Alizadeh Samaneh, Moazen Setareh, Faraji Seyed Nooreddin, Moattari Afagh, Nejatollahi Foroogh
Recombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran.
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver BC, Canada.
Iran J Basic Med Sci. 2021 Jan;24(1):17-23. doi: 10.22038/ijbms.2020.43508.10219.
Development of new antibodies with broad activity would provide anti-influenza prophylaxis and treatment. Human single-chain variable fragments (scFvs) are considered effective agents against viruses. In this study specific human scFvs against highly conserved epitopes in the hemagglutinin (HA) of influenza A viruses were selected and their neutralizing activity was evaluated.
Bioinformatic methods were used to evaluate HA epitopes. The panning process selected specific clones from a scFv library. PCR and DNA fingerprinting differentiated the common patterns. Soluble forms of scFvs were produced and evaluated using Western blot analysis. The neutralizing effects of anti-HA scFvs were assessed by microneutralization assay using MDCK cells. Real-time PCR was done to determine the exact copy number of the virus following neutralization.
Bioinformatic evaluation confirmed the antigenicity and accessibility of the epitopes. Four specific anti-HA scFvs, scFvs I, II, I', and II' were selected. The scFvs neutralized 2009 H1N1 pandemic and 83.34%, 79.17%, 75%, and 62.5% reduction in the virus titers were obtained following treatments with scFv-II', I, I', and II, respectively. Real-time PCR demonstrated 98.6%, 95.7%, 95.26%, and 91.19% reductions in virus numbers following neutralization with scFv-II', I, I', and II, respectively.
Anti-HA scFvs selected against highly conserved HA of influenza A virus with high neutralizing effects, offer novel human antibodies for prophylaxis and treatment of a wide range of influenza viruses including different subtypes of H1N1, H3N2, and H5N1 influenza A virus. The antibodies have the potential to be used for universal therapy.
开发具有广泛活性的新型抗体可用于预防和治疗流感。人单链可变片段（scFv）被认为是对抗病毒的有效制剂。在本研究中，筛选出针对甲型流感病毒血凝素（HA）中高度保守表位的特异性人scFv，并评估其中和活性。
采用生物信息学方法评估HA表位。淘选过程从scFv文库中筛选出特异性克隆。PCR和DNA指纹分析区分常见模式。制备scFv的可溶性形式，并使用蛋白质免疫印迹分析进行评估。使用MDCK细胞通过微量中和试验评估抗HA scFv的中和作用。进行实时PCR以确定中和后病毒的确切拷贝数。
生物信息学评估证实了表位的抗原性和可及性。筛选出四种特异性抗HA scFv，即scFv I、II、I'和II'。这些scFv对2009年H1N1大流行毒株具有中和作用，用scFv-II'、I、I'和II处理后，病毒滴度分别降低了83.34%、79.17%、75%和62.5%。实时PCR显示，用scFv-II'、I、I'和II中和后，病毒数量分别减少了98.6%、95.7%、95.26%和91.19%。
针对甲型流感病毒高度保守的HA筛选出的抗HA scFv具有高效中和作用，可为预防和治疗包括H1N1、H3N2和H5N1甲型流感病毒不同亚型在内的多种流感病毒提供新型人源抗体。这些抗体具有用于通用疗法的潜力。
