PDF(Pubmed)
Abstract：
Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA\'s stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA\'s globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01μM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.
摘要：
流感病毒在全球范围内对人类健康构成重大威胁。病毒表面糖蛋白血凝素(HA)是免疫显性抗原,包含与细胞受体（RBS）结合的位点，它是感染后中和抗体反应的主要目标。我们开发了美洲驼衍生的单链抗体片段（VHHs），对A型流感病毒具有特异性。鉴定并进一步表征了四种VHH。VHHD81结合H1和H5亚型的HA1的C末端区域附近的残基，并显示弱的中和活性，而VHHB33结合在H1，H5和H9亚型的HA茎结构域（HA2）的N末端区域附近的残基，并且没有显示中和活性。最相关的是，VHHsE13和G41识别H1HA球状结构域（HA1）上高度保守的构象表位，并显示出高的病毒中和活性（范围在0.94至0.01μM之间），当针对几种人类H1N1分离株进行测试时。此外,E13显示了一组H1N1毒株的病毒复制，这些毒株跨越了80多年的抗原性漂移，并从人类中分离出来，禽类,猪的起源。有趣的是,E13以低至0.05mg/kg的剂量在体内赋予保护。用E13鼻内处理的小鼠在攻击后第4天导致肺中不可检测的病毒攻击负荷。灭菌泛H1免疫的转移，通过鼻内给予微克范围内的剂量，对于单体VHH具有重要意义，并支持E13作为缓解流感感染的免疫治疗剂的进一步开发。
