Enhancing neutralizing activity against influenza H1N1/PR8 by engineering a single-domain VL-M2 specific into a bivalent form

Abstract

Flu disease, with high mortality and morbidity, is caused by the influenza virus. Influenza infections are most effectively prevented through vaccination, but it requires annual reformulation due to the antigenic shift or drift of hemagglutinin and neuraminidase proteins. Increasing resistance to available anti-influenza drugs was also recently reported. The M2 surface protein of the influenza virus is an attractive target for universal vaccine development as it is highly conserved and multifunctional throughout the viral life cycle. This study aimed to discover a single-chain variable fragment (scFv) targeting the M2 protein of influenza A H1N1/PR8, showing neutralizing activity through plaque inhibition in virus replication. Several candidates were isolated using bio-panning, including scFv and single-domain VLtarget M2 protein, which was displayed on the yeast surface. The scFv/VLproteins were obtained with high yield and high purity through soluble expression inE.coliBL21 (DE3) pLysE strains. A single-domain VL-M2-specific antibody, NVLM10, exhibited the highest binding affinity to influenza virions and was engineered into a bivalent format (NVL2M10) to improve antigen binding. Both antibodies inhibited virus replication in a dose-dependent manner, determined using plaque reduction- and immunocytochemistry assays. Furthermore, bivalent anti-M2 single-domain VLantibodies significantly reduced the plaque number and viral HA protein intensity as well as viral genome (HAandNP) compared to the monovalent single-domain VLantibodies. This suggests that mono- or bivalent single-domain VLantibodies can exhibit neutralizing activity against influenza virus A, as determined through binding to virus particle activity.