RESUMEN
The extracellular domain of influenza A virus matrix protein 2 (M2e) is strongly conserved. Therefore, vaccines based on M2e can induce broad-spectrum immunity against influenza. We have mainly used recombinant virus-like particles derived from Hepatitis B virus core (HBc) as carrier for efficacious presentation of the M2e antigen. Here, we address whether pre-existing HBc-specific immunity interferes with the protective immune response obtained by M2e-HBc vaccination. Anti-HBc antibodies were induced by immunizing mice with unsubstituted HBc virus-like particles in the presence of two different adjuvants. We demonstrate that pre-existing HBc-specific antibodies affect neither the induction of M2e-specific antibody responses to vaccination with M2e-HBc particles, nor the protective efficacy of the resulting response. These results suggest that vaccination with M2e-HBc can induce protective anti-M2e antibodies even in anti-HBc positive individuals. The implications of these findings are discussed in the context of the clinical development of an M2e-based universal influenza vaccine, which recently successfully completed a Phase I trial.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Femenino , Virus de la Influenza A/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Proteínas Recombinantes de Fusión/inmunología , Carga ViralRESUMEN
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.
Asunto(s)
Vacunas contra la Influenza/química , Proteínas de la Matriz Viral/química , Animales , Cromatografía en Gel , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Inmunoglobulina G/química , Espectrometría de Masas/métodos , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Conformación Molecular , Péptidos/química , Estructura Terciaria de ProteínaRESUMEN
M2 is the third integral membrane protein of influenza A. M2e, the extracellular, 23 amino acid residues of M2, has been remarkably conserved in all human influenza A strains. This prompted us to evaluate the use of M2e as a potential broad-spectrum immunogen in a mouse model for influenza infection. Genetic fusion of the M2e and hepatitis B virus core (HBc) coding sequences allowed us to obtain highly immunogenic virus-like particles. This M2e-HBc vaccine induced complete protection in mice against a lethal influenza challenge. Protective immunity was obtained regardless of the position of M2e in the M2e-HBc chimera at the amino-terminus or inserted in the immuno-dominant loop of the HBc protein. Increasing the copy number of M2e inserted at the N-terminus from one to three per monomer (240-720 per particle) significantly enhanced the immune response and reduced the number of vaccinations required for complete protection against a lethal challenge with influenza A virus. A series of M2e-HBc constructs was subsequently combined with CTA1-DD, a recombinant cholera toxin A1 derived mucosal adjuvant, to test its efficacy as an intranasally delivered vaccine. All hybrid VLPs tested with CTA1-DD completely protected mice from a potentially lethal infection and, in addition, significantly reduced morbidity. Overall, increased resistance to influenza challenge in the mice correlated with an enhanced Th1-type M2e-specific antibody response induced by vaccination. These results show that M2e is a valid and versatile vaccine candidate to protect against any strain of human influenza A.