Identification of Antigens for Anti-HIV Broadly Neutralizing Responses

描述（由申请人提供）： HIV疫苗学的一个难以捉摸但基本的目标仍然是鉴定能够引起广泛中和（BN）抗HIV免疫应答的免疫原。然而，已经鉴定了一些具有BN活性的单克隆抗体（Mab）。这些BN单抗中的一些结合gp 41的膜近端外部区域（MPER）中的15-19个氨基酸（aas），但是用MPER肽免疫不引起BN应答，可能是因为MPER肽当不在膜附近的gp 41的背景下表达时不形成BN单抗识别的正确三级结构。我们假设，如果MPER在另一种非免疫原性支架蛋白中表达，我们可以诱导MPER呈现出激发BN反应的形状。然而，目前的技术不能使我们能够专门设计一种MPER-支架蛋白嵌合体，可以驱动MPER进入所需的形状。我们建议，而不是确定一个MPER支架蛋白嵌合体能够引发BN免疫反应，通过使用胃肠道粘膜免疫系统作为一个大规模并行体内筛选装置。在该筛选中，我们提出：1）创建DNA条形码质粒的文库，其包括能够将大量MPR-支架蛋白置于革兰氏阴性细菌表面上的表面表达盒（包括将过客蛋白表达为三聚体的三聚体自转运蛋白表达盒），使得嵌合蛋白的三聚体、多聚体和其他聚集体与膜紧密结合形成。(In在该文库中，每个成员表达不同的变体MPR-支架蛋白嵌合体。2)把图书馆喂老鼠。3)使用PhyloChip微阵列技术（或高通量测序）通过条形码鉴定小鼠粪便中随时间推移相对丰度降低的文库成员，我们将其作为诱导针对丰度降低的MPR-支架嵌合蛋白的粘膜免疫应答的初步证据。我们还将确定是否诱导抗MPER免疫应答。我们将重新筛选最初筛选阳性的克隆，然后单独测试这些克隆引发BN抗HIV免疫应答的能力。因此，所提出的筛选提供了一种创新的、快速的、高通量的方法来鉴定可以引发BN抗HIV免疫应答的潜在免疫原。该方法还有几个额外的优点。该技术可用于比较和评估任何其他潜在的免疫原引发粘膜免疫应答的能力，这将有助于许多疫苗开发工作，并且在筛选中鉴定的任何免疫原都必然会诱导强烈的粘膜免疫应答，这是大多数疫苗，特别是HIV疫苗的有用特征。在该筛选中发现诱导BN抗HIV免疫应答的免疫原将是进一步开发成诱导粘膜免疫的HIV疫苗的良好候选物。

DESCRIPTION (provided by applicant): An elusive, but essential goal of HIV vaccinology remains the identification of an immunogen capable of eliciting a broadly neutralizing (BN) anti-HIV immune response. A few monoclonal antibodies (Mabs) with BN activity have been identified, however. Some of these BN Mabs bind to 15-19 amino acids (aas) in the membrane proximal external region (MPER) of gp41, but immunization with MPER peptides does not elicit a BN response, probably because the MPER peptides do not form the correct tertiary structure recognized by the BN Mabs when not expressed in the context of gp41 near a membrane. We hypothesize that we can induce MPER to assume a shape that elicits a BN response if it is expressed within another, non-immunogenic scaffolding protein. However, current technology does not enable us to specifically design an MPER- scaffolding protein chimera that can drive MPER into the required shape. We propose instead to identify an MPER-scaffolding protein chimera capable of eliciting a BN immune response by using the GI mucosal immune system as a massively parallel in vivo screening device. In this screen we propose to: 1) Create a library of DNA bar-coded plasmids that include surface expression cassettes capable of placing large amounts of MPER-scaffolding proteins on the surface of Gram-negative bacteria (including trimeric autotransporter expression cassettes that express passenger proteins as trimers) so that trimers, multimers, and other aggregates of the chimeric protein form in close association with a membrane. (In this library, each member expresses a different variant MPER-scaffolding protein chimera.) 2) Feed the library to mice. 3) Use PhyloChip microarray technologies (or high throughput sequencing) to identify, via the barcodes, the members of the library that show decreased relative abundance in mouse feces over time, which we would take as initial evidence of the induction of a mucosal immune response directed against MPER-scaffolding chimeric proteins showing decreased abundance. We will also determine whether an anti-MPER immune response is induced. We will rescreen clones that initially screen positive and then test those clones individually for their ability to elicit a BN anti-HIV immune response. This proposed screen therefore offers an innovative, rapid, high- throughput approach to the identification of potential immunogens that can elicit a BN anti-HIV immune response. The approach has several additional advantages. The technology could be used to compare and evaluate any other potential immunogens for their ability to elicit a mucosal immune response, which would be helpful for many vaccine development efforts, and any immunogen identified in the screen would necessarily induce a strong mucosal immune response, a helpful characteristic for most vaccines, and HIV vaccines in particular. Immunogens found to induce a BN anti-HIV immune response in this screen would be good candidates for further development into an HIV vaccine that induced mucosal immunity.