Impact of stabilizing mutations on the antigenic profile and glycosylation of membrane-expressed HIV-1 envelope glycoprotein.
Impact of stabilizing mutations on the antigenic profile and glycosylation of membrane-expressed HIV-1 envelope glycoprotein.
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DOI：
10.1371/journal.ppat.1011452
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发表时间：
2023-08
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
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最近的HIV-1疫苗开发集中在“近天然”可溶性包膜糖蛋白（Env）三聚体上，这些三聚体在横向（在原聚体之间）和顶端（在gp120和gp41之间）被人工稳定。这些突变已被用于膜表达的Env mRNA疫苗，尽管它们在这种情况下的作用尚不清楚。为了解决这个问题，我们使用了293T细胞中产生的病毒样颗粒（VLP）。Uncleaved （UNC）三聚体在膜的温和裂解后横向不稳定。然而，gp120/gp41加工提高了横向稳定性。由于gp120/gp41处理效率低下，UNC被合并到VLPs中。gp120和gp41之间的连接体既不能提高三聚体的稳定性，也不能提高其抗原谱。人工引入的肠激酶切割位点允许gp120/gp41表达后加工，同时增加三聚体的稳定性。Gp41 n -螺旋突变I559P和NT1-5影响了横向三聚体的稳定性，但也减少了gp120/ Gp41的加工和/或影响了V2顶点和界面NAb的结合。I559P持续降低HIV阳性人血浆的识别，进一步支持抗原差异。gp120桥接片的突变不能稳定膜三聚体的预融合构象，也减少了gp120/gp41的加工并暴露了非中和的表位。这些突变的常见副作用是聚糖成熟减少和序列跳变增加。在某些情况下，这可能是由于刚性增加，限制了聚糖加工酶的使用。相比之下，病毒gp120没有表现出聚糖跳过。第二种小种类的高甘露糖gp160不受任何突变的影响，而是绕过正常的折叠和聚糖成熟。包括完整的gp41细胞质尾部导致gp120/gp41加工显著减少，高甘露糖gp160比例大大增加。值得注意的是，单克隆抗体无法与天然蛋白凝胶中的高甘露糖gp160结合。总的来说，我们的研究结果表明，在利用基于核酸的免疫原的稳定突变时要谨慎，以确保它们赋予疫苗使用的有价值的膜三聚体表型。一种能够诱导抗病毒抗体来阻止HIV-1感染的疫苗仍然难以捉摸。为了消除HIV-1感染，抗体必须与病毒表面的真实包膜（Env）糖蛋白结合。然而，Env可以以各种形式存在，其中许多形式相对容易成为无效抗体的靶点。因此，疫苗设计的一个关键挑战是创造不受这些其他形式的Env限制的纯正的真正Env。迄今为止，疫苗研究主要集中在稳定可溶性Env三聚体上，因为这种形式简化了纯化和转化研究。然而，不完整的Env真实性可能会削弱这种方法的有效性。相比之下，在膜上原位表达Env的颗粒疫苗的制造就比较麻烦。在另一种方法中，脂质颗粒可以传递编码膜三聚体的mRNA疫苗，绕过了以前方法的制造挑战。来自可溶性三聚体的稳定突变现在被用于膜三聚体。在这里，我们评估了这些突变的影响。我们的研究结果表明，一些突变改变了Env的构象，因此可能最好从基于核酸的膜Env疫苗中省略。
Recent HIV-1 vaccine development has centered on “near native” soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use. A vaccine that induces virus-fighting antibodies to block HIV-1 infection remains elusive. To ablate HIV-1 infection, antibodies must bind to authentic envelope (Env) glycoprotein on the virus surface. However, Env can exist in various forms, many of which are relatively easy targets for non-effective antibodies. Therefore, a key challenge of vaccine design is to create pure authentic Env that is unfettered by these other forms of Env. Vaccine research to date has focused largely on stabilizing soluble Env trimers, as this format simplifies purification and translational studies. However, incomplete Env authenticity may blunt the efficacy of this approach. By comparison, the manufacture of particle-based vaccines that express Env in situ on membranes is cumbersome. In an alternative approach, lipid particles can deliver mRNA vaccines encoding membrane trimers, bypassing the manufacturing challenges of previous methods. Stabilizing mutations derived from soluble trimers are now being leveraged for membrane trimers. Here, we evaluated the effects of these mutations. Our results show that some mutations alter Env conformation, and therefore might best be omitted from nucleic acid-based membrane Env vaccines.
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影响因子：
5.4
作者：
Castillo-Menendez, Luis R.;Nguyen, Hanh T.;Sodroski, Joseph
通讯作者：
Sodroski, Joseph
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