Evaluation of Novel Human Immunodeficiency Virus Type 1 Gag DNA Vaccines for Protein Expression in Mammalian Cells and Induction of Immune Responses

There is increasing evidence that CD8+ cytotoxic T lymphocytes (CTL) may play an important role in controlling human immunodeficiency virus type 1 (HIV-1) infection. Containment of primary HIV-1 infection in infected individuals correlates with the emergence of virus-specific CTL responses (3, 12, 22). In chronically infected individuals, a high-frequency CTL response against HIV-1 is also correlated with low viral load and slow disease progression (19, 20). An HIV-1-specific CTL response has also been demonstrated in certain highly exposed seronegative individuals (2, 13, 28). Broad, cross-clade CTL responses recognizing conserved epitopes in HIV-1 Gag have been detected in HIV-1-infected individuals (7, 18). It is therefore reasonable to hypothesize that induction of an effective CTL response against conserved internal virion proteins of HIV-1 such as Gag is essential for the development of a safe and effective HIV-1 vaccine. In order to generate an efficient major histocompatibility complex (MHC) class I-restricted cellular immune response to a vaccine, viral proteins must be synthesized endogenously. Efficient production of CTL responses requires endogenous antigen synthesis, usually achieved by using a live, attenuated virus or recombinant virus vectors. Concerns about using a live, attenuated virus vaccine for HIV-1 include potential pathogenic replication and disease development over a longer period of time as well as potential adverse effects of integrated viral DNA. Using recombinant virus-based vectors, it is difficult to achieve repeated boosting because of the strong immune response generated against the viral proteins of the virus vector. Certain virus vectors, such as vaccinia virus, may also inhibit class I MHC-restricted CTL responses (32). Recently, a new approach (DNA vaccination) has been used to express antigens in vivo for the generation of both humoral and cellular immune responses (6). Several groups have used the DNA vaccination approach against HIV-1 (10, 17, 21, 34). Unfortunately, expression of HIV-1 Gag, Pol, and Env proteins by DNA vectors has been hampered by the presence of multiple inhibitory sequences (INS) in the structural genes encoding Gag, Pol, and Env proteins of HIV-1. This makes expression of the structural HIV-1 proteins dependent on the viral regulatory protein Rev, which is responsible for the nuclear export and efficient expression of unspliced HIV-1 mRNAs (5, 8, 23, 24). Rev binds specifically to an RNA site within HIV-1 mRNA named RRE. In the absence of functional Rev/RRE, mRNAs containing INS are either retained in the nucleus or degraded rapidly; therefore, little protein can be expressed from these mRNAs. Furthermore, even with Rev and RRE, expression of HIV-1 Gag, Pol, or Env is very low in certain murine cell lines (10, 33), limiting our ability to study the DNA vaccine-induced immune response against Gag or Pol using a mouse model. It has been reported that type D retroviruses contain a cis-acting posttranscriptional control element (CTE) that can replace the Rev and RRE regulation of HIV-1 (4, 36). When a CTE is inserted into an HIV-1 Rev−/RRE− clone, replication of the CTE-containing clone can approach that of the wild-type HIV-1 clone in cell lines (4, 36). The CTEs of different type D retroviruses are approximately 200 nucleotides in length and form extensive RNA stem-loop structures. They contain binding sites for cellular factors that contain nuclear export signals (9). CTEs function in a wide spectrum of cell types and species (31) and have been identified in both type D simian retroviruses (4, 36) and murine intracisternal-A particles (31). A current hypothesis about the function of INS is that they are binding sites for cellular factors that contribute to mRNA instability (1). Recently, it has been demonstrated that substitutions of AT-rich regions in HIV-1 gag, mostly in third-site positions, without changing the amino acid sequence of the produced Gag protein can result in efficient expression of Gag in a Rev- and RRE-independent fashion (29, 30). In the present study, we have constructed a series of HIV-1 Gag DNA expression vectors and compared the influence of the constitutive transport element (CTE) or of silent-site mutations within HIV-1 gag on Gag expression in a variety of cells of human, simian, and mouse origin. We found that silent-site mutations in HIV-1 gag allowed efficient Gag expression in a species-independent fashion and induced both humoral and CTL responses after naked DNA vaccination in mice. On the other hand, CTE only increased Gag expression moderately in monkey-derived COS cells and negligibly in HeLa (human) or several mouse cell lines. A DNA vaccine containing wild-type gag coding sequence plus CTE did not induce any detectable humoral or CTL response in mice. In contrast, mutated gag not containing INS induced detectable humoral and CTL responses. We have also achieved stable expression of HIV-1 Gag protein in mouse p815 cells and shown that these cells can be used as CTL target cells. Reagents described in this study should facilitate the development and evaluation of HIV-1 DNA vaccine strategies against conserved viral Gag protein in animal models.