title: Dengue 2 PreM-E/LAMP chimera targeted to the MHC class II compartment elicits long-lasting neutralizing antibodies.
abstract: A dengue 2 plasmid DNA vaccine (pD2) expressing the pre-membrane and envelope proteins (preM-E) was modified by replacing the dengue transmembrane and cytoplasmic sequences with those of the mouse lysosome-associated membrane protein (pD2/LAMP). Immunofluorescence and confocal microscopy of human 293, NIH 3T3, and macrophage IC21 cell lines transfected with pD2/LAMP showed that the preM-E/LAMP protein chimera was present in vesicles containing endogenous LAMP and major histocompatability complex class II (MHC II), in contrast to the non-vesicular localization of native preM-E protein lacking the LAMP targeting sequence. Mice immunized with pD2 showed an antigen-specific immunoglobulin response but the neutralizing antibodies titers (plaque reduction neutralization test, PRNT(50)) elicited by the native protein were minimal. In contrast, vaccination with pD2/LAMP resulted in PRNT(50) of 270, 320 and 160 at approximately 1, 3 and 8 months after two immunizations with 50 microg DNA, and approached 100% neutralization at 1:20 dilution. Additional immunization with pD2/LAMP, after 8 months, increased the neutralizing antibody titers to >640. Comparable neutralizing antibody responses were induced by two vector backbones, pVR1012 and pVax-1, at 5 and 50 microg of DNA. The neutralizing responses to the pD2/LAMP chimera were greatly superior to those elicited by pD2 in all conditions. These results underscore the importance of MHC class II presentation of DNA-encoded dengue-virus envelope protein for production of neutralizing antibodies.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Lu
authors.forename: Kanakatte
authors.initials: K
authors.lastname: Raviprakash
authors.forename: Ihid C
authors.initials: IC
authors.lastname: Leao
authors.forename: Priya R
authors.initials: PR
authors.lastname: Chikhlikar
authors.forename: Daniel
authors.initials: D
authors.lastname: Ewing
authors.forename: Azlinda
authors.initials: A
authors.lastname: Anwar
authors.forename: Claire
authors.initials: C
authors.lastname: Chougnet
authors.forename: Gerald
authors.initials: G
authors.lastname: Murphy
authors.forename: Curtis G
authors.initials: CG
authors.lastname: Hayes
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: August
authors.forename: Ernesto T A
authors.initials: ET
authors.lastname: Marques
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D019556:COS Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000276:immunology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000276:immunology*; D058851:GPI-Linked Proteins; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D007279:Injections, Subcutaneous; D051379:Mice; D008807:Mice, Inbred BALB C; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology*; D014709:Vero Cells; D014765:Viral Vaccines / Q000008:administration & dosage / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D015816:Cell Adhesion Molecules, Neuronal; D058851:GPI-Linked Proteins; D000949:Histocompatibility Antigens Class II; D007074:Immunoglobulin G; D019444:Vaccines, DNA; D014765:Viral Vaccines; C095362:limbic system-associated membrane protein