Identification of Antigens for Anti-HIV Broadly Neutralizing Responses

描述(由申请人提供)： 艾滋病毒疫苗学的一个难以捉摸但基本的目标仍然是鉴定一种能够引起广泛中和(BN)抗艾滋病毒免疫反应的免疫原。然而，一些具有BN活性的单抗(MAb)已被鉴定。其中一些BN单抗与gp41膜近端外区(MPER)的15-19个氨基酸(AAS)结合，但MPER多肽免疫不能引起BN反应，可能是因为MPER多肽在gp41膜附近表达时不能形成BN单抗识别的正确三级结构。我们假设我们可以诱导MPER呈现一种形状，如果它在另一种非免疫原性支架蛋白中表达，就会引起BN反应。然而，目前的技术还不能使我们能够专门设计一种能够将MPER驱动成所需形状的MPER-支架蛋白嵌合体。相反，我们建议通过使用GI粘膜免疫系统作为大规模平行的活体筛选设备来鉴定能够引发BN免疫反应的MPER-支架蛋白嵌合体。在这个筛选中，我们建议：1)建立一个DNA条码质粒库，其中包括能够将大量MPER支架蛋白放置在革兰氏阴性细菌表面的表面表达盒(包括以三聚体形式表达乘客蛋白的三聚体自转运体表达盒)，以便嵌合蛋白的三聚体、多聚体和其他聚集体与膜紧密结合。(在这个文库中，每个成员表达不同的变体MPER-支架蛋白嵌合体。)2)将文库喂给小鼠。3)使用PhyloChip微阵列技术(或高通量测序)通过条形码识别在小鼠粪便中显示相对丰度随着时间的推移而减少的文库成员，这将作为针对MPER-支架嵌合蛋白显示丰度降低的黏膜免疫反应的初步证据。我们还将确定是否诱导了抗MPER免疫反应。我们将重新筛选最初筛选呈阳性的克隆，然后分别测试这些克隆是否具有诱导BN抗HIV免疫反应的能力。因此，这一拟议的筛查提供了一种创新的、快速的、高通量的方法来识别可能引起BN抗HIV免疫反应的潜在免疫原。这种方法还有几个额外的优势。这项技术可以用来比较和评估任何其他潜在的免疫原引发粘膜免疫反应的能力，这将有助于许多疫苗开发工作，而且筛查中发现的任何免疫原都必然会诱导强烈的粘膜免疫反应，这对大多数疫苗，特别是艾滋病毒疫苗来说是一个有用的特征。在此筛选中发现的诱导BN抗HIV免疫反应的免疫原将是进一步开发诱导粘膜免疫的HIV疫苗的良好候选者。

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.