Do peptide mimetics of gp41 improve antibody-epitope interactions?

gp41 的肽模拟物是否可以改善抗体-表位相互作用？

• 批准号: 8624504
• 负责人: Vincent Joseph Venditto
• 金额: $ 5.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8624504
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affinity; Alkanesulfonates; Amino Acids; Animals; Antibodies; Antibody Affinity; Antibody Formation; Antigens; Aspartic Acid; Autoimmune Process; Binding; Biological Assay; Cells; Chemicals; Communicable Diseases; Cysteic Acid; Cysteine; Data; Development; Drug Formulations; Enzyme-Linked Immunosorbent Assay; Epidemic; Epitopes; Evaluation; Exhibits; Extravasation; Failure; Freund' s Adjuvant; Future; Glutamic Acid; HIV; HIV Infections; HIV vaccine; HIV-1; Immune Sera; Immune response; Immunity; Immunization; Infection; Inflammation; Infusion procedures; Irrigation; Lead; Lipid Bilayers; Lipids; Liposomes; Mediating; Membrane; Modeling; Modification; Monoclonal Antibodies; Mucosal Immune Responses; Mucosal Immunity; Mus; Mutation; Oryctolagus cuniculus; Patients; Peptides; Phospholipids; Phosphorylation; Population; Positioning Attribute; Post-Translational Protein Processing; Primates; Proteins; Reaction; Sampling; Serine/Threonine Phosphorylation; Serum; Site; Structure-Activity Relationship; Surface; Testing; Tyrosine; Vaccinated; Vaccines; Viral Load result; Virus; Walkers; Work; abstracting; analog; base; clinically relevant; cross reactivity; design; env Gene Products; immunogenic; immunogenicity; immunoreactivity; improved; in vitro Assay; inorganic phosphate; mimetics; monophosphoryl lipid A; neonatal Fc receptor; neutralizing antibody; neutralizing monoclonal antibodies; nitration; novel; phosphonate; polyclonal antibody; prevent; protein aminoacid sequence; receptor binding; success; vaccine development

Do peptide mimetics of gp41 improve antibody-epitope interactions? Abstract In this F32 application I postulate that the broadly HIV neutralizing monoclonal antibodies, 2F5 and 4E10, against the membrane proximal external region (MPR) of gp41 were induced in a small population of donors by MPR epitopes due to modifications in the envelope proteins. My idea is to generate MPR immunogens incorporating unnatural amino acids that may induce stronger immune responses. I believe that these novel peptides will induce stronger affinity toward monoclonal antibodies, 2F5 and 4E10, and potentially induce higher titer polyclonal antibodies in a prime-boost immunization assay. The idea to use unnatural amino acids is unique. MPR immunogen design has focused on eliciting antibody responses that cross-react with phospholipids because the mAbs 2F5 and 4E10 exhibit unusual cross-reactivity with phospholipids. Incorporation of chemically modified amino acids into known epitopes may induce stronger immune responses. I will synthesize a group of MPR-based peptide sequences containing modified residues, which will be used to determine antibody affinity using a BIAcore assay. Structure activity relationships will be derived from these data and will guide subsequent rounds of chemically modified peptides to find sequences that will bind optimally to the monoclonal antibodies. I will then determine the immunogenicity of each peptide in rabbits using a prime-boost immunization strategy. Lipopeptides based on the peptides used in the BIAcore assay will be presented in a liposomal formulation to further enhance immunogenicity by presenting the epitopes in a lipid bilayer. Rabbits will be immunized subcutaneously and intramuscularly and antisera from these animals will be obtained and evaluated in a validated ELISA assay to determine the antibody titer induced by each peptide immunogen. Furthermore, formulations containing the same lipopeptides will be delivered intranasally to evaluate the mucosal immune response. Formulations will be prepared in the presence and absence of a peptide that binds and is transported by the neonatal Fc receptor (FcRn). ELISA assays will be used to determine the antibody responses in serum from SC/IM immunized animals, while mucosal lavage and serum samples will be evaluated from IN immunized animals. Differences in antisera titers from SC/IM immunized animals will be compared to IN immunized animals. Antisera and mucosal lavage samples with high titers in rabbits will be used in traditional inhibition assays to determine the neutralization activity of primary HIV-1 isolates in PBMCs. Success in generating neutralizing antibodies in rabbits will lead to future work to generate monoclonal antibodies. The chemically modified peptides that induce neutralizing antibodies in rabbits could provide a widely applicable epitope design strategy for the development of vaccines to a variety of infectious diseases including HIV.

gp 41肽模拟物能改善抗体-表位相互作用吗？ 摘要 在该F32申请中，我假设针对gp 41的膜近端外部区域（MPR）的广泛HIV中和单克隆抗体2F 5和4 E10在少量供体中由由于包膜蛋白中的修饰而引起的MPR表位诱导。我的想法是产生MPR免疫原，其中包含可能诱导更强免疫反应的非天然氨基酸。我相信这些新的肽将诱导对单克隆抗体2F 5和4 E10的更强亲和力，并在初免-加强免疫测定中潜在地诱导更高滴度的多克隆抗体。使用非天然氨基酸的想法是独一无二的。MPR免疫原设计集中于引发与磷脂交叉反应的抗体应答，因为mAb 2F 5和4 E10表现出与磷脂的不寻常的交叉反应性。将化学修饰的氨基酸并入已知表位可以诱导更强的免疫应答。 我将合成一组含有修饰残基的基于MPR的肽序列，其将用于使用BIAcore测定法测定抗体亲和力。结构活性关系将从这些数据中推导出来，并将指导随后几轮的化学修饰肽，以找到与单克隆抗体最佳结合的序列。然后，我将使用初免-加强免疫策略确定每种肽在兔中的免疫原性。基于BIAcore测定中使用的肽的脂肽将以脂质体制剂形式呈递，以通过在脂质双层中呈递表位来进一步增强免疫原性。将对家兔进行皮下和肌内免疫，并获得这些动物的抗血清，并在经验证的ELISA试验中进行评价，以确定各肽免疫原诱导的抗体滴度。此外，将鼻内递送含有相同脂肽的制剂以评价粘膜免疫应答。将在存在和不存在结合新生儿Fc受体（FcRn）并由新生儿Fc受体（FcRn）转运的肽的情况下制备制剂。ELISA试验将用于测定SC/IM免疫动物血清中的抗体应答，而粘膜灌洗和血清样品将从IN免疫动物中进行评价。将比较SC/IM免疫动物与IN免疫动物的抗血清滴度差异。在传统的抑制试验中，将使用兔中高滴度的抗血清和粘膜灌洗液样本，以测定PBMC中HIV-1原代分离株的中和活性。 在兔子中成功产生中和抗体将导致未来产生单克隆抗体的工作。化学修饰的多肽在兔体内诱导产生中和抗体，为包括HIV在内的多种传染病疫苗的开发提供了一种广泛适用的表位设计策略。