Dityrosine Locked Prefusion F Protein: A Path To A Protective RSV Vaccine

二酪氨酸锁定预融合 F 蛋白：保护性 RSV 疫苗的途径

• 批准号: 8781549
• 负责人: Roberto Mariani
• 金额: $ 29.89万
• 依托单位: AVATAR MEDICAL, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781549
• 关键词: Accounting; Animals; Antibodies; Antibody Formation; Antigens; Binding; Cessation of life; Child; Chimeric Proteins; Complex; Cotton Rats; Development; Disulfides; Elderly; Epitopes; Formalin; Goals; HIV; Hospitalization; Immune response; Immunization; Individual; Infant; Infection; Length; Life; Malaria; Marketing; Medical; Modification; Molecular Conformation; Mus; Mutation; Oryctolagus cuniculus; Phase; Protein Engineering; Proteins; Recombinants; Research; Respiratory Syncytial Virus Vaccines; Safety; Serum; Side; Structure; Technology; Vaccinated; Vaccination; Vaccines; Variant; Virus; Work; base; covalent bond; crosslink; design; dityrosine; immunogenic; immunogenicity; improved; innovation; novel; protein complex; public health relevance; research clinical testing; vaccine development

DESCRIPTION (provided by applicant): The pre-fusion conformation of the RSV F protein (preF) is the primary determinant of neutralization in immunized sera. However, this conformation is unstable and rapidly transitions to the postfusion conformation. A disulfide stabilized prefusion variant with cavity filling mutations (DS-Cav1) has been shown to yield high neutralizing titers in animals; however, it only transiently maintains its native, prefusion conformation. Avatar Medical, LLC has developed a proprietary technology that locks immunogens in native conformations that better present broadly protective epitopes. Locking is accomplished by introducing targeted dityrosine (DT) crosslinks into the fully folded, native protein. We will apply this technology to lock the F protein fully in its pre- fusion conformation (DT-preF) in order to develop a stable, recombinant, preF-based RSV vaccine immunogen. Our minimally modifying, conformational locking technology enzymatically introduces zero-length covalent bonds into proteins and complexes, after the protein has fully folded. Bonds only form between Tyr side-chains in close structural proximity, and thus lock proteins in their native conformation, while preserving structural integrity. DT locking involves 2 steps: (i) expressing and purifying soluble F proteins with targeted, conservative to-Tyr substitutions, and (ii) enzymatically crosslinking the complex in its pre-fusion conformation. Our DT-locked preF immunogens will focus immune responses on potently neutralizing epitopes that are only displayed in the preF conformation, and away from non-or weakly neutralizing, postfusion epitopes. As a result, vaccination with DT-preF should elicit potent and lasting protection against RSV. In this Phase I application, we propose to design and characterize DT-preF variants, and confirm that they maintain the native prefusion conformation, using a panel of preF specific mAbs. We will then demonstrate that our DT-preF immunogen elicits higher neutralizing titers in mice, compared to DS-Cav1. In Phase II we will perform pre- clinical testing in cotton rat challenge studies for efficacy, and in rabbits for safety, with a view toward filing an IND with th FDA. To accomplish the Phase I goals, we will carry out the following Specific Aims: I. Design DT-preF variants in the WT-, disulfide-, and DS-Cav1-stabilized backgrounds, and characterize them antigenically and biochemically by comparison to uncrosslinked and DS-Cav1 controls. (Milestone: Selection of three DT-locked F proteins that retain binding to key preF-specific nAbs.) II. Perform immunogenicity studies in mice, and determine the neutralization titers induced by our DT-preF variants, compared to DS- Cav1, as well as postF, Formalin-inactivated-RSV and live RSV virus controls. (Milestone: Immunization with DT-preF elicits higher neutralization titers than DS-Cav1.)

描述（由申请人提供）：RSV F蛋白（PERF）的融合前构象是免疫血清中和的主要决定因素。但是，这种构象是不稳定的，并迅速过渡到后灌注构象。二硫化物稳定的预融合变体具有腔填充突变（DS-CAV1），已证明可以在动物中产生高中和滴度。但是，它只能瞬时保持其本地的预选构象。 Avatar Medical，LLC开发了一项专有技术，该技术将免疫原子锁定在本地构型中，更好地呈现了广泛的保护性表位。锁定是通过将​​靶向的脱乙糖（DT）交叉链接引入完全折叠的天然蛋白质中来完成的。我们将应用这项技术将F蛋白完全锁定在其预融合构象（DT-PREF）中，以开发出稳定的，重组的，基于PREF的RSV疫苗免疫原。在蛋白质完全折叠之后，我们的最小修饰，构象锁定技术酶促酶酶酶酶的零长度共价键在蛋白质和复合物中引入。仅在近距离结构近端的Tyr侧链之间形成键，从而在其天然构象中锁定蛋白质，同时保留结构完整性。 DT锁定涉及2个步骤：（i）用靶向，保守的TYR取代表达和净化可溶性F蛋白，以及（ii）酶上以其融合前构象的酶线交联。我们的DT锁定PEEF免疫原子会将免疫反应集中在仅在PERF构象中显示的有效中和表位，并远离非或弱中和的后灌注后表位。结果，DT-PREF的疫苗接种应对 RSV。在此I阶段应用中，我们建议设计和表征DT-PREF变体，并使用一组Pref特定的MAB来确认它们保持天然预融合构象。然后，我们将证明与DS-CAV1相比，我们的DT-PREF免疫原中和小鼠的中和滴度更高。在第二阶段，我们将在棉花大鼠挑战研究中进行疗效和兔子的安全性进行临床测试，以期向IND提交FDA。为了实现I阶段目标，我们将执行以下特定目标：I。设计DT-PREF变体，WT-，二硫化物和DS-CAV1稳定背景，并通过与未连接和DS-CAV1控件进行抗原和生物化学表征它们。 （里程碑：选择三种DT锁定F蛋白，它们保留与关键Pref特异性NAB的结合。）II。与DS-CAV1以及PostF，福尔马林灭活的RSV和Live RSV病毒控制相比，在小鼠中进行免疫原性研究，并确定由DT-PREF变体诱导的中和滴度。 （里程碑：使用DT-PREF免疫会引起比DS-CAV1更高的中和滴度。）