Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo.
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.
Pushko
P
P
Virology Division, U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick, Maryland 21702, USA.
Parker
M
M
Ludwig
G V
GV
Davis
N L
NL
Johnston
R E
RE
Smith
J F
JF
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000276:immunology; D000918:Antibody Specificity; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins; D002460:Cell Line; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003673:Defective Viruses / Q000502:physiology*; D004372:Ducks; D004666:Encephalitis Virus, Venezuelan Equine / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics*; D006378:Helper Viruses / Q000502:physiology*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D009980:Influenza A virus / Q000235:genetics / Q000276:immunology*; D007836:Lassa virus / Q000235:genetics / Q000276:immunology*; D008647:Mesocricetus; D051379:Mice; D008807:Mice, Inbred BALB C; D009976:Orthomyxoviridae Infections / Q000517:prevention & control; D012313:RNA / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012093:Replicon; D014611:Vaccination; D014613:Vaccines, Attenuated / Q000276:immunology; D017778:Vaccines, Combined / Q000235:genetics* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000235:genetics* / Q000276:immunology; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology
D000914:Antibodies, Viral; D036022:Capsid Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; C039919:RNA, recombinant; D011993:Recombinant Fusion Proteins; D014613:Vaccines, Attenuated; D017778:Vaccines, Combined; D014614:Vaccines, Synthetic; D015678:Viral Structural Proteins; D014765:Viral Vaccines; C110151:nucleocapsid protein, lassa virus; D012313:RNA