A Novel Mechanism Underlying Antiviral Activity of an Influenza Virus M2-Specific Antibody

Abstract

Protective immunity against influenza A viruses (IAVs) generally depends on antibodies to the major envelope glycoprotein, hemagglutinin (HA), whose antigenicity is distinctive among IAV subtypes. On the other hand, the matrix 2 (M2) protein is antigenically highly conserved and has been studied as an attractive vaccine antigen to confer cross-protective immunity against multiple subtypes of IAVs. However, antiviral mechanisms of M2-specific antibodies are not fully understood. Here, we report the molecular basis of antiviral activity of M2-specific monoclonal antibody (mAb), rM2ss23. We first found that rM2ss23 inhibited A/Aichi/2/1968 (H3N2) (Aichi) but not A/PR/8/1934 (H1N1) (PR8) replication. rM2ss23 altered the cell surface distribution of M2, likely by crosslinking the molecules, and interfered with colocalization of HA and M2 resulting in reduced budding of progeny viruses. However, these effects were not observed for another strain, PR8, despite the binding capacity of rM2ss23 to PR8 M2. Interestingly, HA was also involved in the resistance of PR8 to rM2ss23. We also found that two amino acid residues at positions 54 and 57 in the M2 cytoplasmic tail were critical for the insensitivity of PR8 to rM2ss2. These findings suggest that disruption of the M2-HA colocalization on infected cells and subsequent reduction of virus budding is one of the principal mechanisms of antiviral activity of M2-specific antibodies and that anti-M2 antibody-sensitive and -resistant IAVs have different properties of the interaction between M2 and HA. IMPORTANCE Although the IAV HA is the major target of neutralizing antibodies, most of the antibodies are HA subtype-specific, restricting the potential of HA-based vaccines. On the contrary, the IAV M2 protein has been studied as a vaccine antigen to confer cross-protective immunity against IAVs with multiple HA subtypes since M2 is antigenically conserved. Although a number of studies highlight the protective role of anti-HA neutralizing and non-neutralizing antibodies, precise information on the molecular mechanism of action of M2-specific antibodies is still obscure. In this study, we found that an anti-M2 antibody interfered with the HA-M2 association, which is important for efficient budding of progeny virus particles from infected cells. The antiviral activity was IAV strain-dependent despite the similar binding capacity of the antibody to M2, and interestingly, HA was also involved in the susceptibility to the antibody. Our data provides a novel mechanism underlying antiviral activity of M2-specific antibodies.

A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein

“…It has been reported that M2 proteins are involved in budding off of the viruses in the filamentous form from the infected cells [ 34 , 35 ]. Earlier reported M2-specific antibodies showed inhibition of the filamentous form of the virion as well as disturbed the even distribution of expressed M2 protein on the host cell membrane [ 36 , 37 ].…”

Influenza Viruses: Harnessing the Crucial Role of the M2 Ion-Channel and Neuraminidase toward Inhibitor Design

“…This monoclonal antibody (14c2) recognized the ectodomain of the protein, and it was able to spot M2 on the virions, thus reducing viral growth through the size reduction in lytic plaques [ 161 ]. Manzoor et al in 2020 [ 162 ] examined the anti-viral activity of monoclonal antibody rM22223 and found that rM2ss23 inhibited A/Aichi/2/1968 (H3N2) (Aichi) but not A/PR/8/1934 (H1N1) (PR8) replication. Amino acid residues at positions 54 and 57 in the M2 cytoplasmic tail were also discovered to be important for the sensitivity to rM2ss23.…”

YWHAG inhibits influenza a virus replication by suppressing the release of viral M2 protein

“…Similarly, the M2 protein plays important roles in the viral life cycle ( Chauhan and Gordon, 2022 ). M2 contains 97 amino acid residues and assembles into a homotetramer to constitute three different domains that include a 24-residue N-terminal ectodomain (ED), a single 19-residue transmembrane domain (TM) that forms the pore, and a long 54-residue carboxyterminal cytoplasmic domain called cytoplasmic tail (CT) ( Mezhenskaya et al, 2019 ; Manzoor et al, 2020 ; Okuya et al, 2020 ; Movellan et al, 2021 ), which is highly conserved among viral strains ( Wohlgemuth et al, 2018 ; Kim et al, 2019 ).…”