Ingale Jidnyasa, Tran Karen, Kong Leopold, Dey Barna, McKee Krisha, Schief William, Kwong Peter D, Mascola John R, Wyatt Richard T
Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA.
IAVI Neutralizing Antibody Center at TSRI, La Jolla, California, USA.
J Virol. 2014 Dec;88(24):14002-16. doi: 10.1128/JVI.02614-14. Epub 2014 Sep 24.
The HIV-1 surface envelope glycoprotein (Env) trimer mediates entry into CD4(+) CCR5(+) host cells. Env possesses conserved antigenic determinants, such as the gp120 primary receptor CD4 binding site (CD4bs), a known neutralization target. Env also contains variable regions and protein surfaces occluded within the trimer that elicit nonneutralizing antibodies. Here we engineered additional N-linked glycans onto a cysteine-stabilized gp120 core (0G) deleted of its major variable regions to preferentially expose the conformationally fixed CD4bs. Three, 6, 7, and 10 new NXT/S glycan (G) motifs were engineered into 0G to encode 3G, 6G, 7G, and 10G cores. Following purification, most glycoproteins, except for 10G, were recognized by broadly neutralizing CD4bs-directed antibodies. Gel and glycan mass spectrometry confirmed that additional N-glycans were posttranslationally added to the redesigned cores. Binding kinetics revealed high-affinity recognition by seven broadly neutralizing CD4bs-directed antibodies and low to no binding by non-broadly neutralizing CD4bs-directed antibodies. Rabbits inoculated with the hyperglycosylated cores elicited IgM and IgG responses to each given protein that were similar in their neutralization characteristics to those elicited by parental 0G. Site-specific glycan masking effects were detected in the elicited sera, and the antisera competed with b12 for CD4bs-directed binding specificity. However, the core-elicited sera showed limited neutralization activity. Trimer priming or boosting of the core immunogens elicited tier 1-level neutralization that mapped to both the CD4bs and V3 and appeared to be trimer dependent. Fine mapping at the CD4bs indicated that conformational stabilization of the cores and addition of N-glycans altered the molecular surface of Env sites of vulnerability to neutralizing antibody, suggesting an explanation for why the elicited neutralization was not improved by this rational design strategy.
Major obstacles to developing an effective HIV-1 vaccine include the variability of the envelope surface glycoproteins and its high-density glycan shield, generated by incorporation of host (human) glycosylation. HIV-1 does harbor highly conserved sites on the exposed envelope protein surface of gp120, one of which is the virus receptor (CD4) binding site. Several broadly neutralizing antibodies elicited from HIV patients do target this gp120 CD4 binding site (CD4bs); however, gp120 immunogens do not elicit broadly neutralizing antibodies. In this study, we targeted the CD4bs by conformational stabilization and additional glycan masking. We used the atomic-level structure to reengineer gp120 cores to preferentially present the cysteine-stabilized CD4bs and to mask (by glycan) nonneutralizing determinants. Importantly, glycan masking did successfully focus antibody responses to the CD4bs; however, the elicited CD4bs-directed antibodies did not neutralize HIV or bind to unmodified gp120, presumably due to the structure-guided modifications of the modified gp120 core.
HIV-1表面包膜糖蛋白（Env）三聚体介导病毒进入CD4(+) CCR5(+)宿主细胞。Env具有保守的抗原决定簇，如gp120主要受体CD4结合位点（CD4bs），这是一个已知的中和靶点。Env还包含可变区和三聚体内被遮蔽的蛋白表面，这些区域可引发非中和抗体。在此，我们在去除了主要可变区的半胱氨酸稳定化gp120核心（0G）上设计添加了额外的N-连接聚糖，以优先暴露构象固定的CD4bs。将三个、六个、七个和十个新的NXT/S聚糖（G）基序设计到0G中，以编码3G、6G、7G和10G核心。纯化后，除10G外，大多数糖蛋白都能被广泛中和的CD4bs导向抗体识别。凝胶和聚糖质谱分析证实，额外的N-聚糖是在翻译后添加到重新设计的核心上的。结合动力学显示，七种广泛中和的CD4bs导向抗体具有高亲和力识别，而非广泛中和的CD4bs导向抗体则低结合或无结合。用高糖基化核心免疫的兔子产生了针对每种给定蛋白的IgM和IgG反应，其中和特性与亲本0G引发的反应相似。在引发的血清中检测到位点特异性聚糖遮蔽效应，并且抗血清与b12竞争CD4bs导向的结合特异性。然而，核心引发的血清显示出有限的中和活性。三聚体引发或增强核心免疫原可引发1级水平的中和，该中和作用定位于CD4bs和V3，并且似乎依赖于三聚体。在CD4bs处的精细图谱分析表明，核心的构象稳定和N-聚糖的添加改变了Env易受中和抗体攻击位点的分子表面，这为为什么这种合理设计策略未能改善引发的中和作用提供了一种解释。
开发有效的HIV-1疫苗的主要障碍包括包膜表面糖蛋白的变异性及其通过宿主（人类）糖基化形成的高密度聚糖屏蔽。HIV-1在gp120暴露的包膜蛋白表面确实具有高度保守的位点，其中之一是病毒受体（CD4）结合位点。从HIV患者中引发的几种广泛中和抗体确实靶向该gp120 CD4结合位点（CD4bs）；然而，gp120免疫原不会引发广泛中和抗体。在本研究中，我们通过构象稳定和额外的聚糖遮蔽来靶向CD4bs。我们利用原子水平结构对gp120核心进行重新设计，以优先呈现半胱氨酸稳定化的CD4bs并（通过聚糖）遮蔽非中和决定簇。重要的是，聚糖遮蔽确实成功地使抗体反应集中于CD4bs；然而，引发的CD4bs导向抗体并未中和HIV或与未修饰的gp120结合，这可能是由于修饰的gp120核心的结构导向修饰所致。
