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312 HIV-1 Gag DNA Vaccine Chimera with Expression and Adjuvant Properties of the Lysosomal-Associated Membrane Protein (LAMP) and Dendritic Cell Multi-Lectin Receptor (DC-MLR) in an AAV-ITR Plasmid Vector
Y. Lu*, P. R. Chikhlikar, I. C. Leao, J. T. August, and E. T. A. Marques, Jr.
The Johns Hopkins Sch. of Med., Baltimore, MD, USA
An HIV-1 Gag DNA vaccine chimera encoding a LAMP/Gag protein in an AAV-ITR plasmid has elicited a markedly enhanced immune response of vaccinated mice. We also report a new strategy to increase Gag immunogenicity by replacing the endosomal/lysosomal targeting signal of LAMP with the dendritic cell-specific multilectin endocytic receptor, DC-MLR. This receptor, an alternative pathway of DNA vaccine proteins to the MHC II compartment, is also involved in modulation of APC cytokine responses. RNA from mouse bone marrow cells was used as template for cloning the transmembrane domain and cytoplasmic tail of the DC-MLR, which were then added to the carboxyl end of the lumenal domain of the LAMP/Gag chimera. Human 293 cells transfected with the pITR/LAMP/gag/MLR DNA chimera expressed the Gag antigen in endosomal/lysosomal compartments. All mice immunized twice with 50 mg of LAMP/gag/MLR DNA seroconverted with a strong immunoglobulin response. The IgG1/IgG2a titers 7 days after the second DNA immunization (day 37) were 8,100/900. CD4+-mediated cellular responses were as follows: a 52-fold greater INFg and 3-fold greater IL-2 protein production over that elicited by native Gag DNA; and a 7-fold increase in IL-4 and 5-fold increase in IL-2 mRNA responses over the negative control, as assayed by real-time PCR. Strong cytotoxic T-cell responses followed a DNA prime and vaccinia/Gag boost immunization with 3.5% tetramer binding, 2.5% INFg staining, and 33% cytotoxicity at 50:1 E:T ratio, as measured ex vivo. These results indicate that an HIV-1 Gag DNA vaccine applying the DC-MLR endocytic receptor transmembrane and cytoplasmic domains as a molecular adjuvant elicits enhanced immunogenicity with possible differences in the character of the immune responses as compared to DNA vaccines comprising native Gag or the LAMP/Gag chimera, and lead to new avenues in the design of HIV-1 DNA vaccines.
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