Structure and Immunogenicity of Cleaved, Stabilized HIV-1 Envelope Trimers

描述(由申请人提供)：这一HIVRAD计划融合了领先的HIV-1研究人员的互补专业知识，以提供比引发广泛中和抗体(NAB)更好的HIV-1疫苗的根本性进展。这一创新项目包括两个研究项目：艾滋病毒-1环境疫苗设计(约翰·摩尔博士，康奈尔大学)和艾滋病毒-1三聚体结晶学(伊恩·威尔逊博士，斯克里普斯研究所)。这些项目得到了科学和行政核心的支持(威廉·奥尔森博士，后代制药公司)。该计划利用我们最近在产生稳定的、蛋白水解性成熟的gp140三聚体(SOSIP Gp140)方面的成功，在拓扑和抗原性上模拟病毒粒子相关的环境蛋白。作为免疫原，SOSIP三聚体在动物体内诱导NAB方面优于匹配的gp120。在HIVRAD中，SOSIP模板将被定制用于结构研究和进一步改善动物的免疫原性。我们HIVRAD的总体目标反映在三个主要里程碑中：1)在4A分辨率下确定切割的环境三聚体的结构，2)展示克服HIV-1环境病毒免疫抑制特性的方法，以及3)发现一种能够诱导不同HIV-1毒株的异源中和的SOSIP三聚体疫苗。鉴于目前对三聚体结构的基本知识，原子水平的观点有可能刺激新一代合理设计的Env疫苗的开发，我们对结晶SOSIP三聚体的初步研究为成功提供了高度的热情和谨慎的乐观情绪。平行研究将评估已被修改的SOSIP三聚体，以改善NAB表位的呈现和/或消除Env免疫抑制特征。动物免疫原性研究将与体外研究相补充，比较修饰和未修饰形式的Env在人类DC中诱导免疫抑制反应的能力。除了主要项目和核心，HIVRAD还包括其他领先的学术和企业合作者，他们在疫苗交付(Aldevron，Inc.)、NAB分析(Mongraph Biosciences)、NAB特异性分析(James Binley博士)、体外免疫原性研究(VaxDesign)和探索性免疫遗传学(Sunil Ahuja博士)等领域提供专业知识。我们的共同目标是克服关键的结构和免疫学挑战，开发成功的基于环境的艾滋病毒-1疫苗。 项目1：HIV-1环境疫苗设计(摩尔，J) 项目1说明(由申请人提供)：HIVRAD项目1的目标是基于对HIV-1包膜复合体内结构-功能关系以及包膜蛋白如何在体外和体内与免疫系统细胞相互作用的积累和新出现的知识，设计旨在诱导NAB的疫苗。我们提出了三个相互关联的具体目标。目的1：进一步修饰HFV-1囊膜糖蛋白的氨基酸序列。创造新的形式，提高中和表位的免疫原性，促进结构研究。我们将在SOSIP方法的基础上，通过鉴定和测试稳定gp41-gp41相互作用的额外替换，通过在gp21和/或gp41中引入其他旨在创建或更好地暴露NAB表位的序列变化，以及通过消除Env复合体的免疫抑制区域，来构建稳定的、裂解的GP！40三聚体。后一项研究将与AIM 2中概述的研究相协调，并将导致在AIM 3中创建用于小动物测试的新免疫原，以及在Core B中的模拟系统中。我们还将对旨在降低蛋白质异质性和/或灵活性的环境三聚体进行修改，从而促进Ian Wilson(项目2)进行的X射线结晶学研究。目的：体外研究佐剂和TLR激活剂对gp120免疫抑制作用的影响。我们将使用人类和小鼠基于细胞的系统，特别是树突状细胞(DC)，在体外研究gp120多聚糖的甘露糖部分的免疫抑制作用。然后将测试TLR激活剂和其他佐剂相关分子克服这些不利影响的能力。此外，我们将与Eric Mishkin和Sunil Ahuja领导的小组合作，使用VaxDesign模拟系统(核心B)获得更多的免疫学和遗传信息。这些实验的结果将有助于在目标1中构建新的免疫原，并在目标3和核心B中合理设计免疫方案，以期最大限度地提高对Env的抗体应答。目的：评价修饰的Env三聚体在小动物体内的免疫原性。我们将设计和评估将在Core B内进行的兔和小鼠免疫研究，以确定我们在AIMS 1和2中识别和评估的对Env糖蛋白的各种修改是否可以改变对这些和联合注射的抗原的免疫反应。我们还将比较Env蛋白与其他单独和联合注射的抗原的免疫原性，以确定Env蛋白是否可以抑制或改变对自身和其他抗原(HTV-1来源的或无关的)的免疫反应，包括免疫球蛋白G亚类模式。一些实验的设计还将考虑到在AIM 2中产生的佐剂和TLR激活剂的信息，并通过使用核心B的VaxDesign模拟系统。在迭代过程中，免疫实验本身将指导由VaxDesign使用模拟系统(见核心B)进行的额外研究的设计。这些不同研究的总体结果将有助于在AIM 1中设计额外的Env序列修改，旨在进一步提高免疫原性或消除免疫抑制的原因。

DESCRIPTION (provided by applicant): This HIVRAD program merges the complementary expertise of leading HIV-1 researchers in order to provide a fundamental advance towards an HIV-1 vaccine than can elicit broadly neutralizing antibodies (NAbs). This innovative program comprises two Research Projects: HIV-1 Env Vaccine Design (Dr. John Moore, Cornell University) and HIV-1 Trimer Crystallography (Dr. Ian Wilson, The Scripps Research Institute). The Projects are supported by scientific and administrative Cores (Dr. William Olson, Progenies Pharmaceuticals). This program leverages our recent successes in generating stable, proteolytically mature gp140 trimers (SOSIP gp140s) that mimic virion-associated Env in topology and antigenicity. As immunogens, SOSIP trimers have proven to be superior to matched gp120s in eliciting NAbs in animals. In the HIVRAD, the SOSIP template will be tailored for structural studies and for further improvements in immunogenicity in animals. Our overall goals of the HIVRAD are reflected in three major milestones: 1) determine the structure of cleaved env trimers at <4A resolution, 2) demonstrate methods to overcome HIV-1 Env's immunosuppressive properties, and 3) identify a SOSIP trimer vaccine that elicits heterologous neutralization of diverse HIV-1 isolates. Given the present rudimentary knowledge of trimer structure, an atomic-level view has the potential to spur development of a new generation of rationally designed Env vaccines, and our preliminary studies on crystallizing SOSIP trimers provide high enthusiasm and guarded optimism for success. Parallel studies will evaluate SOSIP trimers that have been modified so as to improve presentation of NAb epitopes and/or remove Env immunosuppressive features. Animal immunogenicity studies will be complemented with in vitro studies that compare modified and unmodified forms of Env for their ability to elicit immunosuppressive responses in human DCs. In addition to the primary Projects and Cores, the HIVRAD includes additional leading academic and corporate collaborators who lend specialized expertise in the areas of vaccine delivery (Aldevron, Inc.), NAb analyses (Monogram Biosciences), NAb specificity analyses (Dr. James Binley), in vitro immunogenicity studies (VaxDesign), and exploratory immunogenetics (Dr. Sunil Ahuja). Our shared goal is to overcome key structural and immunological challenges to developing a successful Env-based HIV-1 vaccine. PROJECT 1: HIV-1 Env Vaccine Design (Moore, J) PROJECT 1 DESCRIPTION (provided by applicant): The goal of Project 1 of the HIVRAD is to design vaccines intended to induce NAbs, based on accumulated and emerging knowledge of structure-function relationships within the HIV-1 Env complex and of how Env proteins interact with cells of the immune system, in vitro and in vivo. We propose three inter-related Specific Aims. Aim 1: To further modify the amino acid sequence of HFV-1 envelope glycoproteins. to create novel forms that improve the immunogenicity of neutralization epitopes and facilitate structural studies. We will build on the SOSIP method for making stable, cleaved gp!40 trimers, by identifying and testing additional substitutions that stabilize gp41-gp41 interactions, by introducing other sequence changes into gp!20 and/or gp41 that are intended to create or better expose NAb epitopes, and by eliminating immunosuppressive regions of the Env complex. The latter studies will be coordinated with research outlined in Aim 2, and will lead to the creation of new immunogens for testing in small animals in Aim 3, and also in the MIMIC system within Core B. We will also make modifications to Env trimers that are intended to reduce protein heterogeneity and/or flexibility, and thereby facilitate X-ray crystallography studies to be conducted by Ian Wilson (Project 2). Aim 2: To study in vitro how to overcome immunosuppressive effects of gp120 by the use of adjuvants and TLR activators. We will study the immunosuppressive effects of the mannose moieties of gp120 glycans in vitro, using both human and murine cell-based systems, particularly dendritic cells (DCs). The abilities of TLR activators and other adjuvant-associated molecules to overcome these adverse effects will then be tested. We will, in addition, collaborate with the groups headed by Eric Mishkin and Sunil Ahuja, to derive additional immunologic and genetic information using the VaxDesign MIMIC system (Core B). The outcome of these experiments will facilitate the construction of new immunogens in Aim 1, and the rational design of immunization regimens in Aim 3 and Core B that are intended to maximize the antibody response to Env. Aim 3: To evaluate the immunogenicity of modified Env trimers in small animals. We will design and evaluate rabbit and mouse immunization studies that will be conducted within Core B, to determine whether the various modifications to Env glycoproteins that we identified and evaluated in Aims 1 and 2, can alter the immune response to these and co-administered antigens. We will also compare the immunogenicity of Envproteins with other antigens, alone and co-administered, to determine whether Env proteins can suppress or modify immune responses, including the IgG subclass pattern, to themselves and other antigens (HTV-1 derived or unrelated). The design of some experiments will also take into account information on adjuvants and TLR activators generated in Aim 2 and by using the VaxDesign MIMIC system in Core B. In an iterative process, the immunization experiments will themselves guide the design of additional studies to be carried out by VaxDesign using the MIMIC system (see Core B). The overall outcome of these various studies will help design additional Env sequence modifications in Aim 1 that are intended to further improve immunogenicity or eliminate the causes of immunosuppression.