ABSTRACT
Influenza virus-like particles (VLPs) are noninfectious and the assembly of influenza VLPs depends on the interactions of M1 proteins and/or other viral surface proteins, such as HA, NA, and M2, with the cellular lipid membranes. In this study we propose that M2 protein can be used as a molecular fabricator without disrupting the assembly of VLPs and while retaining the native structures of HA and NA envelope protein oligomers on the particle surfaces. First, we demonstrated that influenza VLPs can be fabricated by the M2 fusion of enhanced green fluorescent protein for imaging single virus entering A549 cells. Second, we engineered two molecular adjuvants (flagellin and profilin) fused to M2 protein to generate molecular adjuvanted VLPs. Theses molecular adjuvanted VLPs had stimulatory functions, including increasing TNF-α production and promoting the maturation of dendritic cells. Immunization of mice with molecular adjuvanted VLPs also enhanced the response of the neutralizing antibodies against homologous and heterologous H5N1 viruses. The results can provide useful information for imaging single viruses and designing novel vaccines against influenza virus infection.
Subject(s)
Biotechnology/methods , Influenza A Virus, H5N1 Subtype/genetics , Influenza Vaccines/genetics , Influenza Vaccines/metabolism , Technology, Pharmaceutical/methods , Viral Matrix Proteins/genetics , Adjuvants, Immunologic/genetics , Adjuvants, Immunologic/metabolism , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Cell Line , Dendritic Cells/immunology , Flagellin/genetics , Flagellin/metabolism , Genetic Engineering/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Influenza A Virus, H5N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/metabolism , Influenza Vaccines/administration & dosage , Influenza Vaccines/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neuraminidase/chemistry , Neuraminidase/genetics , Neuraminidase/immunology , Neuraminidase/metabolism , Orthomyxoviridae/genetics , Profilins/genetics , Profilins/metabolism , Protein Conformation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Staining and Labeling , Tumor Necrosis Factor-alpha/metabolism , Vaccines, Virosome/administration & dosage , Vaccines, Virosome/genetics , Vaccines, Virosome/immunology , Vaccines, Virosome/metabolism , Viral Matrix Proteins/immunology , Viral Matrix Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolismABSTRACT
We have constructed virus-like particles (VLPs) harboring hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) ,and proton channel protein (M2) using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP) was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.
