Samaneh Alizadeh - Recombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran
Setareh Moazen - Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver BC, Canada
Seyed Nooreddin Faraji - Recombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran
Afagh Moattari - Department of Bacteriology and Virology, Shiraz University of Medical Sciences, Shiraz, Iran
Foroogh Nejatollahi - Recombinant Antibody Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran

Objective(s): 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.

Epitope design, Hemagglutinin, Immunotherapy, Influenza A virus, Neutralizing scFvs