Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.

Human adenoviruses (Ads) are attracting considerable attention because oftheir potential utility for gene transfer and gene therapy, for development of live viral vectored vaccines, and for protein expression in mammalian cells. Engineering Ad vectors for these applications requires a variety of reagents in the form of Ads and bacterial plasmids containing viral DNA sequences and requires different strategies for construction of vectors for different purposes. To simplify Ad vector construction and develop a procedure with maximum flexibility, efficiency, and cloning capacity, we have developed a vector system based on use of AdS DNA sequences cloned in bacterial plasmids. Expanded deletions in early region 1 (3180 bp) and early region 3 (2690 or 3132 bp) can be combined in a single vector that should have a capacity for inserts of up to 8.3 kb, enough to accommodate the majority of cDNAs encoding proteins with regulatory elements. Genes can be inserted into either early region 1 or 3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. To illustrate the flexibility of the system, we have introduced a wild-type early region 3 into the vectors, and to illustrate the high capacity for inserts, we have isolated a vector with two genes totaling 7.8 kb. (17). After 8-10 days, plaques were isolated and expanded, and viral DNA was analyzed by restriction enzyme digestion as described (12, 16). [35S]Methionine labeling, immunoprecipitation, and SDS/PAGE were carried out as described (16,18). Densitometry was performed using the LKB Ultroscan XL enhanced laser densitometer. Construction of adenovirus (Ad

No abstract

Cellular immune responses, particularly those associated with CD3؉ CD8 ؉ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4؉ and CD8 ؉ T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Adenoviral (Ad)-mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and͞or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinasebased system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and ob͞ob (leptindeficient) mice. In contrast to evidence of liver toxicity, inf lammation, and cellular infiltration observed with Adleptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety͞toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications. Adenoviral (Ad) vectors are currently among the most efficient gene transfer vehicles for both in vitro and in vivo delivery, but the utilization of current Ad vectors for many gene therapy applications is limited by the transient nature of transgene expression obtained by these vectors (1-7). Several factors have been shown to contribute to and modulate the duration of Ad-mediated gene expression and the immunogenicity of these vectors, including ''leaky'' viral protein expression and the transgene that is delivered (8-15). The development of Ad vectors that are deleted in all viral protein-coding sequences offers the prospect of a potentially safer, less immunogenic vector with an insert capacity of up to 37 kb (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). This vector is supplied in trans with the structural proteins required for packaging and rescue and is thus helper-dependent (HD) (24).Leptin has been recently identified as a potent modulator of weight and food intake. Daily delivery of recombinant leptin protein was shown to induce weight reduction, suppress appetite, and decrease blood insulin and glucose levels in ob͞ob (leptindeficient) mice (27)(28)(29). It has been shown that delivery of the leptin cDNA by first-generation Ad vectors (Ad-leptin) may substitute for daily recombinant leptin protein treatment, although the effects were transient in both lean and ob͞ob treated mice (7,30). In the present study, we delivered the leptin cDNA using the HD virus (HD-leptin), testing the hypothesis that elimination of the viral protein coding sequences would diminish the vector's cellular immunogenicity and toxicity, and hence support its longevity in vivo. Because both the viral proteins and the trans...