Cell Penetrable Human scFv Specific to Middle Domain of Matrix Protein-1 Protects Mice from Lethal Influenza

Abstract

A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1) is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD) and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN). The PEN-scFv (transbody), when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.

Ribosome display for the rapid generation of high-affinity Zika-neutralizing single-chain antibodies

“…scFvs derived from vaccinated cynomolgus macaques were protective against the Ebola-related Marburg virus when tested in infected mice [ 24 ]. Similarly, a human scFv provided protection to mice infected with influenza virus [ 25 ]. scFvs are also promising candidates for anti-cancer therapeutics [ 26 ].…”

Evolution of Therapeutic Antibodies, Influenza Virus Biology, Influenza, and Influenza Immunotherapy

“…To circumvent this obstacle, several delivery systems including cationic liposome [ 94 ], polyethyleneimine (PEI) [ 95 ], and peptides with cell penetrating capacity, called “Cell penetrating peptides, CPPs” [ 96 , 97 ], have been developed for carrying antibodies/antibody fragments and a variety of other cargoes including proteins, drugs, nucleic acids, plasmids, and siRNAs across the plasma membrane into cytosol and also to different subcellular compartments [ 98 – 102 ]. CPPs have been used as a vehicle for cellular import of therapeutic molecules, both in vitro and in vivo [ 84 , 85 , 100 – 104 ]. Examples of CPPs are (1) protein transduction domains (PTDs) such as penetratin (PEN; synonym antennapedia homeodomain peptide of Drosophila melanogaster ) [ 105 ], HIV-1 Tat peptide: Tat 49–57 [ 106 , 107 ], transportan (a 27 residue-peptide from galanin neuropeptide and mastoparan or wasp venom toxin) [ 97 ], and VP-22 peptide of structural protein of herpes simplex virus [ 108 ]; (2) amphipathic peptides such as noncytotoxic sweet arrow peptide (SAP) which is a proline-rich motif (VRLPPP) [ 105 ], peptide vector named MPG derived from the fusion sequence of HIV-1 gp41, and a hydrophilic domain of SV40 nuclear localization sequence [ 101 ]; and (3) other CPP type such as nonaarginine (R9) and poly-lysine [ 109 ].…”

“…Examples of CPPs are (1) protein transduction domains (PTDs) such as penetratin (PEN; synonym antennapedia homeodomain peptide of Drosophila melanogaster ) [ 105 ], HIV-1 Tat peptide: Tat 49–57 [ 106 , 107 ], transportan (a 27 residue-peptide from galanin neuropeptide and mastoparan or wasp venom toxin) [ 97 ], and VP-22 peptide of structural protein of herpes simplex virus [ 108 ]; (2) amphipathic peptides such as noncytotoxic sweet arrow peptide (SAP) which is a proline-rich motif (VRLPPP) [ 105 ], peptide vector named MPG derived from the fusion sequence of HIV-1 gp41, and a hydrophilic domain of SV40 nuclear localization sequence [ 101 ]; and (3) other CPP type such as nonaarginine (R9) and poly-lysine [ 109 ]. In our laboratory, cell penetrable human scFvs and humanized-camel VHs/VHHs specific to viral proteins and toxins have been prepared by linking the antibody molecules to either penetratin or R9 [ 84 , 85 , 103 , 104 , 110 – 112 ]. These fusion proteins readily entered mammalian cells without causing cytotoxicity and bound to their respective intracellular targets.…”

“…Human scFv phage display library constructed in our laboratory [ 56 ] has been used as a biological tool for providing HuscFv display phage clones that bound to the desired influenza virus targets. Recombinant influenza virus proteins with the inherent functional activities or intact virus adsorbed to cell surface were used as antigens in the phage biopanning process [ 103 , 104 , 243 – 245 ]. The antigen bound phages were then put in nonsuppressor E. coli that could not produce tRNA for the stop codon located between the antibody coding gene (huscfv) and the phage p3 gene.…”

“…The transbody reduced the amounts of virus released from the infected cells compared to the infected nontreated cells [ 103 ]. Because the middle domain of the M1 contains regions that mediate multiple pivotal functions of the influenza virus replication cycle, that is, nuclear localization signal (NLS), NEP binding motif, RNA/RNP-binding site, transcription inhibition motif, self-oligomerization domain, and plasma membrane interactive site for assembly and budding [ 161 , 165 , 166 , 170 , 246 – 251 ], we also produced cell penetrable HuscFv (transbody) to the M1-MD [ 104 ]. This transbody extricated mice from lethal infection with mouse adapted HPAI H5N1 by mitigating symptom severity and reducing lung histopathology of the treated infected mice.…”