Structure-Function Studies of the HIV-1 Envelope Glycoproteins

HIV疫苗的开发受到前所未有的挑战的阻碍，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，具有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关键中和位点的构象掩蔽。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。 我们之前报道了在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），这为HIV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。因此，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，并测试了所得的针对广泛的全球HIV-1毒株的嵌合抗体（n = 208）。FR 3环嵌合增强了几种有效的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有效的bNAb，包括来自巴尔的摩IHV的N49 P家族和来自德国Klein组的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。正在进行临床前研究以评估所选嵌合抗体在人源化小鼠模型中的功效。 2)HIV-1VLP形成mRNA疫苗在猕猴中的临床前评价 研制保护性疫苗仍然是控制艾滋病毒/艾滋病流行病的最高优先事项。我们设计了一种基于信使RNA（mRNA）的新型HIV-1疫苗平台，与Covid-19疫苗使用的技术相同，并在猕猴的临床前模型中进行了测试。这项研究是与Moderna Inc.合作进行的。我们的疫苗设计包括：a）膜锚定的HIV-1包膜（Env）与猿猴免疫缺陷病毒（SIV）Gag蛋白的共表达，以在体内产生非感染性病毒样颗粒（VLP）; B）用接合Env的种系bNA B进行初始引发;和c）用不同的HIV-1进化枝进行多次异源加强免疫。在小鼠中进行的初步实验中，用共配制的Env和Gag mRNA免疫在诱导中和抗体方面上级单独的Env mRNA。在猕猴中，我们测试了一种复杂的方案，包括用缺乏覆盖CD 4结合位点聚糖的关键聚糖（N276）的传递的创始者进化枝B Env mRNA进行初始引发，然后用聚糖修复的自体Env和随后来自不同进化枝（进化枝A和C）的二价异源Env进行多次加强免疫。所有Env与Gag共配制以产生VLP。该方案具有高度免疫原性，并引发针对最流行（第2层）HIV-1毒株的中和抗体，伴有稳健的抗Env CD 4 + T细胞应答。免疫动物在用异源2级猴人类免疫缺陷病毒（SHIV AD 8）重复低剂量粘膜攻击后，每次暴露风险降低79%。因此，我们的多分支envgag VLP mRNA平台代表了开发HIV-1疫苗的有希望的方法。

The development of an 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 conformational masking of key neutralization sites. 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. We previously 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), which opened new perspectives for HIV 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). Thus, 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 several 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 potent bNAbs, including the N49P family from the IHV in Baltimore and 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. Preclinical studies are underway to assess the efficacy of selected chimeric antibodies in humanized mouse models. 2) Pre-clinical evaluation of a VLP-forming mRNA vaccine for HIV-1 in macaques The development of a protective vaccine remains a top priority for the control of the HIV/AIDS pandemic. We have designed a novel vaccine platform for HIV-1 based on messenger RNA (mRNA), the same technology used for the Covid-19 vaccines, and tested in a preclinical model in macaques. The study was conducted in collaboration with Moderna Inc. Our vaccine design includes: a) co-expression of membrane-anchored HIV-1 envelope (Env) with simian immunodeficiency virus (SIV) Gag proteins in order to generate non-infectious virus-like particles (VLPs) in vivo; b) initial priming with a germline bNAb engaging Env; and c) multiple heterologous booster immunizations with different HIV-1 clades. In preliminary experiments conducted in mice, immunization with co-formulated Env and Gag mRNAs was superior to Env mRNA alone in inducing neutralizing antibodies. In macaques, we tested a complex protocol encompassing an initial priming with a transmitted-founder clade-B Env mRNA lacking a critical glycan (N276) that covers the CD4-binding site glycan, followed by multiple booster immunizations with glycan-repaired autologous and subsequently bivalent heterologous Envs from different clades (clades A and C). All Envs were co-formulated with Gag to produce VLPs. This regimen was highly immunogenic and elicited neutralizing antibodies against the most prevalent (tier-2) HIV-1 strains accompanied by robust anti-Env CD4+ T cell responses. Vaccinated animals had a 79% per-exposure risk reduction upon repeated low-dose mucosal challenges with heterologous tier-2 simianhuman immunodeficiency virus (SHIV AD8). Thus, our multiclade envgag VLP mRNA platform represents a promising approach for the development of an HIV-1 vaccine.