的，那么结果将是新的方法，并直接适用于艾滋病毒疫苗的研究。这项研究将提供对人类广泛的艾滋病毒-1中和反应可能性的更深层次的理解，它还将确定一个自然产生的表位(S)，该表位可以成为针对艾滋病毒-1的有效交叉抗体以及相关基因家族的靶标。

 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.