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182 Enhancement of Immune Responses with a DNA Vaccine Construct That Targets APCs
W. Zhang*, Z. Chen, Y. Huang, and D. D. Ho
Aaron Diamond AIDS Res. Ctr., The Rockefeller Univ., New York, NY, USA
Background: DNA vaccination is effective in inducing both humoral and cellular responses in animal models. However, in a limited number of human studies, the immunogenicity achieved by env/rev and gag-pol DNA vaccines for HIV-1 has been weak. Our strategy for enhancing the immunogenicity of DNA vaccination for HIV-1 is 2-fold: to improve the expression of HIV-1 antigens and to target the expressed antigens to APCs utilizing the interaction of CTLA4 and B7 molecules on APCs.
Methods: We used SIV gag as an antigen. To improve the expression level, we "codon optimized" gag. We used the extracellular domain of rhesus CTLA4 as a targeting moiety in the context of a CTLA4-gag fusion gene construct. To further delineate the mechanisms of CTL4-mediated immune modulation, we also made mutations in the CTLA4 binding domain. We expressed CTLA4-gag in 293T cells and studied the expression of p27 and p17 antigen by ELISA and Western blots. We further studied the binding of the fusion proteins to B7.1- and B7.2- expressing CHO cells, monocytes, as well as immature and mature dendritic cells. We immunized mice and assessed humoral and CTL responses.
Results: Our results show that the CTLA4-gag fusion protein binds to B7-expressing cells specifically. The CTLA4 binding domain mutant does not bind to B7-expressing cells. Interestingly, our results also show that the CTLA4 fusion construct enhances the expression level of gag by 50- to 100-fold, a level comparable to that achieved by codon optimization of gag. Our initial results showed that CTLA4 enhanced humoral response to SIV Gag.
Conclusions: CTLA4-gag fusion DNA vaccine enhances the humoral response. This enhancement is CTLA4 binding domain dependent.
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