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186 Immunological Properties of HIV-1 env Gene-Based DNA Vaccines
B. V. Murashev*, I. V. Murasheva, T. Y. Kreslavskiy, A. E. Romanovitch, N. A. Klimov, and A. P. Kozlov
Biomedical Ctr., St. Petersburg, Russia
Objectives: The development of plasmids based on the LAI HIV-1 env gene and differing in the number of encoded cytotoxic epitopes. Investigation of the immune responses evoked by the plasmids as functions of time, vaccine dose, and the number of CTL epitopes expressed.
Methods: Standard methods of DNA cloning were employed. The pNL4-3(NY5/LAI) plasmid and a synthetic DNA sequence containing elements that initiate and terminate translation to result in the expression of particular protein epitopes were used as HIV-1 gene sources. The following methods were used for quality control of plasmids: restriction analysis, agarose gel electrophoresis, Sanger sequencing, Southern hybridization, spectrophotometric scanning, micro-BCA, and the standard LAL-test. The vaccine preparations obtained and tested conform to FDA specifications. BALB/c mice were intramuscularly administered with test DNA vaccines dissolved in saline. Boosting with syngenic fibroblasts constitutively expressing the HIV-1 env gene was performed on day 50 after immunization. The cytotoxic response was assessed with LDH colorimetric cytotoxic test. The results were treated statistically using the two-sided Student’s t-test.
Results: There were constructed plasmids containing the following fragments of the env gene: the complete env gene (pBMC160); part of the env gene coding gp120 (pBMC120); the latter construct with deleted V3 loop (pBMC120?V3); and DNA sequence coding cytotoxic epitope RGPGRAFVTI located within the V3 loop (pBMCV3). According to the Los Alamos HIV-1 database, 3 cytotoxic epitopes are contained in the pBMC160 plasmid, 2 in pBMC120, 1 in pBMC120?V3, and 1 in pBMCV3. All DNA vaccines evoked primary cytotoxic and humoral responses peaking on days 7(10 and 14(21, respectively. Specific cell lysis depended on DNA dose and correlated with the number of CTL epitopes expressed by a particular DNA vaccine. The secondary immune response with all DNA vaccines was higher than the primary 1, cytotoxicity being independent of the number of specific CTL epitopes in the priming DNA vaccine. The results were statistically significant at p < 0.0015.
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