Crystal Structure of the Conserved Amino Terminus of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus Gripped by an Antibody

Abstract

c; Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium dWe report the crystal structure of the M2 ectodomain (M2e) in complex with a monoclonal antibody that binds the amino terminus of M2. M2e extends into the antibody binding site to form an N-terminal ␤-turn near the bottom of the paratope. This M2e folding differs significantly from that of M2e in complex with an antibody that binds another part of M2e. This suggests that M2e can adopt at least two conformations that can elicit protective antibodies. Human influenza viruses can escape antibody-mediated immunity by varying the antigenic sites in their surface antigens that are most prone to virus neutralization. Vaccines that elicit antibody responses against conserved surface-exposed antigens, such as the stem region of influenza hemagglutinin or M2e, can protect against multiple influenza virus subtypes (1). A potential clinical benefit of M2e-based vaccines can be inferred from a challenge study in human volunteers, which showed that injection of human monoclonal antibody (MAb) TCN-032 directed against M2e 1 day after inoculation of an H3N2 influenza virus resulted in reduced clinical signs and virus shedding compared to placebo treatment (2).M2e corresponds to the surface exposed amino-terminal part of M2 and is 23 amino acid residues long (3). The first nine amino acid residues of M2 are extremely conserved across all reported influenza A viruses. Human and murine M2e-specific MAbs that recognize different parts of M2e have been described, but how these interact with M2e is largely unknown (4-6). Here, we report the crystal structure of M2e in complex with a Fab fragment from a MAb that binds to the highly conserved N-terminal part of M2.The ethical committee of Ghent University approved this mouse protection study. We isolated MAb148 from a BALB/c mouse that had been immunized with a recombinant tetrameric protein of M2e using conventional hybridoma technology (7). Intraperitoneal injection of 200 g of this IgG1 MAb protected mice against a potentially lethal challenge with an H1N1 virus (Fig. 1A). To identify the epitope of MAb148, we transfected a series of alanine mutants of M2 into HEK293T cells. Transfected cells were subsequently fixed and used in an enzyme-linked immunosorbent assay (ELISA). This analysis revealed that M2 residues Ser2, Leu3, Leu4, and Thr5 are critical for MAb148 binding (Fig. 1B). A similar N-proximal epitope in M2e was reported for human MAb TCN-031 and -032 (8). ELISA analysis using wells coated with M2e peptide variants showed MAb148 binding was indifferent to most of the sequence variation in the M2e segment spanning residues 9 to 20 (Fig. 1C). However, we noted reduced binding to the M2e peptide derived from A/Hong Kong/485/97 (Fig. 1C). Also a shorter M2e peptide (SLLTEVETPIRN) proved less accessible for MAb148 binding (Fig. 1C), though in solution this peptide competed with MAb148 for binding the coating full-length M2e peptide as efficiently as full-length M2e (Fig. 1D), suggesting that reduced re...

Influenza A Virus M2 Protein: Roles from Ingress to Egress

“…In DMPC bilayers, some residues show a β-strand conformation that shifts to an α-helical conformation in the presence of cholesterol (mimicking the viral membrane) [ 74 , 75 ]. It is worthwhile to mention that crystallography of M2 ED—Fab complex also suggested that M2-ED exists in different conformations supporting the notion that M2-ED is intrinsically disordered [ 76 , 77 ]. The post-APH CT up to residue 71 is largely unstructured with random coil conformation, highly mobile and, unlike the ED, insensitive to the membrane composition [ 75 , 78 ].…”

Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

“…Earlier we reported the crystal structures of M2e in complex with the Fab fragment of two different mouse monoclonal antibodies (MAb 65 and MAb 148) that recognize partially overlapping epitopes of M2e (50,58). In these two crystal structures, M2e adopted two different conformations.…”

M2e-tetramer-specific memory CD4 T cells are broadly protective against influenza infection

“…10 Interestingly, a B-cell epitope in M2e includes this range of aa 7-17, but it appears that differences in aa at certain positions 13, 14, or 18 between different strains do not markedly affect the protective efficacy of M2e immunization, whereas mutations in aa 10, 11, or 16 reduce the antibody-binding affinity. [85][86][87] Considering that M2e-specific CD4 T cells are also protective, some earlier attempts at correlating protective efficacy following M2e immunization to antibody-binding specificities exclusively may have to be revisited. A recently published report from the World Health Organization Product Development for Vaccines Advisory Committee identified 41 universal influenza vaccine candidates at various stages of development; a list that includes our own CTA1-3M2e-DD.…”

“…A tetrameric M2e vaccine formulation has been found to enhance anti-M2e antibody responses, but it would not necessarily be required for the generation of protective memory CD4 T cells, for which a less sophisticated vaccine based on peptides or monomers would be sufficient. [85][86][87] The CTA1-DD adjuvant has previously been shown to be non-toxic in mice and monkeys, it does not drive inflammation at the site of application and, importantly, it does not bind GM1-ganglioside receptors or accumulate in the central nervous tissues following i.n. administration as cholera toxin and other holotoxin adjuvants do.…”