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.

Dendritic cells primed with a chimeric plasmid containing HIV‐1‐ gag associated with lysosomal‐associated protein‐1 (LAMP/ gag ) is a potential therapeutic vaccine against HIV

“…We then investigated the ability of these cells to present the Gag antigen. BALB/c mice were inoculated twice subcutaneously with LAMP/gag plasmid, which generated a significant expansion of Gag-specific T cells, in accordance with our previous studies (30)(31)(32)(33). Lymph node T cells obtained from LAMP/gag-immunized mice (Gagspecific T cells) were cocultured with autologous Lg-mDCs.…”

“…We have demonstrated that association of HIV-1-p55gag (gag, group-specific antigen) with lysosomeassociated membrane protein (LAMP)-1, in the form of naked DNA vaccine (LAMP/gag vaccine) targeted Gag to major histocompatability complex (MHC) II compartments and elicited a diverse, prolonged, and more potent immune response, in mouse and primate experimental models, in comparison to native gag (gag N ) (30)(31)(32)(33)(34). Indeed, immunization with different gag-based DNA vaccines has been shown to induce specific immune response in experimental models (35); however, DNA alone has also shown low immunogenicity in human trials (36).…”

Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens

“…Another approach for targeting antigenic peptides to the MHCII presentation pathways had been demonstrated by Wu et al by fusing the HPV-16 E7 protein to LAMP1 (lysosomal-associated membrane protein 1)-sequences 21 22 23 . That approach targeted the protein into the lysosomal compartments.…”

Molecular mechanisms for enhanced DNA vaccine immunogenicity

“…Subcellular targeting is another strategy for enhancing plasmid-encoded antigen processing and/or presentation. Using E1A targeting to the endoplasmic reticulum or LAMP targeting to lysosome can greatly enhance DNA vaccine efficacy [133,134]. Targeting of the autophagy pathway enhanced the efficacy of tuberculosis DNA vaccines [135][136][137].…”