HIV neutralizing antibodies induced by chemically modified MPR epitopes

描述（由申请人提供）：在该R21申请中，我们假设针对GP 41的膜近端外部区（MPR）的广泛HIV中和单克隆抗体2F 5和4 E10在供体中由已被翻译后修饰的MPR表位诱导。我们的高风险想法是产生MPR免疫原，其中包含化学修饰的氨基酸残基，这些氨基酸残基反映了病毒感染期间可能发生的翻译后修饰。这些修饰紧邻2F 5和4 E10识别的MPR表位。我们相信这些新的免疫原将增强现有表位的效力和/或“揭露”先前隐藏在免疫系统中的诱导中和抗血清的表位。 使用这种含有修饰残基的肽的想法是独特的。MPR免疫原设计集中于引发与磷脂交叉反应的抗体应答，因为广泛中和的mAb 2F 5和4 E10表现出与脂质的异常交叉反应性。这使得许多研究人员将其对病毒膜中的gp 41和磷脂的双重亲和力归因于其效力的来源。然而，这些抗体的“脂质反应性”主要针对带负电荷的脂质头部基团，这些基团在化学上与磷酸化或硝化的氨基酸相似。因此，氨基酸修饰，而不是脂质，可能是2F 5和4 E10不寻常的双重识别特性的真正原因。 我们将合成MPR肽序列含有硝化，磷酸化，或不可水解的电荷等价物，膦酸化或磺化残基，将它们连接到脂质锚，表征其二级结构，并测量其在脂质囊泡中的分配系数。脂肽将配制在免疫原性脂质体中并用于免疫小鼠和兔。将使用经验证的ELISA检测抗血清的抗体滴度，并在临床相关PBMC HIV感染试验中测量中和作用。为了了解病毒模拟三螺旋束中修饰的表位的呈递是否增加免疫原性和/或导致中和抗体的产生，将修饰的肽掺入在相同脂质锚上呈递三个表位的支架中，并如针对脂肽所述进行评价。为了确定修饰的表位的免疫原性是否需要脂质锚，我们将用聚乙二醇替换修饰的表位上的脂质，并重复体外表征和体内免疫研究。来自这些实验的结果将指导随后几轮的表位设计，以优化抗血清滴度和HIV中和活性。成功地从化学修饰的肽表位产生中和抗体将使表位-抗体复合物的结构研究成为可能，并导致在灵长类HIV攻击模型中的保护研究。这种不寻常的方法可以提供一个广泛适用的表位设计策略，用于开发预防感染性疾病（如HIV）的疫苗。 公共卫生相关性：我们将合成MPR免疫原，其包含化学修饰的氨基酸，这些氨基酸反映了HIV感染期间可能发生的翻译后修饰。我们将在预防性HIV疫苗中使用免疫原，在哺乳动物中诱导广泛的HIV中和抗体，这可能是预防艾滋病疫苗的前体。

DESCRIPTION (provided by applicant): In this R21 application we postulate that the broadly HIV neutralizing monoclonal antibodies 2F5 and 4E10, against the membrane proximal external region (MPR) of GP41 were induced in the donors by MPR epitopes that had been post-translational modified. Our high-risk idea is to generate MPR immunogens incorporating chemically-modified amino acid residues that reflect the post-translational modifications that can occur during viral infections. These modifications are immediately adjacent to the MPR epitopes recognized by 2F5 and 4E10. We believe these novel immunogens will enhance the potency of existing epitopes and/or 'unmask' epitopes previously hidden from the immune system that induce neutralizing antisera. The idea to use such peptides containing the modified residues is unique. MPR immunogen design has focused on eliciting antibody responses that cross-react with phospholipids because the broadly neutralizing mAbs 2F5 and 4E10 exhibit unusual cross-reactivity with lipids. This led many investigators to attribute their dual affinity for both gp41 and phospholipids in the viral membrane as the source of their potency. However, the "lipid reactivity" of these antibodies is primarily directed at negatively charged lipid head groups, which are chemically similar to phosphorylated or nitrated amino acids. Thus, amino acid modifications, not lipids, may be the true cause of the unusual dual recognition properties of 2F5 and 4E10. We will synthesize MPR peptide sequences containing nitrated, phosphorylated, or non-hydrolyzable charge equivalent, phosphonated or sulfonated residues, attach them to lipid anchors, characterize their secondary structure and measure their partition coefficient in lipid vesicles. The lipopeptides will be formulated in an immunogenic liposome and used to immunize mice and rabbits. Antisera will be tested for antibody titers using a validated ELISA and neutralization will be measured in a clinically relevant PBMC HIV infection assay. To learn if presentation of the modified epitopes in a viral-mimic triple helix bundle increases immunogenicity and/or results in the generation of neutralizing antibodies, the modified peptides will be incorporated into a scaffold that presents three epitopes on the same lipid anchor and evaluated as described for the lipopeptides. To determine if the immunogenicity of the modified epitopes requires a lipid anchor, we will replace the lipid on the modified epitopes with polyethylene glycol and repeat the in vitro characterization and in vivo immunization studies. Results from these experiments will guide subsequent rounds of epitope design to optimize antisera titers and HIV neutralization activity. Success in generating neutralizing antibodies from chemically-modified peptide epitopes would enable structural studies on the epitope-antibody complex and lead to protection studies in primate HIV challenge models. This unusual approach could provide a widely applicable epitope design strategy for development of vaccines to prevent infectious disease such as HIV. PUBLIC HEALTH RELEVANCE: We will synthesize MPR immunogens that incorporate chemically-modified amino acids which reflect the post- translational modifications that can occur during HIV infections. We will use the immunogens in a prophylactic HIV vaccine to induce broadly HIV neutralizing antibodies in mammals which could be a precursor to a vaccine to prevent AIDS.