Rational and Combinatorial Design of Immunogens to Elicit 2G12-like Antibodies

描述(由申请人提供)： 2G12是一种罕见的广谱中和抗体，它通过识别一簇碳水化合物与HIV包膜糖蛋白gp120结合。该项目的目标是设计一种良好的2G12表位的结构模拟，并测试其激发集中的2G12样抗体反应的能力，从而中和和预防HIV感染。在具体目标1中，我们将从理性设计的角度来处理这个问题。我们设计了一种用于呈现碳水化合物的可调多肽支架，可以用来呈现2-6种碳水化合物，其碳水化合物之间的距离可以系统地变化。我们将用这种方法合成一系列可能的二价、三价和四价碳水化合物簇，并通过ELISA法和表面等离子共振法比较它们与2G12的结合亲和力。这些测量将指导我们设计的改进。我们还将研究某些刚性交联剂对合成抗原结合2G12能力的影响。在具体目标2中，我们将从定向进化的角度来处理这个问题。我们将使用“点击化学”将碳水化合物-叠氮化合物连接到一个随机的mRNA展示文库，该文库包含~1013种不同的非天然炔基肽。然后我们将从这个糖肽文库中挑选出最好的2G12结合子。因为文库中的每个肽都与其编码的RNA相连，所以我们可以通过PCR扩增和/或使选择的获胜者多样化。翻译和点击化学糖基化将产生第二代文库，具有更多的2G12结合子。日益严格的2G12筛选和扩增/多样化循环将产生高亲和力的2G12糖肽结合，这应该是对2G12表位的良好模拟。最好的模拟物将在制备规模上进行测序和合成，以供进一步研究。生物物理测量将评估来自AIMS 1和2的最好的2G12表位模拟体与gp120竞争结合2G12的能力。在具体目标3中，我们将在两个阶段的兔研究中测试最佳Aim1/AIM2抗原的免疫原性。一项小规模的先导性研究将在小范围内测试5种抗原。后续研究将用更大的组、不同的剂量和不同的佐剂重新检查最佳的两种抗原。在这两项研究中，将对动物血清进行筛选，以确定其对一系列艾滋病毒病毒株的中和活性。此外，还将测试血清与gp120以及几种糖肽测试抗原的结合能力。无论是否引发中和抗体反应，这项研究都将揭示2G12-gp120的相互作用。此外，在AIM 2中开发的糖肽进化技术将在细胞信号、癌症生物学和病毒学等领域有许多其他应用，因此将产生跨学科的广泛影响。尽管到目前为止，针对HIV-1的中和抗体疫苗的努力收效甚微，但对艾滋病毒感染人群的研究已经将几个广泛中和抗体的例子带到了聚光灯下。这个项目的重点是应用从这种抗体2G12的研究中获得的知识来设计一种疫苗，通过模仿2G12表位来引起中和抗体反应。

DESCRIPTION (provided by applicant): 2G12 is a rare broadly-neutralizing antibody which binds to the HIV envelope glycoprotein gp120 by recognizing a cluster of carbohydrates. The goal of this project is to design a good structural mimic of the 2G12 epitope and test its ability to elicit a focused 2G12-like antibody response that can neutralize and protect against HIV infection. In Specific Aim 1, we will approach this problem from the standpoint of rational design. We have designed a tunable peptide scaffold for presentation of carbohydrates which can be used to present 2-6 carbohydrates with systematically varied inter-carbohydrate distances. We will synthesize a range of possible di-, tri- and tetravalent carbohydrate clusters this way, and compare their binding affinities for 2G12 by ELISA and surface plasmon resonance. These measurements will guide the refinement of our design. We will also study the effect of certain rigidifying cross-linkers on the synthetic antigen's ability to bind 2G12. In Specific Aim 2, we will approach the problem from the standpoint of directed evolution. We will use "click chemistry" to attach carbohydrate-azides to a random mRNA display library of ~1013 different unnatural alkynyl peptides. We will then select from this glycopeptide library the best binders to 2G12. Because every peptide in the library is attached to its encoding RNA, we can amplify and/or diversify the selection winners by PCR. Translation and click-chemistry glycosylation will yield a second-generation library with an increased population of 2G12 binders. Increasingly stringent cycles of screening with 2G12 and amplification/diversification will give rise to high affinity glycopeptide binders of 2G12, which should be good mimics of the 2G12 epitope. The best mimics will be sequenced and synthesized on preparative scale for further studies. Biophysical measurements will asses the ability of the best 2G12 epitope mimics from Aims 1 and 2 to compete with gp120 for binding to 2G12. In Specific Aim 3, we will test the immunogenicity of the best Aim1/Aim2 antigens in a two-phase rabbit study. A small pilot study will test 5 antigens in small groups. A follow-up study will reexamine the best 2 antigens with larger groups, varying doses, and varying adjuvants. In both studies, animal sera will be screened for neutralization activity against a range of HIV viral strains. Additionally, sera will be tested for the ability to bind gp120, as well as several glycopeptide test antigens. Whether or not a neutralizing antibody response is raised, this research will shed light on the 2G12-gp120 interaction. Moreover, the glycopeptide evolution technique developed in Aim 2 will have numerous other applications in fields such as cell signaling, cancer biology, and virology, and so will have wide- ranging impact across disciplines. Although the effort to create a neutralizing antibody vaccine against HIV-1 has so far met with little success, studies of the HIV-infected population have brought into the spotlight several examples of broadly neutralizing antibodies. The focus of this project is to apply knowledge gained from the study of one such antibody, 2G12, to design a vaccine which elicits a neutralizing antibody response by mimicking the 2G12 epitope.