Dendritic Cell Mediated Delivery of Plasmid DNA Encoding LAMP/HIV-1 Gag Fusion Immunogen Enhances T Cell Epitope Responses in HLA DR4 Transgenic Mice

Abstract

This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA1*0101, DRB1*0401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1×105 immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-γ ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a) the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b) the value of HLA transgenic mice as a model system for the identification and evaluation of epitope-based vaccine strategies, and c) the application of variability analysis across reported sequences in public databases for selection of historically conserved HIV epitopes as vaccine targets.

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

“…Several studies have shown correlation between epitopes identified in HLA TgM and humans [42], [43], [53]. Herein, we have identified a vast repertoire of potential DENV3 T-cell epitopes by use of TgM, with HLA affinity validated for many by use of binding assay, and immunogenicity confirmed for a subset in HLA-matched subjects with history of dengue infection.…”

“…This strategy was applied to HLA transgenic mice, an effective animal model to use for identifying potential epitopes recognized by human T-cells. The repertoire of epitopes identified in these animal models has been correlated with those identified in humans [41], [42], [43], and thus, the strategy was proven to be an effective platform for epitope discovery [44].…”

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

“…However, different from what was observed after immunization with gag N , Lg-DNA vaccine induced a very diverse immune response (33,37), indicating that the strategy is worth testing in the human population.…”

“…Also, initial analysis of the use of primary DCs in HLA-DR transgenic mice indicated that this strategy induces a diverse T-cell response (37). However, we did not address exclude DNA itself as an adjuvant, gag-containing constructs induced a much higher stimulation than naked vector, indicating that the antigen activated the DCs.…”

“…Therefore, we hypothesized that the use of DCs as a delivery system for LAMP/gag DNA (Lg-DNA) would be a promising strategy to further improve the vaccine's efficacy and modulate the immune response. Previous report from our group demonstrated that immunization with DCs transfected with LAMP/gag (Lg-DCs) induced activation of IFN-g-producing T cells against a diverse range of T-cell epitopes in BALB/c and in HLA-DR4 transgenic mice (37), providing proof of concept that the strategy would be efficient in humans. That study motivated further investigations about the immune response elicited by Lg-DCs, including evaluation of systemic and mucosal response mediated by B and T cells and analysis of long-term immunity, in comparison to DCs transfected with gag N or pulsed with inactivated virus.…”