An Influenza A Vaccine Based on Tetrameric Ectodomain of Matrix Protein 2

Abstract

Matrix protein 2 (M2) of influenza A is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain (M2e) has remained nearly invariable since the first human influenza strain was isolated in 1933. By linking a modified form of the leucine zipper of the yeast transcription factor GCN4 to M2e, we obtained a recombinant tetrameric protein, M2e-tGCN4. This protein mimics the quaternary structure of the ectodomain of the natural M2 protein. M2e-tGCN4 was purified, biochemically characterized, and used to immunize BALB/c mice. High M2e-specific serum IgG antibody titers were obtained following either intraperitoneal or intranasal administration. Immunized mice were protected fully against a potentially lethal influenza A virus challenge. Antibodies raised by M2e-tGCN4 immunization specifically bound to the surface of influenza-infected cells and to an M2-expressing cell line. Using a M2e peptide competition enzymelinked immunosorbent assay with M2-expressing cells as target, we obtained evidence that M2e-tGCN4 induces antibodies that are specific for the native tetrameric M2 ectodomain. Therefore, fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies.Influenza has one of the highest infection rates of all human viruses and can kill healthy persons of all ages (1). It is estimated that influenza infection during seasonal epidemics kills 1 in 1000 infected individuals, whereas an unpredictable pandemic is likely to kill millions. In addition, increased hospitalization and absenteeism from school and work are direct consequences of the flu. At present, the best way to protect against influenza is to vaccinate against the ever-mutating strains (2). However, antigenic drift and occasional shift of the two major membrane glycoproteins, hemagglutinin and neuraminidase, make vaccine production cumbersome and necessitate yearly revision of the vaccine seed strains by the World Health Organization.Influenza A also encodes a third integral membrane protein, M2, 2 a homotetramer, the subunit of which has a small external domain (M2e) of 23 amino acid residues (3). Natural M2 protein is present in a few copies in the virus particle but in abundance on virus-infected cells (4). In contrast to hemagglutinin and neuraminidase, M2e is almost nonimmunogenic (5), and its sequence is highly conserved. Capitalizing on these properties, we developed a universal influenza A vaccine by linking the M2e peptide to a virus-like particle based on the hepatitis B virus core (HBc) (6). In this context, M2e is highly immunogenic, and the M2e-HBc vaccine induces antibodies that protect mice against influenza-induced death and morbidity.Oligomeric proteins found in vaccines derived by inactivating or attenuating a pathogen often function as their major antigenic determinant. Conformational epitopes embedded in the quaternary structures may critically con...

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

“…The GCN4 elements used in this study were indeed immunogenic (Fig. 3C and D), consistent with other reports [38]. However, other variants were shown to be not immunogenic [37].…”

“…The first strategy explores GCN4 as flanking structurepromoting elements within the antigen module. GCN4 is well characterized and has been used as an ␣-helical scaffold [35,36] or as a helix promoter [37,38]. We previously reported the inclusion of GCN4 in an antigenic module designed to display Group A Streptococcal antigen on a VLP [9].…”

Structure of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus in Complex with a Protective Monoclonal Antibody

“…Monoclonal antibody 65 (MAb 65) is a mouse IgG2a antibody isolated using conventional hybridoma technology and splenocytes from BALB/c mice immunized with M2e-tGCN4 (27). MAb 65 (3 mg) was treated with papain at a ratio of 1:50 (wt/wt) for 3 h in phosphate-buffered saline (PBS) buffer with 5 mM L-cysteine and 5 mM EDTA at 37°C.…”

New technologies for influenza vaccines

“…Because the transmembrane portion of M2 is tetrameric, it is likely that the extracellular portion also associates into a larger quadrivalent structure. [165][166][167] There is little M2 on an influenza virion, but much more is presented on the surface of infected cells, and infected humans do produce antibodies recognizing M2e. 168 Antibodies against M2e do not neutralize virus but do provide weak protection in rodent models of infection, probably on the basis of antibody-dependent cellular cytotoxicity (ADCC).…”