Joyce Joseph G, Hurni William M, Bogusky Michael J, Garsky Victor M, Liang Xiaoping, Citron Michael P, Danzeisen Renee C, Miller Michael D, Shiver John W, Keller Paul M
Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
J Biol Chem. 2002 Nov 29;277(48):45811-20. doi: 10.1074/jbc.M205862200. Epub 2002 Sep 16.
The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5. Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope. Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for alpha-helical conformation. We have examined DP178 in the context of a model for optimized alpha-helices and show that the native sequence conforms poorly to the model. Solution conformation of DP178 was studied by circular dichroism and NMR spectroscopy and found to be predominantly random, consistent with previous reports. NMR mapping was used to show that the low percentage of alpha-helix present was localized to residues Glu(662) through Asn(671), a region encompassing the 2F5 epitope. Using NH(2)-terminal extensions derived from either GP41 or the yeast GCN4 leucine zipper dimerization domain, we designed peptide analogs in which the average helicity is significantly increased compared with DP178 and show that these peptides exhibit both a modest increase in affinity for 2F5 using a novel competitive solution-based binding assay and an increased ability to inhibit viral entry in a single-cycle infectivity model. Selected peptides were conjugated to carrier protein and used for guinea pig immunizations. High peptide-specific titers were achieved using these immunogens, but the resulting sera were incapable of viral neutralization. We discuss these findings in terms of structural and immunological considerations as to the utility of a 2F5-like response.
合成肽DP178源自人类免疫缺陷病毒1型GP41蛋白的羧基末端七肽重复区域，是一种有效的病毒介导融合抑制剂，包含序列ELDKWA，该序列构成了广泛中和性人类单克隆抗体2F5的识别表位。通过用含有该序列的肽或融合蛋白进行免疫来引发类似2F5免疫反应的努力尚未成功，这可能是因为表位的结构呈现不正确。尽管病毒粒子上羧基末端七肽重复序列的结构尚不清楚，但最近的几份报告表明其有形成α-螺旋构象的倾向。我们在优化α-螺旋模型的背景下研究了DP178，结果表明天然序列与该模型的契合度很差。通过圆二色性和核磁共振光谱研究了DP178的溶液构象，发现其主要为无规结构，这与之前的报告一致。核磁共振图谱显示，存在的低比例α-螺旋定位于Glu(662)至Asn(671)残基，该区域包含2F5表位。利用源自GP41或酵母GCN4亮氨酸拉链二聚化结构域的氨基末端延伸，我们设计了肽类似物，与DP178相比，其平均螺旋度显著增加，并表明这些肽在一种基于溶液竞争的新型结合试验中对2F5的亲和力适度增加，且在单循环感染性模型中抑制病毒进入的能力增强。选择的肽与载体蛋白偶联并用于豚鼠免疫。使用这些免疫原获得了高肽特异性滴度，但所得血清无法中和病毒。我们从结构和免疫学方面讨论了这些关于类似2F5反应效用的发现。
