Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice.

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 1x10(5) 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-gamma 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.

Dendritic cell-lysosomal-associated membrane protein (LAMP) and LAMP-1-HIV-1 gag chimeras have distinct cellular trafficking pathways and prime T and B cell responses to a diverse repertoire of epitopes.

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

DNA vaccine encoding human immunodeficiency virus-1 Gag, targeted to the major histocompatibility complex II compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response.

Antigen presentation by major histocompatibility complex type II (MHC II) molecules and activation of CD4+ helper T cells are critical for the generation of immunological memory. We previously described a DNA vaccine encoding human immunodeficiency virus-1 p55Gag as a chimera with the lysosome-associated membrane protein (LAMP/gag). The LAMP/gag chimera protein traffics to the MHC II compartment of transfected cells and elicits enhanced immune responses as compared to a DNA vaccine encoding native gag not targeted to the MHC II compartment. We have now investigated the long-term responses of immunized mice and show that the LAMP/gag DNA vaccine promotes long-lasting B cell- and CD4+ and CD8+ T-cell memory responses induced by DNA encoding non-targeted Gag decay rapidly and elicit very low or undetectable levels of gag DNA is sufficient to generate T-cell memory. Following this initial priming immunization with LAMP/gag DNA, booster immunizations with native gag DNA or the LAMP/gag chimera are equally efficient in eliciting B- and T-cell secondary responses, results in accordance with observations that secondary expansion of CD8+ cells in the boost phase does not require additional CD4+ help. These findings underscore the significance of targeting DNA-encoded vaccine antigens to the MHC II processing compartments for induction of long-term immunological memory.

Dengue 2 PreM-E/LAMP chimera targeted to the MHC class II compartment elicits long-lasting neutralizing antibodies.

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.

Prognostic significance of biomarkers in squamous cell carcinoma of the tongue: multivariate analysis.

BACKGROUND AND OBJECTIVES: Expression of a panel of biomarkers, such as p53, Bcl-2, Cyclin D1, c-myc, p21ras, c-erb B2, cytokeratin-19 (CK-19), and factor VIII-related antigen (FVIII-RA), was studied together in anterior tongue tumors from the oral cavity and in posterior tongue tumors from the oropharynx of patients with early- and locally advanced-stage disease, to evaluate their prognostic value. METHODS: The expression of the above-mentioned biomarkers was studied by immunohistochemical localization. RESULTS: In this study, 18%, 26%, 62%, 75%, 73%, 50%, and 29% of the tumors exhibited p53, Bcl-2, Cyclin D1, c-myc, p21ras, c-erb B2, and CK-19 expression, respectively. Twenty percent of the tumors had a microvessel count of >0.0. The expression of these biomarkers was also correlated with clinicopathologic parameters. In early-stage patients with a tobacco habit, who showed borderline significance for relapse-free survival by Kaplan-Meier survival analysis, this turned out to be significant, with the general linear model univariate survival analysis. In the total group, disease stage emerged as the most significant prognostic factor, followed by c-myc, when Cox forward stepwise regression and general linear model multivariate survival analysis were performed. However, Cyclin D1, which was significant by Cox forward stepwise regression analysis, lost its significance by general linear model multivariate analysis. In patients with early-stage disease, MVC, which was a significant predictor of disease relapse by Cox forward stepwise regression analysis, lost its significance by general linear model analysis because of small number of patients. In patients with locally advanced tongue cancer, multivariate survival analysis of individual biomarkers by both Cox forward stepwise regression and general linear model analysis indicated c-myc expression to be strongly indicative of poor prognosis. However, multivariate analysis of individual markers along with a combination of markers showed that only by Cox forward stepwise regression analysis did the combined expression of markers c-myc, Cyclin D1, and p21ras emerge as a significant independent prognosticator. CONCLUSIONS: Overall stage emerged as the most significant prognostic indicator of disease outcome. Tobacco habit also affected relapse-free survival in patients with early-stage disease. However, immunostaining of c-myc in the tumors of locally advanced-stage tongue cancer patients might be a potential adjunct to clinical stage in the pathologic evaluation of tongue specimens.