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: 8035414
• 负责人: ABRAHAM PINTER
• 金额: $ 294.27万
• 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/8035414
• 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疫苗预防感染的失败是基于它们无法诱导广泛中和抗体（BNAB）的反应。现在很明显，针对针对最免疫原性结构域（例如V3，CD4-BD或CD4-I表位）的抗体具有有限的对典型临床分离株的活性，主要是由于有效的掩盖机制，而其他保守的靶标（例如B12，MPER，MPER，2G12）不是免疫原性的。在我们自己和其他实验室中的最新研究已经确定了先前未认可的一类保守的表位，这些表位具有有效的主要分离株中和活性，包括经典表位有效地掩盖的那些。这些抗体是针对依赖于天然三聚体env结构的季度中和表位（QNE）的，并且不保留在可溶性Env蛋白中。 QNE特异性抗体经常具有非常有效的中和活动，有些也具有很高的广度。例如，我们已经确定了一名急性感染的患者（CAP256），后者在超级感染后开发了针对针对QNE的亚型C分离株的广泛而有效的BNAB。尽管这些表位的分布很大，但这种抗体很少见，这表明这些四级表位通常不是很免疫原性。该计划的总体目标是学习如何可重复地诱导此类BNAB，最初是通过感染并最终通过疫苗接种。我们将在该队列中确定具有相似中和活性的其他患者，并确定CAP256和其他可能在诱导这些抗体（Morris/Williamson）中发挥作用的ENV变体和表位。这些表位的结构和序列决定因素将被定义，并将它们插入功能性SHIV（Pinter）中。然后将猴子感染这些湿因，并将监测所得的免疫反应以中和宽度和QNE特异性（HU）。还将探讨通过多个序列诱导此类BNAB的感染的要求。将通过从人类和产生BNABS（Robinson）的猴子及其表位特异性，广度和中和效力的中和猴子中的中和筛选来隔离mAb。这些研究将由行政（Pinter），病毒免疫学（Krachmarov）和分子生物学（DE Parseval）核心支持。 相关性：当前的HIV-1疫苗失败了，部分原因是它们无法诱导广泛中和抗体反应。最近的信息表明，针对基于天然第四纪环境结构（QNE）的广泛保守的表位的新型抗体的重要作用。该计划的总体目标是制定有效诱导这种广泛保护性抗体的策略。希望能够诱导此类活动的疫苗可以防止感染。 项目1： 标题：QNE的序列特异性和结构的表征 项目负责人：Pinter，一个 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).该项目的具体目的是：1-定义识别多种QNE的mAb和PAB的最小序列决定因素，包括可用的抗体和新试剂，这些试剂将在此程序中识别出来。将定义VIV2，V3和其他ENV的其他区域中的关键决定因素。 2-表征各种QNE的整体结构和生化特性。这些研究将解决QNE是否以三聚体依赖性方式在三聚体中或单个亚基中形成。将确定“掩盖” ENV构象的表达与QNE形成之间的潜在相关性。 3-表达各种QNE的可溶形式。人为稳定的GP140三聚体能够通过结合测定和中和吸附测定测试来测试由2909和PG09 MAB识别的模型QNE。通过引入亚基间二硫键（SOS构建体）对QNE表达的稳定稳定的影响，如果这些稳定的构建体表达QNE，则将生成可溶性的三聚体的可溶形式。还将尝试通过突变产生表达这些表位的单体分子，从而通过导致GP120结构进行重组，并通过将最小V2和V3决定因素引入人造支架中，从而使这些区域在各种置换下相互作用。 4-将保守的QNE表位引入传染性SHIV结构中。将测试标准病原体分子SHIV（1157IPD3）对CAP256血清中和的灵敏度和MABS PG09和PG16的敏感性。如果这些SHIV不表达表位，则使用AIM 1中获得的信息，将通过小片段交换和位点特异性诱变来引入它们。将测试所得的嵌合SHIVS，以便在猴子PBMC中复制的能力以及对QNE特异性抗体的中和敏感性。拥有这些活动的结构将被选中在项目3中的恒河猕猴中通过。 相关性：该项目将提供有关QNE的序列决定因素和结构的基本信息，并生成本hivrad项目2和3所需的试剂。这些产品将包括可溶性抗原，用于筛查相关抗体（项目2和4）以及免疫接种（项目3），嵌合和突变体环境，用于表位表征（项目2和4），以及向莫尼基（Monkeys）进行感染性和免疫性研究表达QNE的感染性SHIV（Project 3）。