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: 8631034
• 负责人: Vincent Joseph Venditto
• 金额: $ 5.7万
• 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/8631034
• 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.

Gp41的模拟多肽是否改善了抗体-表位的相互作用？ 摘要 在F32的应用中，我假设针对gp41膜近端外区(MPR)的广泛的HIV中和单抗2F5和4E10是由于包膜蛋白的修饰而由MPR表位在一小部分供体中诱导的。我的想法是产生含有非天然氨基酸的MPR免疫原，可能会诱导更强的免疫反应。我相信，这些新的多肽将诱导与单抗2F5和4E10更强的亲和力，并有可能在初始加强免疫试验中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。由于mAb2F5和4E10表现出与磷脂的不同寻常的交叉反应，MPR免疫原设计的重点是激发与磷脂交叉反应的抗体反应。将化学修饰的氨基酸掺入已知的表位可能会诱导更强的免疫反应。 我将合成一组含有修饰残基的基于MPR的多肽序列，这些序列将用于使用Biacore分析来确定抗体的亲和力。结构活性关系将从这些数据中得出，并将指导后续几轮化学修饰的多肽找到与单抗最佳结合的序列。然后，我将使用初始增强免疫策略来确定每种多肽在兔体内的免疫原性。基于Biaccore分析中使用的多肽的脂多肽将以脂质体形式呈现，通过在脂质双层中呈现表位来进一步增强免疫原性。对兔进行皮下和肌肉免疫，从这些动物身上获得抗血清，并在验证的ELISA试验中进行评估，以确定每种多肽免疫原诱导的抗体效价。此外，含有相同脂肽的制剂将经鼻腔给药，以评估粘膜免疫反应。制剂将在存在和不存在由新生儿Fc受体(FcRN)结合和运输的多肽的情况下制备。SC/IM免疫动物的血清抗体反应将用ELISA法测定，IN免疫动物的粘膜灌洗液和血清样本将用来评估。比较SC/IM免疫动物和IN免疫动物抗血清效价的差异。高滴度的兔抗血清和黏膜灌洗液样品将用于传统的抑制试验，以确定HIV-1初级分离株在PBMC中的中和活性。 成功地在兔体内产生中和抗体将导致未来产生单克隆抗体的工作。在兔体内诱导中和抗体的化学修饰多肽可以为包括HIV在内的各种传染病疫苗的开发提供一种广泛适用的表位设计策略。