Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication

Abstract

BackgroundNovel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies.MethodsFully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking.ResultsHuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages.ConclusionsHuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

“…19 Nowadays, recombinant antibodies to any epitope can be rapidly produced in vitro (regardless of the tissue culture facility and immunogenicity and toxicity of the antigen) from E. coli bacteria transfected with the antigen-binding phage selected from an antibody phage display library. [20][21][22][23][24][25] The engineered antibodies can also be made into intact four-chain molecules when the biological activities of the Fc fragment are needed for the therapeutic effect. Alternatively, smaller antibody fragments that are devoid of the Fc fragment or constant domains, e.g., scFv 21,22 or single domain antibodies (nanobodies/VH/ V H H) 13,[23][24][25] can be used to increase the tissue penetrating ability and reduce tissue inflammation.…”

“…A competitive ELISA as described previously 13,22 was used for validating the phage mimotopes and the NS5B epitopes bound by the scFvs. The mimotopic phages (M14, M34/1, M34/2 and M34/3) were propagated in ER2738 E. coli and their titers were determined.…”

“…To assess the mechanisms of the scFv-mediated inhibitions of the RdRp activity and the HCV replication, the regions and residues of the NS5B molecule bound by the scFvs were determined by means of 12 mer-peptide phage mimotope search 21,22 and computerized homology modeling and molecular docking. [31][32][33][34][35][36][37][38] The NS5B of HCV genotype 3a's linear sequence (accession number NP751928) was used in the alignment with the scFv mimotope sequences for tentative epitope identification and the 3D structure of PDB 1C2P was used in the molecular docking with the scFv 3D structures.…”

Influenza virus antigenicity and broadly neutralizing epitopes

“…These antibodies are rare in infected humans but they might be protective if they could be induced by recombinant M2, or amplified in vitro and given therapeutically. Both approaches have been tested and shown to be effective in animal models [8–10]. There is one report of a Phase 2 clinical trial of antibody TCN-032 which showed some reduction in symptoms but no protection from infection in volunteers challenged with influenza virus [11].…”

Autophagy, a new target for disease treatment

“…Autophagy-blocking agents might be useful as prophylactics and therapeutics against human infection by the H5N1 virus [29]. An anti-influenza agent, human monoclonal ScFv, has anti-autophagic function [30]. Recently, a new avian influenza virus, H7N9, emerged as a serious threat to humans.…”