title: West Nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome-associated membrane protein: increased cellular concentration of the transgene product, targeting to the MHC II compartment, and enhanced neutralizing antibody response.
abstract: A genetic vaccine for West Nile virus (WN) has been synthesized with the WN premembrane-envelope (WN preM-E) gene sequences encoded as a chimera with the transmembrane and carboxyl terminal domains of the lysosome-associated membrane protein (LAMP). The LAMP sequences are used to direct the antigen protein to the major histocompatibility class II (MHC II) vesicular compartment of transfected professional antigen-presenting cells (APCs). Vaccine constructs encoding the native WN preM-E and WN preM-E/LAMP chimera were synthesized in pVAX1 and pITR plasmid backbones. Extracts of human fibroblast 293 and monkey kidney COS-7 cells transfected with the WN preM-E/LAMP chimera constructs contained much greater amounts of E than did the cells transfected with constructs encoding the native WN preM-E. This difference in the concentration of native E and the E/LAMP chimera in transfected cells is attributed to the secretion of native E. The amount of preM protein in cell extracts, in contrast to the E protein, and the levels of DNA and RNA transcripts, did not differ between WN preM-E- and WN preM-E/LAMP-transfected cells. Additionally, confocal and immunoelectron microscopic analyses of transfected B cells showed localization of the WN preM-E/LAMP chimera in vesicular compartments containing endogenous LAMP, MHC II, and H2-M, whereas native viral preM-E lacking the LAMP sequences was distributed within the cellular vesicular network with little LAMP or MHC II association. Mice immunized with a DNA construct expressing the WN preM-E/LAMP antigen induced significant antibody and long-term neutralization titers in contrast to the minimal and short-lived neutralization titer of mice vaccinated with a plasmid expressing the untargeted antigen. These results underscore the utility of LAMP targeting of the WN envelope to the MHC II compartments in the design of a genetic WN vaccine.
authors.affiliation: The Johns Hopkins University School of Medicine, Department of Pharmacology and Molecular Sciences, 725 North Wolfe Street, Baltimore, MD 21205, USA. azlinda@hopkins.edu.sg
authors.forename: Azlinda
authors.initials: A
authors.lastname: Anwar
authors.forename: Ananth
authors.initials: A
authors.lastname: Chandrasekaran
authors.forename: Mah Lee
authors.initials: ML
authors.lastname: Ng
authors.forename: Ernesto
authors.initials: E
authors.lastname: Marques
authors.forename: J Thomas
authors.initials: JT
authors.lastname: August
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D014902:West Nile virus / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity
chemical_list: D000914:Antibodies, Viral; D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D014759:Viral Envelope Proteins