ABSTRACT
MicroRNA (miRNA) plays essential roles in regulating gene expression, but miRNA delivery remains a hurdle, thus entailing a vector system for efficient transfer. Baculovirus emerges as a promising gene delivery vector but its inherent transient expression restricts its applications in some scenarios. Therefore, this study primarily aimed to develop baculovirus as a miRNA expression vector for prolonged gene suppression. We constructed recombinant baculoviruses carrying artificial egfp-targeting miRNA sequences within the miR155 backbone, which after expression by the cytomegalovirus promoter could knockdown the enhanced green fluorescent protein (EGFP) expression in a sequence- and dose-dependent manner. By swapping the mature miRNA sequences, the baculovirus miRNA shuttle effectively repressed the overexpression of endogenous TNF-α in arthritic synoviocytes without inducing apoptosis. To prolong the baculovirus-mediated expression, we further developed a hybrid baculovirus vector that exploited the Sleeping Beauty (SB) transposon for gene integration and sustained miRNA expression. The hybrid baculovirus vector that combined the miR155 scaffold and SB transposon effectively repressed the transgene expression for a prolonged period of time, hence diversifying the applications of baculovirus to indications necessitating prolonged gene regulation such as arthritis.
Subject(s)
Baculoviridae/genetics , Biological Products/pharmacology , Drug Carriers , Genetic Vectors , MicroRNAs/pharmacology , Transduction, Genetic , Cell Line , DNA Transposable Elements , Gene Expression Regulation , Genes, Reporter , Genomic Instability , Green Fluorescent Proteins/antagonists & inhibitors , Green Fluorescent Proteins/genetics , Humans , MicroRNAs/genetics , Mutagenesis, Insertional , Recombination, Genetic , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Baculovirus is an enveloped virus that infects insects in nature and has emerged as a novel vaccine vector. We previously constructed a recombinant baculovirus displaying the hemagglutinin protein (HA) of avian influenza virus (AIV) on the viral envelope (Bac-HA64), and demonstrated the induction of humoral responses in immunized mice. To improve the vector design and explore how the vector forms influence the vaccine efficacy, we constructed two more baculoviruses Bac-CHA and Bac-CHA/HA64. Bac-CHA expressed HA after transducing the host cells while Bac-CHA/HA64 not only expressed HA but also displayed HA on the envelope. After administration into BALB/c mice, all three vectors elicited HA-specific humoral (IgG1, IgG2a and hemagglutination inhibition titers), mucosal (IgA titers) and cellular (interferon (IFN)-γ and IL-4 producing T cells and IFN-γ(+)/CD8(+) T cells) immune responses. Intriguingly, the magnitudes and types of responses hinged on the vaccine form and administration route. Via intranasal (i.n.) and subcutaneous (s.c.) inoculation, the HA-displaying vectors Bac-HA64 and Bac-CHA/HA64 triggered stronger humoral and mucosal responses than Bac-CHA, but upon intramuscular (i.m.) injection the HA-expressing vectors (Bac-CHA and Bac-CHA/2HA64) elicited more robust humoral and cellular responses than Bac-HA64. Via either administration route, the dual form vaccine Bac-CHA/HA64 gave rise to superior or at least comparable HA-specific immune responses than the other two vaccine forms, implicating the potential of Bac-CHA/HA64 as a vaccine candidate against AIV infection.
