Novel epitopes that mediate broad neutralization of clade B and C HIV-1 isolates

描述（由申请人提供）：由于无法诱导保护性体液反应，HIV-1疫苗的进展受到阻碍。特征明确的中和表位要么免疫原性较差（例如，b12、2F5、2G12），要么在大多数初级分离株（例如，针对V3、CD4-bd和CD4-i表位的抗体）上被有效掩盖。新的证据表明，即使是高度隐蔽的原代分离株也具有敏感的中和靶点，这些靶点可被自体患者血清识别，偶尔也可被异源血清识别。这表明，绘制所涉及的表位可能会识别出能够诱导广泛中和活性的新靶标。我们已经确定了几种对初级分离物具有广泛中和活性的患者血清，并且我们已经开发了定位目标表位的方法。我们现在建议鉴定介导自体和异源进化枝B和进化枝C Envs中和的表位。具体目的是：1-筛选从北美和非洲队列中获得的大量患者血清的交叉中和活性，并选择具有广泛中和特性的样本进行详细表征。2-通过检测中和敏感和抗性Envs之间形成的嵌合体的中和敏感性来初步定位目标表位，其中关键结构域使用可用的或工程的限制位点进行交换。通过交换更小的区域和关键残基的诱变，将对所涉及的表位进行更精细的定位。同时，在中和试验中，将使用gp120和gp41抗原和表达敏感Envs不同结构域的融合蛋白作为阻断和免疫吸附试剂，对敏感表位进行免疫化学定位。从这些患者和用表达潜在中和靶点的Env蛋白或融合蛋白免疫的小鼠身上获得的ebv转化B细胞培养物中分离出具有广泛中和活性的单克隆抗体（mab）。筛选将通过结合和直接中和试验进行。所得单克隆抗体将用于进一步确定所涉及的表位的结构和分布。最后，用表达新靶点的融合蛋白免疫家兔。这些研究将评估表达序列的免疫原性，并测试针对这些免疫原诱导的抗体的中和性。预计这些研究将确定暴露在典型的中和抗性初级分离株上并负责广泛中和的新靶点，并可能导致基于表达这些表位组合的免疫原的新疫苗方法。

DESCRIPTION (provided by applicant): Progress towards an HIV-1 vaccine has been stymied by the inability to induce a protective humoral response. Well-characterized neutralization epitopes are either poorly immunogenic (e.g., b12, 2F5, 2G12) or effectively masked on the majority of primary isolates (e.g., antibodies to V3, CD4-bd and CD4-i epitopes). New evidence suggests that even highly masked primary isolates possess sensitive neutralization targets that are recognized by autologous patient sera and occasionally by heterologous sera. This suggests that mapping the epitopes involved may identify novel targets that are capable of inducing broadly neutralizing activities. We have identified several patient sera that possess broadly neutralizing activities for primary isolates, and we have developed methods for localizing the target epitopes. We now propose to identify epitopes that mediate neutralization of both autologous and heterologous clade B and clade C Envs. The Specific Aims are: 1- To screen a large number of patient sera obtained from both North-American and African cohorts for cross-neutralizing activities, and select samples with broad neutralizing properties for detailed characterization. 2- To map target epitopes initially by examining the neutralization sensitivities of chimeras formed between neutralization-sensitive and -resistant Envs, in which key domains were exchanged using available or engineered restriction sites. Finer mapping of the epitopes involved will be performed by exchanging smaller regions and by mutagenesis of key residues. In parallel, sensitive epitopes will be mapped immunochemically using gp120 and gp41 antigens and fusion proteins expressing various domains of sensitive Envs as blocking and immunoadsorption reagents in neutralization assays. 3- To isolate monoclonal antibodies (mAbs) with broad neutralizing activities from EBV-transformed B cell cultures obtained from these patients, and from mice immunized with Env proteins or fusion proteins expressing potential neutralization targets. Screening will be performed both with binding and direct neutralization assays. The resulting mAbs will be used to further define the structure and distribution of the epitopes involved. 4- Finally, to immunize rabbits with fusion proteins expressing novel targets. These studies will evaluate the immunogenicity of the expressed sequences and to test the neutralizing properties of antibodies induced against these immunogens. It is anticipated that these studies will define novel targets that are exposed on typical neutralization-resistant primary isolates and responsible for broad neutralization, and could lead to new vaccine approaches based on immunogens expressing combinations of such epitopes.