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

合理组合设计免疫原以引发 2G12 样抗体

• 批准号: 8478035
• 负责人: Isaac Jonathan Krauss
• 金额: $ 39.79万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8478035
• 关键词: Acquired Immunodeficiency Syndrome; Adjuvant; Affinity; Amino Acids; Animals; Antibodies; Antibody Formation; Antigens; Asses; Azides; Binding; Cancer Biology; Carbohydrates; Cessation of life; Chemistry; Coupling; Discipline; Dose; Elements; Enzyme-Linked Immunosorbent Assay; Epitopes; Evaluation; Evolution; Generations; Glycopeptides; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; HIV-1; Immune response; Immunology; Individual; Infection; Knowledge; Libraries; Light; Measurement; Messenger RNA; Oligosaccharides; Oryctolagus cuniculus; Peptide Library; Peptides; Phase; Pilot Projects; Population; RNA; Research; Series; Serum; Signal Transduction; Structure; Surface Plasmon Resonance; Synthetic Antigens; Techniques; Testing; Translations; Vaccines; Viral; Virus; combinatorial; design; directed evolution; falls; follow-up; glycosylation; immunogenicity; meetings; member; neutralizing antibody; polypeptide; scaffold; screening; success; vaccine candidate; vaccine development; virology

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.

描述（由申请人提供）： 2G12 是一种罕见的广泛中和抗体，通过识别碳水化合物簇与 HIV 包膜糖蛋白 gp120 结合。该项目的目标是设计 2G12 表位的良好结构模拟物，并测试其引发集中的 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解决这个问题。我们设计了一种用于呈现碳水化合物的可调肽支架，可用于呈现具有系统变化的碳水化合物间距离的2-6种碳水化合物。我们将通过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 和表面等离子共振比较它们与 2G12 的结合亲和力。这些测量将指导我们设计的完善。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解决这个问题。我们将使用“点击化学”将碳水化合物叠氮化物附着到约 1013 种不同非天然炔基肽的随机 mRNA 展示库上。然后我们将从这个糖肽库中选择 2G12 的最佳结合剂。因为文库中的每个肽都与其编码 RNA 相连，所以我们可以通过 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更多 2G12 结合物数量的第二代文库。越来越严格的2G12筛选和扩增/多样化循环将产生2G12的高亲和力糖肽结合物，其应该是2G12表位的良好模拟物。最好的模拟物将以预备规模进行测序和合成，以供进一步研究。生物物理测量将评估目标 1 和 2 中最佳 2G12 表位模拟物与 gp120 竞争结合 2G12 的能力。在特定目标 3 中，我们将在两阶段兔子研究中测试最佳 Aim1/Aim2 抗原的免疫原性。一项小型试点研究将分小组测试 5 种抗原。后续研究将通过更大的群体、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，将筛选动物血清针对一系列 HIV 病毒株的中和活性。此外，还将测试血清结合 gp120 以及几种糖肽测试抗原的能力。无论是否引发中和抗体反应，这项研究都将揭示 2G12-gp120 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有许多其他应用，因此将在跨学科领域产生广泛的影响。尽管迄今为止，研制针对 HIV-1 的中和抗体疫苗的努力尚未取得什么成功，但对 HIV 感染人群的研究已经引起了人们的关注，一些广泛中和抗体的例子引起了人们的关注。该项目的重点是应用从此类抗体 2G12 研究中获得的知识来设计一种疫苗，通过模仿 2G12 表位来引发中和抗体反应。