Strategies for Eliciting bnAbs against Conserved HIV-1 Quaternary Epitopes

引发针对保守的 HIV-1 四级表位的 bnAb 的策略

• 批准号: 7896926
• 负责人: ABRAHAM PINTER
• 金额: $ 278.53万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7896926
• 关键词: Strategies; Eliciting; bnAbs; against; Conserved

DESCRIPTION (provided by applicant): The failure of HIV-1 vaccines to protect against infection is based in part on their inability to induce a broadly neutralizing antibody (bnAb) response. It is now clear that antibodies directed against the most immunogenic domains (e.g., V3, CD4-bd or CD4-i epitopes) have limited activities against typical clinical isolates, primarily due to effective masking mechanisms, while other conserved targets (e.g., b12, MPER, 2G12) are not immunogenic. Recent studies in our own and other labs have identified the existence of a previously unrecognized class of conserved epitopes that possess potent neutralizing activities for primary isolates, including those in which the classical epitopes are effectively masked. These antibodies are directed against quaternary neutralization epitopes (QNEs) that are dependent on the maintenance of the native trimeric Env structure and are not retained in soluble Env proteins. QNE-specific antibodies frequently possess remarkably potent neutralizing activities, and some have great breadth as well. For example, we have identified an acutely infected patient (CAP256) who after superinfection developed broad and potent bnAbs for subtype C isolates that are directed against QNEs. Despite the broad distribution of these epitopes, antibodies of this sort are rare, suggesting that these quaternary epitopes are usually not very immunogenic. The overall goal of this program is to learn how to reproducibly induce such bnAbs, initially by infection and eventually by vaccination. We will identify additional patients in this cohort that possess similar neutralizing activities, and identify Env variants and epitopes in CAP256 and other patients that may have played a role in inducing these antibodies (Morris/Williamson). The structure and sequence determinants of these epitopes will be defined, and they will be inserted into functional SHIVs (Pinter). Monkeys will then be infected with these SHIVs and the resulting immune responses will be monitored for neutralization breadth and QNE specificity (Hu). The requirement of infection by multiple sequences for induction of such bnAbs will also be explored. MAbs will be isolated via neutralization screens from humans and monkeys producing bnAbs (Robinson) and their epitope specificities, breadth and neutralization potencies characterized. These studies will be supported by Administrative (Pinter), Viral Immunology (Krachmarov) and Molecular Biology (de Parseval) cores. RELEVANCE: Current HIV-1 vaccines have failed, in part due to their inability to induce a broadly neutralizing antibody response. Recent information suggests an important role for a new class of antibodies, directed against broadly conserved epitopes dependent on native quaternary Env structures (QNEs). The overall goal of this program is to develop a strategy for efficiently inducing such broadly protective antibodies. It is hoped that vaccines that are capable of inducing such activities will protect against infection. PROJECT 1: Title: Characterization of Sequence Specificities and Structures of QNEs Project Leader: PINTER, A PROJECT 1 DESCRIPTION (provided by applicant): Key goals of Project 1 will be to map the sequence determinants of known QNEs and novel QNEs that will be identified in studies in Project 2, to define the underlying structure of the Env protein required for QNE formation and expression, to generate these structures as soluble proteins, and to generate chimeric and mutant SHIVs that express QNEs for Infectivity and immunogenicity studies in monkeys (Project 4). The Specific Aims of this Project are: 1- To define minimal sequence determinants for mAbs and pAbs recognizing diverse QNEs, including available antibodies and new reagents that will be identified during this program. The critical determinants in VIV2, V3 and other regions of Env for expression of these epitopes will be defined. 2- To characterize the overall structure and biochemical properties of various QNEs. These studies will address whether QNEs are formed across adjacent protomers in the trimer or in individual subunits in a trimer-dependent manner. The potential correlation between expression of 'masked' Env conformations and formation of QNEs will be determined. 3- To express soluble forms of various QNEs. The ability of artificially stabilized gp140 trimers to express model QNEs recognized by 2909 and PG09 mAbs will be tested by binding assays and neutralization adsorption assays. The effect of stabilizing native trimeric Env structures by introducing inter-subunit disulfide bonds (SOS constructs) on QNE expression will be examined, and if these stabilized constructs express QNEs, soluble forms of the stabilized trimers will be generated. Attempts will also be made to generate monomeric molecules that express these epitopes by mutations that result in reorganization of gp120 structure and by introducing the minimal V2 and V3 determinants into an artificial scaffold that allows these regions to interact under various permutations. 4- To introduce conserved QNEs epitope into infectious SHIV constructs. The sensitivity of a standard pathogenic molecularly cloned SHIV, 1157ipd3, to neutralization by CAP256 serum and mAbs PG09 and PG16 will be tested. If these SHIVs do not express the epitopes, then they will be introduced by small fragment exchanges and site-specific mutagenesis, using information obtained in Aim 1. The resulting chimeric SHIVs will be tested for ability to replicate in monkey PBMCs and for neutralization sensitivity to QNE-specific antibodies. Constructs that possess these activities will be selected for passage in rhesus macaques in Project 3. RELEVANCE: This Project will provide essential information about the sequence determinants and structure of various types of QNEs, and also generate reagents needed by Projects 2 and 3 of this HIVRAD. The products will include soluble antigens that will be used for screening for related antibodies (Projects 2 and 4) and for immunization (Project 3), chimeric and mutant Envs to be used for epitope characterization (Projects 2 and 4), and infectious SHIVs that express QNEs for infectivity and immunogenicity studies in monkeys (Project 3).

描述（由申请人提供）：HIV-1疫苗不能预防感染的部分原因是它们不能诱导广泛中和抗体（bnAb）反应。现在很清楚，针对大多数免疫原性结构域（例如，V3， CD4-bd或CD4-i表位）的抗体对典型临床分离株的活性有限，主要是由于有效的掩蔽机制，而其他保守的靶点（例如，b12， MPER, 2G12）不具有免疫原性。我们和其他实验室最近的研究发现，存在一类以前未被识别的保守表位，它们对初级分离株具有有效的中和活性，包括那些经典表位被有效掩盖的表位。这些抗体针对四元中和表位（QNEs），这些表位依赖于天然三聚体Env结构的维持，并且不保留在可溶性Env蛋白中。qne特异性抗体通常具有非常强的中和活性，有些抗体具有很大的广度。例如，我们已经确定了一名急性感染患者（CAP256），他在重复感染后产生了针对QNEs的C亚型分离物的广泛而有效的bnab。尽管这些表位分布广泛，但这种类型的抗体很少见，这表明这些四级表位通常不具有很强的免疫原性。这个项目的总体目标是学习如何可重复地诱导这种bnAbs，最初是通过感染，最终是通过接种。我们将在该研究中鉴定具有类似中和活性的其他患者，并鉴定CAP256和其他可能在诱导这些抗体中发挥作用的患者的Env变异和表位（Morris/Williamson）。这些表位的结构和序列决定因素将被确定，它们将被插入功能性shiv中（Pinter）。然后将猴子感染这些shiv，并监测由此产生的免疫反应的中和广度和QNE特异性（Hu）。我们还将探讨多序列感染对诱导此类bnAbs的要求。单克隆抗体将通过中和筛选从产生单克隆抗体的人和猴子中分离出来（Robinson），并对其表位特异性、宽度和中和能力进行表征。这些研究将得到行政（Pinter），病毒免疫学（Krachmarov）和分子生物学（de Parseval）核心的支持。