Structure-Function Studies of the HIV-1 Envelope Glycoproteins

HIV-1 包膜糖蛋白的结构功能研究

• 批准号: 10272210
• 负责人: paolo lusso
• 金额: $ 184.77万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272210
• 关键词: AIDS prevention; AIDS/HIV problem; Antibodies; Antigenic Variation; Antigens; Baltimore; Binding; Binding Sites; Biological; CD4 Antigens; CD4 Positive T Lymphocytes; Clinical; Complex; Crystallization; Development; Disulfides; Drug Kinetics; Engineering; Epidemic; Family; Foundations; Framework Regions; Generations; Germany; Glycoproteins; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; HIV-1; Half-Life; Immune Evasion; Immunization; Impairment; Infection; Investigation; Knowledge; Length; Macaca; Macaca mulatta; Mass Spectrum Analysis; Measures; Membrane; Messenger RNA; Modification; Molecular Conformation; Molecular Mimicry; Neutralization Tests; Prevention; Production; Property; Protomer; Reporting; Rodent; Role; SIV; Structure; Structure-Activity Relationship; Surface; Test Result; Therapeutic; Time; Transgenic Mice; Vaccine Design; Vaccines; Virion; Virus-like particle; autoreactivity; base; chimeric antibody; design; disulfide bond; efficacy study; efficacy testing; flexibility; glycosylation; gp160; humanized mouse; immunogenic; immunogenicity; immunoprophylaxis; improved; in vivo; in vivo evaluation; insight; loss of function; mouse model; natural antibodies; neutralizing antibody; novel; prevent; receptor; simian human immunodeficiency virus; vaccine development

The development of a protective and/or therapeutic HIV vaccine has been hampered by unprecedented challenges, primarily due to the unique properties of the HIV-1 envelope (Env) trimer, a cleverly engineered entry machinery that features an extraordinary assortment of immune-evasion tactics, including antigenic variation, heavy glycosylation of exposed surfaces and the so-called "conformational camouflage". Further insights into the complex structure-function relationships in the HIV-1 Env trimer and its protective shield may be critical to guide the rational design of a protective vaccine and other immunoprophylaxis measures. 1) Enhancement of broadly HIV-neutralizing antibodies. Three years ago, we reported the discovery of a second CD4-binding site in the HIV-1 Env trimer, that we defined as CD4-binding site 2 (CD4-BS2), opened new perspectives for vaccine and therapy. We also identified selected anti-CD4-BS antibodies, such as VRC03 and VRC06, which mimic the quaternary-binding mode of CD4 and establish contact with two adjacent gp120 protomers via an extended loop in their heavy chain framework region 3 (FR3). We proved the functional role of this quaternary contact by deleting the FR3 loops of VRC03 and VRC06, which resulted in a near-complete loss of binding and neutralization activity. Since the establishment of quaternary contact appears to bolster the trimer interaction both in CD4 and in selected antibodies, we hypothesized that it might likewise further improve the activity of some of the most potent bNAbs, all of which interact with a single gp120 protomer (e.g., VRC01, VRC07, N6). To validate this assumption, we rationally engrafted the extended FR3 loop of VRC03 onto different CD4-supersite bNAbs and tested the resulting chimeric antibodies against a wide panel of global HIV-1 strains (n = 208). FR3-loop chimerization enhanced the neutralizing activity of potent bNAbs against a majority of global HIV-1 strains. The interactive quaternary surface was delineated by solving the crystal structure of two FR3 loop-chimeric antibodies in complex with a soluble Env trimer. Furthermore, compared to unmodified antibodies, chimeric antibodies displayed a reduced autoreactivity and a prolonged in vivo half-life in both huFcRn transgenic mice and rhesus macaques. Due to their increased neutralizing potency and favorable biological and pharmacokinetic properties, FR3-loop-chimeric bNAbs are being considered for use in HIV prevention and treatment. We have recently extended these studies to other very potent bNAbs, including the N49P family from the IHV in Baltimore and antibody 1-18 from the Klein group in Germany. Interestingly, chimerization of the N49P antibodies was successful resulting in the most potent bNAbs against the CD4-BS ever reported so far. By contrast, modification of antibody 1-18 resulted in a loss of function, confirming that successful chimerization requires specific structural features that ar4 not common to all anti-CD4-BS antibodies. We are currently testing the efficacy of selected chimeric antibodies in humanized mouse models. 2) Structure-guided interdomain stabilization of the HIV-1 envelope trimer abrogates CD4 binding and improves immunogenicity. The inherent flexibility of the HIV-1 Env trimer and its ability to bind the CD4 receptor represent two of the major obstacles to the development of a vaccine capable of eliciting bNAbs. In particular, binding to CD4 not only induces conformational changes that rapidly compromise the native antigenic state of the trimer, but also occludes the CD4-binding site (CD4-BS), which is a critical antigenic target for antibody elicitation, and causes immunogen sequestration by CD4+ T cells, which lack antigen-presenting capabilities. To reduce the trimer flexibility and impair binding to CD4, we used a structure-guided approach to introduce neo-disulfide bonds bridging the inner and outer domains of gp120 (interdomain locks) in a region of molecular mimicry between gp120 and CD4 (SLWDQ). The neo-disulfide bridges effectively formed, as shown by mass-spectrometry analysis, and resulted in trimer stabilization in a native-like pre-fusion configuration. Of note, this design was successfully applied to both soluble trimers (SOSIP) and native full-length gp160 from different HIV-1 strains and clades. Interdomain-locked trimers showed increased thermal stability, reduced spontaneous misfolding, reduced or abrogated binding to non-neutralizing antibodies, enhanced binding to bNAbs and, most importantly, loss of CD4-binding activity. The crystal structure of an interdomain-stabilized trimer confirmed the formation of the expected disulfide bond and provided insights into the structural basis for CD4-binding impairment. 3) In vivo evaluation of an mRNA/VLP vaccine platform in macaques Using the knowledge accrued by structure-guided design of Env-based immunogens, we conducted an efficacy study in rhesus macaques (M. mulatta) using a messenger RNA (mRNA)-based vaccine with the aim of eliciting broadly neutralizing antibodies (bNAbs) against tier-2 HIV-1 strains of different clades of global epidemic relevance. Our vaccine was based on the combination of key technological advances, most notably the use of co-formulated mRNA encoding full-length (membrane-anchored) HIV-1 Env and SIV Gag in order to induce the in vivo production of virus-like particles (VLPs), which provide the best mimic of the native virus particles produced during natural infection. The vaccine was highly immunogenic and we documented for the first time the induction of bona fide tier-2 cross-neutralizing bNAbs, which were associated with protection from tier-2 heterologous challenge with the difficult-to-neutralize strain SHIV-AD8. These results provide a scientific foundation for a transition towards clinical experimentation.

保护性和/或治疗性艾滋病毒疫苗的开发受到前所未有的挑战的阻碍，这主要是由于艾滋病毒-1包膜(Env)三聚体的独特性质，这是一种巧妙设计的进入机制，其特点是具有一系列非同寻常的免疫逃避策略，包括抗原变异、暴露表面的严重糖基化和所谓的“构象伪装”。进一步深入了解HIV-1三聚体及其保护膜中复杂的结构与功能关系，可能对指导保护性疫苗和其他免疫预防措施的合理设计至关重要。 1)加强广泛的艾滋病毒中和抗体。 三年前，我们报道了在HIV-1Env三聚体中发现的第二个CD4结合位点，我们将其定义为CD4结合位点2(CD4-BS2)，为疫苗和治疗开辟了新的视角。我们还鉴定了一些抗CD4-BS抗体，如VRC03和VRC06，它们模拟了CD4的四元结合模式，并通过它们的重链框架区域3(FR3)中的一个延伸环与相邻的两个gp120原核建立联系。我们通过删除VRC03和VRC06的FR3环来证明这种四元接触的功能作用，这导致了几乎完全失去结合和中和活性。由于四元接触的建立似乎支持了CD4和选定抗体中的三聚体相互作用，我们假设它同样可能进一步提高一些最有效的bNAbs的活性，所有这些bNAbs都与单一的gp120原基相互作用(例如，VRC01、VRC07、N6)。为了验证这一假设，我们合理地将VRC03的扩展FR3环嫁接到不同的CD4超位bNAbs上，并测试了产生的针对广泛的全球HIV-1毒株的嵌合抗体(n=208)。FR3-环嵌合增强了有效的bNAbs对大多数全球HIV-1毒株的中和活性。通过求解两个FR3环嵌合抗体与可溶性Env三聚体形成的络合物的晶体结构来描绘相互作用的四元表面。此外，与未经修饰的抗体相比，嵌合抗体在huFcRn转基因小鼠和猕猴体内显示出较低的自身反应性和较长的体内半衰期。由于其增强的中和效力和良好的生物学和药代动力学特性，FR3-环-嵌合bNAbs正被考虑用于HIV的预防和治疗。我们最近将这些研究扩展到其他非常有效的bNAbs，包括巴尔的摩IHV的N49P家族和德国Klein组的抗体1-18。有趣的是，N49P抗体的嵌合化成功，产生了迄今为止报道的最有效的抗CD4-BS的bNAb。相反，抗体1-18的修饰导致功能丧失，证实了成功的嵌合需要AR4的特定结构特征，而AR4并不是所有抗CD4-BS抗体的共同特征。我们目前正在人源化小鼠模型中测试选定的嵌合抗体的有效性。 2)结构导向的HIV-1包膜三聚体结构域间稳定化消除了CD4结合，提高了免疫原性。 HIV-1Env三聚体固有的灵活性及其与CD4受体结合的能力是开发能够诱导bNAbs的疫苗的两个主要障碍。特别是，与CD4的结合不仅引起构象变化，迅速损害三聚体的天然抗原状态，而且还封闭了作为抗体激发关键抗原靶点的CD4结合位点(CD4-BS)，并导致缺乏抗原呈递能力的CD4+T细胞隔离免疫原。为了降低三聚体的灵活性并破坏与CD4的结合，我们使用结构导引的方法在gp120和CD4之间的分子模拟区域(SLWDQ)引入了新的二硫键，将gp120的内部和外部结构域(域间锁定)连接起来。质谱分析表明，新二硫键有效地形成了新的二硫键，并导致了三聚体在类天然融合前构型中的稳定。值得注意的是，这一设计成功地应用于来自不同HIV-1毒株和分支的可溶性三聚体(SOSIP)和天然全长gp160。结构域间锁定的三聚体表现出更高的热稳定性，减少了自发的错误折叠，减少或取消了与非中和抗体的结合，增强了与bNAbs的结合，最重要的是，失去了CD4结合活性。结构域间稳定的三聚体的晶体结构证实了预期的二硫键的形成，并为CD4结合损伤的结构基础提供了见解。 3)猕猴mRNA/VLP疫苗平台的体内评价 利用基于Env的免疫原的结构导向设计所积累的知识，我们使用基于信使RNA(MRNA)的疫苗在恒河猴(M.mulatta)中进行了一项有效性研究，目的是诱导针对不同全球流行分支的第二层HIV-1毒株的广谱中和抗体(BNAbs)。我们的疫苗是基于关键技术进步的组合，最著名的是使用编码全长(膜锚定)HIV-1 Env和SIV Gag的联合配方mRNA来诱导体内产生病毒样颗粒(VLP)，这是对自然感染期间产生的本地病毒颗粒的最佳模拟。该疫苗具有高度的免疫原性，我们首次记录了真正的第2层交叉中和bNAbs的诱导，这与保护免受难以中和的Shiv-AD8株的第2层异源攻击有关。这些结果为向临床实验过渡提供了科学基础。