Pastori Claudia, Clivio Alberto, Diomede Lorenzo, Consonni Roberto, De Mori Giacomo M S, Longhi Renato, Colombo Giorgio, Lopalco Lucia
Infectious Diseases Clinic, San Raffaele Scientific Institute, Milan, Italy.
J Virol. 2008 Apr;82(8):4125-34. doi: 10.1128/JVI.02232-07. Epub 2008 Feb 6.
Antibodies to the first loop (ECL1) of CCR5 have been identified in human immunodeficiency virus (HIV)-exposed uninfected individuals (ESN) and in HIV-positive nonprogressing subjects. Thus, these antibodies may confer resistance against HIV infection. To define which amino acids are involved in antibody binding to CCR5, we performed a peptide-scanning assay and studied the immunogenicity of peptides in animal models. A panel of synthetic peptides spanning the CCR5-ECL1 region and displaying glycine or alanine substitutions was assayed for antibody binding with a pool of natural anti-CCR5 antibodies. We used mice and chickens to study the immunogenicity of mutagenized peptide. Structural characterization by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations were performed to better understand the structural and conformational features of the mutagenized peptide. Amino acid substitutions in positions Ala95 and Ala96 (A(95)-A(96)) increased antibody-peptide binding compared to that of the wild-type peptide (Asp(95)-Phe(96)). The Ala95-96 peptide was shown to induce, in mice and chickens, antibodies displaying biological activity at very low concentrations. Strikingly, chicken antibodies to the Ala95-96 peptide specifically recognize human CCR5 molecules, downregulate receptors from lymphocytes, inhibit CCR5-dependent chemotaxis, and prevent infection by several R5 viruses, displaying 50% inhibitory concentrations of less than 3 ng/ml. NMR spectroscopy and molecular dynamics simulations proved the high flexibility of isolated epitopes and suggested that A(95)-A(96) substitutions determine a slightly higher tendency to generate helical conformations combined with a lower steric hindrance of the side chains in the peptides. These findings may be relevant to the induction of strong and efficient HIV-blocking antibodies.
在暴露于人类免疫缺陷病毒（HIV）但未感染的个体（ESN）以及HIV阳性的非进展性受试者中，已鉴定出针对CCR5第一环（ECL1）的抗体。因此，这些抗体可能赋予对HIV感染的抗性。为了确定哪些氨基酸参与抗体与CCR5的结合，我们进行了肽扫描分析，并在动物模型中研究了肽的免疫原性。检测了一组跨越CCR5-ECL1区域并显示甘氨酸或丙氨酸取代的合成肽与天然抗CCR5抗体池的抗体结合情况。我们使用小鼠和鸡来研究诱变肽的免疫原性。通过核磁共振（NMR）光谱和分子动力学模拟进行结构表征，以更好地了解诱变肽的结构和构象特征。与野生型肽（Asp(95)-Phe(96)）相比，Ala95和Ala96位置（A(95)-A(96)）的氨基酸取代增加了抗体与肽的结合。结果表明，Ala95-96肽在小鼠和鸡中可诱导产生在极低浓度下具有生物活性的抗体。引人注目的是，针对Ala95-96肽的鸡抗体可特异性识别人类CCR5分子，下调淋巴细胞表面的受体，抑制CCR5依赖性趋化作用，并预防几种R5病毒的感染，其50%抑制浓度小于3 ng/ml。NMR光谱和分子动力学模拟证明了分离表位的高灵活性，并表明A(95)-A(96)取代决定了生成螺旋构象的倾向略高，同时肽中侧链的空间位阻较低。这些发现可能与诱导强效和高效的HIV阻断抗体有关。
