Structure-Function Studies of the HIV-1 Envelope Glycoproteins

保护性和/或治疗性HIV疫苗的开发受到前所未有的挑战的阻碍，这主要是由于HIV-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，其特征在于各种各样的免疫逃避策略，包括抗原变异、暴露表面的重糖基化和所谓的“构象伪装”。进一步深入了解HIV-1 Env三聚体及其保护盾中复杂的结构-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)增强广泛的艾滋病毒中和抗体。 三年前，我们报道了在HIV-1 Env三聚体中发现的第二个CD 4结合位点，我们将其定义为CD 4结合位点2（CD 4-BS 2），为疫苗和治疗开辟了新的前景。我们还鉴定了选定的抗CD 4-BS抗体，如VRC 03和VRC 06，其模拟CD 4的四元结合模式，并通过其重链框架区3（FR 3）中的延伸环与两个相邻的gp 120原体建立接触。我们通过删除VRC 03和VRC 06的FR 3环来证明这种四级接触的功能作用，这导致结合和中和活性几乎完全丧失。由于四元接触的建立似乎支持了CD 4和所选抗体中的三聚体相互作用，我们假设它可能同样进一步改善一些最有效的bNAb的活性，所有这些bNAb都与单个gp 120原聚体相互作用（例如，VRC01、VRC07、N6）。为了验证这一假设，我们合理地将VRC 03的延长的FR 3环嫁接到不同的CD 4-超位点bNAb上，并测试了针对广泛的全球HIV-1毒株（n = 208）的所得嵌合抗体。FR 3环嵌合增强了有效bNAb对大多数全球HIV-1毒株的中和活性。通过求解与可溶性Env三聚体复合的两个FR 3环嵌合抗体的晶体结构来描绘相互作用的四级表面。此外，与未修饰的抗体相比，嵌合抗体在huFcRn转基因小鼠和恒河猴中均显示出降低的自身反应性和延长的体内半衰期。由于其增加的中和效力和有利的生物学和药代动力学性质，FR 3-环嵌合bNAb正被考虑用于HIV预防和治疗。我们最近将这些研究扩展到其他非常有效的bNAb，包括来自巴尔的摩IHV的N49 P家族和来自德国Klein小组的抗体1-18。 有趣的是，N49 P抗体的嵌合是成功的，导致迄今为止报道的针对CD 4-BS的最有效的bNAb。 相比之下，抗体1-18的修饰导致功能丧失，证实成功的嵌合需要并非所有抗CD 4-BS抗体共有的特定结构特征。我们目前正在人源化小鼠模型中测试所选嵌合抗体的功效。 2)HIV-1包膜三聚体的结构引导的结构域间稳定消除了CD 4结合并提高了免疫原性。 HIV-1 Env三聚体的固有灵活性及其结合CD 4受体的能力代表了开发能够引发bNAb的疫苗的两个主要障碍。特别地，与CD 4的结合不仅诱导构象变化，其迅速损害三聚体的天然抗原状态，而且封闭CD 4结合位点（CD 4-BS），其是抗体引发的关键抗原靶标，并引起缺乏抗原呈递能力的CD 4 + T细胞的免疫原隔离。为了减少三聚体的灵活性和损害与CD 4的结合，我们使用了结构导向的方法，在gp 120和CD 4之间的分子模拟区域（SLWDQ）中引入桥接gp 120的内部和外部结构域（结构域间锁）的新二硫键。如质谱分析所示，新二硫键有效地形成，并导致三聚体稳定在天然样融合前构型中。值得注意的是，这种设计成功地应用于可溶性三聚体（SOSIP）和来自不同HIV-1毒株和进化枝的天然全长gp 160。结构域间锁定的三聚体显示出增加的热稳定性、减少的自发性错误折叠、减少或消除与非中和抗体的结合、增强与bNAb的结合，并且最重要的是，丧失CD 4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，并提供了深入了解CD 4结合损伤的结构基础。 3)猕猴中mRNA/VLP疫苗平台的体内评价 使用通过基于Env的免疫原的结构指导设计所积累的知识，我们在恒河猴（M. mulatta）使用基于信使RNA（mRNA）的疫苗，目的是引发针对具有全球流行相关性的不同进化枝的2级HIV-1毒株的广泛中和抗体（bNAb）。我们的疫苗基于关键技术进步的组合，最值得注意的是使用编码全长（膜锚定）HIV-1 Env和SIV Gag的共配制mRNA，以诱导病毒样颗粒（VLP）的体内产生，其提供了自然感染期间产生的天然病毒颗粒的最佳模拟物。该疫苗具有高度免疫原性，我们首次记录了真正的2级交叉中和bNAb的诱导，这与保护免受难以中和的SHIV-AD 8株的2级异源攻击有关。这些结果为临床实验的过渡提供了科学基础。

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.