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

描述（由申请人提供）： 2G 12是一种罕见的广泛中和抗体，通过识别一簇碳水化合物与HIV包膜糖蛋白gp 120结合。该项目的目标是设计2G 12表位的良好结构模拟物，并测试其引发集中的2G 12样抗体反应的能力，该抗体反应可以中和和保护免受HIV感染。在具体目标1中，我们将从合理设计的角度来探讨这个问题。我们设计了一种可调的肽支架用于呈递碳水化合物，其可用于呈递具有系统变化的碳水化合物间距离的2-6种碳水化合物。我们将以这种方式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和表面等离子体共振比较它们对2G 12的结合亲和力。这些测量将指导我们的设计的改进。我们还将研究某些硬化交联剂对合成抗原结合2G 12的能力的影响。在具体目标2中，我们将从定向进化的角度来探讨这个问题。我们将使用“点击化学”将碳水化合物-叠氮化物连接到~1013种不同的非天然炔基肽的随机mRNA展示文库。然后，我们将从该糖肽文库中选择2G 12的最佳结合剂。因为文库中的每个肽都与其编码RNA连接，我们可以通过PCR扩增和/或多样化选择获胜者。翻译和点击化学糖基化将产生具有增加的2G 12结合物群体的第二代文库。用2G 12筛选和扩增/多样化的越来越严格的循环将产生2G 12的高亲和力糖肽结合剂，其应该是2G 12表位的良好模拟物。最好的模拟物将测序和制备规模的合成，为进一步的研究。生物物理测量将评估来自目的1和2的最佳2G 12表位模拟物与gp 120竞争结合2G 12的能力。在特定目标3中，我们将在两阶段兔研究中检测最佳Aim 1/Aim 2抗原的免疫原性。一项小型的初步研究将在小组中测试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好的2种抗原。在这两项研究中，将筛选动物血清对一系列HIV病毒株的中和活性。此外，将检测血清结合gp 120以及几种糖肽检测抗原的能力。无论是否产生中和抗体反应，这项研究都将揭示2G 12-gp 120相互作用。此外，Aim 2中开发的糖肽进化技术将在细胞信号传导、癌症生物学和病毒学等领域中具有许多其他应用，因此将在各个学科中产生广泛的影响。尽管研制抗HIV-1中和抗体疫苗的努力迄今为止收效甚微，但对艾滋病毒感染人群的研究使人们注意到几个广泛中和抗体的例子。该项目的重点是应用从研究这种抗体2G 12中获得的知识，设计通过模拟2G 12表位来激发中和抗体应答的疫苗。

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.