Evaluation of novel human immunodeficiency virus type 1 Gag DNA vaccines for protein expression in mammalian cells and induction of immune responses

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) a re an important parameter of host defenses that limit viral replication aft er infection. Induction of effective CTL against conserved viral proteins s uch as Gag may be essential to the development of a safe and effective HIV type 1 (HIV-1) vaccine. DNA vaccination represents a novel strategy for ind ucing potent CD8(+) CTL responses in vivo. However, expression of HIV-1 str uctural proteins by DNA vectors has been hampered by a stringent requiremen t for coexpression with other viral components, such as Rev and RRE. Furthe rmore, even with Rev and RRE present, the level of expression of HIV-1 Gag, pol, or Env is very low in murine cells. These problems have limited our a bility to address the key issue of how to generate effective CTL responses to Gag in a mouse model. To overcome this problem, we compared several nove l DNA expression vectors for HIV-1 Gag protein expression in primate and mo use cells and for generating immune responses in mice after DNA vaccination . A DNA vector containing wild type HIV-1 gag coding sequences did not indu ce detectable Gag expression in any of the cells tested. Attempts to increa se nuclear export of Gag expression RNA by adding the constitutive transpor t element yielded only a moderate increase in Gag expression in monkey-deri ved COS cells and an even lower increase in Gag expression in HeLa cells or several mouse cell lines. In contrast, silent-site mutations in the HIV-1 gag coding sequences significantly increased Gag expression levels in all c ells tested. Furthermore, this construct induced both Gag-specific antibody and CTL responses in mice after DNA vaccination. Using this construct, we achieved stable expression of HIV-1 Gag in the mouse cell line p815, which can now be used as a target cell for measuring HIV-1 Gag-specific CTL respo nses in immunized mice. The DNA vectors described in this study should make it possible to systematically evaluate the approaches for maximizing the i nduction of CTL responses against HIV-1 Gag in mouse and other animal syste ms.