Replicon-Helper Systems from Attenuated Venezuelan Equine Encephalitis Virus: Expression of Heterologous Genesin Vitroand Immunization against Heterologous Pathogensin Vivo

Abstract

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.

Development and preclinical evaluation of an alphavirus replicon vaccine for influenza

“…VRP were produced using a modification of previously described methods [16,17]. Purified DNA plasmids were linearized by NotI endonuclease digestion and used as templates for in vitro RNA transcription using RNA Express T7 kits (Promega, Madison, WI).…”

Development of vaccines for prevention of botulism

“…Pushko et al have demonstrated that when a heterologous gene encoding influenza hemagglutanin (HA) or the Lassa virus nucleocapsid (N) is substituted for the VEE structural proteins followed by transfection into eukaryotic cells, the replicon system can express high levels of HA or N proteins [44]. When packaged into virus-like particles, these replicons have Development of vaccines for prevention of botulismalso been found to induce potent immune responses in inoculated animals against the heterologous proteins, and to protect animals against challenge with the heterologous virus.…”

Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus

“…VRP were produced by electroporation of Vero cells with replicon RNA and two helper RNAs, encoding the Venezuelan equine encephalitis (VEE) virus structural proteins, using modifications of previously described methods [16,17]. VRP concentration, expressed as infectious units (IU) per mL, was determined by an immunofluorescence assay in which serial dilutions of VRP were added to Vero cell monolayers, cultured overnight, reacted with goat anti-gB or anti-pp65 antibody [12] followed by fluorescein isothiocyanate-labeled anti-goat antibody to detect cells expressing the CMV protein.…”