Samaneh Alizadeh1, Setareh Moazen2, Seyed Nooreddin Faraji1, Afagh Moattari3, Foroogh Nejatollahi1,4. 1. Recombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran. 2. Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver BC, Canada. 3. Department of Bacteriology and Virology, Shiraz University of Medical Sciences, Shiraz, Iran. 4. HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran.
Abstract
OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Entities:
Keywords:
Epitope design; Hemagglutinin; Immunotherapy Influenza A virus; Neutralizing scFvs
Authors: Constantinos S Kyriakis; Ming Zhang; Stefan Wolf; Les P Jones; Byoung-Shik Shim; Anna H Chocallo; Jarod M Hanson; MingRui Jia; Dong Liu; Ralph A Tripp Journal: Vet Microbiol Date: 2017-10-18 Impact factor: 3.293