title: Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.
abstract: We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.
authors.affiliation: Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Rodrigo Maciel da Costa
authors.initials: RM
authors.lastname: Godinho
authors.affiliation: Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Flavio Lemos
authors.initials: FL
authors.lastname: Matassoli
authors.affiliation: Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Carolina Gonçalves de Oliveira
authors.initials: CG
authors.lastname: Lucas
authors.affiliation: Laboratorio de Dermatologia e Imunodeficiencias, LIM-56, Departamento de Dermatologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Paula Ordonhez
authors.initials: PO
authors.lastname: Rigato
authors.affiliation: Departamento de Imunologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Jorge Luiz Santos
authors.initials: JL
authors.lastname: Gonçalves
authors.affiliation: Laboratorio de Dermatologia e Imunodeficiencias, LIM-56, Departamento de Dermatologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Maria Notomi
authors.initials: MN
authors.lastname: Sato
authors.affiliation: Enteric Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, Maryland, United States of America; Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Milton
authors.initials: M
authors.lastname: Maciel
authors.affiliation: Departamento de Imunologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ligia Maria Torres
authors.initials: LM
authors.lastname: Peçanha
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: J Thomas
authors.initials: JT
authors.lastname: August
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America; Department of Infectious Diseases and Microbiology, Center for Vaccine Research, Pittsburgh, Pennsylvania, United States of America; Departamento de Virologia, Fiocruz - Pernambuco, Recife, Brazil.
authors.forename: Ernesto Torres de Azevedo
authors.initials: ET
authors.lastname: Marques
authors.affiliation: Departamento de Virologia, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Luciana Barros
authors.initials: LB
authors.lastname: de Arruda
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D011992:Endosomes / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007114:Immunization; D008213:Lymphocyte Activation / Q000276:immunology; D052118:Lysosomal-Associated Membrane Protein 1 / Q000737:chemistry* / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055571:Secretory Pathway; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism
chemical_list: D052118:Lysosomal-Associated Membrane Protein 1; D012333:RNA, Messenger; D054301:gag Gene Products, Human Immunodeficiency Virus