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 enzyme-linked 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.
Highlights
Cine production cumbersome and necessitate yearly revision of the vaccine seed strains by the World Health Organization
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]
Purification and Characterization of M2e-tGCN4—We constructed a bacterial expression vector to produce M2e fused at its C terminus to the GCN4-derived tetramerizing leucine zipper
Summary
Cine production cumbersome and necessitate yearly revision of the vaccine seed strains by the World Health Organization. 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. When producing influenza split vaccines, the hemagglutinin and neuraminidase oligomeric antigens may lose their oligomeric structure during the virus disruption step, or they may form aggregates. By using a competitive ELISA, we provide evidence that this vaccine elicits antibodies that bind to conformational epitopes on the ectodomain of natural M2 protein
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