Characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the CD4-bound state: Antigenicity, biophysics, and immunogenicity
Characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the CD4-bound state: Antigenicity, biophysics, and immunogenicity
复制标题
DOI：
10.1128/jvi.02500-06
复制
发表时间：
2007-06-01
影响因子：
5.4
通讯作者：
Wyatt, Richard
中科院分区：
文献类型：
--
作者：
Dey, Barna;Pancera, Marie;Wyatt, Richard
人类免疫缺陷病毒1型外部gp 120包膜糖蛋白是高度灵活的，这种灵活性可能有助于单体gp 120免疫原不能引起广泛的中和抗体。我们以前表明，S375 W修饰的一个关键的界面腔中心的主要受体结合位点，Phe 43腔，稳定gp 120进入CD 4结合状态。然而，这种改变腔的替代品的免疫效果从未被测试过。随后，我们筛选了其他突变，这些突变沿着S375 W改变，可能进一步稳定CD 4结合状态。在这里，我们定义了一个选定的第二腔改变的替代，T257 S，并分析了在几个gp 120包膜糖蛋白的背景下的双重突变。与最初鉴定的单一S375 W置换相比，具有T257 S-加-S375 W双突变（T257 S + S375 W）的gp 120糖蛋白在被广泛中和的CD 4结合位点抗体b12增强识别方面具有上级抗原谱。等温滴定量热法测量熵的gp 120与CD 4的相互作用表明，双突变体也稳定到CD 4结合状态，增加核心，全长单体，全长三聚体版本的gp 120之间的相对固定。相对于野生型gp 120，还观察到空腔填充突变体的gp 120对CD 4的亲和力显著增加。选择最构象受限的T257 S + S375 W三聚体gp 120蛋白用于兔免疫原性分析，并显示出相对于其野生型对应物在引发中和抗体方面的改善趋势。总之，结果表明，构象稳定化可以提高gp 120引发中和抗体的能力。
The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S+S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S+S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.