Session 53Poster Presentations DNA Vaccines Session Day and Time: Thursday 1:30 - 3:30 pm Room: Hall D
451 Design, Purification, and Immunological Testing of DNA Vaccines against HIV-1 B.V. Murashev*, A. E. Masharsky, I. V. Murasheva, A. E. Romanovich, I. V. Dukhovlinov, M. S. Pavlova, Y. N. Dukhovlinova, Y. S. Dorofeyeva, Y. P. Galatchiants, N. A. Klimov, A. P. Kozlov BioMed Ctr, St Petersburg, Russia
Background: The development of the series of plasmid constructions based on the LAI HIV-1 and Russian region-specific HIV-1 subtype A.
Methods: Standard methods of DNA cloning were employed. The pNL4-3 (NY5/LAI) and plasmids containing Russian region-specific HIV-1 subtype A genes were used. Codon preference of the A subtype sequences was changed to match that used by highly expressed human genes. The cytotoxic activity response was assessed with a colorimetric kit Cytotoxic 96 (Promega Inc., USA). In vitro expression of HIV-1 antigens provided by DNA vaccine plasmids in transformed mouse 3T3 cells was investigated by Northern hybridization and Western-blot analysis. The results were analyzed statistically using the 2-sided Student's t-test.
Results: Plasmids expressing env, gag and nef genes of HIV A and B subtypes were constructed. In vitro expression of HIV-1 antigens by DNA vaccine plasmids in transformed cell cultures was demonstrated. Chromatographic purification was used to obtain large volumes of plasmid DNAs from bacterial cells for in vivo immunization experiments. The levels, dose, and time dependencies of cellular and humoral immune responses in mice immunized by DNA-vaccines were evaluated. DNA vaccines tested evoked primary cytotoxic and humoral responses peaking on days 7-10 and 14-21, respectively. The secondary immune response with DNA vaccines was greater than the primary one. It was shown that boosting with syngenic fibroblasts constitutively expressing the HIV-1 env gene on day 50 after immunization provides high level of secondary immune response.
Conclusions: Candidate DNA vaccines express HIV antigens in transformed cell cultures and induce cytotoxic T-cell and humoral immune responses in immunized mice.
